Chloroquine Induces Lysosomal Membrane Permeability- Mediated Cell Death in Bladder Cancer Cells

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Original Article

Chloroquine induces lysosomal membrane permeability- mediated cell death in bladder cancer cells

Hung‑En Chen1, Ji‑Fan Lin2, Yi‑Chia Lin1,3, Shen‑I Wen2, Shan‑Che Yang2, Te‑Fu Tsai1, Kuang‑Yu Chou1, I‑Sheng Thomas Hwang1,2,3,4 1Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, 2Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, 3School of Medicine, Fu-Jen Catholic University, New Taipei City, 4Department of Urology, Taipei Medical University, Taipei, Taiwan

Abstract Background: Chloroquine (CQ) is recognized as a potent adjuvant when combined with other chemotherapies to treat cancers. However, the effects of a single treatment of CQ on bladder cancer (BC) cells have not been investigated. Methods: The growth and viability of CQ‑treated BC cells were examined. The lysosomal morphology was detected using LysoTracker. The induction of lysosomal membrane permeability (LMP) was detected by acridine orange (AO) translocation, and cathepsin B and D release. The expression of the bid, caspase‑3, and cytosolic cytochrome C (Cyto. C) in CQ‑treated cells was detected by the Western blot. The pepstatin A and E64d were used to attenuate CQ‑induced LMP. Results: A single dose of CQ treatment induced BC cell death, and attenuated by pepstatin A and E64d. The diminishing of fluorescent in CQ‑treated cells stained with LysoTracker, suggesting that CQ targets lysosomal functions. This was further supported by increased AO translocation and the releasing of CatB and CatD into the cytosol. The increased level of cleavage bid and cytosolic Cyto. C indicated mitochondrial outer membrane permeabilization and subsequently leading to apoptosis induction judged by the increased level of activated caspase 3. Conclusion: CQ‑induced LMP that enhances apoptosis and ultimately leading to BC cell death. The study results demonstrated for the first time that single CQ treatment against BC cells by inducing LMP and subsequent mitochondria membrane permeability that trigger apoptosis, making it a potential treatment for BC therapy in the future.

Keywords: Apoptosis, autophagy, bladder cancer cells, chloroquine, lysosomal membrane permeability

Address for correspondence: Dr. I‑Sheng Thomas Hwang, Department of Surgery, Shin Kong WHS Memorial Hospital, Shin Lin District, Taipei City, Taiwan. E‑mail: [email protected] Received: 19-May-2017, Revised: 30-Oct-2017, Accepted: 14‑Jan‑2018

INTRODUCTION urothelial cell carcinomas is accounting for more than 90% of BC s while nearly 70% of bladder tumor present Bladder cancer (BC) is the most common neoplasm in the as superficial (nonmuscle‑invasive) BC.[2] Transurethral urinary tract and has a very high recurrence rate.[1] The resection of bladder tumor (TUR‑BT) is the standard treatment for patients with superficial tumors. However, Supplementary Video Available on: www.e-fjs.org This is an open access journal, and articles are distributed under the terms of the Creative Access this article online Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to Quick Response Code: remix, tweak, and build upon the work non-commercially, as long as appropriate credit Website: is given and the new creations are licensed under the identical terms. www.e-fjs.org For reprints contact: [email protected]

DOI: How to cite this article: Chen HE, Lin JF, Lin YC, Wen SI, Yang SC, Tsai TF, 10.4103/fjs.fjs_83_17 et al. Chloroquine induces lysosomal membrane permeability-mediated cell death in bladder cancer cells. Formos J Surg 2018;51:133-41.

Chen, et al.: CQ‑induced LMP‑mediated apoptosis in bladder cancer cells approximately 60%–70% of these tumors will recur, Hsinchu, Taiwan). The SV‑Huc‑1 cells were cultured in F12 and 25% will continue to progress to a higher stage or medium (Invitrogen). The 5637 and T‑24 cells were cultured grade.[3] Although mitomycin and Bacillus Calmette‑Guerin in RPMI‑1640 medium (Invitrogen, Carlsbad, CA, USA). installation followed by TUR‑BT have shown some evidence These cells have performed STR‑PCR profile at BCRC. Cell

