Peptide Anchor for Folate-Targeted Liposomal Delivery
- Eugénia Nogueira
- Irene C. Mangialavori
- Ana Loureiro
- Nuno G. Azoia
- Marisa P. Sárria
- Patrícia Nogueira
- Jaime Freitas
- Johan Härmark
- Ulyana Shimanovich
- Alexandra Rollett
- Ghislaine Lacroix
- Gonçalo J. L. Bernardes
- Georg Guebitz
- Hans Hebert
- Alexandra Moreira
- Alexandre M. Carmo
- Juan Pablo F. C. Rossi
- Andreia C. Gomes
- Ana Preto
- Artur Cavaco-Paulo
View Author Information
† CBMA − Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Braga 4710-057, Portugal
‡ CEB − Centre of Biological Engineering, University of Minho, Braga 4710-057, Portugal
§ IQUIFIB − Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, 1113 Buenos Aires, Argentina
∥ Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
⊥ IBMC − Instituto de Biologia Molecular e Celular, 4150-180 Porto, Portugal
# Department of Biosciences and Nutrition, The Royal Institute of Technology, School of Technology and Health, Karolinska Institutet, S-14183 Huddinge, Sweden
∇ Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom
○ Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, 3430 Tulln, Austria
◆ INERIS - Institut National de l’Environnement Industriel et des Risques, 60550 Verneuil en Halatte, France
¶ ICBAS − Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4099-003 Porto, Portugal
*Phone: 00351253604409. Fax: 00351253604429. E-mail: [email protected].
Copyright © 2015 American Chemical Society
Abstract
Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.
Introduction
ARTICLE SECTIONS
Liposomes have gained extensive attention as carriers for a wide range of drugs due to properties such as sustained release, altered pharmacokinetics, increased drug stability, ability to overcome drug resistance, and target specific tissues.(1) Folate receptor (FR), one of the most studied molecular targets for directed therapy, is a glycoprotein with high affinity for folate (or folic acid, FA) (Kd ∼ 100 pM).(2, 3) Two main membrane variants of the FR exist, FRα and FRβ. FRα is overexpressed in about 40% of human carcinomas. FRβ, by contrast, is expressed in its functional form on activated macrophages and myelogenous leukemia blasts.(4) Both these FRs are absent in most normal tissues. FR-targeting liposomes, with FA covalently attached via a polyethylene glycol (PEG) linker to a phospholipid or cholesterol (CH) anchor and incorporated into the bilayer during liposome preparation, were previously developed.(5-8) Although these FA conjugates were shown to successfully target FR-overexpressing cancer cells, self-aggregation of FA at the liposome surface has often been observed when using such formulations, leading to reduced FR targeting efficiency.(6) Furthermore, there are concerns over the two negative charges carried by FA-PEG-phospholipid and the use of a carbamate linker in FA-PEG-CH, which has limited hydrolytic stability.(9)
Here, we report the design of a bifunctional peptide, derived from the neck domain of pulmonary surfactant-associated protein D (SP-D),(10) that simultaneously serves as an FA linker and as an anchor. Mammalian pulmonary surfactant proteins can, in nature, promote the self-assembly of phospholipids toward zero potential interface,(11) suggesting that fragments or models of these proteins are capable of recognizing and interacting with liposomal phospholipids. These findings prompted us to investigate the use of pulmonary surfactant proteins for the preparation of targeted liposomes. The α-helical neck region of SP-D shows an affinity for phospholipids and thus might promote the binding of SP-D to these structures,(12) further supporting their potential use as linker and anchor for FA conjugates. We designed several subsets of liposome formulations to show the funtionality of our final system containing the peptide SP-DS3. Their lipid membrane insertion ability was extensively characterized, and tests were performed to evaluate the potential of this formulation as a specific drug delivery system.
Experimental Section
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Materials
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and N-(carbonyl methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-MPEG) were obtained from Lipoid GmbH (Germany), and DSPE-PEG-FA was obtained from Avanti Polar Lipids (USA). CH, fluorescein isothiocyanate (FITC), carbon monoxide-releasing molecule-2 (CORM-2) and methotrexate (MTX) were obtained from Sigma (USA). Celecoxib was obtained from Cadila Pharmaceuticals, Ltd. (India).
Peptide Synthesis
The neck-domain peptides constructed from SP-D were synthesized by JPT Peptide Technologies GmbH (Berlin, Germany) and American Peptide (Sunnyvale, CA, USA). FA was covalently bound at the N-terminus of the peptides via glutamic acid and pteroic acid, using a standard Fmoc-based solid-phase synthesis protocol. Benzotriazole-1-yl-oxy-tris-pyrrolidino phosphoniumhexafluorophosphate was applied as the activating reagent to ensure efficient coupling. Fmoc protecting groups were removed after every coupling step under standard conditions (20% piperidine in DMF). Oregon Green 488 was covalently bound at the C-terminus. Peptides were purified to greater than 75% homogeneity, as determined by analytical high-performance liquid chromatography (Supporting Information Figure S1), and characterized by mass spectrometry (Figure S2).
Liposome Preparation
Liposomes composed of DOPE/CH/DSPE-MPEG were prepared using a thin film hydration method. Briefly, known amounts of DOPE, CH and DSPE-MPEG were dissolved in chloroform in a 50 mL round-bottom flask. In liposomes with DSPE-PEG-FA, this compound was also added in this step. For macrophage internalization studies, FITC was incorporated in the lipidic film, in the hydrocarbon region of the bilayer. The organic solvent was evaporated using a rotary evaporator followed by additional evaporation under reduced pressure by a high-vacuum system to remove remaining traces of chloroform. The resultant dried lipid film was dispersed in phosphate-buffered saline (PBS) buffer containing either peptides. The mixture was vortex-mixed at a temperature greater than the phase-transition temperature (room temperature) to yield multilamellar vesicles, which were then extruded (extruder supplied by Lipex Biomembranes Inc., Vancouver, Canada) through 200 nm pore size polycarbonate filters (Nucleopore) followed by several passages through 100 nm polycarbonate filters (Nucleopore) to form large unilamellar vesicles. The free peptide and drugs that were not incorporated into liposomes were removed from the samples after passage through a gel filtration chromatography column (GE Healthcare), with 5 kDa cutoff (PD-10 Desalting Columns containing 8.3 mL of Sephadex G-25 Medium).
Determination of the Zeta Potential and Size Distribution
The zeta-potential (ζ-potential) values and size distribution of liposomes were determined at pH 7.4 (PBS buffer) and 25.0 °C by electrophoretic laser Doppler anemometry and photon correlation spectroscopy (PCS), respectively, using a Malvern Zetasizer NS (Malvern Instruments). The lipid concentration was held constant at 400 μM. The viscosity and refractive index values were 0.890 cP and 1.330, respectively.
Folic Acid Quantification
The quantification of FA present at the liposomal surface was performed using a RIDASCREEN FAST Folic Acid kit (R-Biopharm, Germany) according to the manufacturer’s instructions. This kit is a competitive enzyme immunoassay and quantifies the folic acid at the liposomal surface.
Fluorescence Spectroscopy
Fluorescence experiments were performed using a Fluorolog spectrofluorometer (JY Horiba) with a 1 × 1 cm quartz cell thermostated at 25.0 °C. The band-pass of the excitation slit was set to 2.0 nm to obtain an optimal signal-to-noise ratio without photodegradation. Fluorescence was excited at 280 nm, and the emission was scanned from 310 to 450 nm at an increment of 1 nm and an integration time of 1 s.
Hydrophobic Photolabeling
The photoactivatable phosphatidylcholine analogue [125I]TID-PC/16 was prepared as described by Mangialavori et al. (2009).(13) A dried film of the photoactivatable reagent [125I]TID-PC/16 was suspended in 0.005% C12E10 and liposomes incorporating SP-DS3. The samples were incubated for 20 min at room temperature in the dark before being irradiated for 20 min with light from a filtered UV source (λ = 366 nm).
Radioactivity and Protein Determination
Electrophoresis was performed according to the Tris-Tricine SDS-PAGE method. Peptide was stained with Coomassie BlueR, the isolated bands were excised from the gel, and the incorporation of radioactivity was directly measured with a gamma counter. The amount of protein was quantified by eluting each stained band, including bovine serum albumin, in each gel for protein quantification. Specific incorporation was calculated as the ratio between the measured radioactivity and the amount of protein determined for each band.
Molecular Dynamics Simulations
The simulations were performed with the GROMACS 4.0.7(14) package using the Gromos ffG43a1 force field. The bond lengths were constrained using LINCS.(15) Nonbonded interactions were calculated using a twin-range method with short- and long-range cut-offs of 0.8 and 1.2 nm, respectively. Neighbor searching was updated every five steps. An integration time step of 2 fs was used. A reaction field correction for the electrostatic interactions was applied using a dielectric constant of 15. Pressure control was implemented using a Berendsen barostat(16) with a reference pressure of 1 bar, a relaxation time of 0.5 ps and an isothermal compressibility of 4.5 × 10–5 bar–1. Temperature control was set at 300 K using a Berendsen thermostat.(16) Each component of the system was included in separate heat baths with temperature coupling constants of 0.1 ps. Five replicate simulations were performed using different initial velocities determined from a Maxwell–Boltzmann distribution at 300 K.
Electron Microscopy Imaging
For scanning transmission electron microscopy (STEM) analysis, a liposomal suspension was dropped onto carbon film over copper grids (400 mesh, 3 mm diameter). The shape and morphology of the liposomes were observed using a NOVA Nano SEM 200 FEI system. For transmission electron microscopy (TEM) analysis, the liposome samples were diluted 1:20 in distilled water and applied to glow-discharged carbon-coated copper grids followed by negative staining with a solution of 1% (w/v) uranyl acetate. Imaging was performed using a JEOL JEM2100F transmission electron microscope operating at an acceleration voltage of 200 kV. Images were recorded with a 4k*4k CCD camera (Tietz Video and Image Processing Systems GmbH, Gauting, Germany) at a magnification of 100 000×.
Cell Culture Conditions
The human colonic epithelial cell line (Caco-2) (ATCC, HTB-37) was obtained from the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 4 mM l-glutamine, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate supplemented with 10% (v/v) of fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin solution, and 1% (v/v) nonessentials amino acids. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Caco-2 cells were routinely subcultured over 7 days, and the culture medium was replaced every 2 days. Caco-2 cells were plated at 106 cells/well in six-well dishes for 10 days. Cells were stimulated by incubation with a mixture of proinflammatory cytokines (Cytomix; 40 ng/mL each of interferon-γ, interleukin-1β and tumor necrosis factor (TNFα) for 16 h.
Cell Viability Assay
Cells were seeded at a density of 10 × 103 cells per 100 μL per well on 96-well TCPS plates (TPP, Switzerland) the day before the experiments and then exposed to different liposome and drug concentrations. At determined time of exposure, cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. A ready-for-use CellTiter 96 Aqueous One solution of MTS (Promega, Madison, WI) was used according to the protocol suggested by the manufacturer. The culture medium was refreshed, and 20 μL of CellTiter 96 Aqueous One solution was added to each well. After 4 h of incubation at 37 °C, the absorbance at 490 nm was measured using a microplate reader (Spectramax 340PC).
Cytometer Analysis
The determination of fluorescence intensity was assessed by flow cytometer analysis, using FACScalibur from BD Biosciences. Briefly, cells were incubated for 1 h with 100 μg/mL liposomes in complete medium at 37 °C. As a negative control, macrophages were incubated just with complete medium. After incubation, cells were washed twice with PBS (pH 7.4) and then resuspended in PBS for fluorescence-activated cell sorting (FACS) analysis. The internalization was determined as the geometrical mean fluorescence intensity of liposomes corrected for the background staining of cells incubated with no nanoparticles (negative control) and normalized by the internalization level of each formulation.
Confocal Laser Scanning Microscopy (CLSM)
Multilamellar vesicles of liposomes incorporating SP-DS3 were used as a model to perform CLSM experiments with a Leica TCS SPE confocal microscope (Leica Microsystems GmbH, Mannheim, Germany). Laser light wavelengths of 488 nm were used for the excitation of Oregon Green 488. The emitted light was detected in the range of 500–556 nm. Every optical field, laser intensity, photomultiplier gain and offset was individually adjusted to optimize the signal/noise ratio. Confocal stacks were acquired with an ACS APO 63.0× 1.30 oil-immersion objective lens while applying a Z-step of 6.01 μm. For liposomal internalization studies, Caco-2 cells were seeded at a density of 5 × 105 cells per 500 μL per well on 22 mm coverslips inserted into the wells of 24-well tissue culture polystyrene (TCPS) plates (TPP, Switzerland). After 10 days of adhesion, with the medium refreshed every 2 days, the medium was removed, and cells were washed twice with PBS. The cells were incubated with FR-targeted liposomes and nontargeted control liposomes, labeled with FITC at a phospholipid concentration of 100 μg/mL and diluted in Hank’s balanced salt solution (HBSS) medium. After 1.5 h of exposure, the medium was removed, and cells were washed three times with acid and basic PBS at 4 °C to remove receptor-bound free folate and not internalized liposomes. The cells were then fixed with 4% paraformaldehyde for 30 min. The cells were further washed three times with PBS. After drying, the coverslips were put on slides coated with 2 μL of Vectashield mounting medium (VECTOR) and sealed. Slides were examined using an inverted Zeiss confocal laser scanning microscope (CLSM, Olympus Fluoview FV1000). Volume rendering and 3D models were created with the software Imaris7.0 (Bitplane, Zurich, Switzerland).
RNA Isolation, cDNA Synthesis and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted using the SV Total RNA Isolation System (Promega) according to the manufacturer’s instructions and quantified with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Reverse transcription (RT) was performed using the iScript cDNA synthesis kit (BioRad) with a unique blended oligo(dT) and random hexamer primers, according to the manufacturer’s protocols. Quantification of gene expression was performed with a CFX96 Real Time PCR Detection System from BioRad using reagents and protocols from the TaqMan Gene Expression Assay Protocol (Applied Biosystems). Each sample was normalized against the expression of mitochondrially encoded ATP synthase (MT-ATP6) as a stable internal control. The assay IDs of the TaqMan Gene Expression Assays were Hs02596862_g1 (MT-ATP6), Hs00153133_m1 (COX-2), HS00174103_m1 (IL-8), and Hs01042796_m1 (MMP7).
Statistical Analysis
Statistical analyses were performed with GraphPad Prism software (version 5.0). Unless otherwise stated, differences were tested for statistical significance by a one-way ANOVA.
Results and Discussion
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Interaction of Peptide with Liposomes
We started by analyzing the interaction of three different peptide sequences (corresponding to amino acids 235–248 of SP-D; Figure 1A) with a liposomal formulation composed of DOPE/CH/DSPE-MPEG. At the N-terminus of the peptide, a hydrophilic bifunctional peptide spacer (DRDD) previously described in the literature, was added to improve the aqueous solubility of the final FA conjugate under physiological conditions.(17, 18) The quantification of FA at the liposomal surface (Figure 1B) demonstrated the utility using of this hydrophilic spacer as liposomes with a peptide linker lacking the spacer DRDD (SP-D3) did not show quantifiable FA at the surface in contrast to liposomes with peptide linkers SP-DS1 and SP-DS3. Furthermore, SP-DS2 presence led to a reduced FA concentration at the surface and thus was excluded in subsequent studies. Tryptophan fluorescence and a photoactivatable phosphatidylcholine analogue were used to discriminate between deep insertion of the peptide in the bilayer and poor adsorption at the membrane surface. A tryptophan residue was included at position 5 from the C-terminus of the peptide sequence. A blue shift in the wavelength of maximum fluorescence emission is known to occur when tryptophan is transferred from a polar medium to a less polar environment. The results demonstrated that both peptide linkers penetrate into the liposomes; however, a pronounced blue shift was observed in the liposomal formulation using SP-DS3 (330 nm vs 342 nm for SP-DS1), suggesting that this peptide penetrates deeper into the non polar hydrocarbon region of the bilayer than does SP-DS1 (Figure 1C). A deep insertion of SP-DS3 prevents their dissociation from the liposome after intravenous injection. Therefore, this peptide was selected for further the studies.
To confirm the deep insertion of SP-DS3 in the bilayer, we used the photoactivatable phosphatidylcholine analogue [125I]TID PC/16, which has been used to identify and characterize regions within membrane proteins that interact with lipids.(19) [125I]TID PC/16 positions itself in the phospholipid milieu and, upon photolysis, reacts with the peptide if present in the vicinity.(20, 21) During the reaction between the photoactivatable group and the peptide, the aryl group is transferred to the peptide and quantified via 125I, demonstrating that SP-DS3 was incorporated in the phospholipid milieu. To further assess the behavior of SP-DS3 in the lipid bilayer, a molecular modeling study was performed using GROMACS software. The resultant model indicated that FA is located at the membrane surface, whereas SP-DS3 is deeply inserted into the lipid bilayer with the tryptophan residue in contact with the hydrocarbon region (Figure 1D). This model is consistent with the experimental results described above. CLSM analysis revealed the homogeneous distribution of SP-DS3 (with Oregon Green 488 dye covalently bound at the C- terminus) in contact with hydrophilic regions both on the liposomal surface of multilamellar vesicles (MLV) and in the aqueous core (data not shown). Using STEM (data not shown) and TEM (Figure 1E) imaging, we showed that nanocapsules of liposomes integrating SP-DS3 have a spherical form with a uniform diameter of approximately 120 nm. This size distribution was confirmed by dynamic light scattering (DLS) measurements (Table S1). Both liposomal formulations exhibited a narrow size distribution (polydispersity index <0.1), indicating that all liposome preparations had similar physicochemical characteristics. After 16 weeks of storage in PBS (pH 7.4) at 4 °C, the particle sizes of either formulations remained unaltered, suggesting a high stability of these liposomes in terms of size distribution. Furthermore, ζ-potential values remained unchanged (close to zero) during this time. To evaluate the cytotoxicity of the liposomal formulations, an important parameter for biological applications, cell viability, was assessed by several assays using immortalized cell lines. Liposomal formulations did not induce toxicity over a wide range of concentrations (up to 750 μg/mL and 72 h; Figure S3). Furthermore, liposomal formulations did not induce hemolysis in rat blood (Figure S4).
