Targeting pro-survival autophagy enhanced GSK-3β inhibition-induced apoptosis and retarded proliferation in bladder cancer cells

doi:10.21203/rs.3.rs-2713018/v1

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Targeting pro-survival autophagy enhanced GSK-3β inhibition-induced apoptosis and retarded proliferation in bladder cancer cells

https://doi.org/10.21203/rs.3.rs-2713018/v1

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Advanced bladder cancer (BC) (local invasive and/or metastatic) is not curable even with cytotoxic chemotherapy, immune checkpoint inhibitors, and targeted treatment. Targeting GSK-3β is a promising novel approach in advanced BC. The induction of autophagy is a mechanism of secondary resistance to various anticancer treatments. Our objectives were to investigate the synergistic effects of GSK-3β in combination with autophagy inhibitors to evade GSK-3β drug resistance. Small molecule GSK-3β inhibitors and GSK-3β knockdown using siRNA promoted the expression of autophagy-related proteins. We further investigated that GSK-3β inhibition induced the nucleus translocation of transcription factor EB(TFEB). Compared to the GSK-3β inhibition alone, its combination with chloroquine (an autophagy inhibitor) significantly reduced BC cell growth. These results provide that targeting autophagy potentiates GSK-3β inhibition-induced apoptosis and retarded proliferation in BC cells.

Biological sciences/Cancer/Urological cancer/Bladder cancer

Biological sciences/Cell biology/Autophagy/Macroautophagy

Bladder cancer (BC) is the 10th most frequent cancer globally, accounting for 573,278 new cases and 212,536 deaths in 2020[1]. Urothelial cancer (UC) is the most common type of BC, accounting for approximately 90% of all cases.

Although non-muscle invasive bladder cancer (NMIBC) reflects a large proportion of all BCs and is commonly associated with a good prognosis, locally advanced or metastatic BCs are mainly associated with death. Although there was no treatment beyond cisplatin-based chemotherapy for a long time, pembrolizumab has been introduced for advanced UC that recurred or progressed after platinum-based chemotherapy. The objective response rate in the international phase III KEYNOTE-045 ClinicalTrials for pembrolizumab was around 21.1%, as compared with 11.4% in the chemotherapy group[2]. Despite currently available multimodality therapy, advanced BC has a very low overall survival (OS) rate of about 6%[3].

Drug pricing is high relative to response rates, and the development of new therapeutic agents is required to improve the clinical outcomes of patients with locally advanced or metastatic BC.

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase which was first described as a component of glycogen synthase (GS) regulation through its phosphorylation[4]. GSK-3 has two isoforms, GSK-3α and GSK-3β[5, 6], which exhibit 97% sequence identity within their catalytic domains[7, 8]. We have previously identified GSK-3 as a potential therapeutic target for BC and reported that the antitumor effects of GSK-3β inhibitors were attributed to enhanced apoptosis. GSK-3β inhibitors also promoted autophagy and the inhibition of autophagy by chloroquine enhanced the antitumor effects of GSK-3β inhibitors[9, 10]. GSK-3β inhibitors have been proven to induce autophagy in other cancer types. Currently, phase Ⅰ and II trials using elraglusib (a novel highly specific GSK-3 inhibitor) are underway [11].

Autophagy is an evolutionary ancient mechanism that culminates with the lysosomal degradation of superfluous or potentially dangerous cytosolic entities[12]. During the initial stage of cancer development, autophagy serves a major role in tumor suppression by maintaining genomic integrity and preventing proliferation and inflammation[13]. However, after the establishment of cancer, cancer cells may utilize autophagy to survive cellular stresses in the adverse microenvironment[14].

The exact mechanism of autophagy induction by GSK-3 inhibition in UC cells is not clear. One of the autophagy signaling pathways is the AMP-AMPK-ULK1 pathway. AMP-activated protein kinase(AMPK) phosphorylates Unc-51 like autophagy activating kinase 1(ULK1), an important initiator of autophagy. Furthermore, AMPK is known to inhibit the mammalian target of rapamycin complex 1(mTORC1) and mTORC1 inhibits the activation of ULK1, but also regulates the transcription of autophagy-related genes[15].

