|Year : 2018 | Volume
| Issue : 4 | Page : 133-141
Chloroquine induces lysosomal membrane permeability-mediated cell death in bladder cancer cells
Hung-En Chen1, Ji-Fan Lin2, Yi-Chia Lin3, Shen-I Wen2, Shan-Che Yang2, Te-Fu Tsai1, Kuang-Yu Chou1, I-Sheng Thomas Hwang4
1 Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, New Taipei City, Taiwan
2 Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, New Taipei City, Taiwan
3 Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital; School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
4 Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital; Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital; School of Medicine, Fu-Jen Catholic University, New Taipei City, Department of Urology, Taipei Medical University, Taipei, Taiwan
|Date of Submission||19-May-2017|
|Date of Decision||30-Oct-2017|
|Date of Acceptance||14-Jan-2018|
|Date of Web Publication||22-Aug-2018|
Dr. I-Sheng Thomas Hwang
Department of Surgery, Shin Kong WHS Memorial Hospital, Shin Lin District, Taipei City
Source of Support: None, Conflict of Interest: None
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
|How to cite this article:|
Chen HE, Lin JF, Lin YC, Wen SI, Yang SC, Tsai TF, Chou KY, Hwang IST. Chloroquine induces lysosomal membrane permeability-mediated cell death in bladder cancer cells. Formos J Surg 2018;51:133-41
|How to cite this URL:|
Chen HE, Lin JF, Lin YC, Wen SI, Yang SC, Tsai TF, Chou KY, Hwang IST. Chloroquine induces lysosomal membrane permeability-mediated cell death in bladder cancer cells. Formos J Surg [serial online] 2018 [cited 2019 Jan 18];51:133-41. Available from: http://www.e-fjs.org/text.asp?2018/51/4/133/239554
| Introduction|| |
Bladder cancer (BC) is the most common neoplasm in the urinary tract and has a very high recurrence rate. The urothelial cell carcinomas is accounting for more than 90% of BC s while nearly 70% of bladder tumor present as superficial (nonmuscle-invasive) BC. Transurethral resection of bladder tumor (TUR-BT) is the standard treatment for patients with superficial tumors. However, approximately 60%–70% of these tumors will recur, and 25% will continue to progress to a higher stage or grade. Although mitomycin and Bacillus Calmette-Guerin installation followed by TUR-BT have shown some evidence of activity against tumor recurrence, their incomplete efficacy and toxicity have limited their use as common chemotherapy agents. Therefore, the development of novel adjuvant agents against BC is warranted.
The chloroquine (CQ) is on the list of essential medicines of the World Health Organization and is classically used to prevent and treat malaria. The CQ treatment was reported to induce apoptotic cell death in melanoma, glicoma, and lung cancer cells.,, In addition, CQ showed lower toxicity to nontumorigenic epithelial cells. Recently, CQ has been demonstrated to be an enhancing agent in cancer therapies when combined with other chemotherapeutic agents., However, the effects of CQ single treatment on BC have not been investigated.
We recently showed that human BC tumor exhibits a high basal level of autophagy, and the autophagic activity is required for the survival of human BC cells. Inhibition of basal autophagy induces apoptotic cell death. CQ has been routinely used to block the fusion of autophagosomes with lysosomes to study the role of drug-induced autophagy in cultured cancer cells. As an effective autophagy inhibitor, CQ has also been tested in several ongoing clinical trials, alone or in combination with other anti-cancer drugs., In the present study, we examined the mechanism involved in CQ-induced cell death focusing on the disruption of lysosomal membrane permeability (LMP), and the subsequent linkage to mitochondria outer membrane permeability (MOMP) that ultimately leading to apoptotic cell death. The study findings provide a new insight of CQ single treatment against human BC cells and the potential use in BC therapy in the future.
| Materials and Methods|| |
The CQ was purchased from Sigma-Aldrich (St Louis, MO, USA) and prepared at a concentration of 100 mM. Aliquots were stored at-20°C. All other chemicals, unless otherwise mentioned, were purchased from Sigma-Aldrich.
