Inhibition of autophagy by chloroquine enhances the antitumor activity of gemcitabine for gallbladder cancer

Fang‑Tao Wang · Hui Wang1 · Qi‑Wei Wang · Mu‑Su Pan1 · Xin‑Ping Li1 · Wei Sun · Yue‑Zu Fan1

Abstract

Gemcitabine (GEM), as an anti-metabolic nucleoside analog, has been shown to have anticancer effects in various tumors, but its chemotherapy resistance is still an important factor leading to poor prognosis of cancer patient. A large number of studies in recent years have shown that autophagy plays an important role in the chemotherapy sensitivity of many tumors, including pancreatic, non-small cell lung, and bladder cancer. However, whether GEM causes autophagy in gallbladder cancer (GBC) and whether it is related to chemotherapy resistance is unknown. In the present study, we demonstrated that GEM induced apoptosis and protective autophagy in GBC cells, which may be related to the AKT/mTOR signaling pathway, and GEM in combination with autophagy inhibitor chloroquine can strengthen the cytotoxic effect of GEM on GBC in vitro and in vivo. These findings showed that both autophagy and AKT/mTOR signals were engaged in GBC cell death evoked by GEM, GBC patients might benefit from this new treatment strategy, and molecular targeted treatment in combination with autophagy inhibitors shows promise as a treatment improvement.

Keywords Gallbladder neoplasm · Gemcitabine · Autophagy · Apoptosis

Introduction

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary tract, and its incidence shows an obvious increasing trend [1, 2]. The early diagnosis of GBC is difficult because of its concealed symptoms. Most patients are diagnosed with advanced tumors and lost the best time for surgery [3]. Even if some patients have the opportunity to undergo surgery, recurrence is very easy after surgery, leading to a poor prognosis of GBC patients. Therefore, for patients with advanced GBC who have lost the opportunity for surgery or recurred after surgery, palliative adjuvant therapy such as chemotherapy is often chosen.
Recently, the National Comprehensive Cancer Network guidelines recommend gemcitabine (GEM) as a first-line chemotherapy drug for the treatment of unresectable hepatobiliary tumors, and also as adjuvant therapy for resectable tumors [4]. GEM is a deoxypyrimidine analog with anti-metabolic effects and inhibition of DNA synthesis. It has been approved as a clinical anticancer drug in many countries [4] and is widely used to treat various advanced malignancies that have lost the opportunity for surgery, including advanced pancreatic cancer, advanced non-small cell lung cancer, and metastatic or local bladder cancer.
[5–7]. Based on recent clinical trials, GEM combined with cisplatin (GEMCIS) has a better effect on biliary tract cancers than GEM alone. The median overall survival (OS) and progression-free survival (PFS) can reach 11.7 months and 8.1 months, respectively [8], so GEMCIS has been recommended as a first-line chemotherapy drug for unresectable biliary tract tumors. However, it has been reported that the prognosis and response to chemotherapy of GBC are poor compared with those of other biliary tract cancer subtypes (extrahepatic bile duct cancer) [9]. A phase II clinical survey involving only unresectable GBC showed that these patients had a median OS and PFS of only 6.2 and 3.1 months, respectively [10]. It has also been reported that GEM combined with tumor angiogenesis inhibitors (bevacizumab or sorafenib) in the treatment of advanced biliary tract cancer had no significant improvement compared with GEM alone [11, 12]. In the clinical observation of adjuvant chemotherapy for resectable biliary tract cancer, compared with GEM alone, GEMCIS did not show a significant advantage, either [13]. Clinical practice has shown that chemotherapy resistance is an important factor affecting the efficacy of GEM in the treatment of advanced tumors [7, 14]. These results also indicate that chemotherapy resistance attenuates the therapeutic effect of GEM on GBC. Therefore, it is urgent to further elucidate the molecular mechanism of chemotherapy resistance in GBC to improve the effect of chemotherapy.
In recent years, a large number of studies have shown that autophagy plays two distinct roles in tumor chemotherapy resistance and chemotherapy sensitization. On the one hand, moderate autophagy can increase the resistance of tumor cells to chemotherapeutic drugs; on the other hand, excessive autophagy can inhibit the formation of drug resistance in tumor cells, thereby accelerating cell death [15]. These results provide a new idea for guiding reasonable clinical chemotherapy. Many studies have pointed out that autophagy can enhance the resistance of pancreatic cancer cells to GEM through various molecular mechanisms [16–22], and GEM-induced autophagy can reduce its proapoptotic effect on lung cancer cells [23] and breast cancer cells [24]. These studies show that GEM can cause cytoprotective autophagy in a variety of tumors, increase tumor chemotherapy resistance, and affect the efficacy of chemotherapy [25]. It is interesting to note that many studies have pointed out that the combined application of autophagy inhibitors such as chloroquine (CQ) or interference with autophagy gene expression can significantly increase the induction of tumor cell apoptosis by GEM [26–28]. Cell autophagy and its autophagy gene suppression or application of autophagy inhibitors may be expected to solve the problem of chemotherapy resistance of tumors. However, it has not been fully investigated whether GEM can cause protective autophagy of GBC cells, whether it is related to the resistance of GBC to GEM, and whether the combined use of autophagy inhibitor CQ can improve the sensitivity of GEM to GBC chemotherapy.
In the present study, we confirmed that GEM can induce apoptosis and protective autophagy in GBC cells, which may be related to the AKT/mTOR signaling pathway. At the same time, GEM in combination with autophagy inhibitor CQ can strengthen the cytotoxic effect of GEM on GBC in vitro and in vivo. These findings provide new ideas and evidence for the study of chemotherapy resistance of GBC and the improvement of its efficacy.

