Targeting Pro-Survival Autophagy Enhanced GSK-3β Inhibition-Induced Apoptosis and Retarded Proliferation in Bladder Cancer Cells

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 are 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 promote 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 suggest that targeting autophagy potentiates GSK-3β inhibition-induced apoptosis and retarded proliferation in BC cells.


Introduction
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 cisplatinbased chemotherapy for a long time, pembrolizumab was introduced for advanced UC that recurred or progressed after platinum-based chemotherapy. The objective response rate in the international phase III KEYNOTE-045 clinical trials 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].
The drug pricing is high relative to the response rates, and the development of new therapeutic agents is required to improve the clinical outcomes of patients with locally advanced or metastatic BC.
The dilution ratios of the primary antibodies were 1:200-1:2000. According to the manufacturer's instructions, nuclear/cytosolic fractionation was carried out using the MinuteTM Cytoplasmic and Nuclear Extraction Kit for Cells (Invent Biotechnologies, Inc., Plymouth, MN, USA, SC-003). α-Tubulin and Histone3 were used as a control for cytosolic and nuclear lysates, respectively.
The cells (70-90% confluent) were grown in 24-well plates and transfected with the plasmid according to the Lipofectamine TM 3000 Reagent protocols. After 24 h of incubation, the cells were treated with 10 µM AR-A014418 for 12, 24, and 48 h, and images were captured with fluorescence microscope (Olympus, Tokyo, Japan, IX71) at 40× 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 (version 1.53t).
The cells (70-90% confluent) were grown in 24-well plates and transfected with the plasmid according to the Lipofectamine TM 3000 Reagent protocols. After 24 h of incubation, the cells were treated with 10 µM AR-A014418 for 24 h, and nuclear translocation of TFEB-EGFP was examined and analyzed via fluorescent microscopy (Olympus, IX71) at 40× 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.

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 software (version 8.0) (GraphPad Software, Inc., San Diego, CA, USA). p < 0.05 was considered to indicate a statistically significant difference.

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. The MTS assay was used to observe the effects of AR-A014418 on cell proliferation. Our results indicate that AR-A014418 inhibits the growth of T24, HT1376, and RT4 cells after 24-72 h of treatment (Figure 1), and that the growth of BC cells is inhibited in a dose-and time-dependent manner. Moreover, AR-A014418 does not 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 h after TDZD-8 treatment, but showed significant differences at 72 h 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 h, and the morphological changes were investigated under an inverted microscope. As shown in Figure 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, including p62, Beclin-1, p-Beclin (S15), p-Beclin (S93), and LC3B ( Figure 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 [28,29], and Beclin-1 is the main autophagy gene associated with cancer, and it was reported that autophagy might inhibit or promote tumorigenesis by focusing on Beclin-1 [30,31]. After treatment with the indicated concentrations of AR-A014418 for 0, 3, and 24 h, the Western blot analysis showed a marked increase in LC3B in a dose-and time-dependent manner except for the 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 h than at 3 h at low concentrations (10 µM), while the expression remained unchanged at high concentrations in T24 and HT1376, and no significant changes were observed in RT4 and HEK293 compared to the controls. No consistent trend was found in p-Beclin.
Similarly, a Western blot analysis was performed with TDZD-8, as shown in Supplementary Figure S2. The level of LC3B increased in a dose-and time-dependent manner except for the normal kidney cells, but no consistent trend was found in p62 and Beclin-1.
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