of activity against tumor recurrence, their incomplete lines were maintained at 37°C under 5% CO2. Media were efficacy and toxicity have limited their use as common supplemented with 10% fetal bovine serum (FBS; Invitrogen), chemotherapy agents.[4] Therefore, the development of 2 mM GlutaMAX‑1 (Invitrogen), 100 units/ml penicillin, novel adjuvant agents against BC is warranted. and 100 µg/ml streptomycin (Invitrogen). Cells were treated with the indicated concentration of CQ, and control cells The chloroquine (CQ) is on the list of essential medicines received an equal volume of DMSO. The final concentration of the World Health Organization and is classically used to of DMSO was <0.1%. prevent and treat malaria.[5] The CQ treatment was reported to induce apoptotic cell death in melanoma, glicoma, and Cell viability assays lung cancer cells.[6‑8] In addition, CQ showed lower toxicity A live cell imaging system (CytoSmart System, Lonza, to nontumorigenic epithelial cells.[9] Recently, CQ has been Visp, Switzerland) was used to record the cell morphology demonstrated to be an enhancing agent in cancer therapies in control or CQ‑treated cells in a real‑time fashion. when combined with other chemotherapeutic agents.[10,11] Control and treated‑cells were constantly and automatically However, the effects of CQ single treatment on BC have monitored with 15 min interval for 72 h. The time‑lapse not been investigated. photos as well as the recorded videos were generated by the build‑in software. The effect of CQ on cell viability We recently showed that human BC tumor exhibits a high at 24–72 h posttreatment was assayed by the WST‑1 basal level of autophagy, and the autophagic activity is reagent (Roche Diagnostics, Mannheim, Germany) as required for the survival of human BC cells. Inhibition of described.[16] In some experiments, 10 μM pepstatin basal autophagy induces apoptotic cell death.[12] CQ has been A (inhibitor for cathepsin D) and 10 μM E64d (inhibitor for routinely used to block the fusion of autophagosomes with cathepsin B/L) was added 1 h before the treatment of CQ lysosomes to study the role of drug‑induced autophagy in to inhibit lysosomal proteases. cultured cancer cells.[13] As an effective autophagy inhibitor, CQ has also been tested in several ongoing clinical trials, Monitoring of lysosomal morphology alone or in combination with other anti‑cancer drugs.[14,15] Alternation of lysosomal morphology in CQ‑treated In the present study, we examined the mechanism involved BC cells was monitored by LysoTracker Red DND‑99 in CQ‑induced cell death focusing on the disruption (Thermo Fisher Scientific, Waltham, MA, USA). In brief, of lysosomal membrane permeability (LMP), and the cells grown in glass chamber slides (BD Biosciences, subsequent linkage to mitochondria outer membrane San Diego, CA, USA) were incubated with indicated permeability (MOMP) that ultimately leading to apoptotic concentrations of CQ. After the CQ treatment, the cells cell death. The study findings provide a new insight of CQ were washed twice with phosphate‑buffered saline (PBS), single treatment against human BC cells and the potential incubated with 100 nM LysoTracker Red DND‑99 use in BC therapy in the future. labeling solution for 30 min at room temperature. When labeling is complete, the solution was removed, and the MATERIALS AND METHODS cells were washed twice with PBS, and then stained with Hoechst 33342 dye (2 μg/ml) at 37°C for 5 min to label Chemicals the nucleus. The labeling solution was quickly removed, The CQ was purchased from Sigma‑Aldrich (St Louis, MO, and the cells were gently washed twice and subjected to USA) and prepared at a concentration of 100 mM. Aliquots imaging. Fluorescent imaging was performed with a Nikon were stored at‑20°C. All other chemicals, unless otherwise inverted microscope‑Eclipse Ti‑E equipped with 130W mentioned, were purchased from Sigma‑Aldrich. fluorescence light source and fitted with Nikon color CMOS Camera‑DS‑Ri2. Images were collected with filter Cell culture bandwidths corresponding to 560–615 nm for red. The human cancer cell lines 5637 (American Type Culture Collection, ATCC#HTB‑9) and T‑24 (ATCC#HTB‑4); and Detection of lysosomal membrane permeability human ureter sumian virus 40 (SV40)‑transformed epithelial To measure lysosomal membrane integrity, cells with or cell line SV‑Huc‑1 (ATCC#CRL‑9520) were obtained without CQ treatment were stained with 10 1 μg/ml from Bioresource Collection and Research Center (BCRC; acridine orange (AO) for 15 min at 37°C, and washed

