Chloroquine and hydroxychloroquine inhibit bladder cancer cell ...

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Kaohsiung Journal of Medical Sciences (2017) xx, 1e9

Available online at www.sciencedirect.com

ScienceDirect journal homepage: http://www.kjms-online.com

ORIGINAL ARTICLE

Chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy and enhancing apoptosis Yi-Chia Lin a,b, Ji-Fan Lin c,*, Sheng-I Wen c, Shan-Che Yang c, Te-Fu Tsai a,b, Hung-En Chen a, Kuang-Yu Chou a,b, Thomas I-Sheng Hwang a,b a

Department of Urology, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan c Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan b

Received 5 September 2016; accepted 16 October 2016

KEYWORDS Apoptosis; Autophagy; Bladder cancer cells; Chloroquine and Hydroxychloroquine

Abstract Chloroquine (CQ) and hydroxychloroquine (HCQ), two antimalarial drugs, are suggested to have potential anticancer properties. in the present study, we investigated the effects of CQ and HCQ on cell growth of bladder cancer with emphasis on autophagy inhibition and apoptosis induction in vitro. The results showed that CQ and HCQ inhibited the proliferation of multiple human bladder cell lines (including RT4, 5637, and T24) in a time- and dosedependent fashion, especially in advanced bladder cancer cell lines (5637 and T24) compared to immortalized uroepithelial cells (SV-Huc-1) or other reference cancer cell lines (PC3 and MCF-7). We found that 24-hour treatment of CQ or HCQ significantly decreased the clonogenic formation in 5637 and T24 cells compared to SV-Huc-1. As human bladder cancer tumor exhibits high basal level of autophagic activities, we detected the autophagic flux in cells treated with CQ and HCQ, showing an alternation in LC3 flux in CQ- or HCQ-treated cells. Moreover, bladder cancer cells treated with CQ and HCQ underwent apoptosis, resulting in increased caspase 3/7 activities, increased level of cleaved poly(ADP-ribose) polymerase (PARP), caspase 3, and DNA fragmentation. Given these results, targeting autophagy with CQ and HCQ represents an effective cancer therapeutic strategy against human bladder cancer. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, No.95, Wenchang Rd., Shilin Dist., Taipei 11101, Taiwan. E-mail address: [email protected] (J.-F. Lin). http://dx.doi.org/10.1016/j.kjms.2017.01.004 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Lin Y.-C, et al., Chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy and enhancing apoptosis, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.01.004

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Introduction Bladder cancer is one of most common urologic neoplastic disorders, which is prevalent in men [1,2]. Organ-confined bladder cancer is operatively manageable by transurethral resection or radical cystectomy [3]. However, advanced or metastatic cancer usually requires systemic chemotherapy. The traditional chemotherapy regimen is a cis-platinumbased chemotherapy, which is known as MVAC (methotrexate, vinblastine, doxorubicin, and cisplatin) [4]. The response rate of chemotherapy for bladder cancer has been reported to be 66% and 77% for MVAC and high-dose MVAC, respectively [5]. Vinflunine, a microtubule inhibitor, has been approved as a second-line treatment for patients with advanced platin-resistant bladder cancer in Europe with a modest effectiveness [6]. Nevertheless, a new treatment modality for bladder cancer is therefore mandatory. Chloroquine (CQ) and hydroxychloroquine (HCQ) are two commonly used antimalarial drugs. They have been demonstrated to exert anticancer activities in several types of cancer [7e9], and have low toxicity to nontumorigenic epithelial cells [8]. In addition, CQ and HCQ have been reported to effectively enhance cancer therapies by suppressing drug-induced autophagy in cancer cells [10]. Several clinical trials have been conducted to investigate the effects of using CQ or HCQ as autophagy inhibitors alone or in combination with other anticancer agents in various types of cancer [11e16]. It is therefore of interest to elucidate the molecular mechanism by which a single treatment of CQ or HCQ inhibits proliferation of bladder cancer cells. Autophagy is now known as a process of survival mechanism for cells encountering difficult environmental challenges such as starvation [17]. A stepwise and complex molecular signaling is involved in the process of autophagy to accomplish this phenomenon [18]. Autophagy is suggested to be involved in not only self-protection but also infection, apoptosis, and cancer behaviors [19]. Manipulation of autophagy in cancer cell has been shown to enhance and promote responses to anticancer treatment [20]. A cell-based study on nonsmall cell lung cancer showed that inhibition of autophagy resulted in enhancement of apoptosis through reactive oxygen species [21]. Several autophagic inhibitors have been investigated in the clinical setting, including CQ and HCQ, which block lysosomal acidification, autophagosome degradation and inactivate autophagy to reduce tumor survival [22,23]. In this study, we examined the effects of CQ and HCQ on proliferation of human bladder cancer cells through autophagy inhibition, and apoptosis induction.