Figure 1
Figure 1. SP-DS3 peptide, derived from pulmonary surfactant-associated protein D, is deeply inserted into the liposomal bilayer with FA at the liposomal surface. (A) Diagram of the analyzed SP-D fragments. The localization in the SP-D sequence is indicated, and numbers correspond to the amino acid positions in the SP-D sequence. (B) Description of the designed peptide linkers and quantification of the FA present at the liposomal surface. (C) Tryptophan fluorescence of the tested peptides. A strong blue shift in the fluorescence emission maximum was observed for SP-DS3. (D) Molecular dynamics simulation of the interaction of SP-DS3 with the PC/CH membrane. The tryptophan residue (yellow) in an apolar environment and the FA (orange) at the membrane surface. (E) Representative TEM images of liposomes. Scale bar (red) represents 200 nm in smaller magnification and 20 nm in higher magnification.
To evaluate the efficiency of FA targeting in liposomes integrating SP-DS3, we used a human colorectal carcinoma derived cell line (Caco-2) overexpressing FRα.(22, 23) Caco-2 cells have also been used not only as an in vitro cancer model but also as a model of chronic inflammation.(24) The images obtained by CLSM showed that FR-targeting liposomes labeled with FITC are well internalized by FR-expressing Caco-2 cells in comparison with nontargeted control liposomes (Figure S5). In our recent study,(25) we measured the uptake of these FR-targeting liposomes in the monocytic cell line THP-1 with and without overexpression of human FRβ. Liposomes with FA were highly internalized by THP-1 cells retrovirally transformed with FRβ in comparison with the wild-type THP-1 cells, which weakly express FRβ, that showed minimal uptake.(25) Furthermore, the in vivo uptake specificity of these new liposomes in a pathological context of rheumatoid arthritis, was also measured. In comparison to liposomes without FA, SP-DS3 liposomes strongly accumulated in the joints of arthritic mice.(25) Furthermore, in vivo evaluation of the clinical benefit of liposomal formulations encapsulating the anti-inflammatory drug MTX, indicated that SP-DS3 liposomes loaded with MTX improved prophylactic efficacy and thus the mice did not show any clinical signs of the disease.(25)
Evaluation of the FA-Tailed Peptide Efficiency
The efficiency of liposomal membrane disruption was compared between the optimized liposomal formulation containing SP-DS3 peptide (Figure 2A) or DSPE-PEG-FA (Figure 2B), a commonly used FA conjugate.(26) In the present study, encapsulated Hoechst 34580 was used to evaluate the efficiency of liposomal membrane disruption, through detection of an eventual increase in blue fluorescence derived from to the specific binding of this dye to DNA. The results clearly indicate that liposomes with FA conjugated to SP-DS3 peptide are more efficiently disrupted in Caco-2 cells, and consequently may quickly deliver the vectorized compounds (Figures 2C,D and S6). Approximately 2-fold more Hoechst-associated fluorescence was detected by flow cytometer analysis with targeted liposomes incorporating SP-DS3, relatively to those prepared with DSPE-MPEG (Figure 2E). We propose that the mechanism of liposomal membrane disruption is more efficient due to the smaller size of the hydrophilic bifunctional peptide spacer (DRDD), as compared to PEG (Figure 2F). This implies that the close contact between liposome and cell can lead to major liposomal membrane deformation in the process of receptor binding and internalization, and consequently faster and more effective disruption.
Figure 2
Figure 2. Liposomes with FA conjugated to SP-DS3 peptide are more efficiently disrupted compared to DSPE-PEG-FA. (A) Schematic representation of liposomes incorporating SP-DS3 peptide and (B) DSPE-PEG-FA. Representative pictures of the Caco-2 cells (FR+) after 1 h of incubation with fluorescent liposomal formulations (Hoechst 34580, blue staining) (C) with SP-DS3 and (D) DSPE-PEG-FA. (E) Efficient membrane disruption of liposomes incorporating SP-DS3 peptide, comparing with DSPE-PEG-FA, by Caco-2 cells. Graphs represent the relative mean fluorescence intensity (MFI) determined by flow cytometry analysis of Hoechst 34580 in Caco-2 cells with internalized liposomes after 1 h of incubation at 37 °C. Values are the mean + SD of 2 experiments. (F) Structures of DSPE-PEG-FA and SP-DS3.
Evaluation of Liposomes Incorporating FA-Tailed Peptide as a Drug Delivery System
We further tested the capacity of SP-DS3 liposomes to encapsulate and specifically deliver drugs into targeted cells. Liposomes loaded with one of two hydrophobic anti-inflammatory drugs, celecoxib and CORM-2, or a hydrophilic antiarthritic and antineoplastic FA analogue, MTX, were produced.(27, 28) Physicochemical characterization was performed, and the respective data, similar to that obtained for unloaded liposomes (Table S1), indicate that drugs are encapsulated inside the liposomes. The concentration of encapsulated drug was determined for each liposomal formulation, and the biological effect was compared with that of the same concentration of free drug. Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) and exerts its effects by inhibiting the synthesis of prostaglandins involved in pain, fever, and inflammation.(29, 30) Previous reports show that celecoxib strongly decreases protein expression of COX-2, which is directly proportional to a decrease of mRNA levels.(31-33) Since (q) PCR is a very sensitive technique, it was considered the best option to monitor this highly regulated pathway. We observed that celecoxib-loaded liposomes decreased the expression of COX-2 (Figure 3A), which is consistent with the fluorescence data showing that these liposomes can be internalized to some extent by Caco-2 cells. This reduction was much more pronounced in liposomes integrating SP-DS3, which showed similar levels to those obtained with celecoxib alone, indicating the effective release of the cargo from the targeted liposomes in vitro.
Similar results were obtained with SP-DS3 liposomes encapsulating CORM-2(34) assessed in Caco-2 cells stimulated with pro-inflammatory cytokines (Cytomix); mRNA levels of interleukin-8 (IL-8) and metalloproteinase-7 (MMP-7), which are crucial for cancer cell proliferation and invasion, were quantified.(24) A previous publication showed that CORM-2 simultaneously decreases both protein and mRNA levels of IL-8 and MMP-7.(24) Both mRNAs were strongly diminished compared with those from cells incubated with liposomes or CORM-2 alone (Figure S7). Of note was the effect of unloaded SP-DS3 liposomes in the expression of these inflammatory mediators as well as of COX-2. Indeed, the presence of FA, vitamin B9, is associated with an attenuation of inflammation and reduction in pro-inflammatory cytokine levels.(35) SP-DS3 liposomes were also tested for the efficient delivery of MTX. Liposomes encapsulating MTX significantly reduced the viability of Caco-2 cells (Figure 3B), whereas empty liposomes had no effect. This cytotoxic effect was enhanced in cells contacting SP-DS3 liposomes, suggesting preferential internalization of these targeted liposomes. Collectively, these data demonstrate that our delivery system is efficient in the encapsulation and specific delivery of both hydrophobic and hydrophilic drugs.
Figure 3
Figure 3. Liposomes integrating SP-DS3 are an efficient drug delivery system to FR-expressing cells. (A) The effects of 250 μg/mL of liposomal formulations with and without 65 μM celecoxib on COX-2 mRNA expression in Caco-2 cells (3 h of contact with liposomes +21 h of medium without liposomes). Values are the mean + SD of 2 experiments. (B) Caco-2 cell viability after 3, 6, and 48 h of contact with 10 μg/mL liposomal formulations encapsulating 1.27 μM methotrexate compared with untreated cells (negative control) and 30% DMSO-treated cells (positive control) as determined by the MTS assay at 48 h. Values are the mean + SD of 2 experiments. Significant differences were detected as shown by an asterisk (P < 0.05) and two asterisks (P < 0.005).
Conclusion
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In conclusion, we have successfully developed novel FR-targeted liposomes by incorporating a bifunctional SP-DS3 peptide consisting of the neck domain present in surfactant proteins, which serves as both a linker and an anchor of the FA to the liposomal membrane. This peptide, linked to a specific hydrophilic peptide spacer, was shown to insert deeply into the lipid bilayer without affecting the integrity of the liposomes. Furthermore, our delivery system was proved to be more efficient than classic systems where the FA is linked to liposomes by PEG. The combination of all complementary characteristics of these tailored liposomes, including their small size, lack of cytotoxicity and their specific targeting of FR-expressing cells, supports their use as specific therapeutic nanodelivery systems as demonstrated here by the biological effect of several drugs. The use of liposomes equipped with the novel bifunctional SP-DS3 peptide linker reported here for FA-mediated delivery opens new opportunities for the treatment of human diseases, including chronic inflammatory diseases and cancer.
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Data of SP-DS3 characterization by analytical high-performance liquid chromatography and mass spectrometry are provided as Supporting Information. Furthermore, physicochemical characterization, cell viability, hemolytic properties and specificity of liposomal formulations, as well the biological effect of liposomes encorporating CORM-2 are included. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00823.
(PDF)
The authors declare the following competing financial interest(s): A.C.P., A.P., E.N. and A.C.G. filed for a patent to use the SP-DS3 peptide in FA-targeted liposomes for specific drug delivery.
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Acknowledgment
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Eugénia Nogueira (SFRH/BD/81269/2011) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundação para a Ciência e a Tecnologia (FCT). Gonçalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioHealth - Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. This work was also supported by FCT I.P. through the strategic funding UID/BIA/04050/2013. We also thank Noëmy Gueriba for her technical assistance in various experiments.
References
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This article references 35 other publications.
- 1Sant, V. P.; Nagarsenker, M. S. AAPS PharmSciTech 2011, 12 (4) 1056– 63 DOI: 10.1208/s12249-011-9674-2[ Crossref], [ PubMed], [ CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFGqtL3E&md5=cbe97dd6433d1fd5dec7624e2052e51cSynthesis of Monomethoxypolyethyleneglycol-Cholesteryl Ester and Effect of its Incorporation in LiposomesSant, Vinayak P.; Nagarsenker, Mangal S.AAPS PharmSciTech (2011), 12 (4), 1056-1063CODEN: AAPHFZ; ISSN:1530-9932. (Springer)The objective of the present study was to synthesize monomethoxypolyethyleneglycol-5000 cholesteryl ester [PEG-CH] as a cost-effective substitute for polyethyleneglycol-phosphatidylethanolamine and to evaluate the influence of its incorporation in liposomal bilayers for surface modification. PEG-CH was synthesized and characterized by IR (IR), proton NMR spectroscopy (1H NMR), and differential scanning calorimetry (DSC) studies. Influence of incorporation of PEG-CH in liposomes was evaluated on various parameters such as zeta potential, DSC, and encapsulation efficiency of a hydrophilic drug pentoxyfylline. Conventional and PEG-CH contg. pentoxyfylline liposomes were formulated and their stability was evaluated at 4°C for 3 mo. PEG-CH could be successfully synthesized with good yields and the structure was confirmed by IR, DSC, and 1H NMR. The incorporation of PEG-CH in liposomes resulted in redn. of the zeta potential and broadening of the DSC endotherm. Furthermore, incorporation of PEG-CH in liposomes decreased the encapsulation efficiency of pentoxifylline in liposomes when compared to conventional liposomes. Conventional and PEG-CH contg. pentoxyfylline liposomes did not show any signs of pentoxyfylline degrdn. when stored at 4°C for 3 mo.
- 2Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Acc. Chem. Res. 2008, 41 (1) 120– 9 DOI: 10.1021/ar7000815[ ACS Full Text
], [ CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXot1Gltbk%253D&md5=20620eb124d19634b10c27a48ed46849Discovery and Development of Folic-Acid-Based Receptor Targeting for Imaging and Therapy of Cancer and Inflammatory DiseasesLow, Philip S.; Henne, Walter A.; Doorneweerd, Derek D.Accounts of Chemical Research (2008), 41 (1), 120-129CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The field of folic-acid-targeted drug delivery has grown rapidly over the past decade, with several applications currently in human clin. trials. Owing to the small size of the vitamin, its high affinity for the folate receptor (Kd = 10-10 M), the limited distribution of its receptor in normal tissues (primarily in kidney proximal tubules), the overexpression of its receptor in pathol. cells (upregulated in cancer cells and activated macrophages/monocytes), and its ease of conjugation to imaging and therapeutic agents, the use of folate as a targeting ligand for selective delivery of attached mols. to tumor tissues and sites of inflammation has attracted considerable interest. This account reviews the discovery of folic acid as a targeting ligand and its current use in the selective targeting of drugs to malignant masses and sites of inflammation. - 3Hilgenbrink, A. R.; Low, P. S. J. Pharm. Sci. 2005, 94 (10) 2135– 2146 DOI: 10.1002/jps.20457[ Crossref], [ PubMed], [ CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVOrurrF&md5=1b1b128980fa656802b23f980f4cd4d3Folate receptor-mediated drug targeting: From therapeutics to diagnosticsHilgenbrink, Andrew R.; Low, Philip S.Journal of Pharmaceutical Sciences (2005), 94 (10), 2135-2146CODEN: JPMSAE; ISSN:0022-3549. (Wiley-Liss, Inc.)A review. Folate targeted drug delivery has emerged as an alternative therapy for the treatment and imaging of many cancers and inflammatory diseases. Due to its small mol. size and high binding affinity for cell surface folate receptors (FR), folate conjugates have the ability to delivery a variety of mol. complexes to pathol. cells without causing harm to normal tissues. Complexes that were successfully delivered to FR expressing cells, to date, include protein toxins, immune stimulants, chemotherapeutic agents, liposomes, nanoparticles, and imaging agents. This review will summarize the applications of folic acid as a targeting ligand and highlight the various methods being developed for delivery of therapeutic and imaging agents to FR-expressing cells.
- 4Low, P. S.; Kularatne, S. A. Curr. Opin. Chem. Biol. 2009, 13 (3) 256– 262 DOI: 10.1016/j.cbpa.2009.03.022[ Crossref], [ PubMed], [ CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXosVWltbY%253D&md5=c6b6b07b0df9d1355e99da7c471a2a4bFolate-targeted therapeutic and imaging agents for cancerLow, Philip Stewart; Kularatne, Sumith AnurasiriCurrent Opinion in Chemical Biology (2009), 13 (3), 256-262CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Cancer therapies that exploit targeting ligands to deliver attached cytotoxic drugs selectively to malignant cells are currently receiving significant attention. While antibody-targeted drugs have been the first to enter the clinic, recent studies demonstrate that the vitamin folic acid can also be used to deliver attached imaging and therapeutic agents selectively to malignant cells in both animal tumor models and human cancer patients. Thus, folate conjugates bind to folate receptors that are overexpressed on ∼40% of human cancers and mediate internalization of their attached drugs by receptor-mediated endocytosis. With the use of proper linkers, folate-targeted drugs can be released inside their target cells where they can perform their desired cytotoxic functions. Based on this strategy, six folate-targeted drugs are currently in human clin. trials.
- 5Gabizon, A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S. Adv. Drug Delivery Rev. 2004, 56 (8) 1177– 1192 DOI: 10.1016/j.addr.2004.01.011[ Crossref], [ PubMed], [ CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjtF2rtLs%253D&md5=b6948331c4bc8ab98a988af1434b5016Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conjugatesGabizon, Alberto; Shmeeda, Hilary; Horowitz, Aviva T.; Zalipsky, SamuelAdvanced Drug Delivery Reviews (2004), 56 (8), 1177-1192CODEN: ADDREP; ISSN:0169-409X. (Elsevier Science B.V.)A review. Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to the outer end of a small fraction of phospholipid-anchored PEG mols. appears to be the most appropriate way to combine long-circulating properties crit. for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a no. of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model, that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and appear to be formulation- and tumor model-dependent. Further studies are required to det. whether folate targeting can confer a clear advantage in efficacy and/or toxicity to liposomal drugs.
- 6Liu, Y.; Xu, S.; Teng, L.; Yung, B.; Zhu, J.; Ding, H.; Lee, R. J. Anticancer Res. 2011, 31 (5) 1521– 1525[ PubMed], [ CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFSrsrk%253D&md5=34c09989ca94b9506eda063047d43266Synthesis and evaluation of a novel lipophilic folate receptor targeting ligandLiu, Ying; Xu, Songlin; Teng, Lesheng; Yung, Bryant; Zhu, Jing; Ding, Hong; Lee, Robert J.Anticancer Research (2011), 31 (5), 1521-1526CODEN: ANTRD4; ISSN:0250-7005. (International Institute of Anticancer Research)Background: Folate receptor (FR)-targeted liposomes have been investigated as delivery vehicles for anticancer drugs. A novel lipophilic FR ligand, folate-glutathione-polyethyleneglycol-distearoyl phosphatidylethanolamine (F-GSH-PEG-DSPE), was synthesized, incorporated into liposomes and evaluated for FR targeting efficiency. These liposomes were then evaluated as carriers of the chemotherapy agent vincristine (VIN). Materials and Methods: F-GSH-PEG-DSPE was synthesized and FR-targeted liposomes loaded with either calcein (F-L-Calcein) or VIN (F-L-VIN) were prepd. by thin film hydration followed by polycarbonate membrane extrusion and, in the case of VIN, by remote loading. To assess liposome stability, the uptake of F-L-VIN in KB (FR+) cancer cells was measured after storage under 4°C for 3 mo. Comparative pharmacokinetic studies were carried out with F-L-VIN and L-VIN (non-targeted control liposomes). Results: F-L-Calcein showed significantly higher cellular uptake in KB cells compared to non-targeted liposomes. In addn., F-L-VIN showed enhanced cytotoxicity in KB cells in vitro compared to control liposomes. Pharmacokinetic parameters indicated that both F-L-VIN and control liposomes had higher area under the curve (AUC), mean residence time (MRT), elimination half life (t1/2-β) and lower total body clearance (CL) than those of free VIN, while there were no significant differences between these liposomal formulations. Conclusion: F-GSH-PEG-DSPE is effective as a novel ligand for the synthesis of FR-targeted liposomes.