The human cancer cell lines 5637 (American Type Culture Collection, ATCC#HTB-9) and T-24 (ATCC#HTB-4); and human ureter sumian virus 40 (SV40)-transformed epithelial cell line SV-Huc-1 (ATCC#CRL-9520) were obtained from Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). The SV-Huc-1 cells were cultured in F12 medium (Invitrogen). The 5637 and T-24 cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA). These cells have performed STR-PCR profile at BCRC. Cell lines were maintained at 37°C under 5% CO2. Media were supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2 mM GlutaMAX-1 (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Cells were treated with the indicated concentration of CQ, and control cells received an equal volume of DMSO. The final concentration of DMSO was <0.1%.
Cell viability assays
A live cell imaging system (CytoSmart System, Lonza, Visp, Switzerland) was used to record the cell morphology in control or CQ-treated cells in a real-time fashion. Control and treated-cells were constantly and automatically monitored with 15 min interval for 72 h. The time-lapse photos as well as the recorded videos were generated by the build-in software. The effect of CQ on cell viability at 24–72 h posttreatment was assayed by the WST-1 reagent (Roche Diagnostics, Mannheim, Germany) as described. In some experiments, 10 μM pepstatin A (inhibitor for cathepsin D) and 10 μM E64d (inhibitor for cathepsin B/L) was added 1 h before the treatment of CQ to inhibit lysosomal proteases.
Monitoring of lysosomal morphology
Alternation of lysosomal morphology in CQ-treated BC cells was monitored by LysoTracker Red DND-99 (Thermo Fisher Scientific, Waltham, MA, USA). In brief, cells grown in glass chamber slides (BD Biosciences, San Diego, CA, USA) were incubated with indicated concentrations of CQ. After the CQ treatment, the cells were washed twice with phosphate-buffered saline (PBS), incubated with 100 nM LysoTracker Red DND-99 labeling solution for 30 min at room temperature. When labeling is complete, the solution was removed, and the cells were washed twice with PBS, and then stained with Hoechst 33342 dye (2 μg/ml) at 37°C for 5 min to label the nucleus. The labeling solution was quickly removed, and the cells were gently washed twice and subjected to imaging. Fluorescent imaging was performed with a Nikon inverted microscope-Eclipse Ti-E equipped with 130W fluorescence light source and fitted with Nikon color CMOS Camera-DS-Ri2. Images were collected with filter bandwidths corresponding to 560–615 nm for red.
Detection of lysosomal membrane permeability
To measure lysosomal membrane integrity, cells with or without CQ treatment were stained with 10 1 μg/ml acridine orange (AO) for 15 min at 37°C, and washed 3 times in PBS to reduce background. LMP was quantified by measuring the reduction of red fluorescence using an Accuri C5 flowcytometer from BD Bioscience.
Immunofluorescence detection of cathepsin B and D release
Cells were grown on glass chamber slides, treated with indicated concentrations of CQ for 24 h, and fixed with 4% paraformaldehyde in PBS for 30 min at 37°C. Cells were permeabilized with 0.1% Triton-X100 in PBS for 30 min and blocked with PBS containing 3% bovine serum albumin at room temperature for 1 h. After incubation with antibodies against cathepsin B or D (Santa Cruz Biotechnology) at 4°C overnight, the cells were washed three times with PBS and treated with the FITC-conjugated goat anti-rabbit secondary antibody for 90 min at 37°C. Cells were washed three times in PBS and counterstained with DAPI to visualize the nuclei before additional washing and mounting with ProLong Gold antifade reagent (Invitrogen). Slides were stored in the dark at 4°C until observation under fluorescent microscopy as described above.
Evaluation of mitochondrial membrane depolarization and permeability
To measure the mitochondrial membrane depolarization, cells were treated with CQ at the indicated concentrations. Mitochondrial membrane depolarization was detected with the mitochondrial membrane potential assay kit with JC-1 dye according to the manufacturer's protocol (Genedirex, Taipei, Taiwan). The data were acquired and analyzed using Accuri C5 flow cytometry as described. The mitochondrial membrane permeability was measured by detecting the release of mitochondrial cytochrome c (Cyto. C) into the cytosol by Western blot. Cytosolic proteins from cells with the same treatment were isolated using Mitochondria Isolation Kit for culture cells (Thermo Fisher Scientific) following the manufacturer's instruction and subsequently used in the Western blot analysis using antibody against Cyto. C. β-actin was detected as a loading control.
Detection of the bid, PARP, and cleaved caspase 3 by the Western blot
Cell lysates from cells treated with indicated concentrations of CQ with or without the pretreatment of lysosomal protease inhibitors (pepstatin A and E64d; PepA/E64d) were prepared for the Western blot analysis, using antibodies against the bid, cleaved bid (c-Bid), PARP, and cleaved caspase-3 (c-Casp3).
