Enhancing Therapeutic Efficacy in Cancer Treatment: Integrating Nanomedicine with Autophagy Inhibition Strategies

The complicated stepwise lysosomal degradation process known as autophagy is in charge of destroying and eliminating damaged organelles and defective cytoplasmic components. This mechanism promotes metabolic adaptability and nutrition recycling. Autophagy functions as a quality control mechanism in cells that support homeostasis and redox balance under normal circumstances. However, the role of autophagy in cancer is controversial because, mostly depending on the stage of the tumor, it may either suppress or support the disease. While autophagy delays the onset of tumors and slows the dissemination of cancer in the early stages of tumorigenesis, numerous studies demonstrate that autophagy promotes the development and spread of tumors as well as the evolution and development of resistance to several anticancer drugs in advanced cancer stages. In this Review, we primarily emphasize the therapeutic role of autophagy inhibition in improving the treatment of multiple cancers and give a broad overview of how its inhibition modulates cancer responses. There have been various attempts to inhibit autophagy, including the use of autophagy inhibitor drugs, gene silencing therapy (RNA interference), and nanoparticles. In this Review, all these topics are thoroughly covered and illustrated by recent studies and field investigations.


INTRODUCTION
1.1.What Is Autophagy?Autophagy is a complex stepwise lysosomal degradation process that is responsible for degrading and removing dysfunctional cytoplasmic materials and damaged organelles, which supports nutrient recycling and metabolic adaptation.Autophagy represents a solid tool that cells employ in order to escape stress, being known as a process that regulates cancer. 1 There are three different kinds of autophagy classified: microautophagy, chaperone-mediated autophagy and macroautophagy, with all focus being centered on the macroautophagy, which would be referred as autophagy throughout the context. 2 The difference between these subtypes of autophagy mainly depends on the way the cargos are transferred into lysosomes. 3In microautophagy, cargos are simply engulfed and internalized directly into lysosomes as a consequence of lysosomal membrane invaginations, 4 whereas chaperone-mediated autophagy requires a specific recognition of the cargos (proteins) that are going to be delivered to lysosomes, usually being marked with the amino acid motif (KFERQ), by another set of heat shock proteins (HSC70) for the degradation to be processed. 5acroautophagy, the main common pathway of autophagy, is characterized by the formation of multiple membrane structures, starting with phagophores formation that is derived mainly from Golgi complex, endosomes, the endoplasmic reticulum (ER), mitochondria, and the plasma membrane. 6agophores mature into autophagosomes which thoroughly enclose the cargo within and then fuse with lysosomes to form structures called autolysosomes.Inside the autolysosomes, cargos got degraded completely by lysosomal enzymes 5 (Figure 1).
Autophagy represents an intricate pathway controlled by the expression of almost 20 autophagy-related genes (ATGs), 7 consisting of five distinguished phases; initiation, elongation, closure, maturation, and degradation. 8Among the ATGs, which have been first specified in yeast Saccharomyces cerevisiae some time ago, 9 ATG5, ATG7, ATG12, and the major markers of autophagic activity; Beclin-1, microtubule associated light chain B (LC3B), one of the LC3 isoforms, and p62/SQSTM1 (hereafter p62) are by far the most investigated and associated with various inflammatory disorders 10,11 as well as cancer. 12−14 LC3 protein plays a crucial function in mammalian autophagy 7 since it enables cargo receipt into autophagosomes and has the distinction of being the only protein to exist on these structures, making it a unique marker of autophagosome formation. 15There are two forms of LC3, the cytosolic LC3-I and the membrane-conjugated form, LC3-II. 16Under varied stresses, a modification of LC3-I to LC3-II by conjugating LC3-I with phosphatidylethanolamine (PE) is being stimulated, which leads LC3-II to bind to the membrane of the autophagosome. 17Becline-1, a Bcl-2-homology (BH)-3only protein found largely in cytoplasmic structures, regulates autophagosome production (size and number) through being involved in class III phosphoinositol 3 phosphate kinase (PI3K) complex. 18Beclin-1, considered as a necessary component for the initiation of autophagy, takes part in the very early stage of autophagosome formation (nucleation phase). 19p62 is an autophagy adapter protein that interacts with ubiquitinated cargo and LC3B to carry out autophagic degradation.In this process, p62 is preferentially taken up by autophagosomes and digested by autolysosomes.As a result, decreased levels of p62 are linked to an active autophagy pathway. 20utophagy is a housekeeping process.Under normal conditions, autophagy serves as a quality control mechanism in cells, helping to maintain homeostasis and redox balance by digesting nonfunctional proteins and damaged organelles, collaborating with the adaptive immune system, 21,22 and enabling survival during malnutrition situations by recycling cellular elements to regenerate energy. 8However, in cases of autophagy failure in immunologic ailments and neurodegenerative illnesses such as Alzheimer's and Parkinson's disease, aberrant, malfunctioning proteins and organelles accumulate, leading to the development of disease. 23,24However, in abnormal conditions like cancer, its role is complex, acting as a double-edged sword, as will be illustrated in the next section. 25,26.2.Role of Autophagy in Cancer.Autophagy is a process that helps cells survive under conditions of metabolic stress, such as nutrient starvation or a hypoxic environment.However, it can also play a role in cell death.The specific way in which autophagy causes cell death is not yet clear; however, when there is high metabolic stress, excessively activated autophagy can result in a type of programmed cell death called autophagic cell death. 27,28Recent studies on the relationship between autophagy and cancer have revealed that autophagy plays a significant role in regulating cancer development and in the response of tumor cells to cancer treatment.However, autophagy can become disrupted or overactivated in cancer cells, which makes it difficult to understand the effects it has on cancer progression and development.
Recent studies have shed light on the intricate relationship between autophagy and cancer, unveiling its profound influence on cancer development and response to therapy.However, the role of autophagy in cancer is far from static, marked by dynamic fluctuations that stem from disruptions or overactivation of autophagic processes within cancer cells.
This dynamic interplay complicates our understanding of autophagy's impact on cancer progression, with its effects varying depending on factors such as the type and stage of the cancer, the unique genetic landscape of the tumor, 29 and the level of autophagy activation, 30 with the tumor stage being a primary determinant. 25,26A moderate level of autophagy protects the tumor from an unfavorable external environment and promotes its growth.−33 Focusing on the consideration of the tumor stage, in the earliest stages of tumorigenesis, autophagy acts as a tumor inhibitor by maintaining homeostasis through the clearance of old, nonfunctional proteins and deteriorated organelles, 30 as well as by inhibiting malignant necrosis and inflammatory responses. 34Studies have shown that Beclin-1 is frequently deleted in breast, ovarian, and prostate malignancies, and its absence leads to a decrement in autophagy and promotes cellular proliferation. 35Bif-1 is another protein that regulates autophagy by interacting with Beclin-1, and its knockout Figure 1.Macroautophagy process overview.The figure illustrates the macroautophagy pathway, showcasing the sequential steps from the formation of phagophores, derived from organelles like the Golgi complex, endosomes, ER, mitochondria, and plasma membrane, to their maturation into autophagosomes enclosing cellular cargo, followed by fusion with lysosomes to form autolysosomes, where cargo undergoes degradation by lysosomal enzymes, facilitating cellular recycling and maintenance.inhibits autophagy, leading to an increase in cancer formation. 36Homozygous deletion of ATG5 causes liver cancer in a high-penetrance animal model. 37However, mutations in ATG5 have been discovered in 135 patient samples with gastric cancer, colorectal cancer, and hepatocellular carcinoma. 38owever, in established cancers, a plurality of data sources suggest the ability of autophagy to promote tumor growth, survival, and colonization by reducing DNA damage, maintaining functional mitochondria, dealing with cellular stresses including hypoxia, nutritional deficiency, and anticancer drug treatments, 39−42 and meeting the high metabolic demands of the tumor in terms of nutritional supply. 43,44The rapid proliferation of tumor cells creates a high demand for nutrients.In the limited nutrient environment of the tumor microenvironment, autophagy can promote interaction between the tumor and the matrix, thereby fostering tumor growth. 30A recent investigation reveals that blocking mitophagy, the selective autophagy of mitochondria, could serve as a tactic to bolster the efficacy of cancer therapies.This research demonstrates that when an anticancer medication is paired with liensinine, a substance known to impede mitophagy, the combined treatment yields synergistic effects in eradicating cancerous cells. 45Altogether, while autophagy seems to be critically important in protecting normal cells from tumorigenesis initiation, inhibiting it is favorable when dealing with established and developed cancers 46−48 (Figure 2).This could aid in blocking autophagy in cancer cells, leading to a decrease in tumor growth, less spread of tumors, overcoming resistance to cancer treatments, and activating apoptosis.