GSK-3 Inhibition Induces Autophagy in Cultured Bladder Cancer Cells
The cells were treated with 10 and 25 µM AR-A014418 for 24 h, and the morphological changes were investigated under an inverted microscope. As shown in Figure 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, including p62, Beclin-1, p-Beclin (S15), p-Beclin (S93), and LC3B ( Figure 2b). LC3B is the most widely used autophagosome marker because the amount of LC3B-II reflects the number of autophagosomes and autophagyrelated 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 [28,29], and Beclin-1 is the main autophagy gene associated with cancer, and it was reported that autophagy might inhibit or promote tumorigenesis by focusing on Beclin-1 [30,31]. After treatment with the indicated concentrations of AR-A014418 for 0, 3, and 24 h, the Western blot analysis showed a marked increase in LC3B in a dose-and time-dependent manner except for the 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 h than at 3 h at low concentrations (10 µM), while the expression remained unchanged at high concentrations in T24 and HT1376, and no significant changes were observed in RT4 and HEK293 compared to the controls. No consistent trend was found in p-Beclin.
Similarly, a Western blot analysis was performed with TDZD-8, as shown in Supplementary Figure S2. The level of LC3B increased in a dose-and time-dependent manner except for the normal kidney cells, but no consistent trend was found in p62 and Beclin-1.
We used a GFP-LC3-RFP-LC3∆G probe to observe the stepwise progression of autophagy. When GFP-LC3-RFP-LC3∆G is expressed in cells, ATG4 cleaves the C-terminus of the wild type LC3, forming GFP-LC3-I, and releasing RFP-LC3-∆G to the cytosol. While RFP-LC3-∆G remains intact in the cytosol, GFP-LC3-I can be conjugated to PE on autophagic vesicle membranes, and a fraction of it is degraded in autolysosomes [32] ( Figure 2c). In short, autophagic activity can be simply and quantitatively estimated by determining the ratio of the GFP and RFP signal intensities. All four cell lines stably expressing GFP-LC3-RFP-LC3∆G were treated with 10 µM AR-A014418 for 0, 12, 24, and 48 h. The GFP/RFP ratio was lower in all the cells treated for 24 h compared to those treated for 12 h. The decline in the GFP/RFP ratio indicates autophagic flux. In HT1376 and HEK293, the cells shrunk, and apoptotic bodies formed after 48 h of treatment (Figure 2d).
To further confirm the mechanism of GSK-3β inhibition to induce autophagy, we used the GSK-3β knockdown using siRNA in BC cells. In these experiments, a scrambled siRNA sequence was used as a negative control, and lipofectamine was only added as a control. The knockdown efficiency of siRNA in all four cell lines was proven 48 h post-siRNA transfection via Western blot analysis (Figure 3a). It should be noted that the efficiency of GSK-3β knockdown was particularly weak in HT1376 compared to the other BC cells, as shown by the densitometry readings/intensity ratio of GSK-3β in Supplementary Figure S4r. GSK-3β knockdown promoted the expression of LC3B and the degradation of p62 in T24, and promoted the expression of LC3B by one of the siRNAs in HEK293. However, the expression of Beclin-1 remained unchanged in all four cells.
Curr. Oncol. 2023, 30 5355 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 is degraded in autolysosomes [32] ( Figure  2c). In short, autophagic activity can be simply and quantitatively estimated by determining the ratio of the 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 h. The GFP/RFP ratio was lower in all the cells treated for 24 h compared to those treated for 12 h. The decline in the GFP/RFP ratio indicates autophagic flux. In HT1376 and HEK293, the cells shrunk, and apoptotic bodies formed after 48 h of treatment (Figure 2d). To further confirm the mechanism of GSK-3β inhibition to induce autophagy, we used the GSK-3β knockdown using siRNA in BC cells. In these experiments, a scrambled siRNA sequence was used as a negative control, and lipofectamine was only added as a control. The knockdown efficiency of siRNA in all four cell lines was proven 48 h post-siRNA transfection via Western blot analysis (Figure 3a). It should be noted that the efficiency of GSK-3β knockdown was particularly weak in HT1376 compared to the other BC cells, as shown by the densitometry readings/intensity ratio of GSK-3β in Supplementary Figure S4r. GSK-3β knockdown promoted the expression of LC3B and the degradation of p62 in T24, and promoted the expression of 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 showed in the previous sections that AR-A014418 suppresses cell proliferation and induces autophagy. Further, we explored whether this autophagy induction is cytoprotective or cytotoxic for BC cells by inhibiting autophagy with chloroquine.
The MTS assay revealed that the combination of AR-A014418 and chloroquine significantly suppressed the viability of T24 (p = 0.001), HT1376 (p = 0.0038), RT4 (p = 0.0007), and HEK293 (p = 0.016), compared with the BC cells treated with AR-A014418 or chloroquine alone (Figure 3b). Next, we observed the morphological changes in the GSK-3β knockdown using siRNA. The siGSK-3β-transfected BC cells treated with chloroquine for