All data were expressed as mean values ± standard deviation. Statistical evaluation was determined using two-tailed Student's t-test. P <0.05 was considered to be statistically significant. All analysis was carried out with the program SigmaPlot Version 10.0 for Windows (Systat Software Inc., Chicago, IL, USA).
The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee of the institute. Informed written consent was obtained from all patients prior to their enrollment in this study.
| Results|| |
Chloroquine inhibits the proliferation of bladder cancer cells
To access the anticancer activity of CQ on human BC, we first investigated the effect of CQ on the proliferation of immortalized urothelial cell line, SV-Huc-1, and two BC cell lines, 5637 and T24. While the cell morphology of SV-Huc-1 was not altered by the treatment of 50 μM CQ, we found both 5637 and T24 cell morphological shrunk and floated with the increased incubation periods [Figure 1]a, and [Supplementary Video 1],[Supplementary Video 2],[Supplementary Video 3]. Consistent with these observations, the cell viability, detected by WST-1 reagents, of 5637 and T24 cells decreased with CQ treatment in a dose- and time-dependent manner [Figure 1]b. On the other hand, decreased cell viability was observed in SV-Huc-1 cells treated with 25 μM CQ for 72 h or 50 and 100 μM CQ for 48 and 72 h [Figure 1]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|
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Chloroquine induces permeabilization of lysosomal membranes
As a lysosomotropic agent, CQ can rapidly diffuse into cells and been trapped in lysosomes. We first labeled lysosomes with LysoTracker red DND-99 and monitored morphological changes of lysosomes associated with CQ treatment. As shown in [Figure 2]a, the LysoTracker-stained control cells displayed a normal punctuate localization in the perinuclear region. The CQ-treated lysosomes swelled, increased, and accumulated in the cytosol with increasing CQ administration. The red fluorescent intensity is then gradually decreased with increased CQ concentration [Figure 2]a. Treatment with CQ produced a gradual and concentration-dependent increase in Lysotracker red fluorescence intensity, suggesting that CQ-induced alterations of lysosomal pH. Furthermore, cells were treated with indicated concentration of CQ for 1, 6, and 24 h and stained with another acidophilic dye, AO. While the increased red fluorescent was monitored in cells treated with an increased concentration of CQ for 1 h, a diminution of the red AO fluorescence was detected at 6 h of CQ treatment and further decreased at 24 h of treatment [Figure 2]b,[Figure 2]c,[Figure 2]d. The results indicated CQ-induced AO translocation which means AO-load cells manifest reduced red fluorescence and increased green fluorescence after LMP.
|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|
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The lysosomal permeabilization was further confirmed in cells treated with CQ by detecting the release of cathepsin B (Cat B) and D (Cat D) into the cytosol [Figure 3]a. To test whether CQ-induced LMP is unique in the BC cells, we treated human immortalized urothelial cell line, SV-Huc-1, with CQ and detected the releasing of Cat B. As shown in [Figure 3]b, the release of Cat B was only detected in T24 cells. These results indicated that CQ mediates the permeabilization of lysosomal membranes predominantly in human BC cells.
|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|
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Chloroquine-induced lysosomal damages trigger mitochondrial membrane depolarization permeability, increased cytochrome c release, and bid activation
The LMP is considered a potentially lethal event because the presence of lysosomal proteases in the cytosol causes digestion of vital protein and the induction of apoptosis through the activation of caspases cascade. For example, proteolytic activation of bid cleaved by lysosomal Cat B and Cat D induces MOMP, resulting in Cyto. C release and apoptosome-dependent caspase activation. We, therefore, investigated the mitochondria membrane potential (MMP) and MOMP in the CQ-treated cells. As shown in [Figure 4]a, mitochondria depolarization was not profound in control cells and in those treated with low concentrations of CQ but was significantly increased in cells treated with relatively high concentrations of CQ (50 and 100 μM), indicated by a decrease in the red/green fluorescence intensity ratio. These results indicated CQ induces the loss of MMP in BC cells. It is possible that CQ-induced lysosomal damages resulted in the release of Cat B and Cat D that activated bid to disrupt mitochondrial function. We, therefore, detected the expression level of the cleaved bid (c-Bid) in CQ-treated cells. As depicted in [Figure 4]b, activation of bid on CQ treatment was observed in a dose-dependent manner. Moreover, the increased level of c-Bid was attenuated when cells pretreated with pepstatin A and E64d that inhibit the protease activity of cathepsins [Figure 4]c. In addition, the release of Cyto. C, activation of caspase-3 and PARP (c-PARP) were also increased in CQ-treated cells [Figure 4]d; and the activation of these pro-apoptotic markers was attenuated by the pretreatment of pepstatin A and E64d [Figure 4]d. These findings suggested that CQ induces LMP that leading to the activation of the bid and subsequently resulted in the release of Cyto. C followed by induction of apoptosis through caspase-3 activation.