Materials and methods Cell culture and chemicals All the chemicals used in this study, including CQ and HCQ, were purchased from Sigma (St. Louis, MO, USA). All cell lines were maintained at 37 C under 5% CO2. The human cancer cell lines RT4 (ATCC#HTB-2), 5637 (ATCC#HTB-9), T24 (ATCC#HTB-4), PC3 (ATCC#CRL-1435), MCF-7 (ATCC#HTB-22), and SV-Huc-1 (ATCC#CRL-9520) were obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan). All cell lines were

Y.-C. Lin et al. cultured in the suggested media as previously described [24,25].

Cell viability assays The cytotoxicity of CQ and HCQ was assayed using the WST1 reagent (Roche Diagnostics, Mannheim, Germany) as described [26]. Bladder cancer cells were treated with indicated doses of CQ or HCQ for 24 hours, 48 hours, and 72 hours and compared to SV-Huc-1 cells. To compare the effect of CQ or HCQ on cell viability in different types of cancer cells, T24 cells along with a prostate cancer cell (PC3) and a breast cancer cell (MCF-7) were treated with 200nM Baf A1, 25mM CQ, or 20mM HCQ for 24 hours. The values are shown as the mean standard deviation of three independent experiments.

Clonogenic assays Cells were plated and cultured in six-well plates at a density of 2000 cells/well overnight prior to the treatment with complete medium in the absence or presence of 25mM CQ or 20mM HCQ for 24 hours, 48 hours, and 72 hours. Cells were cultured in the drug-free complete medium for an additional 14 days followed by staining with crystal violet. Twenty images of colony formation were taken using the GeneFlash gel documentation system (Syngene, Cambridge, UK), and the number of colonies was counted in at least six different fields from two independent experiments.

Autophagy assays Induction of autophagy was determined using the following procedures. (1) Detection of p62 and the processing of microtubule-associated protein 1B light chain 3 (LC3) in cells treated with indicated concentrations of CQ and HCQ for 24 hours, or treated with 25mM CQ for 24 hours, 48 hours, and 72 hours using Western blotting. (2) Immunofluorescence detection of LC3 puncta formation in cells under starvation (serum deprivation) or 5mM 3-MA, 200nM bafilomycin A1 (Baf A1), 12.5mM CQ, or 20mM HCQ treatments. After treatments, cells in chamber slides were fixed and subjected to the first antibody against human LC3, followed by FITC-conjugated second antibody (Green) and counterstained with 40 ,6diamino-2-phenylindole (DAPI) (Blue). Twenty photos of different fields were taken from each condition under fluorescent microscopy, and LC3-positive (>5 bright green punctate) cells were counted. (3) Formation of acidic vesicular organelles (AVOs) detected by acridine orange (AO) staining. Cells plated overnight in six-well plates were treated with 25mM, 50mM CQ or 20mM, 40mM HCQ for 24 hours, and 1 mg/mL AO was added for staining.

Apoptosis assays Apoptosis induction by CQ or HCQ in bladder cancer cells was accessed by (1) activation of caspase 3 activity, (2) detection of caspase 3 and poly(ADP-ribose) polymerase (PARP) cleavage by Western blotting, and (c) DNA fragmentation assay using flow cytometry.

Please cite this article in press as: Lin Y.-C, et al., Chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy and enhancing apoptosis, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.01.004

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Figure 1. Chloroquine (CQ) and hydroxychloroquine (HCQ) significantly reduced the cell viability of bladder cancer cells. Cell viability was detected in SV-Huc-1, RT4, 5637, and T24 cells treated with (A) various doses of CQ or HCQ for 24 hours or (B) 25mM CQ or 20mM HCQ for 24 hours, 48 hours, and 72 hours using WST-1 reagents (Roche Diagnostics, Mannheim, Germany). The values are shown as the mean standard deviation (SD) of three independent experiments. *p < 0.05.