We next focused on transcription factor EB(TFEB). TFEB regulates various cellular processes, including autophagy[16], lysosomal biogenesis[17], and energy metabolism[18]. It is known that mTORC1 inhibits TFEB nuclear translocation[19]. Under normal nutritionally replete conditions, mTORC1 present on the lysosomal membrane phosphorylates TFEB and this phosphorylation inhibits the translocation of TFEB to the nucleus and prevents its function as a transcription factor[19]. Under cellular stress, such as low levels of ATP or glucose, mTORC1 is inhibited and TFEB then translocates to the nucleus to transcribe its target genes[19]. Once in the nucleus, TFEB is responsible for the transcription of autophagy-related genes and genes encoding lysosomal proteins [20]. Once transcribed and translated, the autophagy-related proteins are responsible for the formation of the double membrane autophagosome, in which damaged proteins and organelles are sequestered [21, 22]. The autophagosome loaded with cargo, fuses with the lysosome, resulting in the formation of the autolysosome[23]. The acidic hydrolases from lysosomes degrade the loaded cargo and release nutrients back into the cytoplasm for recycling, correcting the nutritional deficiency[23].

Chloroquine, an anti-malarial drug, is the most frequently used agent for the inhibition of autophagy. Chloroquine and its derivative hydroxychloroquine are the only clinically available autophagy inhibitors approved by the US Food and Drug Administration[24]. A number of published studies have indicated that chloroquine is able to sensitize cancer cells to chemotherapy and radiotherapy, that is to say enhancing the anticancer effect[9, 10, 25]. These findings suggest that autophagy is induced by chemotherapy and radiotherapy as a protective mechanism in BC cells and that inhibition of autophagy is able to enhance therapeutic sensitivity.

GSK-3 inhibits the proliferation of bladder cancer cells and has low cytotoxicity in normal cells

To study the effect of AR-A014418 (GSK-3β inhibitor) on the growth of cells in vitro, we used T24 and HT1376, high grade UC cell lines, RT4, a low grade UC cell line, and human embryonic kidney 293 (HEK293) cells. MTS assay was used to observe the effects of AR-A014418 on cell proliferation. Our results indicated that AR-A014418 inhibited the growth of T24, HT1376, and RT4 cells after 24–72 hours of treatment(Fig. 1) and that the growth of BC cells was inhibited in a dose- and time-dependent manner. Moreover, AR-A014418 didn’t cause toxicity to normal kidney cells comparatively. Similarly, cell proliferation was examined with another GSK-3β inhibitor, TDZD-8, using MTS assay. It showed no significant difference compared to the control up to 48 hours after TDZD-8 treatment, but significant differences at 72 hours after treatment as shown in Supplementary Figure S1.

GSK-3 inhibition induces autophagy in cultured bladder cancer cells

The cells were treated with 10 and 25 µM AR-A014418 for 24 hours and the morphological changes were investigated under an inverted microscope. As shown in Fig. 2a, compared with the control group AR-A014418 treated cells showed an increased accumulation of cytoplasmic vacuoles and the formation of autophagosome-like structures in the cytoplasm.

To determine whether AR-A014418 induces autophagy in BC cells, we examined the expression of autophagy-related proteins; p62, Beclin-1, p-Beclin(S15), p-Beclin(S93), and LC3B(Fig. 2b). LC3B is the most widely used autophagosome marker because the amount of LC3B-Ⅱ reflects the number of autophagosomes and autophagy-related structures. In addition, the degradation of p62 is another widely used marker to monitor autophagic activity because p62 directly binds LC3B and is selectively degraded by autophagy[26, 27]. After treatment with the indicated concentrations of AR-A014418 for 0, 3, and 24 hours, western blot analysis showed a marked increase in LC3B in a dose- and time-dependent manner except for normal kidney cells. Consistent with an increase in autophagy, p62 showed the reverse response to high concentrations (25 µM) of AR-A014418. The levels of Beclin-1 were higher at 24 hours than at 3 hours at low concentrations(10 µM), while expression remained unchanged at high concentrations in T24 and HT1376, and no significant changes were observed in RT4 and HEK293 compared to controls. No consistent trend was found in p-Beclin.

Similarly, western blot analysis was performed with TDZD-8 as shown in Supplementary Figure S2.