|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|
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Chloroquine-induced caspase-dependent and–independent cell death
Cells treated with CQ-induced several hallmarks of apoptosis, including the increased level of cleaved caspase 3 [Figure 4]d, and activation of caspase 3 activity [Figure 5]a. Inhibition of CQ-induced LMP with the pretreatment of lysosomal protease inhibitors (pepstatin A and E64d) or inhibition of CQ-induced apoptosis with pan-caspase inhibitor (Z-Vad-FMK) attenuated the induced apoptosis indicated by the reduced caspase 3 activity [Figure 5]b, but failed to fully recover the reduced viability in CQ-treated cells [Figure 5]c. However, pretreatment of bafilomycin A1 (Baf A1), an inhibitor of the lysosomal vacuolar H + ATPase, inhibited the reduced viability [Figure 6]a and attenuated the activated caspase 3 activity [Figure 6]b. In conclusion, CQ induced LMP, MOMP and z-Vad-FMK-resistant cell death through a pathway that depends on the CQ accumulation in lysosomes.
|Figure 5: Protease inhibitors, PepA/E64d, and pan-caspase inhibitor, Z-Vad-FMK attenuated chloroquine-induced apoptosis but failed to recover loss of cell viability. (a) chloroquine increased caspase 3/7 activity in bladder cancer cells. (b) Pretreatment of PepA/E64d 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 chloroquine decreased cell viability. The values are shown as the mean ± standard deviation of three independent experiments. *P < 0.05|
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|Figure 6: The effects of bafilomycin A1 pretreatment in chloroquine-treated bladder cancer cells. Pretreatment of Baf A1 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|
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| Discussion|| |
In 1964, a report published by Kimura I. and Hiraki K from the Okayama University Medical School describing the use of CQ to treat malignant tumors. They claimed that excellent therapeutic effects were obtained in patients with BC. Their data demonstrated the improvement of the subjective complaints (hematuria, pollakiuria and dysuria), decreases of tumor size, disappearance of the daughter tumors, clearing of the mucous membrane, and histological changes, including the inhibition of the growth of stromal connective tissue, reduction of inflammatory cell infiltration in patients received either 250 mg of CQ diphosphate or 200 mg of CQ diorotate once or twice daily by slow intravenous injection. Although the weakness of this study was the limited number of patients (n = 8), they clearly showed that CQ significantly reduce bladder tumor burden. However, there is no other study using single CQ treatment against BC since this report.
CQ is a cheap and easily obtained drug, and has been widely used as a sensitizer of radiotherapy and chemotherapy. Recent studies demonstrated that CQ is able to significantly enhance the efficiency of traditional chemotherapy drug or tumor targeting drugs against various types of cancer. In our previous study, we showed that CQ, used as autophagy inhibitor, enhanced the apoptosis induced by RAD001 in BC cells. However, the antitumor activities and the related mechanisms of single CQ treatment on BC have not been defined. In the present study, we investigated and validated the efficacy of a single treatment of CQ in human BC cells.