The following sections will delve deeper into the specific mechanisms in which autophagy inhibition enhances tumor therapy and will explore various approaches for inhibiting autophagy in cancer treatment.
Multiple sensors and key regulators, including protooncogenes (PI3K, AKT, mammalian Target of Rapamycin (mTOR), RAS and RAF), tumor suppressors (Beclin-1, TP53, FOXO1 and BCL-2), and microRNAs (miRNAs), have been identified in the analysis of autophagy inhibition or induction. 49,50utophagy suppression has been found to cause tumor cell death, 51 such as in breast cancer. 52As an example, in a polyoma middle T-driven model of breast cancer, a considerable delay in both tumor initiation and progression has been noticed by knocking down the Fip200 autophagy gene. 53−56 Using immunohistochemical analysis, it was revealed that autophagy-related genes are highly expressed in triple-negative breast cancer (TNBC) clinical samples. 57It has also been shown that downregulation of the LC3 gene significantly reduced cell viability and blocked the main cell mechanisms: proliferation, invasion, migration, and resistance to apoptosis in TNBC. 58Tumor growth in pancreatic ductal adenocarcinoma (PDAC) was slowed in one study by suppressing autophagy with autophagy inhibitors and gene silencing tools. 50Another finding indicated that inhibition of autophagy leads to decreased tumor growth in PDAC and suggested that autophagy inhibition could be an effective therapeutic strategy for PDAC, independent of the genetic background of the tumor cells. 59everal studies have shown that autophagy contributes to inflammation in the tumor microenvironment and adjacent cells, promoting tumorigenesis.Toll-like receptors (TLRs), known modulators of inflammatory responses, can influence autophagy activity, impacting tumor progression.Studies have shown that TLR4 activation and TLR9 upregulation can induce autophagy and contribute to cell migration, invasion, and proliferation in cancer. 60,61Similarly, the Receptor for Advanced Glycation End-products (RAGE) has been linked to both inflammation and autophagy activation.A study suggests that promoting autophagy via RAGE expression may contribute to cell survival and protection against oxidative stress-induced damage in pancreatic tumor cells. 62dditionally, Several ATGs, such as ATG5, ATG7, and ATG16L1, are directly involved in regulating the production of pro-inflammatory cytokines and reactive oxygen species (ROS). 63−74 Nevertheless, autophagy has also been correlated to tumor metastasis by promoting cancer's aggressiveness. 75,76Because metastatic tumor cells must adapt to a different milieu than the initial site 77 and overcome a number of obstacles in order to develop colonies, there is strong evidence that autophagy plays a significant role in facilitating this type of adaptation. 78,79−83 According to one study, increased expression of LC3B and ATG17 is related to a considerably lower survival time in TNBC patients. 84Also, upregulated expression of ATG7 in breast cancer tissue is highly related with lower overall survival and distant metastasis-free survival in patients. 85Nonetheless, tumoral expression of autophagy-related proteins has been found to be higher in brain metastasis than in primary breast cancers. 86In human breast cancer, greater punctate staining for LC3B was correlated with lymph node metastases and shorter survival. 82,83In hepatocellular carcinoma, LC3B expression was also associated with metastasis, with increased LC3B staining in metastases compared to primary tumors. 76,87Increased expression of an autophagy gene signature was linked to a more aggressive and invasive phenotype in human glioblastoma. 80−92 And once drug resistance takes place, cancer cells aggressiveness and metastasis became inevitable which bring about failure in the treatment and eventual motility. 93−98 For example, retroviral shRNA suppression of the ETS transcription factor ELK3, a signaling protein involved in autophagy activation, decreased autophagy and improved doxorubicin (DOX) responsiveness, one of the most frequently used drugs in treating breast cancer. 99In another study, miR-520b significantly improved DOX sensitivity in hepatocellular carcinoma (HCC) via suppressing ATG7dependent autophagy. 100Yet, blocking autophagy, as indicated by the cell surface molecule CD24, resulted in a significant increase in sorafenib sensitivity, the first US Food and Medicine Administration-approved drug for targeted HCC treatment. 101It has been demonstrated that autophagy suppression increased chemo-sensitivity and promoted apoptosis in cholangiocarcinoma. 102,103utophagy plays a key role in cell death decisions and has the ability to block apoptosis. 104The most likely causes are autophagy's ability to activate mitophagy and diminish the amount of pro-apoptotic proteins in the cytosol. 105For example, when autophagy is inhibited, the pro-apoptotic protein PUMA is elevated. 106Anoikis, or the lack of extracellular matrix (ECM) attachment, causes cancer cells to die through apoptosis.Nevertheless, autophagy has been proven to help ECM-detached cancer cells evade anoikis and survive. 107It has been demonstrated that inhibiting autophagy reduces glycolytic capability, oxidative phosphorylation, and cell proliferation, while also increasing apoptosis in in vitro and in vivo studies. 108,109According to one study, inhibiting autophagic flux in quiescent breast cancer cells increases the cumulating of damaged mitochondria and ROS, leading to apoptosis. 110Another study found that utilizing the lucanthone drug, a possible sensitizer to chemotherapy and radiation, caused lysosomal dysfunction, autophagy suppression, and apoptosis in cancer cells. 111Nonetheless, suppressing autophagy in cancer cells increased the apoptotic effect of paclitaxel (PTX), an apoptosis inducer used to treat lung adenocarcinoma. 112In a recent study, inhibiting autophagy was found to sensitize apoptosis in cancer cells by FOXO3a Turnover, an autophagy-regulating transcription factor. 104ccordingly, great efforts have been made in order to inhibit autophagy as a mechanism for enhancing cancer treatment therapies 113 using different methods in order to accomplish the inhibition including the use of autophagy inhibitor drugs, knockdown critical ATG genes by RNA interference (RNAi) and utilizing nanoparticles. 114,115

TRADITIONAL MEDICINE FOR AUTOPHAGY