We used a GFP-LC3-RFP-LC3ΔG probe to observe the stepwise progression of autophagy. When GFP-LC3-RFP-LC3ΔG is expressed in cells, ATG4 cleaves the C-terminus of the wild type LC3 forming GFP-LC3-I and releasing RFP-LC3-ΔG to the cytosol. While RFP-LC3-ΔG remains intact in the cytosol, GFP-LC3-I can be conjugated to PE on autophagic vesicle membranes and a fraction of it degraded in autolysosomes[28] (Fig. 2c).

In short, autophagic activity can be simply and quantitatively estimated by determining the ratio of GFP and RFP signal intensities. All four cell lines stably expressing GFP-LC3-RFP-LC3ΔG were treated with 10 µM AR-A014418 for 0, 12, 24, and 48 hours. The GFP/RFP ratio was lower in all cells treated for 24 hours compared to 12 hours. The decline in the GFP/RFP ratio indicates autophagic flux. In HT1376 and HEK293, cells shrunk and apoptotic bodies formed after 48 hours of treatment(Fig. 2d).

To further confirm the mechanism of GSK-3β inhibition to induce autophagy, we used the GSK-3β knockdown using siRNA in BC cells. The knockdown efficiency of siRNA in all four cell lines was proved by western blot analysis(Fig. 3a). GSK-3β knockdown promoted the expression of LC3B and p62 degradation in T24, and LC3B by one of the siRNAs in HEK293. However, the expression of Beclin-1 remained unchanged in all four cells.

Inhibition of autophagy sensitizes the cells to GSK-3 inhibition-induced apoptosis

We explored whether blocking autophagy with chloroquine synergized the inhibitory effects of AR-A014418 on BC cells. MTS assay revealed that combination of AR-A014418 and chloroquine significantly suppressed viability of T24(p = 0.001), HT1376(p = 0.0038), RT4(p = 0.0007), and HEK293(p = 0.016) (Fig. 3b). Next, we observed morphological changes in the GSK-3β knockdown using siRNA. siGSK-3β-transfected BC cells treated with chloroquine for 24 hours showed extensive vacuolation and for 48 hours tend to show nuclear degeneration. However, no such tendency toward cell vacuolation or nuclear degeneration was observed in HEK293(Supplementary Figure S3).

TFEB nuclear translocation is governed by GSK-3 inhibition

The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving the expression of autophagy and lysosomal genes. TFEB modulates the formation of autophagosomes and also the fusion of the autophagosome and lysosome[29].

We analyzed the transcriptional regulatory network of TFEB nucleus translocation upon cell treatment with AR-A014418. We constructed cells expressing the GFP-TFEB fusion proteins and assessed TFEB translocation by fluorescent microscopy. All four cells were incubated in this study with 0(control) and 10 µM AR-A014418 for 24 hours. The intracellular localization of TFEB was demonstrated to be significantly (p < 0.0001) higher in the cytoplasm relative to the nucleus under control conditions. On the other hand, after incubation of cells with AR-A014418, there was an increase in nuclear TFEB and a decrease in cytosolic TFEB expression(Fig. 4a, b). The nucleus translocation was further confirmed by nucleus cytosol separation assay and detected through the western blot analysis in Fig. 4c.

The data in Fig. 4 indicate that the treatment of BC cells with AR-A014418 leads to nuclear translocation of TFEB, the master transcriptional regulator of autophagy.

The role of the AMP-AMPK-ULK1 pathway in GSK-3 inhibition-induced autophagy

Autophagy is elicited in cells through the induction of ULK1 via the phosphorylation of AMPK or the inhibited mTOR[15, 30]. The initial stage of phagophore formation is the most complex step in the process of autophagic flux, in which various functional units are involved, including the ULK1 complex. Elevated p-ULK1 levels are induced with a peak at 3 hours of treatment in T24 and HT1376. However, we observed no consistent trend in RT4 and HEK293(Fig. 5a, b), a low grade UC and normal. One of the GSK3β-specific siRNAs increased the level of p-ULK1 in T24, but conversely, p-ULK1 levels decreased in HT1376(Fig. 5c).

Elevated p-AMPKα levels are induced with a peak at 24 hours of treatment in T24, and with a peak at 12 hours of treatment in HT1376 and RT4 (Fig. 5d). Unlike BC cells, HEK293 had the highest expression level of p-AMPKα in control and decreased after 12 hours of AR-A014418 treatment.