We reported here that CQ exhibited cytotoxicity toward human BC cells in a dose-and time-dependent manner. In addition, the cell morphology was significantly altered in CQ-treated cells. Previous studies have shown that single treatment of CQ exerted an antitumor effect in several types of tumors in a cell type-dependent manner.,,, CQ was reported to induce cell death in a subset of tumor cell lines, however, the underlying mechanism is still not fully understood. We found that treatment with CQ induces several hallmarks of apoptosis, including disrupted MMP, and increased caspase-3 cleavage and activities; which are in accordance with previous studies that CQ induces a genotoxic effect that leading to apoptosis. Further investigation of the mechanism showed that CQ treatment led to LMP which is evident by the loss of LysoTracker and AO staining, suggested that CQ treatment targets to lysosomal functions. These findings were further supported by the observation of cathepsin B and D release into the cytosol. As a substrate of cathepsin B and D, the pro-apoptotic bid was reported to be cleaved and activated in L-leucyl-L-Leucine methyl ester-induced LMP., In the present study, we also found that CQ induces LMP, cathepsins release, cleavage of bid, MOMP-and caspase-dependent apoptosis in human BC cells. In melanoma cells, however, a recent report demonstrated that CQ treatment promoted the apoptosis by stabilizing PUMA in a lysosomal protease-independent manner. In addition, dose-dependent up-regulation of Bin, a member of the BH3-only proapoptotic protein, was reported in CQ-treated liver cancer cells. Indeed, inhibition of LMP using lysosomal protease inhibitors (pepstatin A and E64d) decreased the bid activation but failed to fully recover the reduced viability in CQ-treated BC cells. Furthermore, pretreatment with a pan-caspase inhibitor (Z-Vad-Fmk) also failed to attenuate reduced cell viability in CQ-treated cells. These findings were in consistent with previous reports demonstrating that LMP induced cathepsin release may result in caspase-dependent or independent cell death with or without the involvement of mitochondria., Nevertheless, the CQ- induced LMP was inhibited by Baf A1 suggesting that acidified lysosomes were primary targets of CQ in human BC cells. It was established that the extent of LMP damage determines the cell fate. Limited lysosomal release results in cell death by apoptosis, while massive lysosomal break-down leads to necrosis. Further studies need to be done to determine the level of LMP induced by CQ and other pathways involved in CQ-induced cell death. In addition, patients with rheumatoid arthritis (RA) or systemic lupus erythematosus taking CQ are required to check for maculopathy. CQ may also promote chemotherapy-induced kidney injury through multiple pathways through inhibition of drug-induced autophagy. However, the intravesical therapy could be applied in the case of BC to minimize the systemic side-effects caused by CQ. The translational studies are warrant using CQ installation to treat BC.
Our studies showed that single treatment of CQ effectively suppressed the growth of human BC cells by inducing LMP damages, release of lysosomal proteases (cathepsin B/D), activation of bid that leading to reduced MMP, increased MOMP and ultimately resulted in the induction of apoptosis through activation of caspase cascade in human BC cells.
The research leading to these results has received funding from Shin-Kong WHS Memorial Hospital (grant no. SKH-8302-103-0201 and SKH-8302-103-0202). This project also receives additional support from Ministry of Science and Technology (grant no. NSC102-2314-B-341-003-MY3).
Financial support and sponsorship
The research leading to these results has received funding from Shin-Kong WHS Memorial Hospital (grant no.SKH-8302-103-0201 and SKH-8302-103-0202). This project also receives additional support from Ministry of Science and Technology (grant no. NSC102-2314-B-341-003-MY3).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Babjuk M, Böhle A, Burger M, Capoun O, Cohen D, Compérat EM, et al.
EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: Update 2016. Eur Urol 2017;71:447-61.
Goodwin Jinesh G, Willis DL, Kamat AM. Bladder cancer stem cells: Biological and therapeutic perspectives. Curr Stem Cell Res Ther 2014;9:89-101.
Shen Z, Shen T, Wientjes MG, O'Donnell MA, Au JL. Intravesical treatments of bladder cancer: Review. Pharm Res 2008;25:1500-10.
Gartrell BA, Sonpavde G. Emerging drugs for urothelial carcinoma. Expert Opin Emerg Drugs 2013;18:477-94.
The selection and use of essential medicines (ISBN 978-92-4-121015-7): Report of the WHO Expert Committee, 2017 (including the 20th WHO Model List of Essential Medicines and the 6th WHO Model List of Essential Medicines for Children). Geneva: World Health Organization; 2017 (WHO technical report series; no. 1006).
Lakhter AJ, Sahu RP, Sun Y, Kaufmann WK, Androphy EJ, Travers JB, et al.
Chloroquine promotes apoptosis in melanoma cells by inhibiting BH3 domain-mediated PUMA degradation. J Invest Dermatol 2013;133:2247-54.
Geng Y, Kohli L, Klocke BJ, Roth KA. Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent. Neuro Oncol 2010;12:473-81.