- 7Xiong, S.; Yu, B.; Wu, J.; Li, H.; Lee, R. J. Biomed. Pharmacother. 2011, 65 (1) 2– 8 DOI: 10.1016/j.biopha.2010.10.003[ Crossref], [ PubMed], [ CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXit1ajtr8%253D&md5=3d768f9ede0acb97cd2c670664888c69Preparation, therapeutic efficacy and intratumoral localization of targeted daunorubicin liposomes conjugating folate-PEG-CHEMSXiong, Subin; Yu, Bo; Wu, Jun; Li, Hong; Lee, Robert J.Biomedicine & Pharmacotherapy (2011), 65 (1), 2-8CODEN: BIPHEX; ISSN:0753-3322. (Elsevier Masson SAS)Folate polyethylene glycol-cholesterol hemisuccinate (folate-PEG-CHEMS) is a novel folate ligand firstly synthesized by our group and demonstrated good stability and potential targeting results on KB cells in vitro. Endocytosis mechanisms of liposomes via folate receptor on L1210JF cells and assessed targeted therapeutic efficacy of folate-PEG-CHEMS anchored liposomes loading daunorubicin (F-L-DNR) were studied in vivo. Folate-PEG-CHEMS was synthesized by a modified method. The liposome properties, cell cytotoxicity, intracellular and intratumoral localization, and therapeutic efficacy on a murine tumor model bearing L1210JF cells were evaluated. High encapsulation efficiency (95.1% ± 1.5%) and appropriate particle size (76.0 ± 35.5 nm) and zeta potential (-12.83 ± 1.36 mV) were achieved for F-L-DNR. IC50 of F-L-DNR on L1210JF cells was 2-3-folds lower than that of non-targeted liposomal daunorubicin (L-DNR). Anticancer efficacy on L1210JF tumor model indicated that mice survival time of F-L-DNR group at doses of 5 mg/kg and 10 mg/kg was significantly longer than that of L-DNR or free DNR. Confocal fluorescence photographs of F-L-DNR indicated enhanced endocytosis of liposomes via folate receptor on L1210JF cells, prolonged retaining time in tumors and improved drug release in the tumor site at 24 h post i.v. injection of F-L-DNR. Thus, folate-PEG-CHEMS is an effective ligand for folate-targeted daunorubicin liposomes to achieve increased drug release in tumor and therapeutic efficacy.
- 8Seo, H. J.; Kim, J.-C. Bioconjugate Chem. 2014, 25 (3) 533– 542 DOI: 10.1021/bc400521r[ ACS Full Text], [ CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisFKhsLk%253D&md5=c68f98ef9e5db03c8c9968f9850c82187-Acetoxycoumarin Dimer-Incorporated and Folate-Decorated Liposomes: Photoresponsive Release and in Vitro Targeting and EfficacySeo, Hee Jin; Kim, Jin-ChulBioconjugate Chemistry (2014), 25 (3), 533-542CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Photoresponsive and cancer cell (KB cell)-targetable liposome was developed by incorporating 7-acetoxycoumarin dimer (ACD) in the liposomal membrane and modifying the surface of liposome with folate. The liposomes were prepd. from the dry mixed thin film of egg phosphatidylcholine (EPC), ACD, and folate conjugate (DSPE-PEG2000-folate) by a film hydration method, where the molar ratios of EPC/ACD/DSPE-PEG2000-folate were 10/0/0, 9/1/0, 9/1/0.05, and 9/0/0.05. The liposomal membranes were multilamellar and the diam. was on the order of hundreds of nanometers. The release degrees in 60 min of 5(6)-carboxyfluorescein (CF) from EPC/ACD/DSPE-PEG2000-folate liposome were less than 4% without the irradn. of UV light (λ = 254 nm, 6 W), but more than 20% under the irradn. of UV light (λ = 254 nm, 6W), possibly due to the phototriggered de-dimerization of ACD. Under confocal laser scanning microscopy, KB cells treated with EPC/ACD/DSPE-PEG2000-folate liposomes exhibited CF fluorescence of liposomes at the positions where 4',6-diamidino-2-phenylindole (DAPI) fluorescence of the cell nucleus was shown, indicating the liposomes targeted the cancer cells. In flow cytometric anal., the cancer cells treated with EPC/ACD/DSPE-PEG2000-folate liposomes exhibited much higher fluorescence than the untreated cells did, indicating that there was a specific interaction between the liposome and the cancer cell. With the irradn. of UV light, EPC/ACD/DSPE-PEG2000-folate liposomes markedly promoted the in vitro anti-cancer efficacy of DOX without causing acute in vitro toxicity.
- 9Xiang, G.; Wu, J.; Lu, Y.; Liu, Z.; Lee, R. J. Int. J. Pharm. 2008, 356 (1–2) 29– 36 DOI: 10.1016/j.ijpharm.2007.12.030[ Crossref], [ PubMed], [ CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlsFCks74%253D&md5=839eb82aa5c367f4b8cbbd868fef857cSynthesis and evaluation of a novel ligand for folate-mediated targeting liposomesXiang, Guangya; Wu, Jun; Lu, Yanhui; Liu, Zhilan; Lee, Robert J.International Journal of Pharmaceutics (2008), 356 (1-2), 29-36CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)Folate receptors (FRs) have been identified as cellular surface markers for cancer and leukemia. Liposomes contg. lipophilic derivs. of folate have been shown to effectively target FR-expressing cells. Here, the authors report the synthesis of a novel lipophilic folate deriv., folate-polyethylene glycol-cholesterol hemisuccinate (F-PEG-CHEMS), and its evaluation as a targeting ligand for liposomal doxorubicin (L-DOX) in FR-expressing cells. Liposomes contg. F-PEG-CHEMS, with a mean diam. of 120±20 nm, were synthesized by polycarbonate membrane extrusion and were shown to have excellent colloidal stability. The liposomes were taken up selectively by KB cells, which overexpress FR-α. Compared to folate-PEG-cholesterol (F-PEG-Chol), which contains a carbamate linkage, F-PEG-CHEMS better retained its FR-targeting activity during prolonged storage. In addn., F-PEG-CHEMS contg. liposomes loaded with DOX (F-L-DOX) showed greater cytotoxicity (IC50 = 10.0 μM) than nontargeted control L-DOX (IC50 = 57.5 μM) in KB cells. In ICR mice, both targeted and nontargeted liposomes exhibited long circulation properties, although F-L-DOX (t 1/2 = 12.34 h) showed more rapid plasma clearance than L-DOX (t 1/2 = 17.10 h). These results suggest that F-PEG-CHEMS is effective as a novel ligand for the synthesis of FR-targeted liposomes.
- 10Zhang, P.; McAlinden, A.; Li, S.; Schumacher, T.; Wang, H.; Hu, S.; Sandell, L.; Crouch, E. J. Biol. Chem. 2001, 276 (23) 19862– 19870 DOI: 10.1074/jbc.M100597200[ Crossref], [ PubMed], [ CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXktlKgurg%253D&md5=853a820459759921c1478f5310c801fcThe amino-terminal heptad repeats of the coiled-coil neck domain of pulmonary surfactant protein D are necessary for the assembly of trimeric subunits and dodecamersZhang, Pengnian; McAlinden, Audrey; Li, Shi; Schumacher, Troy; Wang, Hongling; Hu, Shasa; Sandell, Linda; Crouch, ErikaJournal of Biological Chemistry (2001), 276 (23), 19862-19870CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Pulmonary surfactant protein D (SP-D), a lung host defense protein, is assembled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of trimers is required for many of its functions. A peptide contg. the α-helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular assembly of disulfide-cross-linked, trimeric subunits and dodecamers. For the present studies, we synthesized mutant cDNAs with deletions or site-directed substitutions in the neck domain of rat SP-D, and examd. the assembly of the newly synthesized proteins after transfection of CHO-K1 cells. The neck domain contains three "classical" heptad repeat motifs with leucine residues at the "d position," and a distinctive C-terminal repeat previously suggested to drive trimeric chain assocn. Deletion of the highly conserved core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. By contrast, deletion of the entire neck domain or deletion of one or two amino-terminal repeats resulted in defective mol. assembly. The secreted proteins eluted in the position of monomers by gel filtration under nondenaturing conditions. In addn., the neck + carbohydrate recognition domain of SP-D was necessary and sufficient for the trimerization of a heterologous collagen sequence located amino-terminal to the trimeric coiled-coil. These studies provide strong evidence that the amino-terminal heptad repeats of the neck domain are necessary for the intracellular, trimeric assocn. of SP-D monomers and for the assembly and secretion of functional dodecamers.
- 11Kingma, P. S.; Whitsett, J. A. Curr. Opin. Pharmacol. 2006, 6 (3) 277– 283 DOI: 10.1016/j.coph.2006.02.003[ Crossref], [ PubMed], [ CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XksFymsrw%253D&md5=ecaa619b80f055a54c342a05c94f9f74In defense of the lung: surfactant protein A and surfactant protein DKingma, Paul S.; Whitsett, Jeffrey A.Current Opinion in Pharmacology (2006), 6 (3), 277-283CODEN: COPUBK; ISSN:1471-4892. (Elsevier Ltd.)A review. The lung is repeatedly exposed to inhaled particles and pathogens that are cleared by the actions of a multi-component innate host defense system. The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate host defense by binding and clearing invading microbes from the lung. SP-A and SP-D also influence surfactant homeostasis, contributing to the phys. structures of lipids in the alveoli and to the regulation of surfactant function and metab. In addn. to binding and opsonizing infectious pathogens, SP-A and SP-D bind to the surfaces of host defense cells, promoting or inhibiting immune cell activity through multiple cellular pathways. As a consequence of their physiol. functions, SP-A- and SP-D-dependent pathways are targets for clin. therapies designed to limit the proliferation of microorganisms and to ameliorate inflammation following pulmonary infection.
- 12Kishore, U.; Greenhough, T. J.; Waters, P.; Shrive, A. K.; Ghai, R.; Kamran, M. F.; Bernal, A. L.; Reid, K. B. M.; Madan, T.; Chakraborty, T. Mol. Immunol. 2006, 43 (9) 1293– 1315 DOI: 10.1016/j.molimm.2005.08.004[ Crossref], [ PubMed], [ CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhslKjtb4%253D&md5=8eab33323417282245c2d6c1302ca73fSurfactant proteins SP-A and SP-D: Structure, function and receptorsKishore, Uday; Greenhough, Trevor J.; Waters, Patrick; Shrive, Annette K.; Ghai, Rohit; Kamran, Mohammed F.; Lopez Bernal, Andres; Reid, Kenneth B. M.; Madan, Taruna; Chakraborty, TrinadMolecular Immunology (2006), 43 (9), 1293-1315CODEN: MOIMD5; ISSN:0161-5890. (Elsevier B.V.)A review. Surfactant proteins, SP-A and SP-D, are collagen-contg. C-type (calcium dependent) lectins called collectins, which contribute significantly to surfactant homeostasis and pulmonary immunity. These highly versatile innate immune mols. are involved in a range of immune functions including viral neutralization, clearance of bacteria, fungi and apoptotic and necrotic cells, down regulation of allergic reaction and resoln. of inflammation. Their basic structures include a triple-helical collagen region and a C-terminal homotrimeric lectin or carbohydrate recognition domain (CRD). The trimeric CRDs can recognize carbohydrate or charge patterns on microbes, allergens and dying cells, while the collagen region can interact with receptor mols. present on a variety of immune cells in order to initiate clearance mechanisms. Studies involving gene knock-out mice, murine models of lung hypersensitivity and infection, and functional characterization of cell surface receptors have revealed the diverse roles of SP-A and SP-D in the control of lung inflammation. A recently proposed model based on studies with the calreticulin-CD91 complex as a receptor for SP-A and SP-D has suggested an anti-inflammatory role for SP-A and SP-D in naive lungs which would help minimize the potential damage that continual low level exposure to pathogens, allergens and apoptosis can cause. However, when the lungs are overwhelmed with exogenous insults, SP-A and SP-D can assume pro-inflammatory roles in order to complement pulmonary innate and adaptive immunity. This review is an update on the structural and functional aspects of SP-A and SP-D, with emphasis on their roles in controlling pulmonary infection, allergy and inflammation. We also try to put in perspective the controversial subject of the candidate receptor mols. for SP-A and SP-D.
- 13Mangialavori, I.; Giraldo, A. M. V.; Buslje, C. M.; Gomes, M. F.; Caride, A. J.; Rossi, J. P. F. C. J. Biol. Chem. 2009, 284 (8) 4823– 4828 DOI: 10.1074/jbc.M806912200[ Crossref], [ PubMed], [ CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhvVSgurw%253D&md5=cb34ead795ba0f9c2faafdede62ce194A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic ProbeMangialavori, Irene; Giraldo, Ana Maria Villamil; Buslje, Cristina Marino; Gomes, Mariela Ferreira; Caride, Ariel J.; Rossi, Juan Pablo F. C.Journal of Biological Chemistry (2009), 284 (8), 4823-4828CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine (PC) analog 1-palmitoyl-2-[9-[2'-[125I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([125I]TID-PC/16) under different conditions. We detd. the following. (1) Incorporation of [125I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca2+. This decrease in labeling matches qual. the decrease in transmembrane surface exposed to the solvent calcd. from crystallog. data for SERCA structures. (2) Labeling of PMCA incubated with Ca2+ and calmodulin decreases by approx. the same amt. However, incubation with Ca2+ alone increases labeling by more than 50%. Addn. of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [125I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E1 conformations as follows: one that is autoinhibited and is in contact with a higher amt. of lipids (Ca2+ + C28 for SERCA and Ca2+ alone for PMCA), and one in which the enzyme is fully active (Ca2+ for SERCA and Ca2+-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.
- 14Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem. Theory Comput. 2008, 4 (3) 435– 447 DOI: 10.1021/ct700301q[ ACS Full Text], [ CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVSqurc%253D&md5=d53c94901386260221792ea30f151c5fGROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular SimulationHess, Berk; Kutzner, Carsten; van der Spoel, David; Lindahl, ErikJournal of Chemical Theory and Computation (2008), 4 (3), 435-447CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)Mol. simulation is an extremely useful, but computationally very expensive tool for studies of chem. and biomol. systems. Here, we present a new implementation of our mol. simulation toolkit GROMACS which now both achieves extremely high performance on single processors from algorithmic optimizations and hand-coded routines and simultaneously scales very well on parallel machines. The code encompasses a minimal-communication domain decompn. algorithm, full dynamic load balancing, a state-of-the-art parallel constraint solver, and efficient virtual site algorithms that allow removal of hydrogen atom degrees of freedom to enable integration time steps up to 5 fs for atomistic simulations also in parallel. To improve the scaling properties of the common particle mesh Ewald electrostatics algorithms, we have in addn. used a Multiple-Program, Multiple-Data approach, with sep. node domains responsible for direct and reciprocal space interactions. Not only does this combination of algorithms enable extremely long simulations of large systems but also it provides that simulation performance on quite modest nos. of std. cluster nodes.
- 15Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. Comput. Chem. 1997, 18 (12) 1463– 1472 DOI: 10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.3.CO;2-L[ Crossref], [ CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXlvV2nu7g%253D&md5=890f8af0d2ca1f65aa93db5a3a0bacf2LINCS: a linear constraint solver for molecular simulationsHess, Berk; Bekker, Henk; Berendsen, Herman J. C.; Fraaije, Johannes G. E. M.Journal of Computational Chemistry (1997), 18 (12), 1463-1472CODEN: JCCHDD; ISSN:0192-8651. (Wiley)We present a new LINear Constraint Solver (LINCS) for mol. simulations with bond constraints using the enzyme lysozyme and a 32-residue peptide as test systems. The algorithm is inherently stable, as the constraints themselves are reset instead of derivs. of the constraints, thereby eliminating drift. Although the derivation of the algorithm is presented in terms of matrixes, no matrix matrix multiplications are needed and only the nonzero matrix elements have to be stored, making the method useful for very large mols. At the same accuracy, the LINCS algorithm is 3-4 times faster than the SHAKE algorithm. Parallelization of the algorithm is straightforward.
- 16Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81 (8) 3684 DOI: 10.1063/1.448118[ Crossref], [ CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmtlGksbY%253D&md5=5510dc00297d63b91ee3a7a4ae5aacb1Molecular dynamics with coupling to an external bathBerendsen, H. J. C.; Postma, J. P. M.; Van Gunsteren, W. F.; DiNola, A.; Haak, J. R.Journal of Chemical Physics (1984), 81 (8), 3684-90CODEN: JCPSA6; ISSN:0021-9606.In mol. dynamics (MD) simulations, the need often arises to maintain such parameters as temp. or pressure rather than energy and vol., or to impose gradients for studying transport properties in nonequil. MD. A method is described to realize coupling to an external bath with const. temp. or pressure with adjustable time consts. for the coupling. The method is easily extendable to other variables and to gradients, and can be applied also to polyat. mols. involving internal constraints. The influence of coupling time consts. on dynamical variables is evaluated. A leap-frog algorithm is presented for the general case involving constraints with coupling to both a const. temp. and a const. pressure bath.
- 17Vlahov, I. R.; Wang, Y.; Kleindl, P. J.; Leamon, C. P. Bioorg. Med. Chem. Lett. 2008, 18 (16) 4558– 4561 DOI: 10.1016/j.bmcl.2008.07.041[ Crossref], [ PubMed], [ CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1Slu7s%253D&md5=337a292c01e0d2c817f2ea2f69a35cb0Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part II: Folic acid conjugates of tubulysins and their hydrazidesVlahov, Iontcho R.; Wang, Yu; Kleindl, Paul J.; Leamon, Christopher P.Bioorganic & Medicinal Chemistry Letters (2008), 18 (16), 4558-4561CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)Efficient syntheses of folate conjugates of tubulysins and their hydrazides are described. These water sol. folate receptor (FR) targeted conjugates are derivs. of folic acid and the potent cytotoxic agents: tubulysin A, B, or their resp. hydrazides, connected in regioselective manner via a hydrophilic peptide spacer and a reducible disulfide linker.
- 18Leamon, C. P.; Reddy, J. A.; Vlahov, I. R.; Westrick, E.; Parker, N.; Nicoson, J. S.; Vetzel, M. Int. J. Cancer 2007, 121 (7) 1585– 1592 DOI: 10.1002/ijc.22853[ Crossref], [ PubMed], [ CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVSns7zI&md5=3a7b06fc117fca9d88730624d2ce9298Comparative preclinical activity of the folate-targeted Vinca alkaloid conjugates EC140 and EC145Leamon, Christopher P.; Reddy, Joseph A.; Vlahov, Iontcho R.; Westrick, Elaine; Parker, Nikki; Nicoson, Jeffrey S.; Vetzel, MarilynnInternational Journal of Cancer (2007), 121 (7), 1585-1592CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)EC140 is a water sol. folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), which is constructed with an endosome-cleavable acyl hydrazone bond. This agent has proven to be active and specific against well established, s.c. folate receptor (FR)-pos. tumors in multiple animal models. Recent structure-activity and optimization studies have yielded a disulfide bond-contg. counterpart to EC140, herein referred to as EC145. This new conjugate was found to retain high affinity for FR-pos. cells, and it produced specific, dose-responsive activity in vitro. Comparative in vivo efficacy tests confirmed that, like EC140, EC145 displays activity against both syngeneic and xenograft tumor models. However, EC145 was found to be more active and better tolerated than EC140; hence, more durable complete responses were consistently obsd. in EC145-treated tumor-bearing animals. Furthermore, EC145 was not found to be active against a FR-neg. tumor model. Addnl. preclin. studies are therefore warranted to better understand EC145's breadth of activity against FR-pos. tumors.
