Pharmacology Progresses and Applications of Chloroquine in Cancer Therapy

Introduction

Chloroquine (CQ, 4-N-(7-chloroquinolin-4-yl)-1-N,1-N-diethylpentane-1,4-diamine) is recognized as a common antimalarial drug and features in the World Health Organization Standard List of Essential Medicines due to its safety, affordability, and user-friendliness. Beyond its application in malaria treatment,^1,2 CQ is also utilized for lupus erythematosus,³ autoimmune disorders such as rheumatoid arthritis,^4,5 and is being evaluated for its antiviral properties against HIV infection,⁶ chikungunya fever,⁷ and the novel coronavirus SARS-CoV-2.^8–10 Additionally, CQ has also been widely reported as a potential anticancer agent due to its ability to block autophagy.

Recently, modulating autophagy has emerged as a promising therapeutic method for cancer. CQ and its derivative hydroxychloroquine (HCQ) both are recognized autophagy inhibitors that remain the only autophagy inhibitors approved by the Food and Drug Administration (FDA).¹¹ CQ diffuses freely and rapidly across cell and organelle membranes. In lysosomes CQ was protonated and “trapped” in which CQ no longer freely diffused out.¹² It was reported that CQ suppressed autophagy through entering the lysosome and increased its pH which prevented autolysosomal degradation,¹³ while others confirmed that CQ impaired autophagosome-lysosome fusion rather than inhibiting lysosomal degradation to achieve the effect of inhibiting autophagy, resulting in an inability to provide energy through the autophagy pathway,¹⁴ and then inhibited tumor cell growth or induced tumor cell death.¹⁵ Due to its autophagy inhibition, CQ has been used as a supplement agent in clinical trials against cancer. For example, the combination of CQ and cisplatin effectively enhanced the apoptosis of tumor cells induced by cisplatin,¹⁶ and pterostilbene combined with CQ significantly improved its inhibition of autophagy and reduced the activity of tumor cells by down-regulating RAGE/STAT3 and AKT/mTOR signaling pathways.¹⁷ In addition, CQ also effectively improved the inhibition of gemcitabine in gallbladder carcinoma cells and promoted the apoptosis of cancer cells.¹⁸

In tumor suppression, CQ also has activation of apoptosis and necroptosis of cancer cells that are independent of autophagy inhibition. Data have shown that CQ induces lysosome membrane permeability and mitochondrial membrane permeability, triggering caspases cascade and inducing apoptosis of cancer cells, which cannot be achieved by the addition of autophagy inhibitors.^19,20

In this review, we focused on the anticancer properties of CQ, summarized the tumor progression dependent or independent of autophagy inhibition of CQ, and discussed combined pharmacotherapy in cancer therapy. At the end of the review, we also discussed the side effects of CQ, challenges and possible application development in the future hoping to help researchers carry out more in-depth exploration of CQ.

CQ and Its Analogues in Cancer Suppression

CQ was synthesized based on the structure of the natural product quinine. In the 16th century, malaria was widespread in Europe but no effective cure had yet been found. It was not until the 1630s that Indians discovered that drinking water from the bark of the tree which grows in the Andes could cure fever, then the bark was taken to Europe to treat malaria. In 1742, Swedish botanist Carl Linnaeus officially named the tree as “Cinchona tree”. In 1820, French pharmacists Pierre Pelletier and Joseph Caventou isolated the antimalarial ingredient quinine from the “Cinchona tree”, and it was later used to treat malaria. But quinine could not completely cure the malaria, and the side effects were obvious, so scientists had worked to synthesize new antimalarial drugs, and then quinacrine (QC) and CQ had been introduced successively.^21,22 CQ was more effective and less toxic than quinine and QC. However, with the widespread use of CQ, the drug resistance of malaria parasite to it began to appear, and the anti-malaria effect of CQ became worse, especially in Plasmodium falciparum.²³ Since then, scientists have synthesized many other new antimalarial drugs, such as HCQ and mefloquine (MQ).

Originally developed as a treatment for malaria, CQ and its analogues (Figure 1) such as HCQ, Lys05, MQ and QC are now used to treat a variety of other diseases, including cancer, because of their suppression on autophagy or enhancing the sensitivity of chemoradiotherapy drugs.^2,24 Here, we briefly summarized the anticancer types and mechanisms of each analogue above (Table 1).

Table 1 Summary of Chloroquine Analogues and Their Antitumor Types Mentioned in This Article

Figure 1 Chemical structures of CQ and its analogues.

Hydroxychloroquine (HCQ)

While high concentrations of CQ can lead to significant toxicity, its derivative, HCQ, exhibits similar pharmacological actions and mechanisms but is notably less toxic. Recently, HCQ has been clinically evaluated for its efficacy against a variety of cancers, including glioblastoma, bladder, breast and prostate.^25,26,57,58 As an inhibitor of autophagy HCQ enhanced the chemosensitization via altering the lysosomal pH and inducing the transition of tumour-associated macrophages from M2 to the tumor-killing M1 phenotype.²⁷ HCQ also induced the autophagy and apoptosis, enhanced the anticancer effect of indole-3-carbinol in the Ehrlich ascites carcinoma (EAC) model, and induced cycle arrest, restraining cell proliferation in cholangiocarcinoma both in vitro and in vivo.^28,29 In clinical studies, HCQ demonstrated significant effectiveness, notably improving anti-cancer immunity and autophagy inhibition when used in combination with vorinostat, rapamycin, or other chemotherapeutic agents.^59–61 As an adjuvant, HCQ exhibited synergistic antitumor properties, and showed notable anticancer effects even as monotherapy. For instance, HCQ restrained lung adenocarcinoma via increasing the expression of FoxO3a and diminishing the phosphorylation of STAT3, affecting the JAK-STAT and FoxO signaling pathways in lung adenocarcinoma.³⁰ Despite HCQ’s lower toxicity compared to CQ, its clinical application is constrained by the limitations associated with high-concentration autophagy inhibition.