Nowadays, there are a variety of drug agents that block autophagy at its various stages, ranging from autophagy induction and autophagosome formation through lysosomal degradation, 116 headed by chloroquine (CQ), hydroxychloroquine (HCQ), 3-methyladenine (3-MA) and Bafilomycin A1 (BafA1), with CQ and HCQ being the most investigated in clinics 117 (Figure 3).−121 CQ and HCQ are the most commonly used medications for chronic inflammatory disorders such rheumatoid arthritis, 122 systemic lupus erythematosus, 123 and sarcoidosis, 124 and have been used to limit the spread of melanoma, pancreatic cancer, and bladder cancer by blocking autophagy. 116,125They have also been used to make cancer cells in chemo-resistant and radio-resistant tumors more susceptible to various treatments. 119,126Q was developed in 1934 and has primarily been used to treat malaria. 127It has been recently employed as an autophagy inhibitor since it has been shown to impede the functionality of lysosomes by causing a stoppage in the fusion of autophagosomes and lysosomes. 128HCQ, a CQ derivative, suppresses autophagy by manipulating the pH of lysosomes, which prevents autophagosome destruction. 129,130Because HCQ is less hazardous than CQ at peak doses, it has been commonly used in clinical trials over CQ. 129,131 CQ has been utilized in glioma cells to see whether there are any synergistic effects that may be produced by mixing Temozolomide, a medicine used to treat brain malignancies, with autophagy inhibitory agents, and the results reveal that inhibiting autophagy with CQ can help to prevent cancer growth. 118urthermore, by inhibiting autophagy, CQ suppressed tumor growth in PDAC mice models. 56HCQ inhibited autophagy in cancer stem-like cells, which influenced HCC cell invasion and migration, as well as EMT, a critical stage in the progression of cancer metastasis. 132The combination of Thymoquinone, a major bioactive ingredient isolated from black cumin, 133 and CQ triggered apoptosis in cancer cells in glioblastoma. 134In one study, treatment with CQ coupled with artemisinin, an element derived from sweet wormwood, 135 induced lung cancer cells to apoptosis in a synergistic manner. 136Combining the effective autophagy inhibitor, mTOR inhibitor, with HCQ increased six-month median progression-free survival (PFS) in breast cancer patients, according to a phase II trial. 137CQ treatment caused apoptosis in HeLa cells, which was determined by DNA breakage. 138However, combining CQ with cisplatin, a chemotherapy treatment, activated apoptosis by suppressing autophagy, as seen by an accumulation of ubiquitinated misfolded intracellular proteins such as Beclin-1 and LC3-II, which increased the drug's efficacy. 12CQ boosted the anticancer effects of 5-fluorouracil (5-FU), cytotoxic chemotherapeutic drug, on human colorectal cancer cells while inhibiting colony forming abilities, as pretreatment with CQ caused cell cycle arrest at phase G0/G1. 119Furthermore, using CQ in combination with a class of cancer drugs such sunitinib, bevacizumab, and oxaliplatin (OX) boosted apoptosis and sensitivity in colon cancer cells under hypoxic circumstances. 120,121In a recent study, HCC cells were treated with a combined treatment of HCQ and sorafenib, which reduced tumor growth, cellular proliferation, migration, and invasiveness by suppressing autophagy and regulating apoptosis, overcoming drug resistance. 139In a recent study, a nanovesicle known as DC-DIV/C was designed to deliver CQ and DOX-HCl simultaneously to cancer cells that were resistant to the drug.Both in vitro and in vivo experiments showed that this codelivery suppressed autophagy and hence enhanced the antitumor effects of the treatment. 140-MA is an autophagy inhibitor that inhibits the development of autophagosomes by altering PI3K, a critical autophagy's arranger.141 It is been proven that suppressing autophagy with 3-MA improves chemotherapeutic medication efficacy in laryngeal cancer.142,143 In a recent study, treating HCC cells, HepG2, with sorafenib and 3-MA at the same time inhibited cell proliferation while also triggering apoptosis.Thus, by suppressing autophagy, MA may be able to reduce drug resistance and improve drug efficiency.144 In the same manner, in colon cancer, OX resistance has been overcome by blocking autophagy.CT26 cells were cotreated with OX and 3-MA, which increased apoptosis and inhibited tumor growth in vivo, as well as extending animal survival time.145 3-MA enhanced Tocomin apoptosis, an available commercial drug with antitumor characteristics made up of vitamin E components, in breast cancer cells. 146Autophagy provides tumor cells with an adaptability to survive under starving situations, as previously indicated.In a recent study, dendritic mesoporous organosilica nanoparticles (DMONs) loaded with glucose oxidase, a starvation inducer, and 3-MA have been created as a method for improving cancer starvation therapies.
Autophagy inhibition has been shown to improve the efficacy of starvation treatment, resulting in cancer progression control. 147In another study, a nanocarrier system has been developed based on the use of functionalized mesoporous silica nanoparticles and Temozolomide, in which its anticancer activity was further increased when combined with 3-MA. 148afA1, a Streptomyces gresius-derived antibiotic, suppresses autophagy by blocking the acidification of endosomes and lysosomes, which prevents their union. 149By suppressing autophagy in colorectal cancer cells, BafA1 may be able to prevent cell cycle transition and increase cell death. 150,151afA1 also stopped prostate cancer cells from spreading by blocking autophagy. 152BafA1 suppressed the growth of TNBC cells and caused an increase in the levels of p62 and the ratio of LC3-II to LC3-I. 153.In tongue squamous cell carcinoma (TSCC), BafA1 suppressed autophagy and increased the susceptibility of the cells to cisplatin. 154The same effect was shown in small cell lung carcinoma, where the use of BafA1 increased cisplatin cytotoxicity. 155In HCC, BafA1 treatment inhibited cellular proliferation, induced cell cycle arrest, prompted Cyclin D1 turnover, and caused caspase-independent apoptosis in both 2-dimensional and 3-dimensional cultures.All of this was caused by autophagy suppression, which was demonstrated by an increase in LC3 conversion. 156n breast cancer, epirubicin cytotoxicity, a commonly used medication to treat breast cancer, was elevated when MDA-MB-231 and SK-BR-3 cell lines were treated with bafilomycin A1. 157 Furthermore, combining RAD001, an mTOR inhibitor, with BafA1 increased mTOR cytotoxicity, decreased cell viability, and promoted apoptosis in bladder cancer cells. 158oreover, treatment with BafA1 improved the cytotoxicity of cytarabine, a chemotherapeutic medication used to treat leukemia in acute myeloid leukemia (AML) cells. 159tudies on autophagic medicines are summarized in Table 1.