- 19Durrer, P.; Galli, C.; Hoenke, S.; Corti, C.; Glück, R.; Vorherr, T.; Brunner, J. J. Biol. Chem. 1996, 271 (23) 13417– 13421 DOI: 10.1074/jbc.271.23.13417[ Crossref], [ PubMed], [ CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjsVKqtL4%253D&md5=3bba58b02f7917851b562ec6df9321ceH+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil regionDurrer, Peter; Galli, Carmela; Hoenke, Stefan; Corti, Chantal; Glueck, Reinhard; Vorherr, Thomas; Brunner, JosefJournal of Biological Chemistry (1996), 271 (23), 13417-13421CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Fusion of influenza virus with target membranes is induced by acid and involves complex changes in the viral envelope protein hemagglutinin (HA). In a first, kinetically distinct step, the HA polypeptide chain 2 (HA2) is inserted into the target membrane bilayer. Using hydrophobic photolabeling with the phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, we identified the segment within HA2 that interacts with the membrane. The sole part of the HA2 ectodomain that was labeled with the membrane-restricted reagent is the NH2-terminal fusion peptide (residues 1-22). No labeling occurred within the long coiled coil region generated during the acid-induced conformational transition (Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994) Nature 371, 37-43). These data strongly suggest that the coiled coil region of HA2 does not insert into the lipid bilayer. This conclusion is at variance with a recent suggestion that the coiled coil of HA may splay apart and insert into the target membrane, providing a mechanism by which the viral and the target membrane may come in close apposition.
- 20Giraldo, A. M. V.; Castello, P. R.; Flecha, F. L. G.; Moeller, J. V.; Delfino, J. M.; Rossi, J. P. F. C. FEBS Lett. 2006, 580 (2) 607– 612 DOI: 10.1016/j.febslet.2005.12.078[ Crossref], [ PubMed], [ CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmslWkuw%253D%253D&md5=a44be6bd229ea2794eac137b5192c030Stoichiometry of lipid-protein interaction assessed by hydrophobic photolabelingGiraldo, Ana Maria Villamil; Castello, Pablo Raul; Flecha, F. Luis Gonzalez; Moeller, Jesper V.; Delfino, Jose Maria; Rossi, Juan Pablo F. C.FEBS Letters (2006), 580 (2), 607-612CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Here the authors undertook a comparative study of the compn. of the lipid annulus of three ATPases pertaining to the P-type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K-ATPase. The photoactivatable phosphatidylcholine analog [125I]TID-PC/16 was incorporated into mixts. of dimyristoylphosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C12E10. After photolysis, the extent of the labeling reaction was assessed to det. the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na,K-ATPase (α-subunit) and 5.6 mol PC/mol protein for the Na,K-ATPase (β-subunit).
- 21Mangialavori, I. C.; Caride, A. J.; Rossi, R. C.; Rossi, J. P. F. C.; Strehler, E. E. Curr. Chem. Biol. 2011, 5 (2) 118– 129 DOI: 10.2174/2212796811105020118[ Crossref], [ PubMed], [ CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXltF2mtLs%253D&md5=e53f36e77d9391e9aab43d070bf00695Diving into the lipid bilayer to investigate the transmembrane organization and conformational state transitions of P-type ion ATPasesMangialavori, Irene C.; Caride, Ariel J.; Rossi, Rolando C.; Rossi, Juan Pablo F. C.; Strehler, Emanuel E.Current Chemical Biology (2011), 5 (2), 118-129CODEN: CCBUB2; ISSN:1872-3136. (Bentham Science Publishers Ltd.)A review. Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca2+ pump (SERCA) revealing different conformations, and recently, crystal structures of H+-ATPase and (Na+,K+)-ATPase were reported as well. However, there are no at. resoln. structures for other P-type ATPases including the plasma membrane Ca2+-pump ATPase (PMCA), which is integral to cellular Ca2+ signaling. Crystn. of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystn. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an anal. of the lipid-protein interactions. Here, the authors review recent efforts using different hydrophobic photolabeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different exptl. conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in PMCA, (Na+,K+)-ATPase, and other P-type ATPases.
- 22Weitman, S. D.; Lark, R. H.; Coney, L. R.; Fort, D. W.; Frasca, V.; Zurawski, V. R., Jr.; Kamen, B. A. Cancer Res. 1992, 52 (12) 3396– 401[ PubMed], [ CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVGltLo%253D&md5=76668b6af4b0f1e89c3180affcb040b5Distribution of the folate receptor GP38 in normal and malignant cell lines and tissuesWeitman, Steven D.; Lark, Richard H.; Coney, Leslie R.; Fort, Daniel W.; Frasca, Verna; Zurawski, Vincent R., Jr.; Kamen, Barton A.Cancer Research (1992), 52 (12), 3396-401CODEN: CNREA8; ISSN:0008-5472.In some epithelial cells studied in vitro a membrane-bound folate receptor initiates the process for cell accumulation of 5-methyltetrahydrofolic acid. This receptor was found to be GP38, an overexpressed, glycosyl-phosphatidylinositol anchored glycoprotein, recognized by two monoclonal antibodies, designated MOv18 and MOv19. Using immunoblotting with MOv19, RIA with MOv18 and 19, Northern blot anal., and radioligand binding when possible, the present study describes the limited expression of the folate receptor in a large no. of normal tissues from four autopsies. The immunoblot technique detected as little as 40 pg (≈1 fmol) of receptor protein. Choroid plexus consistently had the largest amt. of folate receptor. Other tissues contg. substantial amts. of receptor included lung, thyroid, and kidney. The liver, intestines, muscle, cerebellum, cerebrum, and spinal cord were immunol. nonreactive. Folate receptor gene expression detd. by Northern blot anal. confirmed these observations. Several malignant cell lines express more receptor than normal epithelial cells or fibroblasts. Specifically, malignant cells bound ≥20 pmol [3H]folate/106 cells, whereas normal epithelial cells and fibroblasts bound ≤1 pmol radioligand/106 cells. Four of six brain tumors overexpressed the folate receptor, a cell surface protein which may be a useful immunol. or pharmacol. target for the development of selective cancer therapy.
- 23Roger, E.; Kalscheuer, S.; Kirtane, A.; Guru, B. R.; Grill, A. E.; Whittum-Hudson, J.; Panyam, J. Mol. Pharmaceutics 2012, 9 (7) 2103– 10 DOI: 10.1021/mp2005388[ ACS Full Text], [ CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotVWqu74%253D&md5=d43c838229f4ba609b7e3cbb622c0289Folic Acid Functionalized Nanoparticles for Enhanced Oral Drug DeliveryRoger, Emilie; Kalscheuer, Stephen; Kirtane, Ameya; Guru, Bharath Raja; Grill, Alex E.; Whittum-Hudson, Judith; Panyam, JayanthMolecular Pharmaceutics (2012), 9 (7), 2103-2110CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)The oral absorption of drugs that have poor bioavailability can be enhanced by encapsulation in polymeric nanoparticles. Transcellular transport of nanoparticle-encapsulated drug, possibly through transcytosis, is likely the major mechanism through which nanoparticles improve drug absorption. We hypothesized that the cellular uptake and transport of nanoparticles can be further increased by targeting the folate receptors expressed on the intestinal epithelial cells. The objective of this research was to study the effect of folic acid functionalization on transcellular transport of nanoparticle-encapsulated paclitaxel, a chemotherapeutic with poor oral bioavailability. Surface-functionalized poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles loaded with paclitaxel were prepd. by the interfacial activity assisted surface functionalization technique. Transport of paclitaxel-loaded nanoparticles was investigated using Caco-2 cell monolayers as an in vitro model. Caco-2 cells were found to express folate receptor and the drug efflux protein, p-glycoprotein, to high levels. Encapsulation of paclitaxel in PLGA nanoparticles resulted in a 5-fold increase in apparent permeability (Papp) across Caco-2 cells. Functionalization of nanoparticles with folic acid further increased the transport (8-fold higher transport compared to free paclitaxel). Confocal microscopic studies showed that folic acid functionalized nanoparticles were internalized by the cells and that nanoparticles did not have any gross effects on tight junction integrity. In conclusion, our studies indicate that folic acid functionalized nanoparticles have the potential to enhance the oral absorption of drugs with poor oral bioavailability.
- 24Megías, J.; Busserolles, J.; Alcaraz, M. J. Br. J. Pharmacol. 2007, 150 (8) 977– 986 DOI: 10.1038/sj.bjp.0707184[ Crossref], [ PubMed], [ CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkt1Chs7g%253D&md5=8cc37c766b6a9f77257e1150441e3a8dThe carbon monoxide-releasing molecule CORM-2 inhibits the inflammatory response induced by cytokines in Caco-2 cellsMegias, J.; Busserolles, J.; Alcaraz, M. J.British Journal of Pharmacology (2007), 150 (8), 977-986CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)Recent evidence indicates that carbon monoxide-releasing mols. (CO-RMs) exhibit potential anti-inflammatory properties. Here, the authors have investigated whether tricarbonyl dichloro ruthenium(II) dimer (CORM-2) can control the inflammatory response induced by cytokines in a human colonic epithelial cell line, Caco-2. Caco-2 cells were preincubated with CORM-2 for 30 min and then stimulated with interleukin (IL)-1β, tumor necrosis factor-α, and interferon-γ for different times. Gene expression was analyzed by real-time PCR. Protein expression was investigated by Western blot and ELISA. Transcription factor activation was detd. by the luciferase method. The authors show that CORM-2 decreased the mRNA expression of nitric oxide synthase-2 (NOS-2) and the prodn. of nitrite, in Caco-2 cells stimulated with cytokines. IL-8, IL-6, and metalloproteinase-7 (MMP-7) mRNA and protein were also reduced by CORM-2. Time-course and small interfering RNA studies suggest that inhibition of IL-6 plays a role in the regulation of MMP-7 expression by CORM-2. These effects of CORM-2 can be dependent on the modulation of nuclear factor-κB (NF-κB), activator protein-1, CCAT/enhancer binding protein and the phosphorylated forms of NF-κB inhibitory protein-α, c-Jun N-terminal protein kinase 1/2, p38 and extracellular signal-regulated kinase 1/2. Thus, CORM-2 can regulate a no. of genes relevant in intestinal inflammation and cancer progression. These findings provide new insights into the anti-inflammatory properties and potential applications of this class of compds.
- 25Nogueira, E.; Lager, F.; Le Roux, D.; Nogueira, P.; Freitas, J.; Charvet, C.; Renault, G.; Loureiro, A.; Almeida, C. R.; Ohradanova-Repic, A.; Machacek, C.; Bernardes, G. J. L.; Moreira, A.; Stockinger, H.; Burnet, M.; Carmo, A. M.; Gomes, A. C.; Preto, A.; Bismuth, G.; Cavaco-Paulo, A. J. Biomed. Nanotechnol. 2015, 11, 2243– 2252 DOI: 10.1166/jbn.2015.2170[ Crossref], [ PubMed], [ CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XkslOnuw%253D%253D&md5=1544f7334fcd8bc0d255473e60724fa6Enhancing methotrexate tolerance with folate tagged liposomes in arthritic miceNogueira, Eugenia; Lager, Franck; Le Roux, Delphine; Nogueira, Patricia; Freitas, Jaime; Charvet, Celine; Renault, Gilles; Loureiro, Ana; Almeida, Catarina R.; Ohradanova-Repic, Anna; Machacek, Christian; Bernardes, Goncalo J. L.; Moreira, Alexandra; Stockinger, Hannes; Burnet, Michael; Carmo, Alexandre M.; Gomes, Andreia C.; Preto, Ana; Bismuth, Georges; Cavaco-Paulo, ArturJournal of Biomedical Nanotechnology (2015), 11 (12), 2243-2252CODEN: JBNOAB; ISSN:1550-7033. (American Scientific Publishers)Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biol. therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor β present at the surface of activated macrophages, key effector cells in this pathol. The specificity of our liposomal formulation to target folate receptor β was investigated both in vitro as in vivo using a mouse model of arthritis (colalgen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor β. These liposomal formulations also significantly increase the clin. benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance.
- 26Gabizon, A.; Tzemach, D.; Gorin, J.; Mak, L.; Amitay, Y.; Shmeeda, H.; Zalipsky, S. Cancer Chemother. Pharmacol. 2010, 66 (1) 43– 52 DOI: 10.1007/s00280-009-1132-4[ Crossref], [ PubMed], [ CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkslaltLY%253D&md5=6f519aaa32603eebd767f460ef77d01aImproved therapeutic activity of folate-targeted liposomal doxorubicin in folate receptor-expressing tumor modelsGabizon, Alberto; Tzemach, Dina; Gorin, Jenny; Mak, Lidia; Amitay, Yasmine; Shmeeda, Hilary; Zalipsky, SamuelCancer Chemotherapy and Pharmacology (2010), 66 (1), 43-52CODEN: CCPHDZ; ISSN:0344-5704. (Springer)Purpose The folate receptor (FR) is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anti-cancer agents to FR-expressing tumors. Targeting liposomes to the FR has been proposed as a way to enhance the effects of liposome-based chemotherapy. Methods Folate-polyethylene glycol-distearoyl-phosphatidyl-ethanolamine conjugate was inserted into pegylated liposomal doxorubicin (PLD). The therapeutic activity of folate-targeted (FT-PLD) and non-targeted (PLD) pegylated liposomal doxorubicin was tested in two human tumor models (KB, KB-V) and in one mouse ascitic tumor model (FR-expressing J6456) by the i.v. systemic route in all models, and by the i.p. intracavitary route in the ascitic tumor model only. Results Consistent with previous studies, PLD was clearly superior to free doxorubicin in all tumor models. When targeted and non-targeted liposome formulations were compared, FT-PLD was more effective than PLD in the KB and KB-V xenograft models, and in the J6456 intra-cavitary therapy model. The therapeutic effect was dose-dependent in the KB model and schedule-dependent in the J6456 intra-cavitary therapy model. In some expts., toxic deaths aggravated by folate-depleted diet were a major confounding factor. In a non-FR expressing J6456 model, FT-PLD was as active as PLD indicating that its activity is not limited to FR-expressing tumors. Conclusion Folate-targeting confers a significant albeit modest therapeutic improvement to PLD in FR-expressing tumor models, which appears particularly valuable in intracavitary therapy. The potential clin. added value of this approach has yet to be detd.
- 27Chan, E. S. L.; Cronstein, B. N. Nat. Rev. Rheumatol. 2010, 6 (3) 175– 178 DOI: 10.1038/nrrheum.2010.5[ Crossref], [ PubMed], [ CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXis1ygt7w%253D&md5=ff7c199b2a9a3d86f96b90a2a639d80eMethotrexate-how does it really work?Chan, Edwin S. L.; Cronstein, Bruce N.Nature Reviews Rheumatology (2010), 6 (3), 175-178CODEN: NRRACB; ISSN:1759-4790. (Nature Publishing Group)A review. Methotrexate is a proven and efficacious therapy for inflammatory diseases such as rheumatoid arthritis. The main mechanism of action of methotrexate as an anti-cancer drug, at high doses, involves folate antagonism, but what other mechanisms might be operative in the use of this drug at lower doses as an effective anti-inflammatory agent. Methotrexate remains a cornerstone in the treatment of rheumatoid arthritis and other rheumatic diseases. Folate antagonism is known to contribute to the antiproliferative effects that are important in the action of methotrexate against malignant diseases, but concomitant administration of folic or folinic acid does not diminish the anti-inflammatory potential of this agent, which suggests that other mechanisms of action might be operative. Although no single mechanism is sufficient to account for all the anti-inflammatory activities of methotrexate, the release of adenosine from cells has been demonstrated both in vitro and in vivo. Methotrexate might also confer anti-inflammatory properties through the inhibition of polyamines. The biol. effects on inflammation assocd. with adenosine release have provided insight into how methotrexate exerts its effects against inflammatory diseases and at the same time causes some of its well-known adverse effects. These activities contribute to the complex and multifaceted mechanisms that make methotrexate efficacious in the treatment of inflammatory disorders.
- 28Genestier, L.; Paillot, R.; Quemeneur, L.; Izeradjene, K.; Revillard, J.-P. Immunopharmacology 2000, 47 (2–3) 247– 257 DOI: 10.1016/S0162-3109(00)00189-2[ Crossref], [ PubMed], [ CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXktlKktL8%253D&md5=ea8dcba9e8a0485feb42a058bd14f36fMechanisms of action of methotrexateGenestier, L.; Paillot, R.; Quemeneur, L.; Izeradjene, K.; Revillard, J.-P.Immunopharmacology (2000), 47 (2-3), 247-257CODEN: IMMUDP; ISSN:0162-3109. (Elsevier Science B.V.)A review with ∼110 refs. This review, although not exhaustive, focuses on the most recent or significant mechanisms of action of methotrexate (MTX) as an anti-inflammatory and an immunosuppressive drug.
- 29Kısmet, K.; Akay, M. T.; Abbasoǧlu, O.; Ercan, A. Cancer Detect. Prev. 2004, 28 (2) 127– 142 DOI: 10.1016/j.cdp.2003.12.005[ Crossref], [ PubMed], [ CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkt12jtLs%253D&md5=f15c5fa16f8d152d7b6e0459fcba21e4Celecoxib: A potent cyclooxygenase-2 inhibitor in cancer preventionKismet, Kemal; Akay, M. Turan; Abbasoglu, Osman; Ercan, AyguenCancer Detection and Prevention (2004), 28 (2), 127-142CODEN: CDPRD4; ISSN:0361-090X. (Elsevier Science B.V.)A review. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used therapeutic agents in the treatment of pain, inflammation and fever. They may also have a role in the management of cancer prevention, Alzheimer's disease and prophylaxis against cardiovascular disease. These drugs act primarily by inhibiting cyclooxygenase enzyme, which has two isoforms, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Selective COX-2 inhibitors provide potent anti-inflammatory and analgesic effects without the side effects of gastric and renal toxicity and inhibition of platelet function. Celecoxib is a potent COX-2 inhibitor being developed for the treatment of rheumatoid arthritis and osteoarthritis. Chemoprevention is the use of pharmacol. or natural agents to prevent, suppress, interrupt or reverse the process of carcinogenesis. For this purpose, celecoxib is being used for different cancer types. The effects of NSAIDs on tumor growth remain unclear, but are most likely to be multifocal. In this article, the authors reviewed COX-2 selectivity, the pharmacol. properties of celecoxib, the use of celecoxib for cancer prevention and the mechanisms of chemoprevention.