Figure 5. AMPK-ULK1 signaling pathway through the AR-A014418 induces autophagy. Bladder cancer cells were treated with 10 µM AR-A014418 at the indicated time(0, 3, 6, 12, and 24 hours). (a) Western blot analysis of phosphorylated ULK1 (p-ULK1), total ULK1(ULK1), and β-actin as a loading control. The original blots are presented in Supplementary Figure S4m-p. (b) Relative protein intensity was quantified using Image J. (c) Western blot analysis of p-ULK1 and ULK1 in siGSK-3β-transfected bladder cancer cells. The original blots are presented in Supplementary Figure S4q, r. (d) The expression of phosphorylated AMPKα(p-AMPKα) and total AMPKα(AMPKα) proteins were determined by western blot analysis. The original blots are presented in Supplementary Figure S4s-v.

Autophagy is a promising target for cancer therapy, and induction of autophagy-associated cytotoxic death by blocking autophagy flux has been recognized as a novel cancer therapeutic strategy[31–33]. Previously, we have demonstrated that GSK-3β inhibition had an antitumor effect, and treatment with GSK-3β inhibitor resulted in autophagy in BC cells in our laboratory[34].

The therapeutic resistance process in drug treatment of BC remains to be elucidated, and studies examining GSK-3β inhibitors from the standpoint of autophagy mechanisms have not yet been reported in BC. Therefore, we focused on autophagy, which is thought to contribute to the resistance to existing therapies, and examined whether GSK-3β inhibitors in combination with autophagy inhibitors could be a new therapeutic approach for treating BC.

Marchand B et al. have demonstrated that GSK-3 inhibition(CHIR99021 or SB216763) induces prosurvival signals through increased activity of the autophagy/lysosomal network in pancreatic cancer cells[35]. Russi S et al. have demonstrated that GSK-3 inhibitor(CHIR99021) induces cell cycle arrest, mitotic catastrophe, and autophagic response, resulting in reduced cell proliferation in ES cells[36].

Similarly, we also found that GSK-3β inhibitors (AR-A014418 and TDZD-8) suppressed cell proliferation in T24, HT1376, RT4, and HEK293 as observed by the MTS assay(Fig. 1 and Supplementary Figure S1). Furthermore, we observed the decreased expression of p62 and increased expression of LC3B and Beclin-1 by treating with GSK-3β inhibitor. The genetic depletion of GSK-3β by siRNA also lead to similar results, although the results were less pronounced than for pharmacological inhibition(Fig. 2b and Fig. 3a).

Autophagy has two opposite functions in cancer therapy; a process known as protective autophagy[9, 25, 37] and typeⅡ programmed cell death which is referred to as autophagic cell death[38–42]. Therefore, it is essential to identify how autophagy behaves in each cancer treatment. Many studies over the past decade have shown that autophagy in BC is inhibited by autophagy inhibitors, including chloroquine, 3MA, and RNA interference[37, 10]. Schlutermann et al. treated BC with cisplatin and chloroquine and demonstrated that autophagy enhances cisplatin cytotoxicity[9]. Wang et al. also reported that radiotherapy activates autophagy in BC and that subsequent cytoprotective autophagy is strongly associated with radiation resistance, further suggesting that inhibition of autophagy by chloroquine contributes to enhanced radiosensitivity[25].

The combination of chloroquine or its derivatives and chemotherapeutics has been used in phase Ⅰ and phase Ⅱ clinical trials for a variety of tumors[43, 44]. Also, previous reports showed that GSK-3β inhibitors potentiated the antitumor effect of gemcitabine and cisplatin, in addition, chloroquine potentiated the antitumor effect of GSK-3β inhibitor in BC cells[34]. Our results of the MTS assay showed that GSK-3β inhibition induced autophagy can enhance lethality with chloroquine in BC. These results suggest that GSK-3β inhibition-induced autophagy acts pro-survival in BC.

TFEB can be phosphorylated by GSK-3 at residues S134 and S138 leading to cytoplasmic retention, whereas GSK-3 inhibition led to TFEB nuclear translocation[45]. GSK-3 inhibition leads to TFEB dephosphorylation, which correlates with TFEB dislocation from 14-3-3 chaperones and TFEB nuclear localization in the pancreatic cancer cell[35]. Our study provides partial support for the role of GSK-3β in the regulation of TFEB in the BC cell. Our data showed TFEB-GFP in T24 translocated from cytoplasm to nucleus after GSK-3β inhibitor treatment, but western blot analysis showed that cytoplasmic TFEB protein was unchanged after treatment and nuclear translocation was reversed with a decrease in TFEB protein(Fig. 4c). One possible explanation for this result is the hypothesis that T24 is a high grade BC and therefore retains TFEB in the nucleus from a steady state, but further experiments are needed to prove this.