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Chen, et al.: CQ‑induced LMP‑mediated apoptosis in bladder cancer cells 3 times in PBS to reduce background. LMP was quantified two‑tailed Student’s t‑test. P <0.05 was considered to by measuring the reduction of red fluorescence using an be statistically significant. All analysis was carried out Accuri C5 flowcytometer from BD Bioscience. with the program SigmaPlot Version 10.0 for Windows (Systat Software Inc., Chicago, IL, USA). Immunofluorescence detection of cathepsin B and D release Ethical approval Cells were grown on glass chamber slides, treated with The study was conducted in accordance with the indicated concentrations of CQ for 24 h, and fixed with Declaration of Helsinki and was approved by the local 4% paraformaldehyde in PBS for 30 min at 37°C. Cells ethics committee of the institute. Informed written consent were permeabilized with 0.1% Triton‑X100 in PBS for was obtained from all patients prior to their enrollment in 30 min and blocked with PBS containing 3% bovine serum this study. albumin at room temperature for 1 h. After incubation with antibodies against cathepsin B or D (Santa Cruz RESULTS Biotechnology) at 4°C overnight, the cells were washed three Chloroquine inhibits the proliferation of bladder cancer times with PBS and treated with the FITC‑conjugated goat cells anti‑rabbit secondary antibody for 90 min at 37°C. Cells To access the anticancer activity of CQ on human BC, we were washed three times in PBS and counterstained with first investigated the effect of CQ on the proliferation of DAPI to visualize the nuclei before additional washing and immortalized urothelial cell line, SV‑Huc‑1, and two BC mounting with ProLong Gold antifade reagent (Invitrogen). cell lines, 5637 and T24. While the cell morphology of Slides were stored in the dark at 4°C until observation under SV‑Huc‑1 was not altered by the treatment of 50 μM CQ, fluorescent microscopy as described above. we found both 5637 and T24 cell morphological shrunk and Evaluation of mitochondrial membrane depolarization floated with the increased incubation periods [Figure 1a, and permeability and Supplementary Videos 1-3]. Consistent with these To measure the mitochondrial membrane depolarization, observations, the cell viability, detected by WST‑1 reagents, cells were treated with CQ at the indicated concentrations. of 5637 and T24 cells decreased with CQ treatment in Mitochondrial membrane depolarization was detected with a dose‑ and time‑dependent manner [Figure 1b]. On the mitochondrial membrane potential assay kit with JC‑1 the other hand, decreased cell viability was observed in dye according to the manufacturer’s protocol (Genedirex, SV‑Huc‑1 cells treated with 25 μM CQ for 72 h or 50 and Taipei, Taiwan). The data were acquired and analyzed using 100 μM CQ for 48 and 72 h [Figure 1b]. Accuri C5 flow cytometry as described.[16] The mitochondrial Chloroquine induces permeabilization of lysosomal membrane permeability was measured by detecting the membranes release of mitochondrial cytochrome c (Cyto. C) into the As a lysosomotropic agent, CQ can rapidly diffuse into cytosol by Western blot. Cytosolic proteins from cells cells and been trapped in lysosomes.[17] We first labeled with the same treatment were isolated using Mitochondria lysosomes with LysoTracker red DND‑99 and monitored Isolation Kit for culture cells (Thermo Fisher Scientific) morphological changes of lysosomes associated with CQ following the manufacturer’s instruction and subsequently treatment. As shown in Figure 2a, the LysoTracker‑stained used in the Western blot analysis using antibody against Cyto. control cells displayed a normal punctuate localization C. β‑actin was detected as a loading control. in the perinuclear region. The CQ‑treated lysosomes Detection of the bid, PARP, and cleaved caspase 3 by swelled, increased, and accumulated in the cytosol with the Western blot increasing CQ administration. The red fluorescent Cell lysates from cells treated with indicated concentrations intensity is then gradually decreased with increased CQ of CQ with or without the pretreatment of lysosomal concentration [Figure 2a]. Treatment with CQ produced a protease inhibitors (pepstatin A and E64d; PepA/E64d) gradual and concentration‑dependent increase in Lysotracker were prepared for the Western blot analysis, using red fluorescence intensity, suggesting that CQ‑induced antibodies against the bid, cleaved bid (c‑Bid), PARP, and alterations of lysosomal pH. Furthermore, cells were treated cleaved caspase‑3 (c‑Casp3). with indicated concentration of CQ for 1, 6, and 24 h and stained with another acidophilic dye, AO. While the increased Statistical analysis red fluorescent was monitored in cells treated with an All data were expressed as mean values ± standard increased concentration of CQ for 1 h, a diminution of the deviation. Statistical evaluation was determined using red AO fluorescence was detected at 6 h of CQ treatment