DEVELOPED AND PROMISING THERAPY FOR INHIBITION OF AUTOPHAGY
3.1.Gene Silencing Therapy.RNAi, which was first reported in 2006 and involves the use of microRNAs (miRNAs) or small interfering RNAs (siRNAs) to inhibit the expression of certain genes, is swiftly gaining traction as a powerful technique for gene silencing. 160Both of them are short RNA duplexes that contain an average of 21−23 nucleotides. 161Despite the structural and functional similarities between miRNAs and siRNAs, there are some significant distinctions between them.The primary distinction between the two molecules is found in their modes of action. 162While both act by promoting the inhibition of messenger mRNA (mRNA) expression, siRNAs work by cleaving mRNAs, while miRNAs work by blocking mRNA translation into proposed proteins.In addition to their modes of action, they differ in the way they recognize target mRNA.To have an effect, siRNA must be completely complementary to its target mRNA, allowing it to be used to silence a single target gene.For miRNA, however, a partial interaction between miRNA and its target is sufficient to cause it to act.As a result, it might be utilized to block several genes. 162owadays, these technologies are employed to inhibit autophagy by focusing on key ATG genes implicated in the process as a successful method to boost the antitumor response while lowering cancer progression. 1,163.1.1.miRNA Utilization.miRNAs have a critical role in cancer formation, growth, and metastasis regulation, 164,165 being found that about 50% of miRNA genes are located at fragile sites in the genome or in cancer-related regions. 166−175 MicroRNA-30a (miR-30a) has been shown to regulate rapamycin-induced autophagy in lung and breast cancer cells 176 while also inhibiting autophagy in cancer cells via targeting Beclin-1. 176,177For example, miR-30a mimics boosted the sensitivity of imatinib, a medication that inhibits the BCR-ABL tyrosine kinase responsible for chronic myeloid leukemia (CML) in a targeted manner, and promoted mitochondria-dependent intrinsic apoptosis in vitro and in vivo. 115Similarly, by suppressing autophagy, miR-30a enhanced imatinib sensitivity and elevated apoptosis in CML cells by downregulating BECN1 and ATG5 levels. 178urthermore, delivering miR-30a improved cisplatin's ability to induce cell apoptosis by silencing Beclin-1 gene, 177 where comparable findings were obtained by modulating miR-885− 3p. 179In a recent study, it was discovered that blocking autophagy using miR-30a enhanced cisplatin efficiency in ovarian cancer by regulating the activation of the TGF-β/ Smad4 pathway. 180In another study, miR-30a was found to improve nonsmall-cell lung cancer (NSCLC) outcome after neoadjuvant chemotherapy via suppressing autophagy and accelerating NSCLC cell death. 181Nonetheless, in another study it has been discovered that miR-30a elevates prostate cancer radiosensitivity by suppressing autophagy. 182miR-30a-5p could also slow the growth of lung squamous cell carcinoma by downregulating ATG5 and thereby blocking autophagy in vitro and in vivo. 183−186 miR-101 inhibits the Zeste homologue 2 (EZH2) and myeloid cell leukemia-1 (Mcl-1) genes, causing apoptosis to increase, proliferation to decrease, and metastatic tumor growth to be prevented. 184,186Furthermore, miR-101 suppresses the RAB5A, ATG4C, and ATG4D genes, making it a primary autophagy mediator. 187For example, miR-101 boosted the chemo-sensitivity of tamoxifen in breast cancer cells by inhibiting the autophagic pathway, which improved treatment outcomes. 188HCC cells have been shown to be more sensitized to doxorubicin and fluorouracil therapy when autophagy is inhibited by miR-101. 189Another study found that inhibiting autophagy via miR-101 can improve cisplatin cytotoxicity in HCC cells and hence increase apoptosis population. 190NC51-like kinase 1 (ULK1) is one of the ATGs that participates in the formation of autophagosomes, 191 and when it is inhibited, cancer cells exhibit an effective reduction of autophagy and an increase in apoptosis. 192,193For instance, a reduction in the proliferation, migration, invasion, and autophagy of pancreatic cancer has been seen by inhibiting ULK1 via miR-372. 194Recent research found that miR-373 inhibited ULK1, which in turn promoted apoptosis in cholangiocarcinoma cells via suppressing autophagy.LC3-II/ LC3-I value and Beclin-1 protein level were down regulated while p62 protein was markedly raised, indicating an inhibition of autophagy.
Numerous other research in this area have taken use of various miRNAs that inhibit the expression of genes associated with autophagy, hence decreasing autophagy and improving the effectiveness of treatment.In one study, it was demonstrated that by reducing autophagy activity by suppressing ATG12 through miR-23b, radiotherapy effectiveness was improved in pancreatic cancer cells. 13Additionally, miR-29c inhibited autophagy and elevated gemcitabineinduced apoptosis, a cytotoxic drug used to treat some cancers, in pancreatic cancer by regulating the expression of USP22. 195Similar to this, miR-29a decreased autophagic activity and made pancreatic cancer cells more susceptible to gemcitabine therapy. 196Using miR-410-3p to target high mobility group box 1 (HMGB1), a critical autophagy regulator that influences inflammation, tumor cell motility, and metastasis, 197,198 enhanced chemo-sensitivity by preventing cancer cells from inducing autophagy. 199In another study, targeting HMGB1 specifically by miR-34a enhanced cell death and blocked autophagy in AML. 200By targeting ATG7, miR-375 prevented the conversion of LC3-I to LC3-II in HCC cells, which reduced autophagy and the proliferation of cancer cells. 201Another study found that miR-590-5p targets the autophagy protein ATG3 and blocks autophagy, increasing the radiosensitivity of PDAC cells. 202n a recent study, miR-373-3p suppressed autophagy by targeting AKT1 in cervical cancer, which limited the growth of the tumor both in vitro and in vivo. 203Furthermore, a recent study found that miR-338 might target ATF2 via the mTOR signaling pathway to suppress the proliferation and autophagy of cervical cancer cells, indicating the possible use of miR-338 in the treatment of this disease. 204In a another study, miR-373 was used to target the autophagy-upregulated proteins CD44 and TGFBR2 in glioblastoma multiforme (GBM), which prevented the cancer cells' migration and invasion. 205umerous other miRNAs, such as miR-216a, miR-30d, miR-205, miR-199a-5p, and miR-885-3p, 177,179,206−208 have been used to block autophagy and make cancer cells more sensitive to radiation or chemotherapy.Therefore, it is evident that miRNAs may offer a novel treatment strategy for improving the prognosis of cancer patients by blocking autophagy.
3.1.2.siRNA Utilization.siRNA is a potent tool that has been used widely in cancer treatment in both preclinical (in vitro and in vivo) and clinical studies.It has been approved for use in treating adults with hATTR amyloidosis 209 and acute hepatic porphyria 210 under the brand names ONPATTRO (Patisiran) and GIVLAARITM (Givosiran).Delivering siRNAs that specifically target autophagic genes, primarily ATG5 and ATG7, has been advocated in numerous studies as a way to potentially overcome anticancer medication resistance.
In one study, six of the main ATGs, Beclin-1, ATG-3, ATG-4b, ATG-4c, ATG-5, and ATG-12, have been successfully suppressed utilizing particular target-siRNAs.By employing siRNAs to block these genes, autophagy was suppressed, as was seen by a reduced concentration of autophagosomes.Consequently, radiation-resistant cancer cells become increasingly susceptible to it. 211In one study, using siRNA to reduce Beclin-1 increased PARP breakage, mitochondrial membrane depolarization, and cytosolic cytochrome c levels following doxorubicin therapy.Each and every one of them highlighted the considerable increase in cell apoptosis. 212nother study found that suppressing autophagy by utilizing siRNAs against ATG5 and ATG7 blocked autophagy that is being activated by the anticancer drug anthracycline daunorubicin (DNR).This inhibition increased the effectiveness of DNR in treating myeloid leukemia. 213Additionally, it has been demonstrated that the use of sorafenib resulted in the activation of autophagy as proven by an accumulation of autophagosomes in various HCC cell lines.Inhibiting the drugrelated autophagy, as a result, made cancer cells more sensitive to it. 214Similar results were seen when Beclin-1 or ATG-5 siRNA were used to block autophagy in renal cell carcinoma (RCC) cells, increasing the sensitivity of cancer cells to sorafenib. 215Likewise, it has been discovered that linifanib, an effective drug with significant anticancer actions in a variety of solid tumors, 216 induces a high amount of autophagy in HCC cells.In which the apoptosis generated by linifanib was enhanced and its effectiveness was therefore boosted by inhibiting this agent-associated autophagy with ATG5 and ATG7-siRNAs. 217n one study, reducing autophagy with siRNAs that targeted Beclin-1 or ATG-5 improved cancer immunotherapy as shown by a recovery in the sensitivity of hypoxic tumor cells to Cytolytic T lymphocytes (CTL)-mediated lysis.While in vivo results indicated that blocking autophagy increased cellular apoptosis and slowed cancer progression. 