- 30Cui, W.; Yu, C.-H.; Hu, K.-Q. Clin. Cancer Res. 2005, 11 (22) 8213– 8221 DOI: 10.1158/1078-0432.CCR-05-1044[ Crossref], [ PubMed], [ CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1entr3I&md5=d9848d42856040f8d5ecd98637ad2d35In vitro and in vivo effects and mechanisms of celecoxib-induced growth inhibition of human hepatocellular carcinoma cellsCui, Wei; Yu, Chang-Hong; Hu, Ke-QinClinical Cancer Research (2005), 11 (22), 8213-8221CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Cyclooxygenase-2 (COX-2) inhibitors cause growth inhibition of human hepatocellular carcinoma cells but it remains unclear whether this is both COX-2 dependent and independent. The related mechanisms remain to be detd. The present study was aimed to det. the effect of celecoxib on growth of hepatocellular carcinoma cells and xenografts and the related mechanisms. Both low COX-2 expressing PLC/PRF/5 and high COX-2 expressing HuH7 cells, and nude mice bearing hepatocellular carcinoma xenografts were used to study the effect and mechanisms of celecoxib on hepatocellular carcinoma cell growth. Celecoxib resulted in a comparable growth inhibition of both hepatocellular carcinoma cells that was assocd. with decreased prodn. of prostaglandin E2 and increased peroxisome proliferator-activated receptor γ in both cells. Addn. of prostaglandin E2 only partially counteracted the effect of celecoxib on both cells. Celecoxib resulted in a significant redn. of retinoblastoma phosphorylation and DP1/E2F1 complex in both cells. Celecoxib caused a significant increase of apoptosis and activation of caspase-3 and caspase-9 in both cells. In nude mice inoculated with HuH7 cells, celecoxib resulted in decreased frequency and mean wt. of hepatocellular carcinoma xenografts. The present study showed that celecoxib causes COX-2-dependent and COX-2-independent growth inhibition of hepatocellular carcinoma cells and xenografts by (a) decreased retinoblastoma phosphorylation and DP1/E2F1 complex; (b) increased activation of caspase-3 and caspase-9; and (c) increased expression of proliferator-activated receptor γ. The present study significantly extended our knowledge on the effect and mechanisms of celecoxib-induced inhibition of hepatocellular carcinoma cell growth.
- 31Zhao, S.-p.; Deng, P.; Huang, H.-g.; Xu, Z.-m.; Dai, H.-y.; Hong, S.-c.; Yang, J.; Zhou, H.-n. Clin. Chem. 2005, 51 (11) 2170– 2173 DOI: 10.1373/clinchem.2005.054288[ Crossref], [ PubMed], [ CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFyht7fN&md5=637390a9e410de1782b415b2ff9f1830Expression of COX-2 mRNA in peripheral blood monocytes from patients with acute myocardial infarction and its significanceZhao, Shui-ping; Deng, Ping; Huang, Hong-guang; Xu, Zhu-mei; Dai, Hai-ying; Hong, Shao-cai; Yang, Jun; Zhou, Hong-nianClinical Chemistry (Washington, DC, United States) (2005), 51 (11), 2170-2173CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)In this study, cyclooxygenase-2 (COX-2) mRNA expression was measured in peripheral blood monocytes from patients with acute myocardial infarction (AMI) and it was investigated whether COX-2-specific inhibitor, celecoxib, can lower the secretion of interleukin (IL)-6 and matrix metalloproteinase (MMP)-9. Forty-patients with AMI and 18 individuals with stable coronary heart disease of similar age for the study were recruited. AMI was defined as a history of ischemic chest pain > 30 min, characteristic electrocardiog. changes, and increased cardiac troponin I at least twice the upper limit of normal within 24 h after the onset of pain. Results showed an increased COX-2 expression in peripheral blood monocytes from patients with AMI, and the correlation of COX-2 expression with IL-6 and MMP-9 secretion by monocytes was also strong. These results support the hypothesis of an acute inflammatory response to AMI that, at least in part, is correlated with COX-2 activation in peripheral blood monocytes. It also measured the secretion of IL-6 and MMP-9 by monocytes incubated with different concns. of celecoxib and found that celecoxib significantly reduced the in vitro secretion of IL-6 and MMP-9 in a concn.-dependent manner. These data suggest that COX-2 expression may promote secretion of IL-6 and MMP-9 by monocytes and that celecoxib might reduce acute atherosclerotic inflammation partly by inhibiting secretion of the inflammatory cytokines IL-6 and MMP-9 by peripheral blood monocytes.
- 32Zhang, G. S.; Liu, D. S.; Dai, C. W.; Li, R. J. Am. J. Hematol. 2006, 81 (4) 242– 55 DOI: 10.1002/ajh.20542[ Crossref], [ PubMed], [ CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XktVGrt7s%253D&md5=654966e6976a2a20e4d91b8d4cfdb13cAntitumor effects of celecoxib on K562 leukemia cells are mediated by cell-cycle arrest, caspase-3 activation, and downregulation of Cox-2 expression and are synergistic with hydroxyurea or imatinibZhang, Guang-Sen; Liu, Ding-Sheng; Dai, Chong-Wen; Li, Rui-JuanAmerican Journal of Hematology (2006), 81 (4), 242-255CODEN: AJHEDD; ISSN:0361-8609. (Wiley-Liss, Inc.)Celecoxib, a specific cyclooxygenase-2 (Cox-2) inhibitor, has been shown to possess antitumor activity in a variety of cancer cells. However, the antitumor activity of celecoxib in hematopoietic tumors, esp. in chronic myeloid leukemia (CML), has not been well established. This study was designed to investigate the effect of celecoxib on growth and apoptosis in a human CML cell line (K562 cells) or in primary CML cells, and to examine the synergistic actions of celecoxib and hydroxyurea or imatinib on K562 cell proliferation and apoptosis. Celecoxib significantly inhibited the growth of both K562 and primary CML cells and induced apoptosis in a dose-dependent fashion. The IC50 of celecoxib was 46 μM for inhibition of K562 cell proliferation. The effect of celecoxib on growth inhibition was accompanied by the downregulation of cyclin D1 and cyclin E and p-Rb expression, the upregulation of P16INK4a and P27KIP expression, and a G1-S phase arrest of the cell cycle. The pro-apoptotic effect of celecoxib was detd. to be mediated by caspase-3 activation. When K562 cells were pretreated with DEVD-fmk, a specific inhibitor of caspases, the apoptotic activity of celecoxib was, in part, abrogated. Importantly, we demonstrated for the first time that K562 cells were Cox-2-pos. both at the mRNA and protein levels. We noted the following observations: (i) we detected Cox-2 mRNA in K562 cells by reverse transcription-PCR (RT-PCR) and protein expression by western blot anal.; (ii) Cox-2 expression in K562 cells was stimulated by IL-1β, a specific inducing agent of Cox-2 expression; (iii) primary CML cells from CML patient bone marrow also exhibited Cox-2 protein expression. Furthermore, Cox-2 expression was downregulated at higher doses of celecoxib (80-160 μM), suggesting a Cox-2-dependent mechanism was involved in the drug's effects of growth inhibition and induction of apoptosis. In addn., a synergistic effect was obsd. when cells were exposed to low-dose celecoxib (40 μM) and hydroxyurea (10 mM) or a combination of celecoxib (40 μM) and imatinib (0.2 μM). These findings provide the basis for uncovering the mechanism of celecoxib's antitumor effects and developing a new therapeutic strategy for treating CML.
- 33Dai, Z.-J.; Ma, X.-B.; Kang, H.-F.; Gao, J.; Min, W.-L.; Guan, H.-T.; Diao, Y.; Lu, W.-F.; Wang, X.-J. Cancer Cell Int. 2012, 12 (1) 53 DOI: 10.1186/1475-2867-12-53[ Crossref], [ PubMed], [ CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslCjtbg%253D&md5=c522988fbfe16b58c344d60c46069e90Antitumor activity of the selective cyclooxygenase-2 inhibitor, celecoxib, on breast cancer in Vitro and in VivoDai, Zhi-Jun; Ma, Xiao-Bin; Kang, Hua-Feng; Gao, Jie; Min, Wei-Li; Guan, Hai-Tao; Diao, Yan; Lu, Wang-Feng; Wang, Xi-JingCancer Cell International (2012), 12 (), 53CODEN: CCIACC; ISSN:1475-2867. (BioMed Central Ltd.)Background: Cyclooxygenase-2(COX-2) promotes carcinogenesis, tumor proliferation, angiogenesis, prevention of apoptosis and immunosuppression. Meanwhile, COX-2 over-expression has been assocd. with tumor behavior and prognosis in several cancers. This study investigated the antitumor effects of the selective COX-2 inhibitor, Celecoxib, on breast cancer in vitro and in vivo. Methods: Human breast cancer MCF-7 and MDA-MB-231 cells were cultured with different concn. (10, 20, 40 μmol/L) of celecoxib after 0-96 h in vitro. MTT assay was used to det. the growth inhibition of breast cancer cells in vitro. The expression of COX-2 on mRNA was measured by real-time quantitive PCR anal. Flow cytometry was performed to analyze the cell cycle of MCF-7 cells. Levels of PGE2 were measured by ELISA method. The in vivo therapeutic effects of celecoxib were detd. using rat breast cancer chem. induced by 7,12-dimethylben anthracene (DMBA). Results: The inhibition of proliferation of both MCF-7 and MDA-MB-231 cells in vitro by celecoxib was observerd in time and dose dependent manner. Celecoxib effectively down-regulated the expression of COX-2. The cell cycle was arrested at G0/G1 and rate of cells in S phase was obviously decreased. Levels of PGE2 were inhibited by Celecoxib. The tumor incidence rate of the celecoxib group was lower than that of the control group. In addn., the tumor latency period of the celecoxib group was longer than that of the control group. Conclusions: Celecoxib inhibited the proliferation of breast cancer cell lines in vitro and prevented the occurrence of rat breast cancer chem. induced by DMBA. Therefore, celecoxib exhibits an antitumor activity and seems to be effective in anti-tumor therapy.
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- 35Kolb, A. F.; Petrie, L. Mol. Immunol. 2013, 54 (2) 164– 172 DOI: 10.1016/j.molimm.2012.11.012[ Crossref], [ PubMed], [ CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslWkt7k%253D&md5=c9908b8558b8012543daa09b4284a084Folate deficiency enhances the inflammatory response of macrophagesKolb, Andreas F.; Petrie, LindaMolecular Immunology (2013), 54 (2), 164-172CODEN: MOIMD5; ISSN:0161-5890. (Elsevier)B-vitamin deficiency is a risk factor for vascular disease. The mechanism by which the deficiency impacts on disease risk is unclear. We have analyzed whether the inflammatory response of mononuclear cells can be modified by cellular folate status in vitro. We show that the mouse monocyte cell line RAW264.7 grown under folate restriction displays a decrease in intracellular folate levels and a reduced growth rate. The cells also show a 2- to 3-fold increase in expression of the inflammatory mediators, IL1β, IL6, TNFα and MCP1 at the RNA and protein level (p < 0.01) under conditions of folate deficiency. In contrast the prodn. of the vaso-protective mediator nitric oxide is significantly reduced under these conditions. These metabolic changes are independent of the concn. of homocysteine in the medium and occur in the absence of significant changes in global DNA methylation. Folate deficiency may therefore exacerbate cardiovascular disease by augmenting pro-inflammatory signals in the monocyte-macrophage lineage.
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Abstract
Figure 1
Figure 1. SP-DS3 peptide, derived from pulmonary surfactant-associated protein D, is deeply inserted into the liposomal bilayer with FA at the liposomal surface. (A) Diagram of the analyzed SP-D fragments. The localization in the SP-D sequence is indicated, and numbers correspond to the amino acid positions in the SP-D sequence. (B) Description of the designed peptide linkers and quantification of the FA present at the liposomal surface. (C) Tryptophan fluorescence of the tested peptides. A strong blue shift in the fluorescence emission maximum was observed for SP-DS3. (D) Molecular dynamics simulation of the interaction of SP-DS3 with the PC/CH membrane. The tryptophan residue (yellow) in an apolar environment and the FA (orange) at the membrane surface. (E) Representative TEM images of liposomes. Scale bar (red) represents 200 nm in smaller magnification and 20 nm in higher magnification.
Figure 2
Figure 2. Liposomes with FA conjugated to SP-DS3 peptide are more efficiently disrupted compared to DSPE-PEG-FA. (A) Schematic representation of liposomes incorporating SP-DS3 peptide and (B) DSPE-PEG-FA. Representative pictures of the Caco-2 cells (FR+) after 1 h of incubation with fluorescent liposomal formulations (Hoechst 34580, blue staining) (C) with SP-DS3 and (D) DSPE-PEG-FA. (E) Efficient membrane disruption of liposomes incorporating SP-DS3 peptide, comparing with DSPE-PEG-FA, by Caco-2 cells. Graphs represent the relative mean fluorescence intensity (MFI) determined by flow cytometry analysis of Hoechst 34580 in Caco-2 cells with internalized liposomes after 1 h of incubation at 37 °C. Values are the mean + SD of 2 experiments. (F) Structures of DSPE-PEG-FA and SP-DS3.
Figure 3
Figure 3. Liposomes integrating SP-DS3 are an efficient drug delivery system to FR-expressing cells. (A) The effects of 250 μg/mL of liposomal formulations with and without 65 μM celecoxib on COX-2 mRNA expression in Caco-2 cells (3 h of contact with liposomes +21 h of medium without liposomes). Values are the mean + SD of 2 experiments. (B) Caco-2 cell viability after 3, 6, and 48 h of contact with 10 μg/mL liposomal formulations encapsulating 1.27 μM methotrexate compared with untreated cells (negative control) and 30% DMSO-treated cells (positive control) as determined by the MTS assay at 48 h. Values are the mean + SD of 2 experiments. Significant differences were detected as shown by an asterisk (P < 0.05) and two asterisks (P < 0.005).
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- 1Sant, V. P.; Nagarsenker, M. S. AAPS PharmSciTech 2011, 12 (4) 1056– 63 DOI: 10.1208/s12249-011-9674-2[ Crossref], [ PubMed], [ CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFGqtL3E&md5=cbe97dd6433d1fd5dec7624e2052e51cSynthesis of Monomethoxypolyethyleneglycol-Cholesteryl Ester and Effect of its Incorporation in LiposomesSant, Vinayak P.; Nagarsenker, Mangal S.AAPS PharmSciTech (2011), 12 (4), 1056-1063CODEN: AAPHFZ; ISSN:1530-9932. (Springer)The objective of the present study was to synthesize monomethoxypolyethyleneglycol-5000 cholesteryl ester [PEG-CH] as a cost-effective substitute for polyethyleneglycol-phosphatidylethanolamine and to evaluate the influence of its incorporation in liposomal bilayers for surface modification. PEG-CH was synthesized and characterized by IR (IR), proton NMR spectroscopy (1H NMR), and differential scanning calorimetry (DSC) studies. Influence of incorporation of PEG-CH in liposomes was evaluated on various parameters such as zeta potential, DSC, and encapsulation efficiency of a hydrophilic drug pentoxyfylline. Conventional and PEG-CH contg. pentoxyfylline liposomes were formulated and their stability was evaluated at 4°C for 3 mo. PEG-CH could be successfully synthesized with good yields and the structure was confirmed by IR, DSC, and 1H NMR. The incorporation of PEG-CH in liposomes resulted in redn. of the zeta potential and broadening of the DSC endotherm. Furthermore, incorporation of PEG-CH in liposomes decreased the encapsulation efficiency of pentoxifylline in liposomes when compared to conventional liposomes. Conventional and PEG-CH contg. pentoxyfylline liposomes did not show any signs of pentoxyfylline degrdn. when stored at 4°C for 3 mo.
- 2Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Acc. Chem. Res. 2008, 41 (1) 120– 9 DOI: 10.1021/ar7000815[ ACS Full Text], [ CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXot1Gltbk%253D&md5=20620eb124d19634b10c27a48ed46849Discovery and Development of Folic-Acid-Based Receptor Targeting for Imaging and Therapy of Cancer and Inflammatory DiseasesLow, Philip S.; Henne, Walter A.; Doorneweerd, Derek D.Accounts of Chemical Research (2008), 41 (1), 120-129CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The field of folic-acid-targeted drug delivery has grown rapidly over the past decade, with several applications currently in human clin. trials. Owing to the small size of the vitamin, its high affinity for the folate receptor (Kd = 10-10 M), the limited distribution of its receptor in normal tissues (primarily in kidney proximal tubules), the overexpression of its receptor in pathol. cells (upregulated in cancer cells and activated macrophages/monocytes), and its ease of conjugation to imaging and therapeutic agents, the use of folate as a targeting ligand for selective delivery of attached mols. to tumor tissues and sites of inflammation has attracted considerable interest. This account reviews the discovery of folic acid as a targeting ligand and its current use in the selective targeting of drugs to malignant masses and sites of inflammation.
- 3Hilgenbrink, A. R.; Low, P. S. J. Pharm. Sci. 2005, 94 (10) 2135– 2146 DOI: 10.1002/jps.20457[ Crossref], [ PubMed], [ CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVOrurrF&md5=1b1b128980fa656802b23f980f4cd4d3Folate receptor-mediated drug targeting: From therapeutics to diagnosticsHilgenbrink, Andrew R.; Low, Philip S.Journal of Pharmaceutical Sciences (2005), 94 (10), 2135-2146CODEN: JPMSAE; ISSN:0022-3549. (Wiley-Liss, Inc.)A review. Folate targeted drug delivery has emerged as an alternative therapy for the treatment and imaging of many cancers and inflammatory diseases. Due to its small mol. size and high binding affinity for cell surface folate receptors (FR), folate conjugates have the ability to delivery a variety of mol. complexes to pathol. cells without causing harm to normal tissues. Complexes that were successfully delivered to FR expressing cells, to date, include protein toxins, immune stimulants, chemotherapeutic agents, liposomes, nanoparticles, and imaging agents. This review will summarize the applications of folic acid as a targeting ligand and highlight the various methods being developed for delivery of therapeutic and imaging agents to FR-expressing cells.