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b Figure 1: Treatment of chloroquine decreased cell viability in 5637 and T24 cells but had minimal impact on SV‑Huc‑1. (a) Real‑time monitoring of cell viability in chloroquine‑treated 537, T24 and SV‑Huc‑1 using image recorder CytoSmart system. Cells seeded on 6‑well plate were treated with 25 μM chloroquine for 1 h and the medium was refreshed prior to the recording. Representative time‑lapse images at 0, 12, 24, 36, 48, 60, and 72 h were shown. (b) Cell viability in chloroquine‑treated cells detected by WST‑1 reagents. Cells were seeded in 96‑well plates and were treated with chloroquine for the indicated concentrations and durations. Cell viability was detected using WST‑1 reagents. The values are shown as the mean ± standard deviation of three independent experiments. *P < 0.05

and further decreased at 24 h of treatment [Figure 2b‑d]. means AO‑load cells manifest reduced red fluorescence and The results indicated CQ‑induced AO translocation which increased green fluorescence after LMP.[18]

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b d

Figure 2: Chloroquine‑induced permeabilization of lysosomal membranes. (a) chloroquine altered lysosomal morphology in 5637 and T24 cells. Cells were treated with indicated concentrations of chloroquine for 24 h prior to the detection of lysosome using LysoTracker DND‑99. (b) Increased acidic vesicles in chloroquine‑treated cells with acridine orange staining. 5637 and T24 cells were treated with indicated concentrations of chloroquine for 1 h and stained with 1 μg/ml acridine orange for 15 min. After PBS washing, the samples were then proceeding immediately to image acquisition. (c) Chloroquine treatment resulted in acridine orange translocation, suggesting an increased permeability of lysosomal membranes. T24 cells were treated with indicated concentrations of chloroquine for 6 or 24 h, stained with acridine orange then proceed immediately for imaging. (d) Decreased acridine orange red fluorescent in chloroquine‑treated 5637 and T24 cells at 24 h posttreatment detected by flow cytometry. The values are shown as the mean ± standard deviation of three independent experiments. *P < 0.05

The lysosomal permeabilization was further confirmed in proteolytic activation of bid cleaved by lysosomal Cat B cells treated with CQ by detecting the release of cathepsin and Cat D induces MOMP, resulting in Cyto. C release B (Cat B) and D (Cat D) into the cytosol [Figure 3a]. To and apoptosome‑dependent caspase activation.[18] We, test whether CQ‑induced LMP is unique in the BC cells, we therefore, investigated the mitochondria membrane treated human immortalized urothelial cell line, SV‑Huc‑1, potential (MMP) and MOMP in the CQ‑treated cells. As with CQ and detected the releasing of Cat B. As shown shown in Figure 4a, mitochondria depolarization was not in Figure 3b, the release of Cat B was only detected in profound in control cells and in those treated with low T24 cells. These results indicated that CQ mediates the concentrations of CQ but was significantly increased in permeabilization of lysosomal membranes predominantly cells treated with relatively high concentrations of CQ in human BC cells. (50 and 100 μM), indicated by a decrease in the red/green fluorescence intensity ratio. These results indicated CQ Chloroquine‑induced lysosomal damages trigger induces the loss of MMP in BC cells. It is possible that mitochondrial membrane depolarization permeability, CQ‑induced lysosomal damages resulted in the release of increased cytochrome c release, and bid activation Cat B and Cat D that activated bid to disrupt mitochondrial The LMP is considered a potentially lethal event because function. We, therefore, detected the expression level of the presence of lysosomal proteases in the cytosol causes the cleaved bid (c‑Bid) in CQ‑treated cells. As depicted in digestion of vital protein and the induction of apoptosis Figure 4b, activation of bid on CQ treatment was observed through the activation of caspases cascade. For example, in a dose‑dependent manner. Moreover, the increased