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Figure 2. Clonogenic assay in CQ- or HCQ-treated cells. (A) SV-Huc-1, (B) 5637, and (C) T24 cells were plated in 10-cm dishes, treated with indicated concentrations of DMSO (control), CQ, or HCQ for 24 hours, 48 hours, or 72 hours, and then replaced with complete medium and incubated for 14 days prior to the staining of crystal violet. After staining, 20 photos in different fields were taken using GeneFlash gel documentation system (Syngene) from each plate, and the colonies were counted assisted by the Photoshop software. Data were from three independent experiments with similar results and were presented as mean SD; *p < 0.05. CQ Z chloroquine; DMSO Z dimethyl sulfoxide; HCQ Z hydroxychloroquine; SD Z standard deviation.

Caspase 3/7 activity

Detection of caspase 3 cleavage by Western blotting

Activation of caspase 3 was assayed using the (Z-DEVD)2-R110 substrate. The cells were treated with dimethyl sulfoxide (DMSO; control) or the indicated concentration of CQ or HCQ. Subsequently, the cells were directly lyzed by adding caspase 3 assay buffer containing (Z-DEVD)2-R110 substrates and incubated at 37 C for 1 hour. The fluorescent intensity of R110 was then measured using a plate reader with an excitation of 485 nm and emission of 535 nm. Caspase 3/7 activities were detected at 24 hours or 72 hours after treatment.

Protein concentration was determined using BCA protein assay. Protein samples were subjected to 10e15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to PVDF membranes followed by probing with antibodies (active caspase 3 and b-actin). Subsequent immunoblotting was performed using a chemiluminescence procedure. The intensity of immunoreactive bands was determined. The total protein extracted from

Figure 3. Reduced cell viability in chloroquine (CQ)- or hydroxychloroquine (HCQ)-treated T24 cells were more severe compared to that in PC3 and MCF-7 cells. Cells were treated with 25mM CQ or 20mM HCQ for 24 hours using WST-1 reagents. The values are shown as the mean standard deviation of three independent experiments. *p < 0.05.

Figure 4. Accumulation of p62 and LC3-II autophagic marker protein in CQ- and HCQ-treated cells. The accumulation of p62 and processing of LC3-II was determined in SV-Huc-1, 5637, and T24 cells treated with indicated concentrations of CQ (left panel) and HCQ (middle panel) for 24 hours, or treated with 25mM CQ for 24 hours, 48 hours, and 72 hours (right panel). bActin served as loading control. Representative blots from three independent experiments with similar results are shown. CQ Z chloroquine; HCQ Z hydroxychloroquine.


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CQ, HCQ promote apoptosis by autophagy inhibition cells treated with 0e50mM CQ or 0e40mM HCQ or cells treated with 25mM CQ and 20mM HCQ for 24 hours, 48 hours, and 72 hours was subjected to the detection of cleavage PARP and caspase 3.

5 obtained from 10,000 events and presented as the fold of incorporated fluorescent.

Statistical analysis DNA fragmentation assays T24 cells treated with 25mM, 50 mM CQ or 20 mM, 40 mM HCQ for 24 hours were collected for the DNA fragmentation assay using the APO-Direct apoptosis detection kit (BD Biosciences, San Jose, CA, USA) followed by the detection of fluorescent intensity using flow cytometry. Data were

All experiments were performed at least in triplicate, and data were expressed as means standard deviation. The statistical significance of the difference in the measured variables between the treatment and control groups was determined by Student t test and was considered significant at p < 0.05.