Lys05

Lys05, a dimeric CQ analogue, has shown high potential for autophagy suppression at lower working concentrations compared to HCQ, and has efficient antitumor activity in diverse human tumor xenograft models including melanoma, glioblastoma and colon cancer.⁶² Lys05 was recognized as a sensitizer and enhanced radiosensitivity in the treatment of cancer. In recent years, Lys05 has been reported to exert a sensitizing role in many kinds of cancers, such as glioblastoma in which Lys05 induced lysosomal membrane permeabilization (LMP), caused mitochondrial depolarization, and increased radiosensitivity in antiglioma activity,³¹ clear cell ovarian carcinoma (CCOC) in which Lys05 inhibited the autophagy and potentiated the anticancer property of sunitinib.³² Lys05 also had a great synergistic effect in myeloproliferative neoplasms, which effectively improved the efficiency of ruxolitinib via the inhibition of autophagy.³³ Consequently, as an autophagy inhibitor, Lys05 may be a promising compound in cancer therapy, and more researches need to be displayed to promote its application and development.

Mefloquine (MQ)

MQ is also a derivative of CQ and an FDA-approved agent with highly effective against malaria. A growing body of research showed that MQ significantly counteracted multiple cancers, including glioblastoma, breast cancer, hepatocarcinoma, colorectal cancer and liver cancer.^34–38 MQ exerted its antitumor effects through diverse molecular mechanisms. For example, studies have shown that MQ could inhibit cell proliferation and the PI3K/AKT/mTOR signaling pathway, induced reactive oxygen species (ROS) and trigger apoptosis in human gastric tumor cells.³⁹ The NF‐κB signaling pathway has been reported to be activated in many cancers. Nowadays, researchers found that MQ could serve as an inhibitor of NF‐κB in colorectal cancer, weaken the activity of IκBα kinase, and induced cell cycle arrest and apoptosis of tumor cells both in vitro and in vivo.³⁴ Additionally, MQ has been reported to arrest the growth of prostate cancer cells, caused ROS and induced non-apoptotic cell death, and these influences may involve the AKT, ERK, JNK and AMPK signaling pathways.^40,41 For the inhibition of cervical cancer, MQ destroyed the mitochondrial function through restraining mitochondrial respiration, abolishing membrane potential and reducing ATP levels, meanwhile inhibiting the mTOR signaling pathway in vitro and in vivo.⁴² Furthermore, MQ has been found to trigger mitochondrial autophagy and lysosome disruption in the treatment of esophageal squamous cell carcinoma and chronic lymphocytic leukemia, respectively.^43,44 All the findings above extended the application of MQ, more clinical anti-tumor trials for MQ need to be performed and its clinical application is expected.

Quinacrine (QC)

QC is an acridine derivative and has cytotoxic potential in multiple kind of cancers but with limited toxicity to normalcells.^{45–47,63–66} Studies have demonstrated that QC notably upregulates the expression and activity of cathepsin L, a lysosomal protease, inducing lysosomal membrane permeability and mitochondrial membrane permeability, resulting in the release of cytochrome c and inducing ovarian cancer cell death.⁴⁸ Nowadays, researchers also explained the possible mechanisms of QC anti-ovarian cancer from the transcriptome level, suggesting that QC could restrain the ribosomal biogenesis pathway, trigger nucleolar stress and intensify DNA damage both in vitro and in vivo, which extended the application of QC and the treatment strategy of ovarian cancer.⁶⁷ QC also induced nuclear fragmentation, caused cell cycle G1/S arrest, leaded to cell death in non-small cell lung cancer, and promoted the sensitivity of tumor cells to chemotherapy in endometrial cancer and other types of cancers, such as colorectal cancer, ovarian cancer, upper gastrointestinal cancer and anaplastic thyroid cancer.^49–55 Recently, there was a study that explained the mechanism of QC increased the sensitivity of chemotherapy drugs. The study indicated that QC combined with curcumin could prevent the expression of ABCG2, which was an efflux pump and transported chemotherapy agents out of cells and resulted in cell resistance. The authors also pointed out that ABCG2 increased the expression of angiogenesis factor VEGFA which in turn promoted the angiogenesis, the combination of QC and curcumin suppressed the ABCG2 and inhibited the angiogenesis and migration in breast cancer.⁵⁶ These data above indicated that QC was a promising anticancer agent. Despite QC’s recognized efficacy as an anticancer agent, its rapid intestinal absorption and slow excretion lead to significant cumulative effects and potential toxic reactions. Therefore, innovative studies and breakthroughs are essential before QC can be clinically applied in cancer treatment. For example, Kulkarni et al mentioned that the construction of nanoformulation, which was used in combination with QC and other drugs, could decrease the therapeutic dose and reduce the toxicity and side effects caused by QC to a certain extent.⁵⁵

In addition, there are other CQ analogs like verteporfin and clioquinol, also exhibit potential antitumor properties,²⁴ meriting further exploration of their anticancer mechanisms. While CQ and its derivatives are noted for their anticancer effects, comparative studies assessing the efficacy of these analogs in cancer treatment are sparse. It is necessary to compare the measurement and mode of action of these analogs both in vitro and in vivo simultaneously in the future.

CQ Suppressed Cancer Based on Autophagy-Related Mechanisms

Autophagy is a critical cellular process involving the lysosomal degradation of cytoplasmic proteins and organelles to meet the metabolic needs of cell itself and renew some organelles. Under basic conditions, autophagy is essential for maintaining cell homeostasis and acts as a protein/organelle quality control mechanism. Under stressful conditions such as starvation, hypoxia, and chemotherapy/radiotherapy, it is the basis for cancer cell survival and adaptation to changes in the tumor microenvironment.