Materials and methods

Cell culture and reagents

Human GBC cell lines GBC-SD and NOZ were purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences, and SGC-996 was donated by Professor Yaoqin Yang from the Cancer Cell Institute of Tongji University School of Medicine. The GBC-SD and SGC-996 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, USA), while the NOZ was maintained in DMEM medium supplemented with 10% fetal bovine serum (Gibco, USA), at 37 °C in 5% C O2 (Shanghai Medical Instruments, China). GEM was obtained from Haosen Medicine Co. Ltd. (Jiangsu, China), dissolved in DMSO and diluted with RPMI-1640 or DMEM medium to the desired concentration for in vitro studies. CQ (Sigma, C6628, USA) was dissolved in PBS and diluted with RMPI-1640 or DMEM medium to achieve the desired concentration.

Cell viability assay

The cytotoxicity of GEM on human GBC cells was estimated using the CCK-8 assay (Cell Counting Kit-8, Dojindo, Japan) according to the manufacturer’s protocol. GBC-SD, SGC-996, and NOZ were plated in 96-well plates at 104 cells per well 24 h before the start of treatment. Then, cells were exposed to GEM at 0, 1, 5, 10, 20, 40 and 80 μM for 24, 48 and 72 h. At the test point, 100 μl Cell Counting Kit-8 (CCK8) solution was added into each well, and the plate was incubated at 37 °C for 2 h followed by OD detection using a spectrophotometer. Cell viability was calculated using the following formula: Cell viability (%) = (OD450 sampleOD450 blank)/(OD450 control-OD450 blank) × 100%.

Colony formation assay

For the colony formation assay, 500 cells were plated in 6-well plates and cultured with different treatment at 37 °C for 2 weeks. The number of colonies was visualized and quantified after staining with 0.1% crystal violet in methanol for 15 min. For each experimental condition, colonies were presented as the mean number ± SD from at least three independent experiments.

Apoptosis assay and cell cycle assay

An Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, USA) was used to detect the apoptosis of cells according to the manufacturer’s protocol. In brief, both attached cells and floating cells were harvested after treatment at indicated intervals, then were washed twice with cold PBS and suspended in 100 μL Binding Buffer. Cells were incubated with 5 μL Annexin V-FITC and 10 μL PI at room temperature for 15 min in the dark. Finally, an additional 400 μL of binding buffer was added to each tube. The apoptosis rate was analyzed by flow cytometry using the FACSCalibur system (BD Biosciences, USA).
Cell cycle distribution was further analyzed by flow cytometry. After treatment, cells were harvested and washed twice with cold PBS. Single-cell suspensions were fixed with 70% ice-cold ethanol at 4 °C overnight. Samples were then resuspended with cold PBS and incubated with PI staining solution for 30 min in the dark and finally analyzed by flow cytometer. The percentage of cells at each phase of the cell cycle was quantified using the CELL QUEST software (Becton–Dickinson).

Western blotting

After treatment with GEM and/or CQ, cells were harvested and lysed with RIPA buffer supplemented with PMSF. The total protein concentration was quantified by BCA kit (Beyotime Biotechnology, China). The protein was transferred to PVDF membrane (Millipore, USA) after separated by SDS/ PAGE (Beyotime Biotechnology, China), and blots were probed with anti-p62/SQSTM-1, anti-LC3, anti-PARP (all from Cell Signaling Technology), anti-Bcl-2, anti-Bax, antiβ-actin, anti-AKT, anti-t-AKT, anti-mTOR, anti-t-mTOR (all from Abcam) overnight at 4 °C. The goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated IgG (Beyotime Biotechnology, China) was used as secondary antibodies. Bands were visualized by an enhanced chemiluminescence reagent (Millipore, USA). The gray value and gray coefficient ratio of every band were analyzed and quantified using Image J analysis software (National Institutes of Health, USA).