Figure 5. Blockage of autophagic flux in CQ- and HCQ-treated cells. (A) Immunofluorescent detection of LC3 in 5637 and T24 cells treated with DMSO (Control), serum deprivation (Starvation), or 25mM CQ and 20mM HCQ. After treatment, cells in chamber slides were fixed and incubated with first antibody against human LC3, followed by FITC-conjugated second antibody (Green) and counterstained with DAPI (Blue). Twenty photos of different fields were taken from each condition under fluorescent microscopy, and LC3-positive cells were counted with the assistant of Photoshop software (version 12.1). Cells with more than five bright green punctate staining were considered LC3-positive. (B) Vital acridine orange (AO) staining of acidic vesicles in 5637 and T24 cells with indicated treatment. Cells plated overnight in six-well plates were treated with 25mM, 50mM CQ or 20mM, 40mM HCQ for 24 hours. After treatment, cells were stained with 1 mg/mL AO in complete medium for 15 minutes at 37 C, washed with PBS, and examined under fluorescence microscope. The quantitative results are shown in the lower panels. The values are shown as the mean SD of three independent experiments. *p < 0.05. CQ Z chloroquine; DAPI Z 40 ,6-diamino-2-phenylindole; DMSO Z dimethyl sulfoxide; FITC Z fluorescein isothiocyanate; HCQ Z hydroxychloroquine; PBS Z phosphate-buffered saline.


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Figure 6. Elevated level of apoptosis in CQ- and HCQ-treated cells. (A) Increased caspase 3/7 activity in T24 cells upon treatment of CQ or HCQ compared to SV-Huc-1, PC3 and MCF-7. Cells were seeded in 96-well plates for 16 hours prior to the drug treatment. Caspase 3/7 activity was detected at 24 hours (upper panel) or 72 hours (lower panel) after treatment using CaspaseGlo kit from Promega (Madison, WI, USA) and expressed as percentage of control cells. (B) Dose- and time-dependent increase of caspase 3/7 activity in CQ- or HCQ-treated SV-Huc-1 (upper panel) and T24 (lower panel) cells. Note that caspase 3/7 activity in


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CQ, HCQ promote apoptosis by autophagy inhibition

Results Human bladder cancer cells are sensitive to CQ and HCQ treatment in vitro The cell viability of immortalized uroepithelial cells (SVHuc-1) as well as low-grade (RT4) and high-grade (5637 and T24) bladder cancer cells treated with 0mM to 100mM CQ or HCQ for 24 hours, 48 hours, and 72 hours was evaluated. As shown in Figure 1, the cell viability of SV-Huc-1 exposed to 25mM CQ and 20mM HCQ for 24 hours was 92.49 2.28% and 94.08 3.26%, respectively. However, in bladder cancer cells, it was 91.78 1.72% and 93.41 4.19% for RT4, 79.37 4.17% and 82.84 4.31% for 5637, and 77.35 4.39% and 81.19 3.91% for T24, respectively. Our results showed that CQ and HCQ reduced the cell viability of bladder cancer cells in a dose- and time-dependent manner. To further access the cytotoxic effect of CQ and HCQ on normal and bladder cancer cells, we performed clonogenic assay. Clonogenic formations of bladder cancer cells, 5637 and T24, were significantly inhibited in the presence of CQ or HCQ, whereas SV-Huc-1 treated with CQ or HCQ exhibited a high colony formation rate (Figure 2). In addition, treatment with autophagy inhibitors including CQ or HCQ for 24 hours resulted in a significantly reduced cell viability in T24 and SV-Huc-1 cells compared to PC3 and MCF-7 cells that exhibited low basal autophagic activity (Figure 3). Nevertheless, prolonged exposure to CQ and HCQ led to a reduction in cell viability of other types of cancer cells.

CQ and HCQ block basal autophagy in human bladder cancer cells through inhibition of autophagolysosome degradation Increased accumulation of p62 and LC3-II protein level were observed in SV-Huc-1, 5637 and T24 cells corresponding to increased concentrations of CQ and HCQ. Furthermore, cells treated with 25mM CQ for 24 hours, 48 hours, and 72 hours also showed accumulation of p62 and LC3-II protein (Figure 4). These results suggested that CQ or HCQ blocks autophagic flux, which facilitates the degradation of autophagosome-associated proteins, by inhibition of lysosome fusion. To further elucidate the effects of CQ and HCQ on basal autophagic activity in human bladder cancer cells, we measured the formation of LC3 puncta in cells using immunofluorescence. As shown in Figure 5A, an increase in

7 the number of LC3 puncta was observed in 5637 and T24 cells under starvation. When cells were treated with CQ and HCQ, which block autophagosomeelysosome fusion, the number of LC3 puncta increased significantly. Using AO, a hydrophobic green fluorescent molecule emitting bright red fluorescence in acidic vesicles, we found that the red fluorescent increased dose-dependently in 5637 and T24 cells treated with CQ or HCQ (Figure 5B). These data demonstrated that AO staining may not be suitable to assess autophagy induction through AVO formation in CQ- or HCQ-treated bladder cancer cells. Nevertheless, the red fluorescent accumulation clearly indicated that CQ or HCQ treatment targets lysosomes in human bladder cancer cells.