218Furthermore, S100A8 gene silencing boosted cancer cell apoptosis and the sensitivity to arsenic trioxide (As2O3), a strong environmental cocarcinogen for various human malignancies.As it was found that S100A8 knockdown caused autophagy suppression, which was made clear by a decrease in LC3-II protein levels. 219n addition, it has been demonstrated that ATG7 siRNA can be used to overcome PTX resistance in HeLa cells by suppressing autophagy. 220A demethylated derivative of cantharidin called norcantharidin (NCTD) shows anticancer properties in a number of malignancies.In one study, it was investigated whether combining autophagy inhibition with NCTD would boost the medication's effectiveness in HepG2 cells.Based on this, autophagy inhibition was carried out using ATG5 siRNA and was verified by a reduction in the expression of the LC3-II protein.Cell apoptosis increased as a result of autophagy suppression, as shown by an increase in the expression of Bax, cytochrome c, cleaved caspase-3, caspase-9, and PARP. 221The same results were attained when human cholangiocarcinoma cells were treated with NCTD and autophagy was prevented by siRNA-mediated downregulation of ATG-5. 222nother study demonstrated that boosting apoptosis by suppressing autophagy with ATG7 siRNA made cells more sensitive to Temozolomide and curcumin either alone or in combination. 223In one study, epirubicin, which has been shown to promote autophagy, was combined with gene therapy using ATG-7 and ATG-5 siRNA to treat TNBC cells.Inhibiting autophagy has been demonstrated to significantly reduce the viability of cancer cells and increase apoptosis. 88Similar findings were found whereby blocking autophagy with ATG-7 siRNA caused epirubicin's cytotoxicity to rise in a number of breast cancer cells, along with an increase in caspase-9-and caspase-3-dependent apoptosis. 157n a recent study, downregulation of LC3 gene by delivering siLC3 significantly reduced cell viability and blocked the main cell mechanisms; proliferation, invasion, migration, and resistance to apoptosis in the metastatic MDA-MB-231 cell line, which represents a simulation of TNBC. 58A naturally occurring kava chalcone called flavokawain B (FKB) has a potent anticancer effect on a number of cancer types that also activate autophagy.Blocking ATG5 or ATG7 expression using siRNAs prevented FKB from inducing autophagy in GBM cells, which led to the cells' transition from senescence to death. 224Another recent study demonstrated that the agent Enzalutamide (ENZ), currently being studied to treat bladder cancer, boosted apoptosis when ATG5 was knocked down by siRNA. 225In the PTX-resistant NCI-H23, an NSCLC cell line, knocking down Beclin-1 by siRNA simultaneously decreased multidrug resistance protein 7 (ABCC10) and P-glycoprotein (P-gp), which functions as a drug efflux pump, and restored the sensitivity to PTX in vitro.In vivo, decreasing autophagy caused tumor sizes to decrease. 226Another study discovered that lung adenocarcinoma cells were more susceptible to the effects of Vismodegib, the first inhibitor of Sonic Hedgehog, 227 a crucial target in cancer therapy, when autophagy was inhibited by ATG5 or ATG7-siRNAs. 228In recent studies, siRNA-mediated LC3 knockdown dramatically increased the efficacy of GBM treatments. 229,230Another study found that inhibiting autophagy by using siRNA to silence the ATG-7 gene increased the effectiveness of cisplatin as it was associated with increased apoptosis, decreased cell survival rate, decreased measurements of cell density, altered morphology of the cells, and decreased measurements of mitochondrial membrane potential in ovarian cancer SKOV3 cells. 231.2.Nanoparticles.Nanoparticles (NPs) are particles with dimensions on the nanometer scale, typically between 1 and 100 nm. 232They can be made of a wide variety of materials, including metals, ceramics, polymers, and lipids, and can have unique properties due to their small size and high surface areato-volume ratio.Recently, nanoparticles are widely used as delivery vehicles for genetic material such as DNA and RNA. 233These particles can protect the genetic material from degradation, increase its stability, and target it to specific cells or tissue.The nanoparticles are engineered to have certain properties such as size, surface charge, and surface chemistry that allow them to interact with the cell membrane and deliver the genetic material into the cell.Nanoparticles can also play a role in inhibiting autophagy and cause cancer cell death through various mechanisms, such as by directly targeting and disrupting the autophagic machinery within the cell, 234 by targeting signaling pathways that regulate autophagy, such as the mTOR pathway, or by inducing oxidative stress. 235.2.1.Nanoparticles as Delivery Systems.Genetic therapeutic agents must be successfully introduced into cells in order to inhibit autophagy utilizing gene therapy.The quick degradation of miRNAs and siRNAs once they enter the circulation, as well as their negative charge, which prevents them from entering the cell membrane, are two of the challenges they face.Viral-based delivery systems were developed and employed for this purpose, but they were shown to be unsafe, causing significant immunological reactions as well as carcinogenic potential. 236,237In order to transport genetic materials and meet the many obstacles in the area, a variety of nonviral gene vectors, nanoparticles, have been used.A summary of the most commonly used vectors is shown in Figure 4.
With a median particle size in the nanometer scale, generally between 10 and 400 nm, nanocrystals made up entirely of drugs are carrier-free colloidal drug delivery vehicles. 238−241 In a recent study, a drug-delivering-drug (DDD) platform was developed, comprised of antitumor-drug nanorods, rod-like nanocrystals of PTX, which served as a vehicle for miR-101 delivery to cancer cells.In vivo, this administration resulted in an inhibition of autophagy, as evidenced by a decrease in LC3-II mRNA and an increase in p62 mRNA.The drug's capacity to kill cancer cells was facilitated when it was combined with miR-101, as evidenced by a considerable increase in apoptotic cell death.Furthermore, when compared to administering the nanorods alone, this combination resulted in an 80% reduction in tumor volume. 242olymers have been crucial to the development of drug delivery technology because they provide cyclic dosage, adjustable release of both hydrophilic and hydrophobic medicines, and controlled release of nanomedicines in constant doses over long periods of time. 243In one study, Beclin-1 siRNA and DOX were delivered to cancer cells simultaneously using 1,3-diol-rich hyperbranched polyglycerol (HBPO) and phenylboronic acid-tethered hyperbranched oligoethylenimine (OEI600-PBA).It has been observed that when these two hyperbranched polymers are joined together, a core−corona nanoconstruction arises by itself.This nanostructure provided enhanced siRNA affinity, additional anticancer drug loading capability, facilitated cellular transport, and acidity-responsive payload release.When siRNA and DOX were delivered together using this nanostructure, Beclin-1 gene could be successfully silenced, autophagy that is caused by DOX has been suppressed, and cell death in cultivated malignant cells was significantly increased.Comparing the in vivo combinational treatment to the DOX alone treatment, it was found to significantly increase safety while increasing the tumor's sensitivity to DOX chemotherapy. 244Pullulan, a naturally occurring nonionic and linear homopolysaccharide, has gained increasing attention as a gene delivery system due to its outstanding biocompatibility, low viscosity, and preferable water solubility. 245One study used a pullulan-based copolymer delivery system to deliver both DOX and Beclin-1 siRNA simultaneously.Multiple components have been used in the delivery system known as FPDP in order to produce high outputs.In a study, pullulan was modified with lipophilic desoxycholic acid to produce micelles, and Beclin-1 siRNA was delivered using PEI, with folate (FA) employed for targeted delivery of the system to cancer cells.As a result, the delivery system showed excellent DOX and shBeclin1 loading capacities, practical storage stability, and a sustained drug release profile.The codelivery of DOX and Beclin-1 siRNA produced synergistic cell death, according to in vitro and in vivo results. 246lectrostatic interactions enable negatively charged siRNAs to be incorporated with cationic polymers such polyethylenimine (PEI), polyamidoamine dendrimer (PAMAM), 247,248 and chitosan, which can then be formed into nanoparticles with excellent encapsulation. 249PAMAMs are a category of synthetic polymers which have been exploited as nanocarriers due to their distinctive qualities, including their highly branched structure, water solubility, high charge densities, and abundance of amine groups for additional functionalization. 250For instance, a PAMAM-based nanocarrier (PPP) has been designed in recent research in order to transfer miR-34a into gastric carcinoma cancer cells.Phenylboronic acid was used to modify the surface of PAMAM in the creation of the nanocarrier, enabling targeted distribution of the nanocarrier to cancer cells and improving endocytosis effectiveness.The results demonstrated that delivering miRNA-34a with this nanocarrier counteracted Notch-1, a signaling pathway crucial in autophagy, which resulted in inducing apoptosis and suppressing cell migration and invasion, as well as decreasing tumor growth in vivo. 247he "Golden Standard" nonviral gene delivery vector with respectable transfection potential and moderate immunogenicity is usually regarded as being PEI, a highly assessable synthetic polymer. 