In vivo studies

Male BALB/c nude mice (3–4 weeks old) were purchased from the Institute of Zoology, Chinese Academy of Sciences of Shanghai. All animal procedures were carried out in accordance with institutional guidelines after Tongji University School of Medicine Animal Care and Use Committee approved the study protocol. A suspension containing 2 × 106 SGC-996 cells in 200 μL PBS was subcutaneously injected into the right flanks of each nude mice. After 1 week, when the maximum diameter of the transplanted tumors reached approximately 5 mm, the mice were randomly divided into control (PBS only), CQ (CQ 60 mg/kg), GEM (GEM 20 mg/kg) and GEM + CQ (GEM 20 mg/kg + CQ 60 mg/kg) 4 groups, 4 mice in each group. Mice were administered intraperitoneally twice per week with these compounds. Tumor length and width were measured every 2 days, and the volume was calculated using the formula: volume = V = π/6 × (W2 × L). All mice were sacrificed after 22 days and tumors were removed and weighed.

Statistical analyses

SPSS 22.0 software (IBM Corp.) was used for statistical analysis. Data were expressed as x̄ ± SD. Student’s t test and one-way ANOVA were used for comparison among groups. Statistical significance was determined as P < 0.05.

Results

GEM inhibits the proliferation and colony formation of GBC cells in vitro

To determine the effect of GEM on the proliferation of GBC cells, a series of different concentrations of GEM (0, 1, 5, 10, 20, 40, and 80 μM) were used to treat GBC cell lines (GBC-SD, SGC-996, and NOZ) for 24 h, 48 h and 72 h. As shown in Supplementary Fig. 1, GEM significantly inhibited the in vitro proliferation of the above three GBC cell lines, and the activity of GBC cells decreased significantly with increasing drug concentration and prolonged action (Supplementary Fig. 1a), with the half inhibitory concentrations (IC50): 20.52 µM, 52.69 µM, 2.53 µM of GEM for GBC-SD, SGC-996, and NOZ for 72 h, respectively (Supplementary Fig. 1b). Plate clone formation assay was also performed to test for the contact-dependent proliferation of GBC cell lines. After GEM treatment, GBC-SD, SGC-996, and NOZ cell clone formation (cloning clusters) were significantly reduced, and the reduction of clone formation was more obvious as the drug concentration increased, showing a dose–response relationship (Supplementary Fig. 1c, d). Taken together, these data suggested that GEM inhibited the in vitro proliferation of GBC cells in a time- and dose-dependent manner.

GEM induces apoptosis and cycle arrest in GBC lines in vitro

Induction of apoptosis is the main cytotoxic pattern of many chemotherapy drugs. Studies have shown that GEM can induce apoptosis in a variety of tumor cells [29, 30]. In this experiment, flow cytometry was used to detect the apoptosisinducing effect of GEM on GBC cells. After 48 h of GEM treatment of GBC cells, compared with the control group, the apoptosis rate in the GEM (20 μM) group was significantly increased (GBC-SD: 21.94 ± 1.16% vs. 49.21 ± 2.14%, P < 0.01; SGC-996: 15.47 ± 0.87% vs. 23.86 ± 0.60%, P < 0.05; NOZ: 6.46 ± 0.29% vs. 11.46 ± 0.75%, P < 0.05; Supplementary Fig. 2a). Furthermore, western blotting was used to detect the effect of GEM on apoptosis-related proteins in GBC cells. Compared with the control group, the expressions of anti-apoptotic proteins Bcl-2 and PARP in the GBC-SD, SGC-996, and NOZ cells treated with GEM were significantly reduced, and the expressions of pro-apoptotic proteins BAX were significantly increased, which were related to the concentration of GEM, indicating that GEM induced GBC cells apoptosis in a dose-dependent manner (Supplementary Fig. 2b, c).
To further confirm the effect of GEM on the proliferation and apoptosis of GBC cells, this experiment also examined the effect of GEM on the cycle of GBC cells by flow cytometry. After GEM (20 μM) treatment of GBC cells for 48 h, compared with the control group, G0/G1 phase cells in GBC-SD and SGC-996 cells increased significantly (GBC-SD: 67.67 ± 1.45% vs. 83.33 ± 0.88%, P < 0.01; SGC-996: 69.33 ± 0.88% vs. 86.00 ± 1.16%, P < 0.01), S-phase cells were significantly reduced (GBCSD: 25.33 ± 0.88% vs. 16.67 ± 0.88%, P < 0.05; SGC-996: 23.00 ± 0.58% vs. 14.00 ± 1.16%, P < 0.05); G0/G1 phase cells in NOZ cells were significantly reduced (68.00 ± 1.00% vs. 48.00 ± 1.53%, P < 0.01), S-phase cells increased significantly (24.00 ± 0.58% vs. 45.33 ± 1.45%, P < 0.01; Supplementary Fig. 2d). These data revealed that GEM could effectively induce the cycle arrest of GBC cells.