Inhibition of basal autophagy by CQ and HCQ induces apoptosis in human bladder cancer cells As shown in Figure 6A, caspase 3/7 activity was increased in T24 cells treated with 25mM CQ or 20mM HCQ for 24 hours, whereas caspase 3/7 activity started to increase in SV-Huc1, PC3, and MCF-7 cells when the exposure duration was extended to 72 hours. Caspase 3/7 activity at 24 hours and 72 hours after treatment was higher in T24 cells treated with 25mM CQ and 20mM HCQ than in SV-Huc-1 cells (Figure 6B). We next evaluated the expression of apoptotic markers in the presence of CQ and HCQ. Levels of cleaved PARP (c-PARP) and cleaved caspase 3 (c-Cas3) were increased in bladder cancer cells with increased CQ or HCQ doses and exposure duration (Figures 6C and 6D). However, in SV-Huc-1 cells, it can only be detected when exposed to 25mM CQ for 48 hours and to 20mM HCQ for 72 hours. We further detected the degrees of DNA fragmentation to monitor apoptosis induction in T24 cells exposed to CQ or HCQ for 24 hours. The results showed an increased level of apoptosis in T24 cells upon CQ and HCQ treatment (Figure 6E). Collectively, these results demonstrated that CQ or HCQ alone inhibits basal autophagy and induces apoptosis in human bladder cancer cells.

Discussion Both CQ and HCQ as antimalaria drugs have historically been utilized successfully for the treatment of systemic lupus erythematosus (SLE) and rheumatoid arthritis. HCQ is a preferred drug for SLE, where it has a better control of the dermatological complications, lowers the incidence of gastrointestinal adverse reactions, and lowers the risk of ocular adverse effects compared to CQ. The lysosomotropic

drug-treated T24 cells was more profound in each condition than that of SV-Huc-1 cells. The values are shown as the mean SD of three independent experiments; *p < 0.05. (C and D), Increased level of cleaved PPAR (c-PPAR) and cleaved caspase 3 (c-CAS3) in CQ- and HCQ-treated cells. Total protein extracted from (C) cells treated with 0e50mM and CQ or 0e40mM of HCQ or (D) cells treated with 25mM CQ and 20mM HCQ for 24 hours, 48 hours, and 72 hours was subjected to the detection of cleaved PPAR and caspase 3. b-Actin served as loading control. Representative blots from three independent experiments with similar results were shown. (E) Increased DNA fragmentation in CQ- or HCQ-treated T24 cells. T24 cells treated with 25mM, 50mM CQ or 20mM, 40mM HCQ, for 24 hours were collected for the DNA fragmentation assay (BD) followed by the detection of fluorescent intensity using flow cytometry. Data were obtained from 10,000 events and presented as the fold of incorporated fluorescent. The values are shown as the mean SD of three independent experiments; *p < 0.05. Representative flow cytometry histograms are shown on the right panel. CQ Z chloroquine; HCQ Z hydroxychloroquine; PARP Z poly (ADP-ribose) polymerase; SD Z standard deviation. Please cite this article in press as: Lin Y.-C, et al., Chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy and enhancing apoptosis, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.01.004