251In a recent study, a PEI-based delivery system was used to deliver DOX and an antiautophagy siRNA simultaneously.The resulting delivery system (PEI/Si-D) containing mirror RNAs was subsequently coated with hyaluronic acid (HA), a naturally occurring polymer, to mask the surface charge of PEI (HP/Si-D).In this procedure, DOX was first loaded into a scrambled siRNA and then condensed by PEI along with an antiautophagy siRNA.Due to its ability to preferentially connect with the cluster of differentiation 44 (CD44) receptor, which is overexpressed on a number of cancer cells, 252 HA provided active targeting of the delivery system to tumor cells.By transfecting the cells with this system, the target cells' autophagy level was downregulated.This resulted in a further reduction in ATP supply, which improved drug retention and cell cycle arrest.The findings, in particular, showed that autophagy and DOX combined effects caused synergistically stronger anticancer outcomes in vitro and in vivo than each treatment alone. 253hitosan is a mucopolysaccharide that has a main amine in the glucosamine residue's C-2 position, making it simple to be functionalized. 254Chitosan NPs are readily prepared, stable, biocompatible, and capable of controlling the release of active ingredients. 255In one study, gefitinib, a medication used to treat certain types of cancer, and short hairpin ATG-5 (shAtg-5) were delivered via a polymer-based delivery system that utilized chitosan nanoparticles.The findings demonstrated that the codelivery of gefitinib and shAtg-5 boosted cytotoxicity, significantly increased apoptosis by suppressing autophagy, and considerably reduced tumor growth. 256 plain sheet of carbon atoms forms the hexagonally, twodimensional (2D) crystal structure known as graphene.The most significant graphene derivative is GO, which is the oxidized version of the material and has outstanding water processability, amphiphilicity, and surface functionalization capability. 257Stathmin1 gene, a key regulator of autophagy that has been discovered to be up-regulated in various malignancies, 258−260 could also be repressed by miR-101. 261ased on that, a recent study based on the use of cationized graphene oxide (GO) as a delivery system to provide improved delivery of miR-101and enhanced photothermal therapy has been conducted.In this study GO surface has been functionalized with amine polyethylene glycol (PEG) and Poly-L-arginine (P-L-Arg) to improve particle stability and biocompatibility, as well as to facilitate the contact with target cells.The obtained results indicated that GO-PEG-(P-L-Arg) would be a promising targeted delivery strategy for miR-101 transfection that could suppress autophagy and convey thermal stress to trigger apoptotic cascades when combined with photothermal therapy. 262Another study demonstrated the use of GO functionalized with polyethylenimine, PEI, (PEI-GO) to codeliver Bcl-2 siRNA and DOX to cancer cells.The combination of GO and PEI was predicted to aid in the electrostatic adsorption of siRNA and the stacking of aromatic anticancer medications onto GO sheets.The findings highlighted that the codelivery of siRNA and DOX enhanced the efficiency of the cytotoxic drug by synergistically promoting apoptosis. 263icelles are a family of amphiphilic copolymers and surfactants that function as core−shell nanocarriers. 264They help encapsulate hydrophobic chemotherapeutic medicines and hydrophilic gene drugs.Notably, micelles have great solubility, stability, and biodistribution, and because of their increased penetration and retention (EPR) effect, they can passively accumulate in tumor tissues. 265In a recent study, a peptide micelle system (Co-PMs) was developed using arginine and histidine copolymers to codeliver Beclin-1 siRNA and 6-maleimidocaproylhydrazone DOX derivative, DOX-EMCH.Histidine was shown to assist nanomicelles in escaping from endosomes through the proton sponge effect, whereas arginine was employed to compress RNA through electrostatic interactions.In accordance with in vitro findings, Co-PMs were successful in achieving siRNA endosomal escape, demonstrated greater cytotoxicity in PC3 cells than DOX alone, and consequently, increased the susceptibility of the cells to DOX.Furthermore, the Co-PMs demonstrated a 3 times stronger tumor inhibitory capacity in vivo compared to DOX or Beclin-1 siRNA therapy alone, indicating a considerable antitumor activity. 266n a recent study, a polymer-based micelle delivery system was developed to codeliver docetaxel (DTX) with ATG-7 siRNA, which inhibits autophagy, simultaneously in vitro and in vivo.Where it has been demonstrated that DTX induces autophagy, which is accountable for drug resistance.The hydrophobic drug, DTX, and the ATG-7 siRNA were enclosed in the synthesized PP6iRGD micelles.In order to create PP6iRGD, PEI was first reacted with the triblock copolymer Pluronic P123.The resultant compound, PP6, was then further coupled with the iRGD (CRGDK/RGPD/EC) peptide.The vehicle was given effective cationic characteristics for binding the gene due to the presence of PEI.While iRGD successfully targeted tumor by binding to integrins that are overexpressed on the endothelium of tumor arteries.Results revealed that ATG-7 knockdown reduced autophagy, which improved the effects of DTX treatment as seen by a considerable rise in cellular apoptosis. 267In a related study, a unique approach for treating breast cancer was demonstrated using codelivery of DTX and ATG-7 siRNA in a cross-linked, reducible, polypeptide micellar system.The core components of the system were lipoic acid (LA) and cytosol localization and internalization peptide 6 (CL), which may self-assemble into micelles and then be cross-linked to form cross-linked micelles.With exceptional biocompatibility and serum stability, CL is a novel cell-penetrating peptide (CPP) that may readily penetrate cellular membranes and transfer payload to the cytosol through using nonendosomal pathways.While LA has antioxidant properties since the human body naturally produces it.The findings demonstrated that ATG-7 siRNA might inhibit cells' capacity to activate autophagy by silencing ATG-7 gene, hence reducing the survival of human MCF-7 cancer cells after chemotherapy.That was clear from the fact that the combined effects of DTX and siATG7 on cancer cells were 2.5 and 1.7 times greater than those of DTX alone in terms of cytotoxicity and apoptosis, respectively. 268onetheless, a peptide-based micellar delivery system has been developed to accomplish a codelivery of adenosine monophosphate-activated protein kinase (AMPK) activator narciclasine (Narc) with unc-51-like kinase 1 (ULK1) siRNA into HCC cells.As it has been established that activating AMPK regulates mTOR-dependent signaling pathways to inhibit HCC. 269While ULK1 knockdown inhibits autophagy and, as a result, increases apoptosis.The utilization of lipidmodified cell-penetrating peptides served as the foundation for the development of the self-assembling, biocompatible, pHsensitive micellar system.A markedly disturbed autophagy was seen in tumor cells when the micelles were transfected into HCC cells in vitro.Furthermore, when the micelles were administered to mice with HCC xenografts, apoptosis was induced, tumor growth was decreased, and autophagy was prevented.These results demonstrate that the synergistic administration of Narc and siULK1 in biocompatible micelles can safely reduce tumor growth and autophagy. 270iposomes are phospholipid vesicles that include aqueous gaps inside one or more concentric lipid bilayers.They are biocompatible and biodegradable, and they can recognize and target cancers. 271They can readily be coated with hydrophilic polymers, like PEG, to lengthen the in vivo circulation period. 272For instance, one study used PEGylated liposomes to deliver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA and PTX simultaneously.As it has been demonstrated that blocking GAPDH will result in lower ATP levels, autophagy, and multidrug resistance (MDR) in hypoxic cancerous cells.The liposome delivery system included a cationic inner monolayer lipid vesicle in which GAPDH siRNA was inserted, a PEG-coated outer layer to shield the nanocarrier, and PTX sandwiched in between the two layers.Results obtained in vitro demonstrated that the designed approach had great specificity in GAPDH suppression and synergistically increased the cytotoxicity of PTX in tumor cells (HeLa and MCF-7) in a hypoxic environment.Additionally, in vivo studies indicated that the liposomes improved the PTX chemotherapeutic activity while also gradually increasing drug concentrations in tumors.When GAPDH siRNA was codelivered with PTX utilizing liposomes, tumor cells became more susceptible to PTX and thus treatment outcomes were improved. 