- 4Low, P. S.; Kularatne, S. A. Curr. Opin. Chem. Biol. 2009, 13 (3) 256– 262 DOI: 10.1016/j.cbpa.2009.03.022[ Crossref], [ PubMed], [ CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXosVWltbY%253D&md5=c6b6b07b0df9d1355e99da7c471a2a4bFolate-targeted therapeutic and imaging agents for cancerLow, Philip Stewart; Kularatne, Sumith AnurasiriCurrent Opinion in Chemical Biology (2009), 13 (3), 256-262CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Cancer therapies that exploit targeting ligands to deliver attached cytotoxic drugs selectively to malignant cells are currently receiving significant attention. While antibody-targeted drugs have been the first to enter the clinic, recent studies demonstrate that the vitamin folic acid can also be used to deliver attached imaging and therapeutic agents selectively to malignant cells in both animal tumor models and human cancer patients. Thus, folate conjugates bind to folate receptors that are overexpressed on ∼40% of human cancers and mediate internalization of their attached drugs by receptor-mediated endocytosis. With the use of proper linkers, folate-targeted drugs can be released inside their target cells where they can perform their desired cytotoxic functions. Based on this strategy, six folate-targeted drugs are currently in human clin. trials.
- 5Gabizon, A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S. Adv. Drug Delivery Rev. 2004, 56 (8) 1177– 1192 DOI: 10.1016/j.addr.2004.01.011[ Crossref], [ PubMed], [ CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjtF2rtLs%253D&md5=b6948331c4bc8ab98a988af1434b5016Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conjugatesGabizon, Alberto; Shmeeda, Hilary; Horowitz, Aviva T.; Zalipsky, SamuelAdvanced Drug Delivery Reviews (2004), 56 (8), 1177-1192CODEN: ADDREP; ISSN:0169-409X. (Elsevier Science B.V.)A review. Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to the outer end of a small fraction of phospholipid-anchored PEG mols. appears to be the most appropriate way to combine long-circulating properties crit. for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a no. of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model, that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and appear to be formulation- and tumor model-dependent. Further studies are required to det. whether folate targeting can confer a clear advantage in efficacy and/or toxicity to liposomal drugs.
- 6Liu, Y.; Xu, S.; Teng, L.; Yung, B.; Zhu, J.; Ding, H.; Lee, R. J. Anticancer Res. 2011, 31 (5) 1521– 1525[ PubMed], [ CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvFSrsrk%253D&md5=34c09989ca94b9506eda063047d43266Synthesis and evaluation of a novel lipophilic folate receptor targeting ligandLiu, Ying; Xu, Songlin; Teng, Lesheng; Yung, Bryant; Zhu, Jing; Ding, Hong; Lee, Robert J.Anticancer Research (2011), 31 (5), 1521-1526CODEN: ANTRD4; ISSN:0250-7005. (International Institute of Anticancer Research)Background: Folate receptor (FR)-targeted liposomes have been investigated as delivery vehicles for anticancer drugs. A novel lipophilic FR ligand, folate-glutathione-polyethyleneglycol-distearoyl phosphatidylethanolamine (F-GSH-PEG-DSPE), was synthesized, incorporated into liposomes and evaluated for FR targeting efficiency. These liposomes were then evaluated as carriers of the chemotherapy agent vincristine (VIN). Materials and Methods: F-GSH-PEG-DSPE was synthesized and FR-targeted liposomes loaded with either calcein (F-L-Calcein) or VIN (F-L-VIN) were prepd. by thin film hydration followed by polycarbonate membrane extrusion and, in the case of VIN, by remote loading. To assess liposome stability, the uptake of F-L-VIN in KB (FR+) cancer cells was measured after storage under 4°C for 3 mo. Comparative pharmacokinetic studies were carried out with F-L-VIN and L-VIN (non-targeted control liposomes). Results: F-L-Calcein showed significantly higher cellular uptake in KB cells compared to non-targeted liposomes. In addn., F-L-VIN showed enhanced cytotoxicity in KB cells in vitro compared to control liposomes. Pharmacokinetic parameters indicated that both F-L-VIN and control liposomes had higher area under the curve (AUC), mean residence time (MRT), elimination half life (t1/2-β) and lower total body clearance (CL) than those of free VIN, while there were no significant differences between these liposomal formulations. Conclusion: F-GSH-PEG-DSPE is effective as a novel ligand for the synthesis of FR-targeted liposomes.
- 7Xiong, S.; Yu, B.; Wu, J.; Li, H.; Lee, R. J. Biomed. Pharmacother. 2011, 65 (1) 2– 8 DOI: 10.1016/j.biopha.2010.10.003[ Crossref], [ PubMed], [ CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXit1ajtr8%253D&md5=3d768f9ede0acb97cd2c670664888c69Preparation, therapeutic efficacy and intratumoral localization of targeted daunorubicin liposomes conjugating folate-PEG-CHEMSXiong, Subin; Yu, Bo; Wu, Jun; Li, Hong; Lee, Robert J.Biomedicine & Pharmacotherapy (2011), 65 (1), 2-8CODEN: BIPHEX; ISSN:0753-3322. (Elsevier Masson SAS)Folate polyethylene glycol-cholesterol hemisuccinate (folate-PEG-CHEMS) is a novel folate ligand firstly synthesized by our group and demonstrated good stability and potential targeting results on KB cells in vitro. Endocytosis mechanisms of liposomes via folate receptor on L1210JF cells and assessed targeted therapeutic efficacy of folate-PEG-CHEMS anchored liposomes loading daunorubicin (F-L-DNR) were studied in vivo. Folate-PEG-CHEMS was synthesized by a modified method. The liposome properties, cell cytotoxicity, intracellular and intratumoral localization, and therapeutic efficacy on a murine tumor model bearing L1210JF cells were evaluated. High encapsulation efficiency (95.1% ± 1.5%) and appropriate particle size (76.0 ± 35.5 nm) and zeta potential (-12.83 ± 1.36 mV) were achieved for F-L-DNR. IC50 of F-L-DNR on L1210JF cells was 2-3-folds lower than that of non-targeted liposomal daunorubicin (L-DNR). Anticancer efficacy on L1210JF tumor model indicated that mice survival time of F-L-DNR group at doses of 5 mg/kg and 10 mg/kg was significantly longer than that of L-DNR or free DNR. Confocal fluorescence photographs of F-L-DNR indicated enhanced endocytosis of liposomes via folate receptor on L1210JF cells, prolonged retaining time in tumors and improved drug release in the tumor site at 24 h post i.v. injection of F-L-DNR. Thus, folate-PEG-CHEMS is an effective ligand for folate-targeted daunorubicin liposomes to achieve increased drug release in tumor and therapeutic efficacy.
- 8Seo, H. J.; Kim, J.-C. Bioconjugate Chem. 2014, 25 (3) 533– 542 DOI: 10.1021/bc400521r[ ACS Full Text], [ CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisFKhsLk%253D&md5=c68f98ef9e5db03c8c9968f9850c82187-Acetoxycoumarin Dimer-Incorporated and Folate-Decorated Liposomes: Photoresponsive Release and in Vitro Targeting and EfficacySeo, Hee Jin; Kim, Jin-ChulBioconjugate Chemistry (2014), 25 (3), 533-542CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Photoresponsive and cancer cell (KB cell)-targetable liposome was developed by incorporating 7-acetoxycoumarin dimer (ACD) in the liposomal membrane and modifying the surface of liposome with folate. The liposomes were prepd. from the dry mixed thin film of egg phosphatidylcholine (EPC), ACD, and folate conjugate (DSPE-PEG2000-folate) by a film hydration method, where the molar ratios of EPC/ACD/DSPE-PEG2000-folate were 10/0/0, 9/1/0, 9/1/0.05, and 9/0/0.05. The liposomal membranes were multilamellar and the diam. was on the order of hundreds of nanometers. The release degrees in 60 min of 5(6)-carboxyfluorescein (CF) from EPC/ACD/DSPE-PEG2000-folate liposome were less than 4% without the irradn. of UV light (λ = 254 nm, 6 W), but more than 20% under the irradn. of UV light (λ = 254 nm, 6W), possibly due to the phototriggered de-dimerization of ACD. Under confocal laser scanning microscopy, KB cells treated with EPC/ACD/DSPE-PEG2000-folate liposomes exhibited CF fluorescence of liposomes at the positions where 4',6-diamidino-2-phenylindole (DAPI) fluorescence of the cell nucleus was shown, indicating the liposomes targeted the cancer cells. In flow cytometric anal., the cancer cells treated with EPC/ACD/DSPE-PEG2000-folate liposomes exhibited much higher fluorescence than the untreated cells did, indicating that there was a specific interaction between the liposome and the cancer cell. With the irradn. of UV light, EPC/ACD/DSPE-PEG2000-folate liposomes markedly promoted the in vitro anti-cancer efficacy of DOX without causing acute in vitro toxicity.
- 9Xiang, G.; Wu, J.; Lu, Y.; Liu, Z.; Lee, R. J. Int. J. Pharm. 2008, 356 (1–2) 29– 36 DOI: 10.1016/j.ijpharm.2007.12.030[ Crossref], [ PubMed], [ CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlsFCks74%253D&md5=839eb82aa5c367f4b8cbbd868fef857cSynthesis and evaluation of a novel ligand for folate-mediated targeting liposomesXiang, Guangya; Wu, Jun; Lu, Yanhui; Liu, Zhilan; Lee, Robert J.International Journal of Pharmaceutics (2008), 356 (1-2), 29-36CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)Folate receptors (FRs) have been identified as cellular surface markers for cancer and leukemia. Liposomes contg. lipophilic derivs. of folate have been shown to effectively target FR-expressing cells. Here, the authors report the synthesis of a novel lipophilic folate deriv., folate-polyethylene glycol-cholesterol hemisuccinate (F-PEG-CHEMS), and its evaluation as a targeting ligand for liposomal doxorubicin (L-DOX) in FR-expressing cells. Liposomes contg. F-PEG-CHEMS, with a mean diam. of 120±20 nm, were synthesized by polycarbonate membrane extrusion and were shown to have excellent colloidal stability. The liposomes were taken up selectively by KB cells, which overexpress FR-α. Compared to folate-PEG-cholesterol (F-PEG-Chol), which contains a carbamate linkage, F-PEG-CHEMS better retained its FR-targeting activity during prolonged storage. In addn., F-PEG-CHEMS contg. liposomes loaded with DOX (F-L-DOX) showed greater cytotoxicity (IC50 = 10.0 μM) than nontargeted control L-DOX (IC50 = 57.5 μM) in KB cells. In ICR mice, both targeted and nontargeted liposomes exhibited long circulation properties, although F-L-DOX (t 1/2 = 12.34 h) showed more rapid plasma clearance than L-DOX (t 1/2 = 17.10 h). These results suggest that F-PEG-CHEMS is effective as a novel ligand for the synthesis of FR-targeted liposomes.
- 10Zhang, P.; McAlinden, A.; Li, S.; Schumacher, T.; Wang, H.; Hu, S.; Sandell, L.; Crouch, E. J. Biol. Chem. 2001, 276 (23) 19862– 19870 DOI: 10.1074/jbc.M100597200[ Crossref], [ PubMed], [ CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXktlKgurg%253D&md5=853a820459759921c1478f5310c801fcThe amino-terminal heptad repeats of the coiled-coil neck domain of pulmonary surfactant protein D are necessary for the assembly of trimeric subunits and dodecamersZhang, Pengnian; McAlinden, Audrey; Li, Shi; Schumacher, Troy; Wang, Hongling; Hu, Shasa; Sandell, Linda; Crouch, ErikaJournal of Biological Chemistry (2001), 276 (23), 19862-19870CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Pulmonary surfactant protein D (SP-D), a lung host defense protein, is assembled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of trimers is required for many of its functions. A peptide contg. the α-helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular assembly of disulfide-cross-linked, trimeric subunits and dodecamers. For the present studies, we synthesized mutant cDNAs with deletions or site-directed substitutions in the neck domain of rat SP-D, and examd. the assembly of the newly synthesized proteins after transfection of CHO-K1 cells. The neck domain contains three "classical" heptad repeat motifs with leucine residues at the "d position," and a distinctive C-terminal repeat previously suggested to drive trimeric chain assocn. Deletion of the highly conserved core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. By contrast, deletion of the entire neck domain or deletion of one or two amino-terminal repeats resulted in defective mol. assembly. The secreted proteins eluted in the position of monomers by gel filtration under nondenaturing conditions. In addn., the neck + carbohydrate recognition domain of SP-D was necessary and sufficient for the trimerization of a heterologous collagen sequence located amino-terminal to the trimeric coiled-coil. These studies provide strong evidence that the amino-terminal heptad repeats of the neck domain are necessary for the intracellular, trimeric assocn. of SP-D monomers and for the assembly and secretion of functional dodecamers.
- 11Kingma, P. S.; Whitsett, J. A. Curr. Opin. Pharmacol. 2006, 6 (3) 277– 283 DOI: 10.1016/j.coph.2006.02.003[ Crossref], [ PubMed], [ CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XksFymsrw%253D&md5=ecaa619b80f055a54c342a05c94f9f74In defense of the lung: surfactant protein A and surfactant protein DKingma, Paul S.; Whitsett, Jeffrey A.Current Opinion in Pharmacology (2006), 6 (3), 277-283CODEN: COPUBK; ISSN:1471-4892. (Elsevier Ltd.)A review. The lung is repeatedly exposed to inhaled particles and pathogens that are cleared by the actions of a multi-component innate host defense system. The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), play important roles in innate host defense by binding and clearing invading microbes from the lung. SP-A and SP-D also influence surfactant homeostasis, contributing to the phys. structures of lipids in the alveoli and to the regulation of surfactant function and metab. In addn. to binding and opsonizing infectious pathogens, SP-A and SP-D bind to the surfaces of host defense cells, promoting or inhibiting immune cell activity through multiple cellular pathways. As a consequence of their physiol. functions, SP-A- and SP-D-dependent pathways are targets for clin. therapies designed to limit the proliferation of microorganisms and to ameliorate inflammation following pulmonary infection.
- 12Kishore, U.; Greenhough, T. J.; Waters, P.; Shrive, A. K.; Ghai, R.; Kamran, M. F.; Bernal, A. L.; Reid, K. B. M.; Madan, T.; Chakraborty, T. Mol. Immunol. 2006, 43 (9) 1293– 1315 DOI: 10.1016/j.molimm.2005.08.004[ Crossref], [ PubMed], [ CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhslKjtb4%253D&md5=8eab33323417282245c2d6c1302ca73fSurfactant proteins SP-A and SP-D: Structure, function and receptorsKishore, Uday; Greenhough, Trevor J.; Waters, Patrick; Shrive, Annette K.; Ghai, Rohit; Kamran, Mohammed F.; Lopez Bernal, Andres; Reid, Kenneth B. M.; Madan, Taruna; Chakraborty, TrinadMolecular Immunology (2006), 43 (9), 1293-1315CODEN: MOIMD5; ISSN:0161-5890. (Elsevier B.V.)A review. Surfactant proteins, SP-A and SP-D, are collagen-contg. C-type (calcium dependent) lectins called collectins, which contribute significantly to surfactant homeostasis and pulmonary immunity. These highly versatile innate immune mols. are involved in a range of immune functions including viral neutralization, clearance of bacteria, fungi and apoptotic and necrotic cells, down regulation of allergic reaction and resoln. of inflammation. Their basic structures include a triple-helical collagen region and a C-terminal homotrimeric lectin or carbohydrate recognition domain (CRD). The trimeric CRDs can recognize carbohydrate or charge patterns on microbes, allergens and dying cells, while the collagen region can interact with receptor mols. present on a variety of immune cells in order to initiate clearance mechanisms. Studies involving gene knock-out mice, murine models of lung hypersensitivity and infection, and functional characterization of cell surface receptors have revealed the diverse roles of SP-A and SP-D in the control of lung inflammation. A recently proposed model based on studies with the calreticulin-CD91 complex as a receptor for SP-A and SP-D has suggested an anti-inflammatory role for SP-A and SP-D in naive lungs which would help minimize the potential damage that continual low level exposure to pathogens, allergens and apoptosis can cause. However, when the lungs are overwhelmed with exogenous insults, SP-A and SP-D can assume pro-inflammatory roles in order to complement pulmonary innate and adaptive immunity. This review is an update on the structural and functional aspects of SP-A and SP-D, with emphasis on their roles in controlling pulmonary infection, allergy and inflammation. We also try to put in perspective the controversial subject of the candidate receptor mols. for SP-A and SP-D.
- 13Mangialavori, I.; Giraldo, A. M. V.; Buslje, C. M.; Gomes, M. F.; Caride, A. J.; Rossi, J. P. F. C. J. Biol. Chem. 2009, 284 (8) 4823– 4828 DOI: 10.1074/jbc.M806912200[ Crossref], [ PubMed], [ CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhvVSgurw%253D&md5=cb34ead795ba0f9c2faafdede62ce194A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic ProbeMangialavori, Irene; Giraldo, Ana Maria Villamil; Buslje, Cristina Marino; Gomes, Mariela Ferreira; Caride, Ariel J.; Rossi, Juan Pablo F. C.Journal of Biological Chemistry (2009), 284 (8), 4823-4828CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine (PC) analog 1-palmitoyl-2-[9-[2'-[125I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([125I]TID-PC/16) under different conditions. We detd. the following. (1) Incorporation of [125I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca2+. This decrease in labeling matches qual. the decrease in transmembrane surface exposed to the solvent calcd. from crystallog. data for SERCA structures. (2) Labeling of PMCA incubated with Ca2+ and calmodulin decreases by approx. the same amt. However, incubation with Ca2+ alone increases labeling by more than 50%. Addn. of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [125I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E1 conformations as follows: one that is autoinhibited and is in contact with a higher amt. of lipids (Ca2+ + C28 for SERCA and Ca2+ alone for PMCA), and one in which the enzyme is fully active (Ca2+ for SERCA and Ca2+-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.
- 14Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem. Theory Comput. 2008, 4 (3) 435– 447 DOI: 10.1021/ct700301q[ ACS Full Text], [ CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVSqurc%253D&md5=d53c94901386260221792ea30f151c5fGROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular SimulationHess, Berk; Kutzner, Carsten; van der Spoel, David; Lindahl, ErikJournal of Chemical Theory and Computation (2008), 4 (3), 435-447CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)Mol. simulation is an extremely useful, but computationally very expensive tool for studies of chem. and biomol. systems. Here, we present a new implementation of our mol. simulation toolkit GROMACS which now both achieves extremely high performance on single processors from algorithmic optimizations and hand-coded routines and simultaneously scales very well on parallel machines. The code encompasses a minimal-communication domain decompn. algorithm, full dynamic load balancing, a state-of-the-art parallel constraint solver, and efficient virtual site algorithms that allow removal of hydrogen atom degrees of freedom to enable integration time steps up to 5 fs for atomistic simulations also in parallel. To improve the scaling properties of the common particle mesh Ewald electrostatics algorithms, we have in addn. used a Multiple-Program, Multiple-Data approach, with sep. node domains responsible for direct and reciprocal space interactions. Not only does this combination of algorithms enable extremely long simulations of large systems but also it provides that simulation performance on quite modest nos. of std. cluster nodes.