The AMPK pathway is an important upstream signal, as a key cellular energy sensor, of autophagy activation[46]. When the cellular AMP/ATP ratio increases, AMPK is activated and mTORC1 is suppressed[47]. mTORC1 lies upstream of the ULK1 complex and negatively regulates this complex. AMPK activation and mTORC1 suppression lead to the phosphorylation of ULK1, resulting in phagophore and autophagosome formation[48]. In addition, previous studies reported that treating cells with GSK-3 inhibitors inhibited mTORC1 activity and increased autophagic flux[49]. (Fig. 5e).

Our data showed that p-ULK1/ULK1 protein levels increased as early as 3 hours after GSK-3β inhibitor (AR-A014418) treatment in high and intermediate grade BC cells and decreased with time, whereas no such trend was observed in low grade BC cells or normal kidney cells. In the present study, from an autophagy perspective, treatment with GSK-3β inhibitor potentiate apoptosis and proliferation in the BC cell.

Finally, phase Ⅰ and Ⅱ clinical trials of GSK-3β inhibitor are being conducted overseas for pancreatic cancer and gliomas. If clinically applied in the future, it is expected to contribute significantly to BC treatment.

Our results suggest that autophagy inhibition can potentiate the effect of GSK-3β inhibition by abrogating pro-survival autophagy activation. Thus, combination therapy with these two small molecules can be advantageous in BC patients.

Cells and cell culture

Bladder cancer cell lines T24, HT1376, RT4, and human embryonic kidney 293 (HEK293) cells were purchased from the American Type Culture Collection(ATCC). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% MEM Non-Essential Amino Acids (Gibco; Thermo Fisher Scientific, Inc.), 1% MEM sodium pyruvate solution 100 mM (Gibco; Thermo Fisher Scientific, Inc.) and 90 µg/ml kanamycin in a 37°C incubator containing 5% CO₂.

Passaging was achieved using 5 ml 0.05% Trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) when cells were fused to 80%. AR-A014418 and TDZD-8(GSK-3 inhibitor) were purchased from Abcam.

Cell viability assay

The cells were seeded in 96-well plates at 1–2×10^3–4 cells/well with 100 µl medium for 24 hours and then treated with AR-A014418 at the indicated concentrations (0, 0.5, 1, 5, 10, and 25 µM) for 24, 48, and 72 hours. Following incubation, 10 µl CellTiter 96 Aqueous One Solution Regent(Promega Corporation) was added into each well and incubated for 2 hours. The absorbance was measured at 490 nm by the iMark™ 96-well microplate reader (Bio-Rad Laboratories, Inc.). Chloroquine(phosphate) (14194) was purchased from Cayman chemical company.

Protein Extraction and Western blot analysis

Subconfluent cell cultures were washed with cold PBS and lysed in lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 µg/ml).

Following clarification of the lysates by centrifugation at 15,000 × g for 30 min at 4°C, protein concentration was detected by the Bradford method, and 20–30 µg of each protein was electrophoretically separated on a 4–15% or 10 or 7.5% SDS-polyacrylamide gel and transblotted to a PVDF membrane. Immunoblots were blocked with 10% skimmed milk in TBS followed by incubation with primary antibodies. Horseradish peroxidase-labeled ECL™ Anti-mouse IgG (1:5,000) and ECL™ Anti-rabbit IgG (1:10,000) from GE Healthcare were used as secondary antibodies and detected using Clarity Max Western ECL Substrate from Bio-Rad Laboratories, Inc. according to the manufacturer's instructions. The expression of β-actin(Cell Signaling Technology, Inc., 12262) was used as a loading control. The images were analyzed using Ez-Capture MG (Atto Corporation). The following primary antibodies were purchased from Cell Signaling Technology, Inc.: SQSTM1/p62(8025), Beclin-1(3738), p-Beclin-1(S15)(84966), p-Beclin-1(S93)(14717), LC3(3868), GSK3β(12456), TFEB(4240), α-Tubulin(9099), Histone3(12648), p-ULK1(14202), ULK1(8054), p-AMPKα(2535), AMPKα(5831).

