Exploration of Antiproliferative Activity and Apoptosis Induction of New Nickel(II) Complexes Encompassing Carbazole Ligands

To attest the effectiveness of nickel complexes as anticancer drug candidates with minimum side effects, the present investigation describes the facile synthesis and anticancer activities of nickel(II) complexes enriched with three derivatives of carbazolone-based benzhydrazone ligands(L) having a [Ni(L)2] composition. Analytical and spectral techniques were used to characterize the synthesized Ni(II) complexes. The single-crystal X-ray diffraction performed for complex 4 confirmed the square planar geometry with a [Ni(κ2-N,O-L)2] arrangement. The MTT assay was carried out for the complexes to determine in vitro cytotoxicity against cancerous human-cervical carcinoma, human-colon carcinoma, and non-cancerous L929 (fibroblast) cells. All three complexes exhibited good toxicity against the cancer cells with a low IC50 concentration. Complex 4, containing −OCH3 fragment, exhibits high lipophilicity and revealed exceptional cytotoxicity against cancer cells. AO-EB fluorescent staining indicated apoptosis-associated cell morphological changes after exposure to complex 4. The apoptosis induction was further confirmed by a HOECHST-33342 fluorescent staining technique via chromosomal condensation and nuclear fragmentation. Further, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) mechanistic studies revealed that complex 4 can raise ROS levels and reduce MMP and promote mitochondrial dysfunction-mediated apoptotic cell death. Further, stimulation of late apoptosis by complex 4 in cervical cancer cells was quantitatively differentiated through the staining of phosphatidylserine externalization by flow cytometry. Furthermore, the ELISA analysis confirmed that complex 4 induced apoptosis through caspase activation.

Best commercial grade reactants and solvents were used for all the reactions.
Nickel(II) acetate tetrahydrate and benzhydrazide derivatives were purchased from Merck and Aldrich Chemicals and used as received. 2,3,4,9-tetrahydro-1H-carbazol-1-one was prepared from the procedures as specified in the literature. 1  single crystal of suitable size was covered with Paratone oil, mounted on the top of a glass fibre, and transferred to a Bruker AXS Kappa APEX II single crystal X-ray diffractometer using monochromated MoK α radiation (λ = 0.71073). Data were collected at 293 K. The structure was solved by direct methods using SIR-97 and was refined by the full matrix least-squares method on F2 with SHELXL-97. 2 Nonhydrogen atoms were refined with anisotropy thermal parameters. All hydrogen atoms were geometrically fixed and collected to refine using a riding model. Frame integration and data reduction were performed using the Bruker SAINT Plus (Version 7.06a) software. The multi-scan absorption corrections were applied to the data using SADABS software. 3 Figure 3 was drawn with ORTEP and the structural data have been deposited at the Cambridge Crystallographic Data Centre: CCDC 2113259. S3

Experimental Procedures
Stability studies UV-visible time dependent spectral method has been used to examine the stability of the complexes. Complexes were dissolved in a minimum amount of 1% DMSO and then diluted with PBS buffer to 1×10 −3 M concentration. The hydrolysis profiles of the complexes were monitored by their electronic spectra over 72 h.

MTT assay
The monolayer cell culture has been trypsinized and the cell count was altered to 1.0 x 10 5 cells/mL using respective media comprising 10% FBS. 100 µL of the diluted cell suspension (50,000 cells/well) was added to each well of the 96 well microtiter plate. When a partial monolayer formed after 24 hours, the supernatant was swiped off and the monolayer has been washed with the medium. 100 µL of various concentrations of complexes have been added on to the partial monolayer in microtiter plates. Then, the plates have been incubated at 37 o C for 24 hours in 5% carbon dioxide atmosphere. After incubation, the test solutions in the wells have been discarded and 100 µL of MTT (5 mg/10 ml of MTT in PBS) was added to each well. The plates have been incubated for four hours at 37 o C in 5% carbon dioxide atmosphere. The supernatant has been removed and 100 µL of DMSO was applied to dissolve the formed formazan. The absorbance was recorded using a microplate reader at 590 nm. The % growth inhibition has been determined using the

Reactive Oxygen Species (ROS) Assay
To quantify the intracellular ROS, 5 × 10 5 HeLa cells have been seeded on a 6-well plate comprising cover slip and incubated overnight for attachment. After incubation, the cells have been treated using fresh medium with IC 50 concentration of the complex 4 and incubated further for 24 hours. After that, the cover slip has been removed from the culture plate, and stained with 40 µM of 2',7'-dichlorofluorescein-diacetate (DCFHDA) dye for half an hour. The stained cover slip has been rinsed using PBS solution and imaged under fluorescence microscope.

Mitochondrial Membrane Potential (MMP)
Culture cells on cover slips in 6-well cell culture plates at a density around 5 x 10 5 cells/mL overnight in an incubator (5% carbon dioxide, 37 °C). Complex 4 (IC 50 μM) has been incubated with HeLa cells for 24 hours.

Annexin V-FITC/PI staining by flow cytometry method
HeLa ells have been seeded in a 6-well plate (10 5 cells/well)) and cultured at 37 °C for 24 hours. The cells have been treated with complex 4 (IC 50 concentration) and incubated for 24 hours. Then, the cells were trypsinized, washed with PBS and stained with annexin V-FITC/PI according to the annexin V-FITC apoptosis detection kit. Finally, apoptosis induction has been assessed using a flow cytometer (SYSMEX, Japan), and the data have been analyzed by Flow Jo software. The cells that were not treated were employed as a control group.
About 50 ml of supernatant was incubated with specific substrate (at 37C) for 2 h in a water bath. The absorbance of the cleaved substrate was measured at 405 nm using a microtiter plate reader (BioRad, UK).