- 15Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J. Comput. Chem. 1997, 18 (12) 1463– 1472 DOI: 10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.3.CO;2-L[ Crossref], [ CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXlvV2nu7g%253D&md5=890f8af0d2ca1f65aa93db5a3a0bacf2LINCS: a linear constraint solver for molecular simulationsHess, Berk; Bekker, Henk; Berendsen, Herman J. C.; Fraaije, Johannes G. E. M.Journal of Computational Chemistry (1997), 18 (12), 1463-1472CODEN: JCCHDD; ISSN:0192-8651. (Wiley)We present a new LINear Constraint Solver (LINCS) for mol. simulations with bond constraints using the enzyme lysozyme and a 32-residue peptide as test systems. The algorithm is inherently stable, as the constraints themselves are reset instead of derivs. of the constraints, thereby eliminating drift. Although the derivation of the algorithm is presented in terms of matrixes, no matrix matrix multiplications are needed and only the nonzero matrix elements have to be stored, making the method useful for very large mols. At the same accuracy, the LINCS algorithm is 3-4 times faster than the SHAKE algorithm. Parallelization of the algorithm is straightforward.
- 16Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81 (8) 3684 DOI: 10.1063/1.448118[ Crossref], [ CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmtlGksbY%253D&md5=5510dc00297d63b91ee3a7a4ae5aacb1Molecular dynamics with coupling to an external bathBerendsen, H. J. C.; Postma, J. P. M.; Van Gunsteren, W. F.; DiNola, A.; Haak, J. R.Journal of Chemical Physics (1984), 81 (8), 3684-90CODEN: JCPSA6; ISSN:0021-9606.In mol. dynamics (MD) simulations, the need often arises to maintain such parameters as temp. or pressure rather than energy and vol., or to impose gradients for studying transport properties in nonequil. MD. A method is described to realize coupling to an external bath with const. temp. or pressure with adjustable time consts. for the coupling. The method is easily extendable to other variables and to gradients, and can be applied also to polyat. mols. involving internal constraints. The influence of coupling time consts. on dynamical variables is evaluated. A leap-frog algorithm is presented for the general case involving constraints with coupling to both a const. temp. and a const. pressure bath.
- 17Vlahov, I. R.; Wang, Y.; Kleindl, P. J.; Leamon, C. P. Bioorg. Med. Chem. Lett. 2008, 18 (16) 4558– 4561 DOI: 10.1016/j.bmcl.2008.07.041[ Crossref], [ PubMed], [ CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1Slu7s%253D&md5=337a292c01e0d2c817f2ea2f69a35cb0Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part II: Folic acid conjugates of tubulysins and their hydrazidesVlahov, Iontcho R.; Wang, Yu; Kleindl, Paul J.; Leamon, Christopher P.Bioorganic & Medicinal Chemistry Letters (2008), 18 (16), 4558-4561CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)Efficient syntheses of folate conjugates of tubulysins and their hydrazides are described. These water sol. folate receptor (FR) targeted conjugates are derivs. of folic acid and the potent cytotoxic agents: tubulysin A, B, or their resp. hydrazides, connected in regioselective manner via a hydrophilic peptide spacer and a reducible disulfide linker.
- 18Leamon, C. P.; Reddy, J. A.; Vlahov, I. R.; Westrick, E.; Parker, N.; Nicoson, J. S.; Vetzel, M. Int. J. Cancer 2007, 121 (7) 1585– 1592 DOI: 10.1002/ijc.22853[ Crossref], [ PubMed], [ CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVSns7zI&md5=3a7b06fc117fca9d88730624d2ce9298Comparative preclinical activity of the folate-targeted Vinca alkaloid conjugates EC140 and EC145Leamon, Christopher P.; Reddy, Joseph A.; Vlahov, Iontcho R.; Westrick, Elaine; Parker, Nikki; Nicoson, Jeffrey S.; Vetzel, MarilynnInternational Journal of Cancer (2007), 121 (7), 1585-1592CODEN: IJCNAW; ISSN:0020-7136. (Wiley-Liss, Inc.)EC140 is a water sol. folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), which is constructed with an endosome-cleavable acyl hydrazone bond. This agent has proven to be active and specific against well established, s.c. folate receptor (FR)-pos. tumors in multiple animal models. Recent structure-activity and optimization studies have yielded a disulfide bond-contg. counterpart to EC140, herein referred to as EC145. This new conjugate was found to retain high affinity for FR-pos. cells, and it produced specific, dose-responsive activity in vitro. Comparative in vivo efficacy tests confirmed that, like EC140, EC145 displays activity against both syngeneic and xenograft tumor models. However, EC145 was found to be more active and better tolerated than EC140; hence, more durable complete responses were consistently obsd. in EC145-treated tumor-bearing animals. Furthermore, EC145 was not found to be active against a FR-neg. tumor model. Addnl. preclin. studies are therefore warranted to better understand EC145's breadth of activity against FR-pos. tumors.
- 19Durrer, P.; Galli, C.; Hoenke, S.; Corti, C.; Glück, R.; Vorherr, T.; Brunner, J. J. Biol. Chem. 1996, 271 (23) 13417– 13421 DOI: 10.1074/jbc.271.23.13417[ Crossref], [ PubMed], [ CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjsVKqtL4%253D&md5=3bba58b02f7917851b562ec6df9321ceH+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil regionDurrer, Peter; Galli, Carmela; Hoenke, Stefan; Corti, Chantal; Glueck, Reinhard; Vorherr, Thomas; Brunner, JosefJournal of Biological Chemistry (1996), 271 (23), 13417-13421CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Fusion of influenza virus with target membranes is induced by acid and involves complex changes in the viral envelope protein hemagglutinin (HA). In a first, kinetically distinct step, the HA polypeptide chain 2 (HA2) is inserted into the target membrane bilayer. Using hydrophobic photolabeling with the phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, we identified the segment within HA2 that interacts with the membrane. The sole part of the HA2 ectodomain that was labeled with the membrane-restricted reagent is the NH2-terminal fusion peptide (residues 1-22). No labeling occurred within the long coiled coil region generated during the acid-induced conformational transition (Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994) Nature 371, 37-43). These data strongly suggest that the coiled coil region of HA2 does not insert into the lipid bilayer. This conclusion is at variance with a recent suggestion that the coiled coil of HA may splay apart and insert into the target membrane, providing a mechanism by which the viral and the target membrane may come in close apposition.
- 20Giraldo, A. M. V.; Castello, P. R.; Flecha, F. L. G.; Moeller, J. V.; Delfino, J. M.; Rossi, J. P. F. C. FEBS Lett. 2006, 580 (2) 607– 612 DOI: 10.1016/j.febslet.2005.12.078[ Crossref], [ PubMed], [ CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmslWkuw%253D%253D&md5=a44be6bd229ea2794eac137b5192c030Stoichiometry of lipid-protein interaction assessed by hydrophobic photolabelingGiraldo, Ana Maria Villamil; Castello, Pablo Raul; Flecha, F. Luis Gonzalez; Moeller, Jesper V.; Delfino, Jose Maria; Rossi, Juan Pablo F. C.FEBS Letters (2006), 580 (2), 607-612CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)Here the authors undertook a comparative study of the compn. of the lipid annulus of three ATPases pertaining to the P-type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K-ATPase. The photoactivatable phosphatidylcholine analog [125I]TID-PC/16 was incorporated into mixts. of dimyristoylphosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C12E10. After photolysis, the extent of the labeling reaction was assessed to det. the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na,K-ATPase (α-subunit) and 5.6 mol PC/mol protein for the Na,K-ATPase (β-subunit).
- 21Mangialavori, I. C.; Caride, A. J.; Rossi, R. C.; Rossi, J. P. F. C.; Strehler, E. E. Curr. Chem. Biol. 2011, 5 (2) 118– 129 DOI: 10.2174/2212796811105020118[ Crossref], [ PubMed], [ CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXltF2mtLs%253D&md5=e53f36e77d9391e9aab43d070bf00695Diving into the lipid bilayer to investigate the transmembrane organization and conformational state transitions of P-type ion ATPasesMangialavori, Irene C.; Caride, Ariel J.; Rossi, Rolando C.; Rossi, Juan Pablo F. C.; Strehler, Emanuel E.Current Chemical Biology (2011), 5 (2), 118-129CODEN: CCBUB2; ISSN:1872-3136. (Bentham Science Publishers Ltd.)A review. Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca2+ pump (SERCA) revealing different conformations, and recently, crystal structures of H+-ATPase and (Na+,K+)-ATPase were reported as well. However, there are no at. resoln. structures for other P-type ATPases including the plasma membrane Ca2+-pump ATPase (PMCA), which is integral to cellular Ca2+ signaling. Crystn. of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystn. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an anal. of the lipid-protein interactions. Here, the authors review recent efforts using different hydrophobic photolabeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different exptl. conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in PMCA, (Na+,K+)-ATPase, and other P-type ATPases.
- 22Weitman, S. D.; Lark, R. H.; Coney, L. R.; Fort, D. W.; Frasca, V.; Zurawski, V. R., Jr.; Kamen, B. A. Cancer Res. 1992, 52 (12) 3396– 401[ PubMed], [ CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XksVGltLo%253D&md5=76668b6af4b0f1e89c3180affcb040b5Distribution of the folate receptor GP38 in normal and malignant cell lines and tissuesWeitman, Steven D.; Lark, Richard H.; Coney, Leslie R.; Fort, Daniel W.; Frasca, Verna; Zurawski, Vincent R., Jr.; Kamen, Barton A.Cancer Research (1992), 52 (12), 3396-401CODEN: CNREA8; ISSN:0008-5472.In some epithelial cells studied in vitro a membrane-bound folate receptor initiates the process for cell accumulation of 5-methyltetrahydrofolic acid. This receptor was found to be GP38, an overexpressed, glycosyl-phosphatidylinositol anchored glycoprotein, recognized by two monoclonal antibodies, designated MOv18 and MOv19. Using immunoblotting with MOv19, RIA with MOv18 and 19, Northern blot anal., and radioligand binding when possible, the present study describes the limited expression of the folate receptor in a large no. of normal tissues from four autopsies. The immunoblot technique detected as little as 40 pg (≈1 fmol) of receptor protein. Choroid plexus consistently had the largest amt. of folate receptor. Other tissues contg. substantial amts. of receptor included lung, thyroid, and kidney. The liver, intestines, muscle, cerebellum, cerebrum, and spinal cord were immunol. nonreactive. Folate receptor gene expression detd. by Northern blot anal. confirmed these observations. Several malignant cell lines express more receptor than normal epithelial cells or fibroblasts. Specifically, malignant cells bound ≥20 pmol [3H]folate/106 cells, whereas normal epithelial cells and fibroblasts bound ≤1 pmol radioligand/106 cells. Four of six brain tumors overexpressed the folate receptor, a cell surface protein which may be a useful immunol. or pharmacol. target for the development of selective cancer therapy.
- 23Roger, E.; Kalscheuer, S.; Kirtane, A.; Guru, B. R.; Grill, A. E.; Whittum-Hudson, J.; Panyam, J. Mol. Pharmaceutics 2012, 9 (7) 2103– 10 DOI: 10.1021/mp2005388[ ACS Full Text], [ CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XotVWqu74%253D&md5=d43c838229f4ba609b7e3cbb622c0289Folic Acid Functionalized Nanoparticles for Enhanced Oral Drug DeliveryRoger, Emilie; Kalscheuer, Stephen; Kirtane, Ameya; Guru, Bharath Raja; Grill, Alex E.; Whittum-Hudson, Judith; Panyam, JayanthMolecular Pharmaceutics (2012), 9 (7), 2103-2110CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)The oral absorption of drugs that have poor bioavailability can be enhanced by encapsulation in polymeric nanoparticles. Transcellular transport of nanoparticle-encapsulated drug, possibly through transcytosis, is likely the major mechanism through which nanoparticles improve drug absorption. We hypothesized that the cellular uptake and transport of nanoparticles can be further increased by targeting the folate receptors expressed on the intestinal epithelial cells. The objective of this research was to study the effect of folic acid functionalization on transcellular transport of nanoparticle-encapsulated paclitaxel, a chemotherapeutic with poor oral bioavailability. Surface-functionalized poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles loaded with paclitaxel were prepd. by the interfacial activity assisted surface functionalization technique. Transport of paclitaxel-loaded nanoparticles was investigated using Caco-2 cell monolayers as an in vitro model. Caco-2 cells were found to express folate receptor and the drug efflux protein, p-glycoprotein, to high levels. Encapsulation of paclitaxel in PLGA nanoparticles resulted in a 5-fold increase in apparent permeability (Papp) across Caco-2 cells. Functionalization of nanoparticles with folic acid further increased the transport (8-fold higher transport compared to free paclitaxel). Confocal microscopic studies showed that folic acid functionalized nanoparticles were internalized by the cells and that nanoparticles did not have any gross effects on tight junction integrity. In conclusion, our studies indicate that folic acid functionalized nanoparticles have the potential to enhance the oral absorption of drugs with poor oral bioavailability.
- 24Megías, J.; Busserolles, J.; Alcaraz, M. J. Br. J. Pharmacol. 2007, 150 (8) 977– 986 DOI: 10.1038/sj.bjp.0707184[ Crossref], [ PubMed], [ CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkt1Chs7g%253D&md5=8cc37c766b6a9f77257e1150441e3a8dThe carbon monoxide-releasing molecule CORM-2 inhibits the inflammatory response induced by cytokines in Caco-2 cellsMegias, J.; Busserolles, J.; Alcaraz, M. J.British Journal of Pharmacology (2007), 150 (8), 977-986CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)Recent evidence indicates that carbon monoxide-releasing mols. (CO-RMs) exhibit potential anti-inflammatory properties. Here, the authors have investigated whether tricarbonyl dichloro ruthenium(II) dimer (CORM-2) can control the inflammatory response induced by cytokines in a human colonic epithelial cell line, Caco-2. Caco-2 cells were preincubated with CORM-2 for 30 min and then stimulated with interleukin (IL)-1β, tumor necrosis factor-α, and interferon-γ for different times. Gene expression was analyzed by real-time PCR. Protein expression was investigated by Western blot and ELISA. Transcription factor activation was detd. by the luciferase method. The authors show that CORM-2 decreased the mRNA expression of nitric oxide synthase-2 (NOS-2) and the prodn. of nitrite, in Caco-2 cells stimulated with cytokines. IL-8, IL-6, and metalloproteinase-7 (MMP-7) mRNA and protein were also reduced by CORM-2. Time-course and small interfering RNA studies suggest that inhibition of IL-6 plays a role in the regulation of MMP-7 expression by CORM-2. These effects of CORM-2 can be dependent on the modulation of nuclear factor-κB (NF-κB), activator protein-1, CCAT/enhancer binding protein and the phosphorylated forms of NF-κB inhibitory protein-α, c-Jun N-terminal protein kinase 1/2, p38 and extracellular signal-regulated kinase 1/2. Thus, CORM-2 can regulate a no. of genes relevant in intestinal inflammation and cancer progression. These findings provide new insights into the anti-inflammatory properties and potential applications of this class of compds.
- 25Nogueira, E.; Lager, F.; Le Roux, D.; Nogueira, P.; Freitas, J.; Charvet, C.; Renault, G.; Loureiro, A.; Almeida, C. R.; Ohradanova-Repic, A.; Machacek, C.; Bernardes, G. J. L.; Moreira, A.; Stockinger, H.; Burnet, M.; Carmo, A. M.; Gomes, A. C.; Preto, A.; Bismuth, G.; Cavaco-Paulo, A. J. Biomed. Nanotechnol. 2015, 11, 2243– 2252 DOI: 10.1166/jbn.2015.2170[ Crossref], [ PubMed], [ CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XkslOnuw%253D%253D&md5=1544f7334fcd8bc0d255473e60724fa6Enhancing methotrexate tolerance with folate tagged liposomes in arthritic miceNogueira, Eugenia; Lager, Franck; Le Roux, Delphine; Nogueira, Patricia; Freitas, Jaime; Charvet, Celine; Renault, Gilles; Loureiro, Ana; Almeida, Catarina R.; Ohradanova-Repic, Anna; Machacek, Christian; Bernardes, Goncalo J. L.; Moreira, Alexandra; Stockinger, Hannes; Burnet, Michael; Carmo, Alexandre M.; Gomes, Andreia C.; Preto, Ana; Bismuth, Georges; Cavaco-Paulo, ArturJournal of Biomedical Nanotechnology (2015), 11 (12), 2243-2252CODEN: JBNOAB; ISSN:1550-7033. (American Scientific Publishers)Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biol. therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor β present at the surface of activated macrophages, key effector cells in this pathol. The specificity of our liposomal formulation to target folate receptor β was investigated both in vitro as in vivo using a mouse model of arthritis (colalgen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor β. These liposomal formulations also significantly increase the clin. benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance.
- 26Gabizon, A.; Tzemach, D.; Gorin, J.; Mak, L.; Amitay, Y.; Shmeeda, H.; Zalipsky, S. Cancer Chemother. Pharmacol. 2010, 66 (1) 43– 52 DOI: 10.1007/s00280-009-1132-4[ Crossref], [ PubMed], [ CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkslaltLY%253D&md5=6f519aaa32603eebd767f460ef77d01aImproved therapeutic activity of folate-targeted liposomal doxorubicin in folate receptor-expressing tumor modelsGabizon, Alberto; Tzemach, Dina; Gorin, Jenny; Mak, Lidia; Amitay, Yasmine; Shmeeda, Hilary; Zalipsky, SamuelCancer Chemotherapy and Pharmacology (2010), 66 (1), 43-52CODEN: CCPHDZ; ISSN:0344-5704. (Springer)Purpose The folate receptor (FR) is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anti-cancer agents to FR-expressing tumors. Targeting liposomes to the FR has been proposed as a way to enhance the effects of liposome-based chemotherapy. Methods Folate-polyethylene glycol-distearoyl-phosphatidyl-ethanolamine conjugate was inserted into pegylated liposomal doxorubicin (PLD). The therapeutic activity of folate-targeted (FT-PLD) and non-targeted (PLD) pegylated liposomal doxorubicin was tested in two human tumor models (KB, KB-V) and in one mouse ascitic tumor model (FR-expressing J6456) by the i.v. systemic route in all models, and by the i.p. intracavitary route in the ascitic tumor model only. Results Consistent with previous studies, PLD was clearly superior to free doxorubicin in all tumor models. When targeted and non-targeted liposome formulations were compared, FT-PLD was more effective than PLD in the KB and KB-V xenograft models, and in the J6456 intra-cavitary therapy model. The therapeutic effect was dose-dependent in the KB model and schedule-dependent in the J6456 intra-cavitary therapy model. In some expts., toxic deaths aggravated by folate-depleted diet were a major confounding factor. In a non-FR expressing J6456 model, FT-PLD was as active as PLD indicating that its activity is not limited to FR-expressing tumors. Conclusion Folate-targeting confers a significant albeit modest therapeutic improvement to PLD in FR-expressing tumor models, which appears particularly valuable in intracavitary therapy. The potential clin. added value of this approach has yet to be detd.