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b Figure 3: Chloroquine increased the release of cathepsin B and D from lysosome into cytosol. Immunofluorescence detection of cathepsin B (Cat B) and D (Cat D) in 5637 and T24 cells. (b) Immunofluorescence detection of Cat B in chloroquine‑treated T24 and SV‑Huc‑1 cells. The number of cells with diffused green fluorescent in the cytosol were count from 40 randomly selected photos in each condition, and the statistic results were shown in the right panel. The values in the histograms are mean ± standard deviation from three independent experiments. *P < 0.05

level of c‑Bid was attenuated when cells pretreated with A and E64d) or inhibition of CQ‑induced apoptosis with pepstatin A and E64d that inhibit the protease activity of pan‑caspase inhibitor (Z‑Vad‑FMK) attenuated the induced cathepsins [Figure 4c]. In addition, the release of Cyto. apoptosis indicated by the reduced caspase 3 activity [Figure 5b], C, activation of caspase‑3 and PARP (c‑PARP) were also but failed to fully recover the reduced viability in CQ‑treated increased in CQ‑treated cells [Figure 4d]; and the activation cells [Figure 5c]. However, pretreatment of bafilomycin A1 (Baf of these pro‑apoptotic markers was attenuated by the A1), an inhibitor of the lysosomal vacuolar H + ATPase, pretreatment of pepstatin A and E64d [Figure 4d]. These inhibited the reduced viability [Figure 6a] and attenuated the findings suggested that CQ induces LMP that leading to activated caspase 3 activity [Figure 6b]. In conclusion, CQ the activation of the bid and subsequently resulted in the induced LMP, MOMP and z‑Vad‑FMK‑resistant cell death release of Cyto. C followed by induction of apoptosis through a pathway that depends on the CQ accumulation in through caspase‑3 activation. lysosomes.

Chloroquine‑induced caspase‑dependent DISCUSSION and–independent cell death Cells treated with CQ‑induced several hallmarks of In 1964, a report published by Kimura I. and Hiraki K apoptosis, including the increased level of cleaved from the Okayama University Medical School describing caspase 3 [Figure 4d], and activation of caspase 3 activity the use of CQ to treat malignant tumors.[19] They claimed [Figure 5a]. Inhibition of CQ‑induced LMP with the that excellent therapeutic effects were obtained in patients pretreatment of lysosomal protease inhibitors (pepstatin with BC. Their data demonstrated the improvement of

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a b

c d Figure 4: Disruption of lysosomal membrane permeability by chloroquine resulted in increased MMP, Bid activation and cytochrome C release. (a) Chloroquine‑induced MMP in bladder cancer cells. Cells were treated with indicated concentrations of chloroquine for 24 h, then the MMP was detected by JC‑1 staining using a flow cytometer. Representative flow histograms of each condition were shown in the right panel. The values are shown as the mean ± standard deviation of three independent experiments. *P < 0.05. (b) Chloroquine increased cleaved bid (c‑Bid) expression in bladder cancer cells. (c) Pretreatment of Pepstatin A/E64d (PepA/E64d) attenuated the activation of Bid in chloroquine‑treated bladder cancer cells. Cells were treated with 50 μM chloroquine with or without 2 h pretreatment of PepA/E64d for 24 h. The 5637 and T24 cells were treated with indicated concentrations of chloroquine or 50 μM chloroquine with or without the 2 h pretreatment of PepA/E64d for 24 h. Total proteins from each sample were extracted and subjected to the detection of Bid and c‑Bid by Western blot. β‑actin serves as loading control. Representative blots from three experiments with similar results were shown. Quantitative analysis of c‑Bid expression was performed using ImageJ software, and the results were shown in the lower histograms. The values in the histograms are mean ± standard deviation from three independent experiments. *P < 0.05. (d) Pretreatment of PepA/E64d attenuated chloroquine‑induced cytochrome C release and expression of pro‑apoptotic proteins, cleaved PARP (c‑PARP) and cleaved caspase 3 (c‑Casp3). Cells were treated with 50 μM chloroquine with or without the 2 h pretreatment of Pep/E64d for 24 h. Cytosolic proteins were isolated in each sample and subjected to the detection of cytochrome C, PARP and c‑Casp3 as described in the materials and methods