There are three main types of autophagy: microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy that depending on the ways of intracellular substrates entering the lysosomal cavity (Figure 2).⁶⁸ Microautophagy is a process in which lysosome membrane directly invaginates to encase and degrade cytoplasmic substances.⁶⁹ Different from microautophagy, CMA does not require the involvement of membrane structures. Cytoplasmic proteins with special motifs (KFERQ) are recognized by chaperones like heat shock 70 kDa (HSC70) and are unfolded by chaperones. After that substrates are delivered to the lysosomal membrane and bind to the lysosome-related membrane protein Lamp2A which is a special receptor on the lysosome membrane, then go into lysosome and enter the degradation process.⁷⁰ Different from the previous two, macroautophagy refers to the formation of autophagosome by the endoplasmic reticulum, Golgi apparatus or plasma membrane which enclose the cytoplasmic substances and then fuse with lysosome and degrade its contents.⁷¹ Macroautophagy is commonly known as we call autophagy which occurs rarely under normal conditions, helps cells maintain normal physiological functions by specifically degrading damaged or redundant organelles. When under stressful conditions, such as nutritional or energy starvation, macroautophagy can be further induced to degrade cytoplasmic material into metabolites that can be used in biosynthetic processes or energy production, allowing cells to survive. In this sense, macroautophagy is mainly a cell protective mechanism. However, excessive self-degradation is harmful and autophagy dysfunction has been linked to a variety of human diseases, such as neurodegeneration, diabetes and cancer.⁷²

Figure 2 Classifications and mechanisms of autophagy.

CQ is probably the most widely used autophagy inhibiting agent in vitro, and its effect on cancer cell death has been attributed to its inhibition of autophagy when used together with other chemotherapy agents.^73,74 It was indicated that CQ could prevent the fusion between autophagosomes and lysosomes through interfering with the recruitment of autophagosomal SNARE protein SNAP29, and further inhibited autophagy.¹⁴ CQ has been reported to play an antitumor role by suppressing autophagy in a variety of tumors occurring in different parts of the body, such as breast, lung, colorectal, ovary and bladder (Figure 3). In this review, we summarized the tumor types, concentrations and specific mechanisms of CQ based on autophagy inhibition published in the past ten years (Table 2), aiming to help researchers better engage in the anti-tumor research of CQ.

Table 2 The Classification is Summarized According to the CQ Different Antitumor Types

Figure 3 The distribution of CQ antitumor types in humans and its related mechanisms.

Breast Cancer

Breast cancer ranks as one of the most prevalent malignancies and the leading cause of cancer-related deaths among women worldwide. Recently, CQ has been reported to have a significant protective effect against breast cancer, including triple-negative breast cancer, primarily through its inhibition of autophagy.¹¹⁵ According to Hu et al, a combination of 20 μM CQ and isorhamnetin (IH) decreased the MDA-MB-231 and BT549 cell viability but not MCF-7 in vitro, and 40 mg/kg CQ together with 20 mg/kg IH dramatically reduced tumor weight in vivo via inhibition of autophagy.⁷⁵ CQ also played synergistic effects on the PI3K/AKT inhibitors taselisib and ipatasertib across various breast cancer cells, including MDA-MB-231, MDA-MB-468, MCF-7 and SKBR3.⁷⁶ In addition, CQ played anti-breast cancer effect via modulating the tumor microenvironment. The research of Zhang et al revealed that only low dose of CQ (IC₅₀ < 1 µM for 48 h treatment) dramatically restrained the growth and induced the apoptosis of 4T1 in vitro, and suppressed the expression of TGF-β, increased CD8⁺ T cells and decreased macrophages in tumor microenvironment in vivo.⁷⁷ Besides, the modified CQ also displayed inhibition of breast cancer. Joshi et al used gold nanoparticles with a diameter of 7 nm as the delivery system for CQ, the pharmaceutical effect was detected in MCF-7, and the IC₅₀ for 24 h treatment was 30 ± 5 μg/mL. The nanoparticle packaged CQ displayed an obvious necrosis in MCF-7 which was mediated by autophagy. This research provided new ideas for anti-tumor combination therapy, but more data such as comparing the efficacy with CQ alone and other anti-tumor drugs need to be further explored.¹¹⁶ Moreover, there was data indicated that CQ blocked the potassium channels, such as Kv10.1, which abundantly expressed in MDA-MB-231 cells and controlled the migration of MDA-MB-231. The inhibition of CQ on Kv10.1 significantly reduced the migration of breast cancer cells.⁷⁸ However, some evidence suggested that CQ inhibition on autophagy in breast cancer cells related to the epithelial–mesenchymal transition (EMT) which is important for tumor cells invasion and metastasis. The treatment of CQ on MCF7 and T47D cells reduced the expression of E-cadherin, triggered the EMT-related transcription factor and caused ROS, leading to cell migration.¹¹⁷ This study reminds researchers and healthcare professionals to be cautious when conducting anti-autophagy studies of CQ or targeting autophagy inhibition in breast cancer treatment and to consider the double-sided nature of CQ.

Lung Cancer

CQ inhibited the lung cancer through a variety of mechanisms as mono- or combination therapy. There was proof that when used as monotherapy CQ could control A549 cells proliferation via blocking the PI3K/AKT signaling pathway, mediated mitochondrial apoptosis and inhibited autophagy.⁸³ For non-small cell lung carcinoma (NSCLC), CQ also did effective inhibition in H1299 cells. It was reported that most NSCLC patients harbored p53-R273H mutation which could accelerate tumorigenesis, and further analysis revealed a tight relationship exist between p53-R273H and autophagy regulation. So the authors restrained NSCLC cells with autophagy inhibitor CQ, a high dose of CQ (50 µM) decreased transcriptional activity and disrupted the localization of p53-R273H.⁷⁹ The combination of CQ and lidamycin (LDM) also had a significant effect on NSCLC. CQ enhanced the sensitivity of LDM to both H460 and A549 cells in which the IC₅₀ values of CQ were 55.6 ± 12.5 and 71.3 ± 6.1 µM, respectively. The cell apoptosis was significantly increased after combined CQ with LDM and the content of key proteins such as PARP and caspase changed obviously. At the same time, cytoprotective autophagy was significantly inhibited.⁸⁴ Moreover, CQ enhanced the inhibition of anlotinib against NSCLC. Anlotinib induced Calu-1 and A549 cells apoptosis and autophagy, blocking the JAK2/STAT3/VEGFA signaling pathway, the addition of CQ suppressed autophagy and amplified these effects.⁸⁵ These findings demonstrate that CQ, whether utilized independently or in combination with other agents, exhibits a significant inhibitory impact on lung cancer, particularly NSCLC, suggesting its potential selective use in lung cancer treatment.

Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer in the world following breast cancer and lung cancer, with its incidence and mortality rates rising in recent years. CQ has emerged as a significant player in CRC treatment, attributed to its autophagy inhibition properties. There were few studies of CQ on the inhibition of CRC as monotherapy, and most studies focused on the combination with other drugs for CRC treatment. 5-Fluorouracil (5-FU) is the priority drug for CRC, but resistance has emerged in recent years, researchers have found that combined with a high dose of CQ (80 μM) effectively enhanced the anti-tumor effect of 5-FU.⁸⁷ CQ combined with selumetinib which was a MEK inhibitor also exhibited significant enhancement effect in CRC and reinforced its efficiency on apoptosis induction.⁸⁸ The results of the study by Lu et al revealed that CQ treatment in CRC increased the expression of PD-1, which was a key immune checkpoint and inhibitors and siRNA based on PD-1 have played a vital role in tumor therapy. Therefore, the combination of CQ and therapies targeting PD-1 such as PD-1 siRNA-related agents is a promising option for CRC treatment.⁸⁹ Another innovative approach to enhance the effectiveness of chemotherapy involves drug-loaded nano-systems. Researchers found that combination of CQ and artesunate, a renowned antimalarial agent, exhibited significant inhibition on cancer cells proliferation, and altered the phenotype of tumor-associated macrophages. These drugs were accurately targeted to the tumor tissue by using a PLGA-based biomimetic nanoparticle drug-delivery system.⁹⁰ This method significantly improved the efficacy of drugs, marking it as a promising avenue for future tumor therapy research.

Ovarian Cancer

CQ also has been studied as a potential treatment for ovarian cancers. Low IC₅₀ of CQ were obtained in four ovarian cancer cells (IGROV-1, OVCAR-8, SKOV-3 and A2780), and with combination CQ could effectively promote the anti-tumor ability of panobinostat, causing ROS and DNA damage and inducing apoptosis in vitro dependent or independent of its autophagy inhibition.⁹⁴ CQ also facilitated the inhibitors of poly(adenosine diphosphate ribose) polymerase (PARP), a key enzyme that recognizes DNA single-strand breaks, to suppress ovarian cancers. Recently, the application of PARP inhibitors against ovarian cancer has brought new benefits to patients. PARP inhibitors such as olaparib and niraparib triggered autophagy during ovarian cancer therapy, and their combination with CQ specifically inhibited autophagy and also caused ROS and increased the expression of γ-H2AX which was a biomarker of DNA strand breaks both in vitro and in vivo.⁹⁵ And because of the induction of γ-H2AX, CQ also displayed synergistic effect on the chemotherapy agent cisplatin in epithelial ovarian cancer, and increased the expression of p21^WAF1/CIP1, causing cell cycle arrest and cell death.⁹⁶ Given CQ’s capability to induce DNA damage in ovarian cancer, its combination with DNA damage repair inhibitors presented a compelling treatment strategy. Ovejero-Sánchez et al revealed that CQ combined with the DNA repair inhibitor nonhomologous end joining (NHEJ) had efficiently induced DNA damage and apoptosis in multiple ovarian cancer cell lines, such as A2780, OVCAR-8, and SK-OV-3, and which provided new therapeutic regimen for ovarian cancer.⁹⁷

Bladder Cancer

Bladder cancer represents a significant challenge in the field of oncology, with high recurrence rates and the necessity for innovative treatment strategies. Recent years have witnessed substantial researches into the use of CQ as a potential therapeutic option in bladder cancer because of its autophagy inhibition properties. There were data verified that CQ and its derivative HCQ produced alternation in LC3 flux and inhibited autophagy obviously in multiple human bladder cell lines and induced apoptosis.²⁵ CQ was detected to accelerate the expression of PDL-1 which is the ligand of PD-1 in bladder cancer, the underlying mechanism was realized on the ERK-JNK-c-Jun pathway, indicating that the combination of CQ and PD-1/PDL-1 inhibitors is also effectively applicable to the treatment of bladder cancer.⁹⁸ GSK-3β is an important therapeutic target in cancers but its inhibitors can cause obvious autophagy in tumor cells when used alone. By combined with CQ, GSK-3β inhibitors substantially inhibited the proliferation of bladder cancer cells, such as T24, HT1376 and RT4.⁹⁹ The results of Tong et al indicated that CQ inhibited the epithelial–mesenchymal transition (EMT) which was triggered by starvation and could promote migration and invasion of bladder cancer cells T24 and 5637. The use of CQ in this study not only inhibited autophagy but also reduced the expression of TGF‐β1 and phosphorylated Smad3, both of which were induced in starvation.¹⁰⁰ Moreover, CQ was used in combination with other drugs, such as RAD001 (an inhibitor of mTOR signaling pathway), Lapatinib and Gefitinib (inhibitors of epithelial growth factor receptor (EGFR)), and Enzalutamide (an androgen receptor inhibitor) in treatment of advanced bladder cancer because of its autophagy inhibition properties.^25,101–103 In conclusion, recent advancements in understanding the molecular mechanisms of CQ’s anti-bladder cancer properties have illuminated novel approaches in the treatment of this challenging malignancy, offering exciting prospects for combination therapies in the fight against bladder cancer. While the prospects are promising, further preclinical and clinical studies are warranted to optimize the use of CQ in bladder cancer treatment.

Other Types of Cancers

In addition to the aforementioned cancer types, CQ also had inhibitory effect on other cancers, such as hepatocellular carcinoma (HCC), pancreatic cancer, melanoma, glioma, among others. Song et al investigated that autophagy could improve adaptability of liver cancer stem cells (LCSCs) which can drive and sustain the growth, metastasis, and recurrence of HCC under adverse conditions like hypoxia and nutrient deficiency. Their findings revealed that CD133⁺ cells which hold LCSCs properties were significantly enriched after hypoxia and starvation, higher autophagy level, higher survival and less apoptosis were detected in CD133⁺ cells. However, treatment with autophagy inhibitor CQ had significantly reversed these effects and dramatically impaired the colony-forming capacity of CD133⁺ cells and increased cells apoptosis.¹⁰⁵ In addition, the results of Xu et al demonstrated that in HepG2 cells, inhibition of autophagy with CQ decreased the degradation of lipid droplets by inhibiting autophagic flux and reducing ATP production, thereby hindering cell proliferation.¹⁰⁶ For pancreatic cancer, CQ dramatically facilitated the efficacy of pterostilbene, suppressing cell viability and downregulating the AKT/mTOR signaling pathway both in vitro and in vivo.¹⁷ Recently, CQ has been found to inhibit the neutrophil extracellular traps which was related to the hypercoagulability occurance in murine pancreatic cancer, the finding provided fresh insights into the anti-pancreatic cancer potential of CQ.¹⁰⁷ Autophagy also was induced in chemotherapeutics in melanoma and glioma, there was no doubt that CQ played important synergistic role in the chemotherapy of these tumors.^110,111,113 All these data affirmed the importance of autophagy for tumor cells, and autophagy inhibitor CQ was effective therapeutic strategy of human cancers.