Inhibition of Autophagy Sensitizes the Cells to GSK-3 Inhibition-Induced Apoptosis
We showed in the previous sections that AR-A014418 suppresses cell proliferation and induces autophagy. Further, we explored whether this autophagy induction is cytoprotective or cytotoxic for BC cells by inhibiting autophagy with chloroquine.
The MTS assay revealed that the combination of AR-A014418 and chloroquine significantly suppressed the viability of T24 (p = 0.001), HT1376 (p = 0.0038), RT4 (p = 0.0007), and HEK293 (p = 0.016), compared with the BC cells treated with AR-A014418 or chloroquine alone (Figure 3b). Next, we observed the morphological changes in the GSK-3β knockdown using siRNA. The siGSK-3β-transfected BC cells treated with chloroquine for 24 h showed extensive vacuolation, and for 48 h, they tended to show nuclear degeneration. However, no such tendency toward cell vacuolation or nuclear degeneration was observed in HEK293 (Supplementary Figure S3). It was shown that AR-A014418 induces autophagy in BC cells, and its inhibition suppresses cell proliferation. In other words, this form of autophagy is cytoprotective and reduces cell death.

TFEB Nuclear Translocation Is Governed by GSK-3 Inhibition
Apart from autophagy initiation mediated by four signal-sensing kinases, mTORC1, ULK1, AMPK, and protein kinase B (AKT), autophagy is also regulated by transcriptional and epigenetic mechanisms. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinates this program by driving the expression of autophagy and lysosomal genes. TFEB modulates the formation of autophagosomes and the fusion of the autophagosome and lysosome [33].
We analyzed the transcriptional regulatory network of the TFEB nucleus translocation upon cell treatment with AR-A014418. We constructed cells expressing the GFP-TFEB fusion proteins and assessed the TFEB nuclear translocation via fluorescent microscopy. All four GFP-TFEB expressed cells were incubated in this study with 0 (control) and 10 µM AR-A014418 for 24 h. Based on the fluorescent microscopy analysis, TFEB is more abundant in the cytoplasm relative to the Hoechst 33342-stained nucleus in the control condition. On the other hand, treatment with AR-A014418 resulted in aggregates of granular puncta of TFEB in the nucleus, overlapping with Hoechst staining (Figure 4a). The intracellular localization of the TFEB intensity was demonstrated to be significantly (p < 0.0001) higher in the cytoplasm relative to the nucleus under the control conditions. On the other hand, after treatment with AR-A014418, there was an increase in the nuclear TFEB intensity and a decrease in the cytosolic TFEB intensity (Figure 4b). The nucleus translocation was further confirmed via nucleus cytosol separation assay and detected through the Western blot analysis in Figure 4c.
24 h showed extensive vacuolation, and for 48 h, they tended to show nuclear degeneration. However, no such tendency toward cell vacuolation or nuclear degeneration was observed in HEK293 (Supplementary Figure S3). It was shown that AR-A014418 induces autophagy in BC cells, and its inhibition suppresses cell proliferation. In other words, this form of autophagy is cytoprotective and reduces cell death.

TFEB Nuclear Translocation Is Governed by GSK-3 Inhibition
Apart from autophagy initiation mediated by four signal-sensing kinases, mTORC1, ULK1, AMPK, and protein kinase B (AKT), autophagy is also regulated by transcriptional and epigenetic mechanisms. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinates this program by driving the expression of autophagy and lysosomal genes. TFEB modulates the formation of autophagosomes and the fusion of the autophagosome and lysosome [33].
We analyzed the transcriptional regulatory network of the TFEB nucleus translocation upon cell treatment with AR-A014418. We constructed cells expressing the GFP-TFEB fusion proteins and assessed the TFEB nuclear translocation via fluorescent microscopy. All four GFP-TFEB expressed cells were incubated in this study with 0 (control) and 10 µM AR-A014418 for 24 h. Based on the fluorescent microscopy analysis, TFEB is more abundant in the cytoplasm relative to the Hoechst 33342-stained nucleus in the control condition. On the other hand, treatment with AR-A014418 resulted in aggregates of granular puncta of TFEB in the nucleus, overlapping with Hoechst staining (Figure 4a). The intracellular localization of the TFEB intensity was demonstrated to be significantly (p < 0.0001) higher in the cytoplasm relative to the nucleus under the control conditions. On the other hand, after treatment with AR-A014418, there was an increase in the nuclear TFEB intensity and a decrease in the cytosolic TFEB intensity (Figure 4b). The nucleus translocation was further confirmed via nucleus cytosol separation assay and detected through the Western blot analysis in Figure 4c.
The data in Figure 4 indicate that the treatment of BC cells with AR-A014418 leads to the nuclear translocation of TFEB, the master transcriptional regulator of autophagy.