The dilution ratios of the primary antibodies were 1:200-1:2,000.

Nuclear/cytosolic fractionation was carried out by the Minute^™ Cytoplasmic and Nuclear Extraction Kit for Cells(Invent Biotechnologies, Inc., SC-003) according to the manufacturer's instructions. α-Tubulin and Histone3 were used as a control for cytosolic and nuclear lysates respectively.

Estimation of autophagic flux

pMRX-IP-GFP-LC3-RFP-LC3ΔG(plasmid # 84572) was purchased from Addgene, Inc. The plasmid was transformed into NEB stable Competent E.coli (New England Biolabs, Inc., C3040H) and plasmid DNA was extracted and purified by PureLink™ HiPure Plasmid Midiprep Kit(Invitrogen; Thermo Fisher Scientific, Inc.).

The cells(70–90% confluent) were grown in 24-well plates and transfected with the plasmid according to the Lipofectamine™ 3000 Reagent protocols. After 24 hours incubation, the cells were treated with 10µM AR-A014418 for 12, 24, and 48 hours, and images were captured by fluorescence microscope(Olympus, IX71) at 40x magnification. The absorption filters used were BA510IF for green fluorescence and BA575IF for red fluorescence. The fluorescence intensity was quantified using the ImageJ imaging software program.

Nuclear translocation of TFEB-EGFP

pEGFP-N1-TFEB (plasmid#38119) was purchased from Addgene, Inc. The plasmid was transformed into competent high DH5α(TOYOBO CO., LTD., DNA-903) and plasmid DNA was extracted and purified by PureLink™ HiPure Plasmid Midiprep Kit(Invitrogen; Thermo Fisher Scientific, Inc.).

The cells(70–90% confluent) were grown in 24-well plates and transfected with the plasmid according to the Lipofectamine™ 3000 Reagent protocols. After 24 hours incubation, the cells were treated with 10 µM AR-A014418 for 24 hours and nuclear translocation of TFEB-EGFP was examined and analyzed by fluorescent microscopy(Olympus, IX71) at 40x magnification. The absorption filters used were BA510IF for green fluorescence. The nuclei were stained with Hoechst 33342(Bio-Rad Laboratories, Inc., 1351304) 5 µg/ml for 10 min at room temperature.

siRNA transfection

For GSK-3β silencing, the cells were transfected with specific human siRNAs against GSK-3β by using Lipofectamine RNAiMAX(Invitrogen, Thermo Fisher Scientific Inc.) according to the manufacturer’s recommendations. siRNA targeting human GSK-3β(s6239, s6241) and control siRNA with a scrambled sequence(AM4611) were purchased from Ambion, Thermo Fisher Scientific, Inc. Targeting sequence of siRNA are as follows: GSK-3β(s6239); CUCAAGAACUGUCAAGUAAtt, GSK-3β(s6241); GCUAGAUCACUGUAACAUAtt. The knockdown efficiency was then measured by immunoblotting.

Statistical analysis

Continuous variables are presented as the mean ± SD. All continuous variables in this study met the criteria for normal distribution and were assumed to be parametric. Data were analyzed using one-way ANOVA with Dunnett's test for multiple comparisons. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc.). P < 0.05 was considered to indicate a statistically significant difference.

Author contributions

Y.S., V.B. and Y.T. designed the study. Y.S. performed the majority of experiments and analyzed the data. Y.S., V.B., T.A., H.K., A.K., M.M., K.H. and Y.T. discussed and assessed the data. Y.S. and V.B. wrote the main manuscript text. Y.S. designed the figures. V.B. and Y.T. supervised the project. All authors read the manuscript, provided critical feedback and approved the final version of the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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Targeting pro-survival autophagy enhanced GSK-3β inhibition-induced apoptosis and retarded proliferation in bladder cancer cells

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Abstract

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Introduction

Results

GSK-3 inhibits the proliferation of bladder cancer cells and has low cytotoxicity in normal cells

GSK-3 inhibition induces autophagy in cultured bladder cancer cells

Inhibition of autophagy sensitizes the cells to GSK-3 inhibition-induced apoptosis

TFEB nuclear translocation is governed by GSK-3 inhibition

The role of the AMP-AMPK-ULK1 pathway in GSK-3 inhibition-induced autophagy

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Competing interests

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