Although numerous studies have documented that CQ induces cancer cell death via the inhibition of autophagy, the underlying mechanisms remain to be fully elucidated. Masud et al, using Primary Effusion Lymphoma (PEL) cells to identify the mechanisms of CQ-induced cancer cell death. They found that CQ could induce endoplasmic reticulum (ER) stress obviously and cause caspase-dependent apoptosis in vitro and in vivo through autophagy inhibition. The treatment of CQ did not affect the protein markers expression of NF-ĸB, JAK/STAT, and PI3K/AKT signaling pathways, indicating that CQ-induced cell death is independent of these pathways in PEL cells.¹⁸ More elaborate mechanisms were discovered, for example, the discovery of palmitoyl-protein thioesterase 1 (PPT1) which was the target of CQ and its derivatives in lysosomes and was highly expressed in most cancer cell lines. CQ derivatives have been used as therapeutic agents for decades, but no protein targets have been found, so the appearance of PPT1 was a major breakthrough. Knockout of PPT1 in cancer cells abrogated the enhancement of lysosomal deacidification and autophagy regulation of CQ derivatives.¹¹⁸ Recently, many new inhibitors for PPT1 have been designed, which greatly promoted the development of cancer treatment strategies.

CQ Suppressed Cancer with Autophagy-Independent Mechanisms

In tumor suppression, CQ also has activation of apoptosis and necroptosis of cancer cells that independent of autophagy inhibition. Apoptosis refers to the autonomic and orderly death of cells controlled by genes in order to maintain the stability of internal environment. Different from cell necrosis which is a passive process, apoptosis is an active process that adapts to the living environment.^119,120 Abnormal apoptosis may lead to cancer. There are two main apoptosis signaling pathways, one is the death receptor pathway, which activates the intracellular caspases through extracellular signals, and the other is the mitochondrial-mediated apoptosis pathway that activates caspases through the release of cytochrome c into cytosol.^121–123 In addition, lysosomal mediated apoptosis also plays an important role in cell death. Nowadays, more and more attentions have been paid to the role of lysosomes in regulating apoptosis. Lysosomes contain a variety of proteolytic enzymes, such as cathepsins and other hydrolases, and the release of these enzymes from the lysosomal lumen to the cytosol can promote the release of mitochondrial cytochrome c, activating caspases and eventually leading to apoptosis.^124–128

PI3K/AKT/mTOR signaling pathway is crucial for cell survival, proliferation and differentiation, especially in tumor cells. Studies have indicated that inhibitors against this pathway have become a focus in cancer treatment research. It was reported that CQ could enhance the effects of PI3K/AKT/mTOR inhibitors in mouse breast cancer cells 67NR and 4T1 in vitro, and which could not be imitated through knockdown or deletion of key autophagy-related genes.⁸⁰ Seitz et al revealed an underlying mechanism in which CQ and the PI3K/mTOR inhibitor BEZ235 cooperated to induce apoptosis via initiating LMP and lysosome-mediated apoptosis in neuroblastoma cells. The authors investigated that treatment with CQ alone led to the accumulation of CQ in lysosomes which initiated LMP, and inhibition of CQ in lysozymes markedly reduced LMP and apoptosis, which was independent of autophagy inhibition. The authors also found that CQ and BEZ235 cooperated to trigger Bax activation and loss of mitochondrial membrane potential (MMP), indicating that the combination treatment with CQ and BEZ235 could mediate mitochondrial outer membrane permeabilization (MOMP) and induce apoptosis. Otherwise, they further explored the correlation between LMP and MOMP through adding CA-074-Me, an inhibitor of lysosomal enzyme cathepsin B, which significantly reduced the loss of MMP and ultimately pointed that CQ and BEZ235 could synergize to trigger apoptosis via a lysosomal-mitochondrial cross-talk.¹⁹ Moreover, Harhaji-Trajkovic et al investigated that CQ induced apoptosis in serum-starved cancer cells in vitro and in vivo, and this effect could not be mimicked by autophagy inhibitors or LC3II shRNA, indicating a mechanism independent of autophagy inhibition. The authors found that CQ mediated lysosomal dysfunction in nutrient-deprived cancer cells which brought about oxidative stress, accompanied by loss of MMP and finally led to the activation of caspase cascades and apoptosis of cancer cells.²⁰ In CRC treatment, CQ induced ROS and also mediated the loss of LMP and MMP, facilitating the effect of SN-38 which is a chemotherapy agent in vitro and in vivo.⁹¹

The property of CQ that induces LMP categorizes it as an enhancer in drugs, genes or siRNA delivery. Studies have shown that CQ can markedly enhance the transfection activity and promote the expression of exogenous genes.^86,112 Furthermore, it plays a pivotal role in the field of nanotechnology for the treatment of cancer. CQ can promote the release of drugs, genes or siRNA from lysosomes by inducing LMP, improving the efficiency of drug delivery system, which strengthens the anti-tumor effect of agents. Bhattarai et al co-loaded CQ and plasmid DNA/siRNA into polycation- and PEG-coated mesoporous silica nanoparticles and significantly improved transfection efficiency, providing a powerful tool for tumor targeted therapy.¹⁰⁸ CQ co-packaged with cisplatin into hyaluronan (HA) nanogel facilitated the escape of cisplatin from lysosomal and enhanced its anti-tumor efficacy in breast cancer cells. The CQ/cisplatin HA nanogel also decreased toxicity compared with the combination of free CQ and cisplatin. In conclusion, CQ can not only play an active anti-tumor role by inducing LMP itself but also exert a synergistic role via assisting other drugs delivery.