273evertheless, other nanocarriers have been also utilized in delivering autophagy-related siRNAs and miRNAs into cancer cells.For example, cisplatin and Beclin-1 siRNA were simultaneously delivered to lung cancer cells using a peptidebased nanoprodrug delivery system.Three primary elements made up the delivery system.The prodrug complex (Pt(IV)peptide-bis(pyrene)) was first created by conjugating tetravalent cisplatin (Pt(IV)) to a cationic peptide and then functionalizing it onto the hydrophobic polymer backbone.This cisplatin functionalization ensured great drug loading efficiency (up to 95%) and aided in the delivery of Beclin-1 siRNA into the cytosol by providing cationic charge for the complexation.To further strengthen and stabilize the system's biocompatibility, the DSPE-PEG molecule was introduced.Finally, to provide targeted administration, cRGD was fixed onto the DSPE-PEG molecule.The system caused the Beclin-1 gene to be suppressed, hence blocking autophagy.As a result, the medicine's effectiveness was improved, drug resistance was overcome, and tumor growth in a xenograft mice model was inhibited. 274Another investigation used lipid-coated calcium carbonate nanoparticles to deliver sorafenib and miR-375 in combination for the treatment of HCC.The lipid had a positive charge that was used for electrostatically complexing with the miRNA.With the help of this approach, autophagy has been successfully inhibited.According to in vitro data, the nanoparticles displayed high cytotoxicity and pH-dependent drug release.Whereas in vivo results indicated that the chemotherapeutic drug's efficiency was greatly increased as demonstrated by a reduction in tumor sizes. 275In our current research group work, a "smart" nanocarrier has been developed as the carrier of LC3 siRNA to deliver combination of DOX and LC3 siRNA for cancer therapy in TNBC.The "smart" nanoparticle system was based on the use of FDA -approved βcyclodextrin (β-CD) that consists of two sides: a primary side and a secondary side.Half of the primary side were functionalized with three different moieties: 2-(dimethylamino) ethyl methacrylate (DMAEMA) monomers, which are pH sensitive, hydrophobic hexyl methacrylate (HMA) and cationic trimethylaminoethyl methacrylate iodide (TMAEMA) monomers, while the other half was PEG bound.Results showed that this polymer was able to detect changes in the environmental pH, disrupt the endosomal membrane, and achieve electrostatic complexation between its cationic amine groups and the anionic phosphate groups of LC3 siRNA.We demonstrated that utilizing this delivery approach could efficiently suppress the autophagy-related gene LC3, inhibit cellular autophagy and exhibit improved anticancer effects.Furthermore, in the TNBC cell line, MDA-MB-231, coadministration of siLC3 and DOX was more effective than either agent alone in treating breast cancer.The inhibition of cell growth, colony formation, and migration in the cells demonstrated this.Furthermore, our combination caused an increase in the apoptotic population, as shown by an increase in the sub-G1 population, as well as induction in G2/M cell cycle arrest.

Nanoparticles as Autophagy Inhibitors.
Several NPs have the ability to modulate autophagy on their own, either by enhancing or suppressing the autophagic flux.Lysosomal dysfunction is the predominant way through which autophagy got manipulated since lysosomes are involved in the process's final stages.Autophagosome accumulation is the main effect of this lysosome dysfunction.−287 Unfortunately, some of the studies mentioned above did not take into account the relationship between the number of autophagosomes and the rates at which they are produced and degraded at any given time.Since accumulation of autophagosomes may result from either autophagy blockage or autophagy stimulation.In this part, we highlighted a number of studies where cells exposed to naked NPs resulted in autophagy dysfunction.
Carbon nanotubes (CNTs) have several distinctive characteristics that allow them to appeal for usage in a variety of nanomedicine applications, including intravascular use.One study examined the impact of two systems of carbon nanomaterials on autophagy, particularly in cultured murine peritoneal macrophages.These systems were acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) and graphene oxides (GOs).By examining autophagosomes, lysosomes, and the amount of p62, it has been demonstrated that both systems were capable of causing an autophagy blockage.p62 was dose-dependently accumulated by AF-SWCNTs and GOs, which indicates that autophagy is being inhibited.Additionally, AF-SWCNTs and GOs accumulated in macrophage lysosomes and caused lysosome membrane instability, which in turn resulted in an increase of autophagosomes, suggesting impaired autophagic breakdown. 288Another study examined the impact of exposure to carboxylated multiwalled carbon nanotubes (MWCNTs) on autophagy in human umbilical vein endothelial cells (HUVECs).It was shown that using MWCNTs led to an accumulation of autophagosomes that was brought on by the blocking of the autophagic flux instead of through the activation of autophagy.Measuring the expression of p62, when no decline in its level was seen, demonstrated this. 289In a different investigation, pristine multiwall carbon nanotubes (PMWCNT) demonstrated an inhibition in autophagic flux demonstrated by an induction in pulmonary autophagy accumulation in normal mouse lung as well as an elevation in the level of p62 expression. 290One study used graphite carbon nanofibers (GCNFs), a possible substitute for carbon nanotubes (CNT), to block autophagy, which caused the activation of apoptosis in human lung cancer cells.The capacity of GCNFs to destabilize lysosomes and produce ROS resulted in an accumulation of autophagosomes that blocked the autophagic flux. 291ne study found that AgNPs might cause lysosomal degradation and autophagy disruption in HepG2 cells, which led to the induction of apoptosis.It has been demonstrated that AgNPs could disrupt the autophagy−lysosomal pathway, hence activating pro-inflammatory caspase-1-dependent signaling. 292In another study, utilizing AgNPs resulted in dysfunctional autophagy by impairing autophagosome-lysosome fusion.By evaluating LC3 conversion and the level of p62 expression, the suppression of autophagy was demonstrated.In which a rise in LC3-I to LC3-II conversion as well as a dosedependent buildup of p62 expression have been observed. 293n another study, silica nanoparticles (SiNPs) were exposed to the HepG2 hepatocellular carcinoma cell line, and the influence on autophagy was examined.SiNPs have been found to have the potential to impede autophagic flux by affecting lysosome function.The findings demonstrated that SiNPs are being taken up by endocytosis and building up in lysosomes, resulting in an overload on lysosomes that causes their edema and malfunction.In turn, autophagosomes and lysosomes were blocked from fusing, which prevented the breakdown of autophagic cargo.This was emphasized by AO staining, which showed increased lysosomal membrane permeability and lysosome breakdown.Yet, the level of p62 expression has also been studied, and its time-dependent increment highlighted the disruption of autophagic flux. 234t is worth noting that the NPs' shape influences how they affect the autophagy pathway, lysosome activity, and toxicity profiles of the nanoparticles.According to one study, tubular carbon nanoparticles (CNT) restricted the autophagy flow in RAW 264.7 murine macrophages as contrast to spherical carbon nanoparticles, whose use had no effect on autophagy.It has been demonstrated that CNT prevented the fusion of the autophagosome with the lysosome and imposed autophagy suppression as a result of lysosome dysfunction and subsequent downregulation of the expression of the SNAP-associated protein (SNAPIN), a crucial coordinator of late endocytic transport and lysosomal maturation. 294In a different study, the impact of the two types of gold nanoparticles most frequently utilized in biomedical fields�gold nanospheres and nano-rods�on autophagy was examined.In contrast to what was seen when Au nanorods were used, it has been found that Au nanospheres impede the lysosomal function by accumulating in autolysosomes.Lysosome dysfunction caused autophagic flow to be inhibited, which was suggested by the accumulation of autophagosomes, as demonstrated by an increase in LC3-II and an increase in p62. 295t is also interesting to note that altering the lipid content of the autophagosomal or lysosomal membranes hinders autophagosome-lysosome fusion, which lowers autophagic activity.In one in vitro study, it was discovered that exposing autophagosomes and lysosomes to the potent β-CD derivative, methyl-β-cyclodextrin (MBCD), decreased the cholesterol levels in the membranes by 25 and 70%, respectively.This change in the content of the membranes led to a 40−50% decrease in their fusion efficiency, which had an impact on the overall activity of autophagy. 296In a different investigation, in vivo effects of another β-CD derivative 2-hydroxypropyl-βcyclodextrin (CYCLO) were examined.It has been demonstrated that CYCLO delayed autophagosome maturation and impeded autophagosome-lysosome fusion by increasing the accumulation of LC3-II and p62, as well as a buildup of autophagosomes. 297dditionally, a "smart" nanoparticle based on the utilization of β-CD has been created, and its endosomal escape mechanism is accomplished by both DMAEMA, which expresses proton-sponge influence, and the hydrophobic moiety HMA, which tears up the endosome membranes. 298,299t is thought that as the "smart" nanoparticle enters the lysosomes and autophagosomes as well, it bursts their membranes via the same mechanism, interrupting the autophagy pathway.