the subjective complaints (hematuria, pollakiuria and against various types of cancer.[20] In our previous study, we dysuria), decreases of tumor size, disappearance of the showed that CQ, used as autophagy inhibitor, enhanced the daughter tumors, clearing of the mucous membrane, and apoptosis induced by RAD001 in BC cells.[21] However, the histological changes, including the inhibition of the growth antitumor activities and the related mechanisms of single of stromal connective tissue, reduction of inflammatory CQ treatment on BC have not been defined. In the present cell infiltration in patients received either 250 mg of CQ study, we investigated and validated the efficacy of a single diphosphate or 200 mg of CQ diorotate once or twice daily treatment of CQ in human BC cells. by slow intravenous injection. Although the weakness of this study was the limited number of patients (n = 8), they We reported here that CQ exhibited cytotoxicity toward clearly showed that CQ significantly reduce bladder tumor human BC cells in a dose‑and time‑dependent manner. In burden. However, there is no other study using single CQ addition, the cell morphology was significantly altered in treatment against BC since this report. CQ‑treated cells. Previous studies have shown that single CQ is a cheap and easily obtained drug, and has treatment of CQ exerted an antitumor effect in several been widely used as a sensitizer of radiotherapy and types of tumors in a cell type‑dependent manner.[22‑25] CQ chemotherapy.[17] Recent studies demonstrated that was reported to induce cell death in a subset of tumor cell CQ is able to significantly enhance the efficiency of lines, however, the underlying mechanism is still not fully traditional chemotherapy drug or tumor targeting drugs understood. We found that treatment with CQ induces

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b Figure 6: The effects of bafilomycin A1 pretreatment in chloroquine‑treated bladder cancer cells. Pretreatment of Baf A1 c attenuated (a) chloroquine reduced cell viability and (b) activation of caspase 3/7. Cells were treated with 50 μM chloroquine with or without 2 h pretreatment of 200 nM bafilomycin A1 for 24 h, then the cell viability and caspase 3/7 activity were detected as described in the materials and methods. The values are shown as the mean ± standard deviation of three independent experiments. *P < 0.05

manner.[6] In addition, dose‑dependent up‑regulation of Bin, a member of the BH3‑only proapoptotic protein, was reported in CQ‑treated liver cancer cells.[25] Indeed, inhibition Figure 5: Protease inhibitors, PepA/E64d, and pan‑caspase inhibitor, of LMP using lysosomal protease inhibitors (pepstatin A and Z‑Vad‑FMK attenuated chloroquine‑induced apoptosis but failed to recover loss of cell viability. (a) chloroquine increased caspase E64d) decreased the bid activation but failed to fully recover 3/7 activity in bladder cancer cells. (b) Pretreatment of PepA/E64d the reduced viability in CQ‑treated BC cells. Furthermore, or Z‑Vad‑Fmk for 2 h attenuated chloroquine‑ induced apoptosis. (c) Pretreatment of PepA/E64d or Z‑Vad‑Fmk for 2 h did not rescue pretreatment with a pan‑caspase inhibitor (Z‑Vad‑Fmk) chloroquine decreased cell viability. The values are shown as the also failed to attenuate reduced cell viability in CQ‑treated mean ± standard deviation of three independent experiments. *P < 0.05 cells. These findings were in consistent with previous reports demonstrating that LMP induced cathepsin release may result in several hallmarks of apoptosis, including disrupted MMP, caspase‑dependent or independent cell death with or without the and increased caspase‑3 cleavage and activities; which are in involvement of mitochondria.[18,29] Nevertheless, the CQ‑ induced accordance with previous studies that CQ induces a genotoxic LMP was inhibited by Baf A1 suggesting that acidified lysosomes [26] effect that leading to apoptosis. Further investigation of were primary targets of CQ in human BC cells. It was established the mechanism showed that CQ treatment led to LMP that the extent of LMP damage determines the cell fate. Limited which is evident by the loss of LysoTracker and AO staining, lysosomal release results in cell death by apoptosis, while massive suggested that CQ treatment targets to lysosomal functions. lysosomal break‑down leads to necrosis.[30] Further studies need These findings were further supported by the observation to be done to determine the level of LMP induced by CQ of cathepsin B and D release into the cytosol. As a substrate and other pathways involved in CQ‑induced cell death. In of cathepsin B and D, the pro‑apoptotic bid was reported addition, patients with rheumatoid arthritis (RA) or systemic to be cleaved and activated in L‑leucyl‑L‑Leucine methyl lupus erythematosus taking CQ are required to check for ester‑induced LMP.[27,28] In the present study, we also found maculopathy.[31] CQ may also promote chemotherapy‑induced that CQ induces LMP, cathepsins release, cleavage of bid, kidney injury through multiple pathways through inhibition MOMP‑and caspase‑dependent apoptosis in human of drug‑induced autophagy.[11] However, the intravesical BC cells. In melanoma cells, however, a recent report therapy could be applied in the case of BC to minimize the demonstrated that CQ treatment promoted the apoptosis systemic side‑effects caused by CQ. The translational studies by stabilizing PUMA in a lysosomal protease‑independent are warrant using CQ installation to treat BC.