ER stress can lead to proteotoxicity and induce apoptosis, becoming another breakthrough point in cancer therapy. Lopiccolo et al revealed that the combination of CQ and nelfinavir dramatically induced ER stress and increased the cell apoptosis of NSCLC in vitro and in vivo, in an autophagy-independent pattern.¹²⁹ Furthermore, research indicated that CQ mediated tumor vessel normalization.¹⁰⁹ In this study, the authors investigated that CQ could improve tumor perfusion and oxygenation, reduce invasion and intravasation of cancer cells, and promote tumor vessel normalization in metastatic melanoma models. However, these events were not simulated with ATG5 (an essential autophagy factor) knockdown, even though ATG5 silence decreased autophagy in cancer cells and increased tumor cell death. Therefore, the effect of CQ on normalizing tumor vessels was not due to its inhibition of tumor autophagy but an autophagy-independent mechanism.¹⁰⁹ CQ was also reported to reduce cancer stem cells (CSC) in pancreatic cancer and decrease tumorigenicity in vivo, this was contributed to its suppression on the CXCL12/CXCR4 signaling pathway.¹¹⁴

These researches strongly suggested that CQ could induce cancer cell apoptosis through a pathway independent of autophagy inhibition (Figure 4). Many other researches also amply demonstrated this opinion.

Figure 4 Mechanisms of CQ induced apoptosis in tumor cells independent of autophagy inhibition.

CQ Combined with Chemoradiotherapy for Tumor Treatment

CQ Combined with Chemotherapy Drugs in Cancer Therapy

Deregulation of autophagy is believed to play a key pathogenic role in cancer cells. Therefore, CQ, as an autophagy inhibitor combined with other chemotherapy drugs, is widely used in the treatment of various cancers, making cancer cells more sensitive to chemotherapy. In recent years, many studies have reported on the combination with CQ. Cisplatin is widely used as one of the most effective chemotherapeutic agents for the treatment of cancers¹³⁰ and has been shown to increase apoptosis in human lung cancer cells (A549 and H460) and epithelial ovarian cancer cells (SKOV3 and hey) when combined with CQ, via autophagy inhibition.^16,131 Chen et al revealed that pterostilbene combined with CQ significantly improved autophagy inhibition, decreased cell viability and increased apoptosis in pancreatic ductal adenocarcinoma cells via the downregulation of the RAGE/STAT3 and AKT/mTOR pathways.¹⁷ Moreover, Monma et al found CQ could enhance TRAIL-induced apoptosis in two human pancreatic cancer cell lines: MiaPaCa-2 and Panc-1. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which can deliver death signals via the extrinsic apoptosis pathway and induce cancer cell death, was reported to encounter resistance in tumor cells. The authors investigated that CQ could improve the sensitivity of cancer cells to TRAIL, contributing to apoptosis via partially downregulating the anti-apoptotic proteins and inducing cell cycle arrest at the G2/M phase.⁸¹ Therefore, the research further proved that combination with CQ in the treatment of pancreatic cancer was assuredly promising. Wang et al combined CQ with gemcitabine which is an anti-metabolic nucleoside analog, aiming at gallbladder cancer (GBC) and found that CQ significantly enhanced the inhibition of proliferation and colony formation, facilitated the induction of apoptosis and cell cycle arrest.⁸² Nelfinavir is a human immunodeficiency virus protease inhibitor, currently being repositioned as an anticancer drug for its inhibition of cancer cell proliferation and induction of apoptosis.^92,129 Johnson et al revealed that CQ could promote nelfinavir-induced ER stress and cell death due to its cytotoxic action in an autophagy-independent manner.⁹³

CQ Used as Sensitizer in Radiotherapy Against Cancer

In addition to chemotherapy, CQ has been shown to enhance the sensitivity of radiation therapy for cancer. Findings showed that a low dose of CQ (10 μM) was sufficient to enhance radiosensitization and induce cell death without producing significant cytotoxicity in breast cancer cells. The authors also revealed that CQ-mediated radiosensitization attributed to the destabilization of lysosomal membrane and increased cell necrosis.¹⁰⁴ In mouse model, a low dose of CQ (40 mg/kg) significantly restrained the invasion and migration of triple negative breast tumor cells, which were enhanced by radiotherapy. The underlying mechanism was that CQ decreased the levels of interleukin-1β, interleukin-6, and cyclooxygenase-2, which are inflammatory factors.¹³² In colorectal cancer, CQ compared with temsirolimus (a mTOR inhibitor) dramatically induced apoptosis and increased the efficiency of ionizing radiation in vitro and in vivo. CQ alone or combined with 5-FU enhanced the sensitivity of HCT-116 and HT-29 cells to radiotherapy in vitro.^133,134 CQ was also detected to enhance the sensitivity of radiation in bladder cancer cells.¹³⁵

Clinically, CQ has shown significant sensitization to radiotherapy or chemotherapy, as verified by Briceno et al. In their research, CQ was used as an adjunct therapeutic agent to treat glioblastoma multiforme. The average survival time of patients treated with CQ was significantly longer than those treated with conventional therapy.¹³⁶ The analysis of clinical data also confirmed this view, indicating that the effect of the combined autophagy inhibitor CQ was significantly better than that of traditional therapies.^137,138 The sensitization of CQ to conventional cancer therapies, such as chemotherapy and radiotherapy, makes CQ a potential new adjuvant drug for cancer treatment. Further exploration of new properties of CQ is anticipated.