The Role of the AMP-AMPK-ULK1 Pathway in GSK-3 Inhibition-Induced Autophagy
We investigated the effects of AR-A014418 on ULK1 and AMPK, the signal-sensing kinases involved in autophagy initiation. Autophagy is elicited in cells through the induction of ULK1 via the phosphorylation of AMPK or the inhibited mTOR [17,34]. 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. In the present study, elevated p-ULK1 levels are induced with a peak at 3 h of treatment in T24 and HT1376. However, we observed no consistent trend in RT4 and HEK293 (Figure 5a,b), a low grade UC and normal cells, respectively. The GSK3β-specific siRNAs increased the level of p-ULK1 in T24, and the p-ULK1 levels did not change in HT1376 compared to the negative control (Figure 5c).
Then, we also investigated the phosphorylation of AMPK. Elevated p-AMPKα levels are induced with a peak at 24 h of treatment in T24, and a peak at 12 h of treatment in HT1376 and RT4. Unlike the BC cells, HEK293 had the highest expression level of p-AMPKα in the control, and it decreased after 12 h of AR-A014418 treatment (Figure 5d). . AR-A014418 results in the nuclear translocation of TFEB. (a) Immunofluorescence analysis was performed to study the co-localization of TFEB relative to Hoechst 33342-stained nucleus. Scale bar = 50 µm. (b) Quantitative analysis of TFEB intensity in (a) using ImageJ. Columns, mean; bars, SD. p values are indicated above the pairwise brackets. * p < 0.05, ** p < 0.01, *** p < 0.001, nsnot significant; compared to control cells. Statistical analysis was performed using Student's t-test. (c) Cytoplasmic and nuclear fractions were analyzed using Western blot analysis to study the levels of TFEB, α-Tubulin (cytoplasmic control), and Histone3 (nuclear control). The original blots are presented in Supplementary Figure S4i The data in Figure 4 indicate that the treatment of BC cells with AR-A014418 leads to the nuclear translocation of TFEB, the master transcriptional regulator of autophagy.

The Role of the AMP-AMPK-ULK1 Pathway in GSK-3 Inhibition-Induced Autophagy
We investigated the effects of AR-A014418 on ULK1 and AMPK, the signal-sensing kinases involved in autophagy initiation. Autophagy is elicited in cells through the induction of ULK1 via the phosphorylation of AMPK or the inhibited mTOR [17,34]. 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. In the present study, elevated p-ULK1 levels are induced with a peak at 3 h of treatment in T24 and HT1376. However, we observed no consistent trend in RT4 and HEK293 (Figure 5a,b), a low grade UC and normal cells, respectively. The GSK3β-specific siRNAs increased the level of p-ULK1 in T24, and the p-ULK1 levels did not change in HT1376 compared to the negative control (Figure 5c).
Then, we also investigated the phosphorylation of AMPK. Elevated p-AMPKα levels are induced with a peak at 24 h of treatment in T24, and a peak at 12 h of treatment in HT1376 and RT4. Unlike the BC cells, HEK293 had the highest expression level of p-AMPKα in the control, and it decreased after 12 h of AR-A014418 treatment (Figure 5d). These data are in consensus with previous reports showing AMPK activation and autophagy after treatment with structurally different GSK-3 inhibitors [35,36].