Fan C, Wang W, Zhao B, Zhang S, Miao J. Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorg Med Chem 2006;14:3218-22.
Rahim R, Strobl JS. Hydroxychloroquine, chloroquine, and all-trans retinoic acid regulate growth, survival, and histone acetylation in breast cancer cells. Anticancer Drugs 2009;20:736-45.
Liang X, Tang J, Liang Y, Jin R, Cai X. Suppression of autophagy by chloroquine sensitizes 5-fluorouracil-mediated cell death in gallbladder carcinoma cells. Cell Biosci 2014;4:10.
Kimura T, Takabatake Y, Takahashi A, Isaka Y. Chloroquine in cancer therapy: A double-edged sword of autophagy. Cancer Res 2013;73:3-7.
Lin YC, Lin JF, Wen SI, Yang SC, Tsai TF, Chen HE, et al.
Inhibition of high basal level of autophagy induces apoptosis in human bladder cancer cells. J Urol 2016;195:1126-35.
Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A. Life and death partners: Apoptosis, autophagy and the cross-talk between them. Cell Death Differ 2009;16:966-75.
Zhou M, Wang R. Small-molecule regulators of autophagy and their potential therapeutic applications. ChemMedChem 2013;8:694-707.
Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ. Potential therapeutic applications of autophagy. Nat Rev Drug Discov 2007;6:304-12.
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.
Solomon VR, Lee H. Chloroquine and its analogs: A new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol 2009;625:220-33.
Boya P, Kroemer G. Lysosomal membrane permeabilization in cell death. Oncogene 2008;27:6434-51.
Hiraki K, Kimura I. Studies on the treatment of malignant tumors with fibroblast-inhibiting agent 3. Effects of chloroquine on human cancers. Acta Med Okayama 1964;18:71-85.
Zhang Y, Liao Z, Zhang LJ, Xiao HT. The utility of chloroquine in cancer therapy. Curr Med Res Opin 2015;31:1009-13.
Lin JF, Lin YC, Yang SC, Tsai TF, Chen HE, Chou KY, et al.
Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells. Drug Des Devel Ther 2016;10:1501-13.
Balic A, Sørensen MD, Trabulo SM, Sainz B Jr., Cioffi M, Vieira CR, et al.
Chloroquine targets pancreatic cancer stem cells via inhibition of CXCR4 and hedgehog signaling. Mol Cancer Ther 2014;13:1758-71.
Zheng Y, Zhao YL, Deng X, Yang S, Mao Y, Li Z, et al.
Chloroquine inhibits colon cancer cell growth in vitro
and tumor growth in vivo
via induction of apoptosis. Cancer Invest 2009;27:286-92.
Kim EL, Wüstenberg R, Rübsam A, Schmitz-Salue C, Warnecke G, Bücker EM, et al.
Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells. Neuro Oncol 2010;12:389-400.
Hu T, Li P, Luo Z, Chen X, Zhang J, Wang C, et al.
Chloroquine inhibits hepatocellular carcinoma cell growth in vitro
and in vivo
. Oncol Rep 2016;35:43-9.
Farombi EO. Genotoxicity of chloroquine in rat liver cells: Protective role of free radical scavengers. Cell Biol Toxicol 2006;22:159-67.
Uchimoto T, Nohara H, Kamehara R, Iwamura M, Watanabe N, Kobayashi Y, et al.
Mechanism of apoptosis induced by a lysosomotropic agent, L-leucyl-L-leucine methyl ester. Apoptosis 1999;4:357-62.
Cirman T, Oresić K, Mazovec GD, Turk V, Reed JC, Myers RM, et al.
Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of bid by multiple papain-like lysosomal cathepsins. J Biol Chem 2004;279:3578-87.
Kirkegaard T, Jäättelä M. Lysosomal involvement in cell death and cancer. Biochim Biophys Acta 2009;1793:746-54.
Johansson AC, Appelqvist H, Nilsson C, Kågedal K, Roberg K, Ollinger K, et al.
Regulation of apoptosis-associated lysosomal membrane permeabilization. Apoptosis 2010;15:527-40.
Geamănu Pancă A, Popa-Cherecheanu A, Marinescu B, Geamănu CD, Voinea LM. Retinal toxicity associated with chronic exposure to hydroxychloroquine and its ocular screening. Review. J Med Life 2014;7:322-6.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]