Clinical Trials and Adverse Reactions of CQ and Its Analogues

Autophagy is activated in tumor patients during chemoradiotherapy, serving as a survival mechanism for tumor cells. Therefore, as autophagy inhibitors, CQ and its analogues are widely used as adjuvant agents in chemoradiotherapy. Among the analogues, mainly CQ and HCQ are used in clinical trials (Table 3). CQ has been used in glioblastoma multiforme treatment accompanied with chemotherapy or radiotherapy of all patients. The results showed that patients receiving CQ displayed lower death rate compared to those in the control group, which may be due to the ability of CQ to enhance the cytotoxicity caused by conventional therapy or prevent the mutagenicity of tumor cells.^136,139 A Phase II trial was carried out to assess the efficacy of CQ combined with taxane or taxane-like drugs in treatment of patients with advanced or metastatic breast cancer who are resistant to anthracycline chemotherapy drugs. The results indicated that the combination was more effective than chemotherapy alone and without significant toxicity.¹⁴⁰ The trial of Horne et al presented an obvious enhancement of the combination of HCQ and imatinib compared with imatinib used alone in chronic-phase chronic myeloid leukaemia, but a common diarrhea was happened with one case displayed cardiac rhythm disorder, dyspnoea and heart failure.¹⁴¹ High dose HCQ (1800 mg/d) combined with gemcitabine/nab-paclitaxel revealed improved pathological tumor response and serum biomarker response in pancreatic cancer, and HCQ did not increase the toxicity of chemotherapy.¹⁴² However, the other study displayed a dose-limiting toxicity of HCQ (the maximum tolerated dose (MTD) was 600 mg/d), and at the MTD dose, HCQ did not show obvious improvement in the survival of glioblastoma patients, which remind us that more low-toxicity autophagy inhibitors need to be developed in tumor adjuvant therapy.¹⁴³ In addition, the combinations of HCQ in clinical trials of other tumor types, such as non-small cell lung cancer, melanoma, colorectal cancer and prostate, have also demonstrated that it can significantly improve treatment response of chemotherapy agents and possess great anti-tumor potential.^144–150

Table 3 Clinical Trials and Adverse Reactions of CQ and Its Analogues

Although CQ and its analogues can improve the efficacy of chemotherapy or radiotherapy, safety concerns have arisen, with severe side effects observed in some cases. Rustogi et al noted in their medical work that CQ could cause skin desquamation when used alongside radiotherapy.¹⁵¹ Because of the double-edged properties of autophagy, CQ’s inhibition of autophagy sensitized not only cancer cells but also normal cells to chemotherapy, potentially causing acute kidney damage while increasing chemotherapy sensitivity in cancer cells.^153,154 Furthermore, combination with CQ in chemotherapy also caused damage to other organs, such as the liver, heart, brain and hematopoietic cells, due to the critical role of autophagy in these organs.^155–159 Additionally, Angel et al evaluated the effect of CQ alone in breast tumor patients before their surgery and found that compared with placebo, treatment with CQ had no significant effects on breast cancer cellular proliferation but was associated with toxicity.¹⁵² These findings highlight the side effects of CQ, underscoring the need for more clinical trials to further verify its pharmacological properties.

Nanotechnology Improve the Safety of CQ

The side effects of CQ limit its usage, but the emergence of nanotechnology, a novel technique that has already contributing to a huge impact in the biomedical sciences, particularly in the fields of cancer treatment and diagnosis, has effectively reduced the side effects of CQ and greatly improved its efficacy in cancer therapy. CQ wrapped in gold nanoparticles displayed an obvious necrosis in MCF-7 and reduced its side effects compared with CQ along.¹¹⁶ Drug delivery systems play a vital role in inhibiting CSCs. CQ was reported to reduce CSCs in pancreatic cancer and decrease tumorigenicity in vivo, nanoparticles carrying CQ accurately and efficiently delivered CQ to the tumor site, suppressing tumor development.^114,160,161 Sun et al encapsulated CQ and other chemotherapeutic agents, such as DOX, DTXL into poly(ethylene glycol)-block-poly(D, L-lactide) (PEG-b-PLA) along or together, forming nanoparticles about 110 nm in diameter, which significantly increased drug delivery efficiency. The nanoparticles contained CQ and other drugs effectively enhanced autophagy inhibition, reduced CSCs and improved the efficacy of chemotherapeutic drugs against breast cancer in vitro.¹⁶² These studies indicate that nanoparticle formulations of CQ are a promising strategy with strong prospects for application.

Application of CQ in Other Diseases

In addition to its anti-malaria and anti-tumor properties, CQ also exerts inhibitory effects on rheumatic autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sjögren’s syndrome. Moreover, it possesses antiviral activity, including the inhibition of HIV-1 and the severe acute respiratory syndrome (SARS) coronavirus. Here, we provide a brief overview and discussion of these properties of CQ (Figure 5).

Figure 5 Functional classifications of CQ.

Inhibition of Rheumatic Autoimmune Diseases

Rheumatic autoimmune diseases, such as RA, SLE and Sjögren’s syndrome, are types of diseases caused by the immune system mistakenly attacking its own tissues, including joints.¹⁶³ CQ and its analogue HCQ have been reported to be effective in treatment of rheumatic autoimmune diseases because of their anti-inflammatory and immunomodulatory properties.¹⁶⁴ The possible mechanisms are related to the inhibition of autophagy, lysosomal acidification, suppression of toll-like-receptors (TLRs), prevention of the interaction between TLRs and their ligands, and restraint of calcium allocation.¹⁶³ TLRs are transmembrane proteins and expressed in various types of cells, like macrophages, monocytes, T/B lymphocytes, and dendritic cells, act as the host’s innate immune defense sensors and are associated with the activation of adaptive immunity. TLRs can recognize pathogenic microorganisms or endogenous second messengers (such as nucleic acids), inducing the production of pro-inflammatory cytokines, causing immune response and even inflammatory. Pro-inflammatory cytokines like TNF-α, IL-1 and IL-6 are essential in mediating the inflammatory response in RA, SLE and Sjögren’s syndrome.^165–167 More than 10 TLRs have been identified in mammals and humans, with TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 expressed on the plasma membrane, and TLR3, TLR7, TLR8 and TLR9 expressed in endosomes and involved in identifying nucleic acid components of microorganisms.¹⁶⁸ Studies have shown that CQ can inhibit TLR3, TLR7 and TLR9, impairing the TLR signaling via blocking the binding of TLR to ligands, disturbing endosomal acidification or other mechanisms as described above, thus suppressing the secretion of pro-inflammatory cytokines.¹⁶³ Therefore, the inhibition of the TLR signaling pathway by CQ plays an important role in the treatment of rheumatic autoimmune diseases.