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Chen, et al.: CQ‑induced LMP‑mediated apoptosis in bladder cancer cells Our studies showed that single treatment of CQ effectively 10. Liang X, Tang J, Liang Y, Jin R, Cai X. Suppression of autophagy by suppressed the growth of human BC cells by inducing LMP chloroquine sensitizes 5‑fluorouracil‑mediated cell death in gallbladder carcinoma cells. Cell Biosci 2014;4:10. damages, release of lysosomal proteases (cathepsin B/D), 11. Kimura T, Takabatake Y, Takahashi A, Isaka Y. Chloroquine in cancer activation of bid that leading to reduced MMP, increased therapy: A double‑edged sword of autophagy. Cancer Res 2013;73:3‑7. MOMP and ultimately resulted in the induction of 12. Lin YC, Lin JF, Wen SI, Yang SC, Tsai TF, Chen HE, et al. Inhibition apoptosis through activation of caspase cascade in human of high basal level of autophagy induces apoptosis in human bladder cancer cells. J Urol 2016;195:1126‑35. BC cells. 13. Eisenberg‑Lerner A, Bialik S, Simon HU, Kimchi A. Life and death partners: Apoptosis, autophagy and the cross‑talk between them. Cell Acknowledgment Death Differ 2009;16:966‑75. The research leading to these results has received funding 14. Zhou M, Wang R. Small‑molecule regulators of autophagy and their potential therapeutic applications. ChemMedChem 2013;8:694‑707. from Shin‑Kong WHS Memorial Hospital (grant no. 15. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ. Potential SKH‑8302‑103‑0201 and SKH‑8302‑103‑0202). This project therapeutic applications of autophagy. Nat Rev Drug Discov also receives additional support from Ministry of Science 2007;6:304‑12. and Technology (grant no. NSC102‑2314‑B‑341‑003‑MY3). 16. Lin JF, Tsai TF, Liao PC, Lin YH, Lin YC, Chen HE, et al. Benzyl isothiocyanate induces protective autophagy in human prostate cancer cells via inhibition of mTOR signaling. Carcinogenesis 2013;34:406‑14. Financial support and sponsorship 17. Solomon VR, Lee H. Chloroquine and its analogs: A new promise of The research leading to these results has received an old drug for effective and safe cancer therapies. Eur J Pharmacol funding from Shin‑Kong WHS Memorial Hospital (grant 2009;625:220‑33. no.SKH‑8302‑103‑0201 and SKH‑8302‑103‑0202). 18. Boya P, Kroemer G. Lysosomal membrane permeabilization in cell death. Oncogene 2008;27:6434‑51. This project also receives additional support from 19. Hiraki K, Kimura I. Studies on the treatment of malignant tumors Ministry of Science and Technology (grant no. with fibroblast‑inhibiting agent 3. Effects of chloroquine on human NSC102‑2314‑B‑341‑003‑MY3). cancers. Acta Med Okayama 1964;18:71‑85. 20. Zhang Y, Liao Z, Zhang LJ, Xiao HT. The utility of chloroquine in cancer therapy. Curr Med Res Opin 2015;31:1009‑13. Conflicts of interest 21. Lin JF, Lin YC, Yang SC, Tsai TF, Chen HE, Chou KY, et al. 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