CHALLENGES AND FUTURE PERSPECTIVES
In our comprehensive review of autophagy modulation in cancer therapy, we aim to elucidate the benefits and drawbacks of various therapeutic strategies while exploring the potential of nanoparticles in regulating autophagy.Traditional drug agents targeting autophagy pathways, such as chloroquine and hydroxychloroquine, offer the advantage of established clinical use.However, their efficacy may be limited by off-target effects and dose-dependent toxicity. 300,301Gene silencing therapy, utilizing RNA interference techniques, presents a promising avenue for precise modulation of autophagy-related genes.Yet, challenges persist in achieving efficient delivery and avoiding immune responses.Nanoparticles, with their unique properties and tunable characteristics, offer a promising platform for targeted delivery of therapeutic agents, including RNA-based therapies, to cancer cells.By encapsulating drugs or genetic material, nanoparticles can enhance drug stability, prolong circulation time, and facilitate cellular uptake, potentially overcoming the limitations of conventional drug delivery methods.Moreover, nanoparticles can be engineered to modulate autophagy directly through various mechanisms, such as targeting autophagic machinery or signaling pathways.However, issues such as biocompatibility, biodistribution, and scalability remain to be addressed for widespread clinical translation. 302Despite these challenges, the versatility of nanoparticles holds great promise for advancing precision cancer therapies by enabling the timely and accurate regulation of autophagy in tumor cells.As research in this field progresses, continued efforts to optimize nanoparticle design and delivery strategies will be essential for realizing the full therapeutic potential of autophagy modulation in cancer treatment.
In conclusion, the intricate relationship between autophagy and cancer presents a dual role for this cellular process in tumorigenesis and treatment response.While autophagy serves as a crucial mechanism for maintaining cellular homeostasis and adapting to metabolic stress, its dysregulation can contribute to tumor progression and resistance to therapy.
Our review has highlighted the therapeutic potential of autophagy inhibition in improving cancer treatment outcomes, emphasizing various strategies such as drug agents, gene silencing therapy, and nanoparticle-based approaches.Through targeting key autophagy-related genes and modulating the autophagic flux, these interventions hold promise for enhancing the efficacy of traditional cancer therapies and overcoming drug resistance mechanisms.However, challenges remain in understanding the precise mechanisms underlying the dual role of autophagy in cancer and optimizing the delivery and efficacy of autophagy-targeting agents.
Furthermore, we have provided a comprehensive overview of key points, mechanisms, and challenges in autophagy and cancer therapy in Table 2, which summarizes the current understanding and ongoing efforts in this field.This table serves as a valuable resource for researchers and clinicians seeking to elucidate the complex interplay between autophagy and cancer and develop novel therapeutic strategies to combat this disease effectively.

CONCLUSION
A substantial body of current research indicates that autophagy inhibition has been reported to cause tumor cell death, generates inflammation in the tumor microenvironment and cancer-adjacent cells, which promote carcinogenesis.Autophagy, however, has also been linked to tumor spreading as it increases the aggressiveness of malignancy.Recently, autophagy has also been highlighted as a potential factor in the emergence and development of anticancer drug resistance.Autophagy is a crucial factor in cell death decisions.The ability of autophagy to trigger mitophagy and decrease the concentration of pro-apoptotic proteins in the cytosol are the most plausible explanations.CQ, HCQ, 3-MA, and BafA1 are among the drug agents that have been designed to prevent autophagy at its various phases.Far from autophagic medicines, other methods for blocking autophagy have also been explored.Gene silencing tools, specifically siRNA and miRNA, are being used to suppress autophagy by focusing on critical ATG genes implicated in the process.However, because siRNA and miRNA quickly degrade once they reach

Autophagy Mechanisms
• Macroautophagy: The primary pathway involved in autophagy, characterized by the formation of autophagosomes that engulf cellular components for degradation.• Microautophagy: Direct engulfment of cytoplasmic material by lysosomes.
• Chaperone-mediated autophagy: Selective degradation of proteins recognized by chaperone proteins.

Role of Autophagy in Cancer
• Dual nature: Autophagy can both suppress and promote cancer, depending on tumor stage and context.
• Tumor suppression: Delays tumor onset and slows tumor dissemination in early stages.
• Tumor promotion: Promotes tumor development, spread, and resistance to anticancer drugs in advanced stages.
• Nanoparticles: Serving as delivery vehicles for genetic material (siRNA, miRNA) to inhibit autophagy or modulate autophagic flow.

Delivery Matters
• Delivery challenges: Overcoming hurdles in delivering RNA-based therapies due to degradation and cell membrane penetration issues.• Optimizing nanoparticle design: Engineering nanoparticles with optimal properties for efficient delivery and autophagy modulation.
the bloodstream and because of their negative charge, which prevents them from entering cell membranes, various nanoparticles have been used to successfully deliver them into tumor cells.In addition to their function as carriers of genetic material, nanoparticles have also been designed to modulate autophagy on their own by inhibiting the autophagic flow through accumulating autophagosomes.Accordingly, as one recent review highlighted, numerous efforts and studies are currently being made in an effort to improve the effectiveness of cancer treatment by utilizing various strategies to block autophagy.

Figure 2 .
Figure 2. Summary of the key impacts of autophagy inhibition on cancer cells, as represented by tumor growth suppression, tumor metastasis reduction, overcoming anticancer treatment resistance, and apoptosis activation.

Figure 3 .
Figure 3.The most common autophagy inhibitors work to inhibit autophagy.3-MA prevents the formation of the autophagosomes, whereas CQ, HCQ and BafA1 work by inhibiting autophagosome−lysosome fusion, leading to accumulation of autophagosomes.

Table 1 .
Summary of the Studies on Autophagic Medicines

Table 2 .
Key Points, Mechanisms, and Challenges in Autophagy and Cancer Therapy Drug resistance: Autophagy inhibition may lead to the development of resistance mechanisms in cancer cells.