Discussion
Autophagy is a promising target for cancer therapy, and the induction of autophagyassociated cytotoxic death by blocking autophagy flux has been recognized as a novel cancer therapeutic strategy [37][38][39]. Previously, we demonstrated that GSK-3β inhibition had an antitumor effect, and treatment with GSK-3β inhibitors resulted in autophagy in BC cells in our laboratory [10].
The therapeutic resistance process in the 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. demonstrated that GSK-3 inhibition (CHIR99021 or SB216763) induces pro-survival signals through the increased activity of the autophagy/lysosomal network in pancreatic cancer cells [40]. Russi S. et al. demonstrated that GSK-3 inhibitor (CHIR99021) induces cell cycle arrest, mitotic catastrophe, and autophagic response, resulting in reduced cell proliferation in ES cells [41].
Similarly, we also found that GSK-3β inhibitors (AR-A014418 and TDZD-8) suppress cell proliferation in T24, HT1376, RT4, and HEK293 as observed by the MTS assay ( Figure 1 and Supplementary Figure S1). Furthermore, we observed the decreased expression of p62 and increased expression of LC3B and Beclin-1 via treatment with GSK-3β inhibitors. The genetic depletion of GSK-3β by siRNA also lead to similar results, although the results were less pronounced than for pharmacological inhibition (Figures 2b and 3a).
Autophagy has two opposite functions in cancer therapy, a process known as protective autophagy [11,27,42], and type II programmed cell death, which is referred to as autophagic cell death [43][44][45][46][47]. 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 [12,42]. Schlutermann et al. treated BC with cisplatin and chloroquine and demonstrated that autophagy enhances cisplatin cytotoxicity [11]. 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 the inhibition of autophagy by chloroquine contributes to enhanced radiosensitivity [27].
The combination of chloroquine or its derivatives and chemotherapeutics has been used in phase I and II clinical trials for a variety of tumors [48,49]. Additionally, 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 inhibitors in BC cells [10]. Our results of the MTS assay show that GSK-3β inhibition-induced autophagy can enhance lethality with chloroquine in BC. These results suggest that GSK-3β inhibition-induced autophagy acts as a pro-survival signal in BC.
TFEB can be phosphorylated by GSK-3 at residues S134 and S138, leading to cytoplasmic retention, whereas GSK-3 inhibition leads to TFEB nuclear translocation [50]. 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 [40]. Our study provides partial support for the role of GSK-3β in the regulation of TFEB in the BC cell. Our data showed that TFEB-GFP in T24 translocated from the cytoplasm to the nucleus after GSK-3β inhibitor treatment, but the Western blot analysis showed that the cytoplasmic TFEB protein was unchanged after treatment, and the nuclear translocation was reversed with a decrease in the TFEB protein (Figure 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 [51]. When the cellular AMP/ATP ratio increases, AMPK is activated and mTORC1 is suppressed [52]. 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 [53]. In addition, previous studies reported that treating cells with GSK-3 inhibitors inhibited mTORC1 activity and increased autophagic flux [54] (Figure 5e).
Our data showed that the p-ULK1/ULK1 protein levels increased as early as 3 h after GSK-3β inhibitor (AR-A014418) treatment in the high and intermediate grade BC cells and decreased with time, whereas no such trend was observed in the low grade BC cells or normal kidney cells. In the present study, from an autophagy perspective, treatment with GSK-3β inhibitors potentiate apoptosis and proliferation in the BC cell. However, the in vitro culture cannot completely simulate the in vivo internal environment. Therefore, in vivo experiments will be required to be conducted in a future study. In addition, bioinformatic analysis such as BCG and IL-2 models for bladder cancer treatment [55] could also be useful. The results of our study and observation are in concordance with similar studies [56][57][58], and this approach presents a rationale to overcome drug resistance from an autophagy perspective.
Finally, phase I and II clinical trials of GSK-3β inhibitor are being conducted for pancreatic cancer and gliomas. If this is 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.

Conclusions
We found that GSK-3β inhibitor induces pro-survival autophagy, and targeting autophagy potentiates GSK-3β inhibition-induced apoptosis and retarded proliferation in BC cells. This indicates that GSK-3β inhibitor can be used as an autophagy-targeted drug, and autophagy blockade has the potential to be an effective interventional strategy for addressing cancer progression and overcoming therapeutic resistance in addition to existing treatment. Despite there being only a few clinical trials targeting autophagy in BC or using GSK-3β inhibitors, they are expected to make a significant contribution to BC treatment if clinically applied in the future.