GEM induces cell autophagy in GBC cell lines in vitro

Autophagy is involved in cell survival and cell death processes and can be induced by many cytotoxic compounds. Among them, the conversion of LC3-I to LC3-II is an important step in cell autophagy, and the expression level of LC3-II represents the overall level of autophagosome formation, so LC3-II is a good marker of autophagosome formation, and p62/SQSTM-1 is a known autophagydegrading scaffold protein. When cells undergo autophagy, LC3-II expression increases and p62/SQSTM-1 expression decreases [31]. To determine whether GEM induces autophagy of GBC cells, this experiment examined the effects of GEM on the expression of autophagosomeforming marker LC3-II and autophagy-degrading scaffold protein p62/SQSTM-1 in GBC cells. As the GEM concentration increased, the expression of LC3-II in GBC-SD, SGC-996, and NOZ cells increased significantly (LC3-II expression was positively correlated with GEM concentration), and p62/SQSTM-1 expression was significantly reduced (P62/SQSTM-1 expression is inversely related to GEM concentration) (Fig. 1a, b). Therefore, it was confirmed that GEM induced autophagy flux in GBC cells in vitro.

Autophagy inhibitor CQ synergistically enhances the inhibition of GEM on the proliferation and colony formation in GBC cells in vitro

Autophagy promotes cell survival and also induces cell death. To further observe the effect of GEM-induced autophagy on GBC cells, in this study we applied a late autophagy inhibitor CQ, which can prevent autophagy by blocking autophagosomal–lysosomal fusion events [32]. As shown in Fig. 4, when CQ concentration is 10–40 μM, its effect on cell viability is not obvious, but the concentration is greater than 40 μM, the cytotoxicity is significantly increased (Fig. 2a). The CQ working concentration is thus set to 10 μM, so as not to cause significant cell death. Moreover, because autophagy is a dynamic process, upregulation of autophagy and high-level inhibition of late autophagy can cause autophagy to accumulate, thereby increasing the expression level of autophagosome-forming marker LC3-II [33], the expression of LC3-II and p62/ SQSTM-1 proteins were, therefore, detected. It is shown that the expression of LC3-II and p62/SQSTM-1 in GBCSD, SGC-996, and NOZ cells in the GEM combined CQ group was significantly increased compared with GEM alone (Fig. 2b, c).
To observe the effect of autophagy inhibition in combination with GEM on the proliferation of GBC cells, CQ (10 μM) was then used to block the autophagy of GBC cells in this study. The results showed that the proliferation (GBC-SD: 36.34 ± 0.93% vs. 18.49 ± 1.61%, P < 0.01; SGC-996: 62.31 ± 1.84% vs. 52.49 ± 0.81%, P < 0.05; NOZ: 59.42 ± 3.41% vs. 47.91 ± 2.73%, P < 0.05; Fig. 3a) and clone formation (GBC-SD: 39.32 ± 3.17% vs. 4.13 ± 0.39%, P < 0.01; SGC-996: 36.56 ± 2.38% vs. 7.36 ± 1.45%, P < 0.01; NOZ: 66.37 ± 1.48% vs. 8.70 ± 0.89%, P < 0.01; Fig. 3b, c) of GBC cells in the GEM + CQ group were significantly inhibited compared with the single GEM group. These results revealed that GEM could induce protective autophagy of GBC cells, and inhibition of autophagy could significantly enhance the growth inhibitory effect of GEM on GBC cells.