- 27Chan, E. S. L.; Cronstein, B. N. Nat. Rev. Rheumatol. 2010, 6 (3) 175– 178 DOI: 10.1038/nrrheum.2010.5[ Crossref], [ PubMed], [ CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXis1ygt7w%253D&md5=ff7c199b2a9a3d86f96b90a2a639d80eMethotrexate-how does it really work?Chan, Edwin S. L.; Cronstein, Bruce N.Nature Reviews Rheumatology (2010), 6 (3), 175-178CODEN: NRRACB; ISSN:1759-4790. (Nature Publishing Group)A review. Methotrexate is a proven and efficacious therapy for inflammatory diseases such as rheumatoid arthritis. The main mechanism of action of methotrexate as an anti-cancer drug, at high doses, involves folate antagonism, but what other mechanisms might be operative in the use of this drug at lower doses as an effective anti-inflammatory agent. Methotrexate remains a cornerstone in the treatment of rheumatoid arthritis and other rheumatic diseases. Folate antagonism is known to contribute to the antiproliferative effects that are important in the action of methotrexate against malignant diseases, but concomitant administration of folic or folinic acid does not diminish the anti-inflammatory potential of this agent, which suggests that other mechanisms of action might be operative. Although no single mechanism is sufficient to account for all the anti-inflammatory activities of methotrexate, the release of adenosine from cells has been demonstrated both in vitro and in vivo. Methotrexate might also confer anti-inflammatory properties through the inhibition of polyamines. The biol. effects on inflammation assocd. with adenosine release have provided insight into how methotrexate exerts its effects against inflammatory diseases and at the same time causes some of its well-known adverse effects. These activities contribute to the complex and multifaceted mechanisms that make methotrexate efficacious in the treatment of inflammatory disorders.
- 28Genestier, L.; Paillot, R.; Quemeneur, L.; Izeradjene, K.; Revillard, J.-P. Immunopharmacology 2000, 47 (2–3) 247– 257 DOI: 10.1016/S0162-3109(00)00189-2[ Crossref], [ PubMed], [ CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXktlKktL8%253D&md5=ea8dcba9e8a0485feb42a058bd14f36fMechanisms of action of methotrexateGenestier, L.; Paillot, R.; Quemeneur, L.; Izeradjene, K.; Revillard, J.-P.Immunopharmacology (2000), 47 (2-3), 247-257CODEN: IMMUDP; ISSN:0162-3109. (Elsevier Science B.V.)A review with ∼110 refs. This review, although not exhaustive, focuses on the most recent or significant mechanisms of action of methotrexate (MTX) as an anti-inflammatory and an immunosuppressive drug.
- 29Kısmet, K.; Akay, M. T.; Abbasoǧlu, O.; Ercan, A. Cancer Detect. Prev. 2004, 28 (2) 127– 142 DOI: 10.1016/j.cdp.2003.12.005[ Crossref], [ PubMed], [ CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkt12jtLs%253D&md5=f15c5fa16f8d152d7b6e0459fcba21e4Celecoxib: A potent cyclooxygenase-2 inhibitor in cancer preventionKismet, Kemal; Akay, M. Turan; Abbasoglu, Osman; Ercan, AyguenCancer Detection and Prevention (2004), 28 (2), 127-142CODEN: CDPRD4; ISSN:0361-090X. (Elsevier Science B.V.)A review. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used therapeutic agents in the treatment of pain, inflammation and fever. They may also have a role in the management of cancer prevention, Alzheimer's disease and prophylaxis against cardiovascular disease. These drugs act primarily by inhibiting cyclooxygenase enzyme, which has two isoforms, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Selective COX-2 inhibitors provide potent anti-inflammatory and analgesic effects without the side effects of gastric and renal toxicity and inhibition of platelet function. Celecoxib is a potent COX-2 inhibitor being developed for the treatment of rheumatoid arthritis and osteoarthritis. Chemoprevention is the use of pharmacol. or natural agents to prevent, suppress, interrupt or reverse the process of carcinogenesis. For this purpose, celecoxib is being used for different cancer types. The effects of NSAIDs on tumor growth remain unclear, but are most likely to be multifocal. In this article, the authors reviewed COX-2 selectivity, the pharmacol. properties of celecoxib, the use of celecoxib for cancer prevention and the mechanisms of chemoprevention.
- 30Cui, W.; Yu, C.-H.; Hu, K.-Q. Clin. Cancer Res. 2005, 11 (22) 8213– 8221 DOI: 10.1158/1078-0432.CCR-05-1044[ Crossref], [ PubMed], [ CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1entr3I&md5=d9848d42856040f8d5ecd98637ad2d35In vitro and in vivo effects and mechanisms of celecoxib-induced growth inhibition of human hepatocellular carcinoma cellsCui, Wei; Yu, Chang-Hong; Hu, Ke-QinClinical Cancer Research (2005), 11 (22), 8213-8221CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)Cyclooxygenase-2 (COX-2) inhibitors cause growth inhibition of human hepatocellular carcinoma cells but it remains unclear whether this is both COX-2 dependent and independent. The related mechanisms remain to be detd. The present study was aimed to det. the effect of celecoxib on growth of hepatocellular carcinoma cells and xenografts and the related mechanisms. Both low COX-2 expressing PLC/PRF/5 and high COX-2 expressing HuH7 cells, and nude mice bearing hepatocellular carcinoma xenografts were used to study the effect and mechanisms of celecoxib on hepatocellular carcinoma cell growth. Celecoxib resulted in a comparable growth inhibition of both hepatocellular carcinoma cells that was assocd. with decreased prodn. of prostaglandin E2 and increased peroxisome proliferator-activated receptor γ in both cells. Addn. of prostaglandin E2 only partially counteracted the effect of celecoxib on both cells. Celecoxib resulted in a significant redn. of retinoblastoma phosphorylation and DP1/E2F1 complex in both cells. Celecoxib caused a significant increase of apoptosis and activation of caspase-3 and caspase-9 in both cells. In nude mice inoculated with HuH7 cells, celecoxib resulted in decreased frequency and mean wt. of hepatocellular carcinoma xenografts. The present study showed that celecoxib causes COX-2-dependent and COX-2-independent growth inhibition of hepatocellular carcinoma cells and xenografts by (a) decreased retinoblastoma phosphorylation and DP1/E2F1 complex; (b) increased activation of caspase-3 and caspase-9; and (c) increased expression of proliferator-activated receptor γ. The present study significantly extended our knowledge on the effect and mechanisms of celecoxib-induced inhibition of hepatocellular carcinoma cell growth.
- 31Zhao, S.-p.; Deng, P.; Huang, H.-g.; Xu, Z.-m.; Dai, H.-y.; Hong, S.-c.; Yang, J.; Zhou, H.-n. Clin. Chem. 2005, 51 (11) 2170– 2173 DOI: 10.1373/clinchem.2005.054288[ Crossref], [ PubMed], [ CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFyht7fN&md5=637390a9e410de1782b415b2ff9f1830Expression of COX-2 mRNA in peripheral blood monocytes from patients with acute myocardial infarction and its significanceZhao, Shui-ping; Deng, Ping; Huang, Hong-guang; Xu, Zhu-mei; Dai, Hai-ying; Hong, Shao-cai; Yang, Jun; Zhou, Hong-nianClinical Chemistry (Washington, DC, United States) (2005), 51 (11), 2170-2173CODEN: CLCHAU; ISSN:0009-9147. (American Association for Clinical Chemistry)In this study, cyclooxygenase-2 (COX-2) mRNA expression was measured in peripheral blood monocytes from patients with acute myocardial infarction (AMI) and it was investigated whether COX-2-specific inhibitor, celecoxib, can lower the secretion of interleukin (IL)-6 and matrix metalloproteinase (MMP)-9. Forty-patients with AMI and 18 individuals with stable coronary heart disease of similar age for the study were recruited. AMI was defined as a history of ischemic chest pain > 30 min, characteristic electrocardiog. changes, and increased cardiac troponin I at least twice the upper limit of normal within 24 h after the onset of pain. Results showed an increased COX-2 expression in peripheral blood monocytes from patients with AMI, and the correlation of COX-2 expression with IL-6 and MMP-9 secretion by monocytes was also strong. These results support the hypothesis of an acute inflammatory response to AMI that, at least in part, is correlated with COX-2 activation in peripheral blood monocytes. It also measured the secretion of IL-6 and MMP-9 by monocytes incubated with different concns. of celecoxib and found that celecoxib significantly reduced the in vitro secretion of IL-6 and MMP-9 in a concn.-dependent manner. These data suggest that COX-2 expression may promote secretion of IL-6 and MMP-9 by monocytes and that celecoxib might reduce acute atherosclerotic inflammation partly by inhibiting secretion of the inflammatory cytokines IL-6 and MMP-9 by peripheral blood monocytes.
- 32Zhang, G. S.; Liu, D. S.; Dai, C. W.; Li, R. J. Am. J. Hematol. 2006, 81 (4) 242– 55 DOI: 10.1002/ajh.20542[ Crossref], [ PubMed], [ CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XktVGrt7s%253D&md5=654966e6976a2a20e4d91b8d4cfdb13cAntitumor effects of celecoxib on K562 leukemia cells are mediated by cell-cycle arrest, caspase-3 activation, and downregulation of Cox-2 expression and are synergistic with hydroxyurea or imatinibZhang, Guang-Sen; Liu, Ding-Sheng; Dai, Chong-Wen; Li, Rui-JuanAmerican Journal of Hematology (2006), 81 (4), 242-255CODEN: AJHEDD; ISSN:0361-8609. (Wiley-Liss, Inc.)Celecoxib, a specific cyclooxygenase-2 (Cox-2) inhibitor, has been shown to possess antitumor activity in a variety of cancer cells. However, the antitumor activity of celecoxib in hematopoietic tumors, esp. in chronic myeloid leukemia (CML), has not been well established. This study was designed to investigate the effect of celecoxib on growth and apoptosis in a human CML cell line (K562 cells) or in primary CML cells, and to examine the synergistic actions of celecoxib and hydroxyurea or imatinib on K562 cell proliferation and apoptosis. Celecoxib significantly inhibited the growth of both K562 and primary CML cells and induced apoptosis in a dose-dependent fashion. The IC50 of celecoxib was 46 μM for inhibition of K562 cell proliferation. The effect of celecoxib on growth inhibition was accompanied by the downregulation of cyclin D1 and cyclin E and p-Rb expression, the upregulation of P16INK4a and P27KIP expression, and a G1-S phase arrest of the cell cycle. The pro-apoptotic effect of celecoxib was detd. to be mediated by caspase-3 activation. When K562 cells were pretreated with DEVD-fmk, a specific inhibitor of caspases, the apoptotic activity of celecoxib was, in part, abrogated. Importantly, we demonstrated for the first time that K562 cells were Cox-2-pos. both at the mRNA and protein levels. We noted the following observations: (i) we detected Cox-2 mRNA in K562 cells by reverse transcription-PCR (RT-PCR) and protein expression by western blot anal.; (ii) Cox-2 expression in K562 cells was stimulated by IL-1β, a specific inducing agent of Cox-2 expression; (iii) primary CML cells from CML patient bone marrow also exhibited Cox-2 protein expression. Furthermore, Cox-2 expression was downregulated at higher doses of celecoxib (80-160 μM), suggesting a Cox-2-dependent mechanism was involved in the drug's effects of growth inhibition and induction of apoptosis. In addn., a synergistic effect was obsd. when cells were exposed to low-dose celecoxib (40 μM) and hydroxyurea (10 mM) or a combination of celecoxib (40 μM) and imatinib (0.2 μM). These findings provide the basis for uncovering the mechanism of celecoxib's antitumor effects and developing a new therapeutic strategy for treating CML.
- 33Dai, Z.-J.; Ma, X.-B.; Kang, H.-F.; Gao, J.; Min, W.-L.; Guan, H.-T.; Diao, Y.; Lu, W.-F.; Wang, X.-J. Cancer Cell Int. 2012, 12 (1) 53 DOI: 10.1186/1475-2867-12-53[ Crossref], [ PubMed], [ CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjslCjtbg%253D&md5=c522988fbfe16b58c344d60c46069e90Antitumor activity of the selective cyclooxygenase-2 inhibitor, celecoxib, on breast cancer in Vitro and in VivoDai, Zhi-Jun; Ma, Xiao-Bin; Kang, Hua-Feng; Gao, Jie; Min, Wei-Li; Guan, Hai-Tao; Diao, Yan; Lu, Wang-Feng; Wang, Xi-JingCancer Cell International (2012), 12 (), 53CODEN: CCIACC; ISSN:1475-2867. (BioMed Central Ltd.)Background: Cyclooxygenase-2(COX-2) promotes carcinogenesis, tumor proliferation, angiogenesis, prevention of apoptosis and immunosuppression. Meanwhile, COX-2 over-expression has been assocd. with tumor behavior and prognosis in several cancers. This study investigated the antitumor effects of the selective COX-2 inhibitor, Celecoxib, on breast cancer in vitro and in vivo. Methods: Human breast cancer MCF-7 and MDA-MB-231 cells were cultured with different concn. (10, 20, 40 μmol/L) of celecoxib after 0-96 h in vitro. MTT assay was used to det. the growth inhibition of breast cancer cells in vitro. The expression of COX-2 on mRNA was measured by real-time quantitive PCR anal. Flow cytometry was performed to analyze the cell cycle of MCF-7 cells. Levels of PGE2 were measured by ELISA method. The in vivo therapeutic effects of celecoxib were detd. using rat breast cancer chem. induced by 7,12-dimethylben anthracene (DMBA). Results: The inhibition of proliferation of both MCF-7 and MDA-MB-231 cells in vitro by celecoxib was observerd in time and dose dependent manner. Celecoxib effectively down-regulated the expression of COX-2. The cell cycle was arrested at G0/G1 and rate of cells in S phase was obviously decreased. Levels of PGE2 were inhibited by Celecoxib. The tumor incidence rate of the celecoxib group was lower than that of the control group. In addn., the tumor latency period of the celecoxib group was longer than that of the control group. Conclusions: Celecoxib inhibited the proliferation of breast cancer cell lines in vitro and prevented the occurrence of rat breast cancer chem. induced by DMBA. Therefore, celecoxib exhibits an antitumor activity and seems to be effective in anti-tumor therapy.
- 34Romao, C. C.; Blattler, W. A.; Seixas, J. D.; Bernardes, G. J. L. Chem. Soc. Rev. 2012, 41 (9) 3571– 3583 DOI: 10.1039/c2cs15317c[ Crossref], [ PubMed], [ CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xlt1yntrc%253D&md5=dacf963a490bd088eacaefbf93f2b7b8Developing drug molecules for therapy with carbon monoxideRomao, Carlos C.; Blaettler, Walter A.; Seixas, Joao D.; Bernardes, Goncalo J. L.Chemical Society Reviews (2012), 41 (9), 3571-3583CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. The use of Carbon Monoxide (CO) as a therapeutic agent has already been tested in human clin. trials. Pre-clin., CO gas administration proved beneficial in animal models of various human diseases. However, the use of gaseous CO faces serious obstacles not the least being its well-known toxicity. To fully realize the promise of CO as a therapeutic agent, it is key to find novel avenues for CO delivery to diseased tissues in need of treatment, without concomitant formation of elevated, toxic blood levels of carboxyHb (COHb). CO-releasing mols. (CO-RMs) have the potential to constitute safe treatments if CO release in vivo can be controlled in a spatial and temporal manner. It has already been demonstrated in animals that CO-RMs can release CO and mimic the therapeutic effects of gaseous CO. While demonstrating the principle of treatment with CO-RMs, these first generation compds. are not suitable for human use. This tutorial review summarizes the biol. and chem. behavior of CO, the current status of CO-RM development, and derives principles for the creation of the next generation of CO-RMs for clin. applications in humans.
- 35Kolb, A. F.; Petrie, L. Mol. Immunol. 2013, 54 (2) 164– 172 DOI: 10.1016/j.molimm.2012.11.012[ Crossref], [ PubMed], [ CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhslWkt7k%253D&md5=c9908b8558b8012543daa09b4284a084Folate deficiency enhances the inflammatory response of macrophagesKolb, Andreas F.; Petrie, LindaMolecular Immunology (2013), 54 (2), 164-172CODEN: MOIMD5; ISSN:0161-5890. (Elsevier)B-vitamin deficiency is a risk factor for vascular disease. The mechanism by which the deficiency impacts on disease risk is unclear. We have analyzed whether the inflammatory response of mononuclear cells can be modified by cellular folate status in vitro. We show that the mouse monocyte cell line RAW264.7 grown under folate restriction displays a decrease in intracellular folate levels and a reduced growth rate. The cells also show a 2- to 3-fold increase in expression of the inflammatory mediators, IL1β, IL6, TNFα and MCP1 at the RNA and protein level (p < 0.01) under conditions of folate deficiency. In contrast the prodn. of the vaso-protective mediator nitric oxide is significantly reduced under these conditions. These metabolic changes are independent of the concn. of homocysteine in the medium and occur in the absence of significant changes in global DNA methylation. Folate deficiency may therefore exacerbate cardiovascular disease by augmenting pro-inflammatory signals in the monocyte-macrophage lineage.
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ARTICLE SECTIONSData of SP-DS3 characterization by analytical high-performance liquid chromatography and mass spectrometry are provided as Supporting Information. Furthermore, physicochemical characterization, cell viability, hemolytic properties and specificity of liposomal formulations, as well the biological effect of liposomes encorporating CORM-2 are included. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00823.
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