Antiviral Activity

Apart from its anti-inflammatory activity, CQ also possesses antiviral properties with a broad spectrum of virus types.^169,170 Studies have shown that CQ can inhibit HIV-1/AIDS virus in vitro no matter the treatment is executed before or after the infection of cells with the virus, and this effect may be attributed to the ability of CQ to inhibit the glycosylation of the virus receptor on the cell, thereby reducing HIV-1/AIDS infection.^171–173 CQ also had a braking effect on the SARS coronavirus, where it suppressed the replication of the SARS coronavirus and decreased the glycosylation of SARS coronavirus receptor ACE2 that expressed on the human cell surface.^174,175 Moreover, Yiwu et al have found that the avian influenza A H5N1 virus infection leads to acute lung injury through triggering autophagic cell death of alveolar epithelial. As an autophagy inhibitor, CQ significantly reduced lung injury and prolonged the lifespan of mice infected with the avian influenza A H5N1 virus.¹⁷⁶ The massive outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 claimed many lives, there was no definitive treatment in the early phase, but scientists have found that the old drug CQ and its derivative HCQ have an inhibitory effect on this novel coronavirus which are similar to the SARS coronavirus and use ACE2 as a receptor to enter cells.^177,178 However, some people are skeptical about the conclusion that CQ is effective in treating the SARS-CoV-2 and note its significant side effects.¹⁷⁹ But the research on CQ against SARS-CoV-2 is continuing and its potential prophylactic and therapeutic effects remain to be determined.

Existing researches have shown CQ has a broad spectrum antiviral activity or can be used to treat complications caused by certain vital infections. More studies on the antiviral mechanism of CQ are expected to expand its medicinal value and scope of application.

Conclusion

CQ is a widely used antimalarial drug that has received much attentions for its potential use in cancer therapy recently. Early studies have found that CQ can induce apoptosis of tumor cells by inhibiting autophagy, thus inhibiting tumor growth and diffusion.^99,105,106 Studies also have shown that CQ enhances the efficacy of certain chemotherapeutic drugs by blocking autophagy, thus increasing the accumulation of damaged proteins and organelles, and sensitizing cancer cells to chemotherapy-induced cell death.

While autophagy is a well-accepted survival mechanism during cancer treatment with different chemotherapy agents, and inhibiting autophagy with CQ increases the sensitivity of anticancer drugs, there are still people challenge this view and test it. Maycotte et al suggested that combination treatment with CQ in cancers should consider the possibility that CQ may act through mechanisms other than inhibition autophagy, since in their research they found that CQ could decrease the viability of cancer cells treated with chemotherapy but this effect could not be mimicked with autophagy associated proteins knockdown or autophagy inhibitors treatment. Hence, the authors concluded that CQ mediated chemotherapy sensitization was an autophagy-independent event in tumor cells.⁸⁰ Beyond the autophagy-independent mechanisms above CQ was always thought to induce tumor cell apoptosis via activating the p53 pathway. Kim et al found that CQ treatment stabilized p53 protein prominently and increased the expression of p53 target genes simultaneously in the glioma lines expressing wild type p53, compared with the cells lacking functional p53. They also reported a mitochondrial dysfunction apoptosis pathway that is independent of p53 effect which may be consistent with those described above.¹⁸⁰ In summary, we draw a conclusion that CQ has many other autophagy-independent mechanisms in tumor therapy and one should put the mechanisms independent of autophagy inhibition into account when treated with CQ in their studies (Figure 6).

Figure 6 The pharmacological mechanisms of CQ with autophagy dependent or independent in cancer therapy. CQ inhibited autophagy and induced ROS, leading to the loss of LMP. CQ also promoted the activation of Bax, which further led to the release of cytochrome c by mitochondria and mediated the apoptosis of tumor cells. CQ also blocked the PI3K/AKT/mTOR signaling pathway and inhibited tumor cell proliferation. In addition, CQ decreased the expression of TGF-β and inhibited EMT. CQ also inhibited the TLRs and thus played its anti-inflammatory properties on rheumatic autoimmune diseases. Abbreviations: CQ, Chloroquine; ER, Endoplasmic reticulum; ROS, Reactive oxygen species; LMP, Lysosomal membrane permeabilization; MOMP, Mitochondrial outer membrane permeabilization; EMT, Epithelial–mesenchymal transition; TLRs, Toll-like-receptors.

In addition, CQ can inhibit tumor growth through various mechanisms, including inducing tumor cell necrosis and preventing angiogenesis. In recent years, more and more studies have shown that CQ not only has anti-autophagy effect but also can affect tumor immunity and remodel tumor microenvironment. Research showed that CQ could act as an antitumor immunomodulator to the tumor-associated macrophages (TAMs), which could be switched from M2 phenotype to tumor killing M1 phenotype. The specific mechanism was that CQ induced the activation of p38 and NF-κB via increasing the pH of macrophage lysosomes and leading to the release of Ca²⁺, thus polarizing TAMs into the M1 phenotype. Resetting macrophages to improve the tumor immune microenvironment raised anti-tumor T cell immunity and enhanced the body’s ability to attack cancer cells.^181,182 Therefore, CQ has extensive antitumor activity and potential therapeutic application value.

Except its anti-malaria and anti-tumor properties, CQ also has inhibitory effects on rheumatic autoimmune diseases, such as RA, SLE and Sjögren’s syndrome, and also possesses antiviral activity, like the inhibition of HIV-1 virus and SARS coronavirus. More studies on these properties and its underlying mechanisms are expected to expand the medicinal value and scope of application of CQ.

Although CQ has shown promise in antitumor research, there are still many problems to be solved. For example, the antitumor mechanism of CQ is not fully understood, necessitating further investigation. Clinical trials are still ongoing to determine its safety and efficacy in humans. In addition, there are certain side effects associated with clinical use of CQ, which need to be better understood and managed. Moreover, the exploration of CQ’s potential in combination with other antitumor agents and its applicability across various tumor types also warrants further study.

Looking ahead, the antitumor research of CQ is poised for further development, including exploring more therapeutic application scenarios, improving dosage forms and medication regimens, and developing new CQ analogues. In general, the antitumor research of CQ holds broad application prospects and potential therapeutic value, and future research endeavors will continue to advance its development and clinical therapy applications.