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8 property has been used to redefine CQ and its derivatives as late-phase inhibitors of autophagy. CQ blocks autophagy by inhibiting lysosomal proteases and preventing autophagosomeelysosome fusion; hence, it has become the most widely used drug to inhibit autophagy in basic research fields [27,28]. Several clinical trials have been conducted to examine the effectiveness of CQ or HCQ against cancer other than the existing anticancer therapies. In the present study, we assessed and validated the efficacy of a single treatment of CQ or HCQ on bladder cancer cells in vitro. These agents had a profound effect on bladder cancer cell viability by inhibiting cancer cell growth, blocking basal autophagy, and promoting apoptosis in high-grade 5637 and T24 cells. Autophagy is an evolutionarily conserved process that functions to remove damaged organelle/proteins, and limit cell growth and genomic instability [29]. It plays a crucial role as a cell survival mechanism [30]. Because autophagy activation is closely related to various stress conditions, dysfunction of autophagy is linked to various human diseases, including cancer [31]. High basal level of autophagic activity has been involved in tumor growth such as human lung and pancreatic cancers [21,32]. It has been reported that bladder cancer also exhibits high basal levels of autophagic activities [25,33]. Therefore, targeting basal autophagy in human bladder cancer by autophagic inhibitors seems to be a reasonable therapeutic approach. In eukaryotic cells, basal constitutive autophagy is involved in the clearance of protein aggregates that form in cells as a consequence of aging, oxidative stress, and other alternations [34]. In CQ- and HCQ-treated bladder cancer cells, p62 accumulation and LC3-II processing were increased dose- and time-dependently. The accumulation of LC3 puncta was observed in cells treated with CQ and HCQ. These results indicated that these autophagy inhibitors efficiently block autophagy by preventing autophagosomeelysosome fusion, leading to inhibition of p62 and LC3-II degradation. Monitoring of red fluorescentlabeled AVO formation by AO staining is shown to be an ideal method to determine autophagy induction in cancer cells [24]. When cells are cotreated with chemotherapeutic drugs that induce autophagy with autophagy inhibitors such as Baf A1, a specific inhibitor of the vacuolar type HþATPase (V-ATPase) that also blocks the autophagosomeelysosome fusion as CQ and HCQ do, the number of red fluorescence-labeled AVOs should be decreased [24,35]. In the present study, we found that the number of red fluorescence-labeled AVOs increased in 5637 and T24 cells exposed to CQ and HCQ. CQ treatment raises the lysosomal pH and consequently inhibits autophagosome fusion with lysosome and lysosomal protein degradation, suggesting that red fluorescence-labeled AVOs is not associated with increased acidification of lysosome [36]. One reasonable explanation is that CQ and HCQ treatment increased the lysosomal compartment in the bladder cancer cells, resulting in more AO accumulation in the expanded lysosomes. However, further investigations are necessary to explore the exact mechanism by which CQ and HCQ affect the AO-stained AVOs in cells. Our results demonstrated that evaluation of AVO increment by AO vital staining in cultured cells is not suitable when using CQ or HCQ as autophagy inhibitors. Nevertheless, these data indicated that CQ and

Y.-C. Lin et al. HCQ did target lysosomal functions and block autophagy in human bladder cancer cells. We found that CQ and HCQ slightly reduced cell viability in a dose- and time-dependent manner in SV-Huc-1 cells, and they exerted higher cytotoxicities in bladder cancer cells. The therapeutic plasma dose of CQ is reported to be about 2.5 mL/L to 5.0 mL/L, which is equivalent to 4.85mM to 9.69mM. The lethal dose was 11 mg/L in several handbooks. According to our results, achieving the most effective dose of CQ can be lethal to human patients. However, an intravesical instillation may be considered to maintain high concentrations in the bladder to avoid systematic side effects as described previously in SLE and rheumatoid arthritis treatment. Further investigation on the combination of moderate dose of CQ or HCQ with other drugs is another direction to avoid toxicity. A novel autophagic inhibitor with lower toxicity is mandatorily needed.

Conclusion CQ and HCQ effectively inhibited basal autophagy in bladder cancer cells that, in turn, reduced viability in cellbased model. The effects of CQ and HCQ were especially prominent in bladder cancer cells. The cell viability loss was possibly mediated through lysosomal dysfunction and ultimately causing apoptosis of the cancer cells. Clinical application can be expected as a primary or an adjunct treatment option.

Acknowledgments This research has received funding from Shin-Kong WHS Memorial Hospital (grant no. SKH-8302-103-0201 and SKH8302-103-0202). This project receives also additional support from Ministry of Science and Technology (grant no. NSC102-2314-B-341-003-MY3). The authors disclose no potential conflicts of interest.

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