Autophagy inhibitor CQ synergistically enhances the induction of apoptosis and cycle arrest of GBC cells by GEM in vitro

To further observe the effect of autophagy inhibition in combination with GEM on the growth and GEM-induced autophagy of GBC cells, the apoptosis of GBC cells was detected by flow cytometry and western blotting. As shown in Fig. 4, the apoptosis rates in the GEM + CQ group (GBCSD: 54.34 ± 1.12% vs. 42.15 ± 2.10%, P < 0.05; SGC996: 14.31 ± 0.87% vs. 12.49 ± 0.96%, P < 0.05; NOZ: 11.42 ± 1.35% vs. 9.91 ± 1.57%, P < 0.05; flow cytometry; Fig. 4a, b) were significantly increased when compared with the single GEM group. Moreover, the expression of anti-apoptotic protein Bcl-2 and PARP was significantly decreased and the expression of pro-apoptotic protein BAX was increased in the GEM + CQ group compared with the single GEM group (Fig. 4c, d). Furthermore, we analyzed the effect of GEM in combination with CQ on the cell cycle of GBC cells by flow cytometry. As expected that compared with the single GEM, the cell cycle arrest in the GEM + CQ group was obvious: the percentage of G1/G0 phase cells in the GBC-SD and SGC-996 increased significantly (GBC-SD: 82.83 ± 1.73% vs. 93.5 ± 1.45%, P < 0.05; SGC-996: 82.16 ± 1.86% vs. 92.5 ± 0.88%, P < 0.05), and the percentage of S-phase cells decreased significantly (GBC-SD: 17.21 ± 1.45% vs. 6.87 ± 1.45%, P < 0.05; SGC996: 16.87 ± 1.45% vs. 7.87 ± 0.88%, P < 0.05); in NOZ, the percentage of cells in the S phase of the GEM + CQ group increased significantly (42.32 ± 1.86% vs. 61.99 ± 1.53%, P < 0.05), and the percentage of cells in the G1/G0 phase decreased significantly (48.5 ± 1.20% vs. 29.84 ± 0.88%, P < 0.01) (Fig. 4e, f). The results above suggested that inhibition of autophagy effectively enhanced the apoptosisinducing effect of GEM on GBC cells and significantly postponed the cell cycle of GBC cells following GEM application.

AKT/mTOR signaling pathway is involved in GEM‑induced autophagy in GBC cells in vitro

mTOR signaling pathway is a key signaling pathway for cell metabolism, growth, proliferation and survival. Recent studies have shown that the mTOR signaling pathway is also closely related to cell autophagy. The upstream AKTtargeted mTOR signaling pathway can inhibit cell autophagy [34]. AKT/mTOR signaling pathway is autophagy and negative regulatory mechanisms of apoptosis [35, 36]. To explore the molecular mechanism of autophagy induced by GEM in GBC cells, we here detect the expression levels of AKT and mTOR in GBC cells: GBC-SD, SGC-996, NOZ cells after GEM treatment using western blotting. The results showed that as the GEM concentration increased, the expression of p-AKT and p-mTOR in GBC-SD, SGC-996, and NOZ cells decreased significantly, despite the unchanged total amount of AKT and mTOR proteins (Fig. 5a, b). And as mentioned before, GEM induced autophagy and apoptosis in GBC cells. Thus, we believed that GEM may induce autophagy and apoptosis in GBC cells through inhibiting the AKT/mTOR signaling pathway.

Tumor suppression induced by GEM is enhanced by CQ in vivo

Based on the results above, xenograft models were used to further confirm the synergistic inhibitory effect of GEM in combination with autophagy inhibitor CQ on tumor growth in vivo. After SGC-996 cells were injected subcutaneously into the right back, nude mice were randomly divided into 4 groups: PBS (blank control group), CQ, GEM and GEM + CQ groups, the growth of subcutaneous GBC xenografts in nude mice in each group was observed for 3 weeks. After 3 weeks, the tumor volumes in CQ group did not show a significant difference compared with the control group. The GEM + CQ group exerted greater antitumor effects in GBC xenograft models compared with the drugs independently. As expected, the tumor xenografts in GEM + CQ group were significantly smaller than those in the GEM group (207.9 ± 46.86 mm3 vs. synergistically enhanced the inhibitory effect of GEM on GBC cells. a After treatment with GEM in the presence or absence of 10 µM CQ for 48 h, the cell viability of GBC cells was measured by CCK-8 assay. Results were expressed as mean ± SD representing at least three independent experiments. b, c The colony formation assay was used to assess the proliferation capacity of GBC cells treated with GEM in the presence or absence of 10 µM CQ for 48 h. Values were given as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group 453.3 ± 70.1 mm3, P < 0.05; Fig. 6a, b). Consistent with the results of tumor volume, tumor weight could be suppressed by GEM monotherapy or CQ + GEM combination therapy. GEM in combination with CQ showed a more effective inhibition in GBC xenografts of nude mice (0.1667 ± 0.03333 g vs. 0.6 ± 0.05774 g, P < 0.05, Fig. 6c). Altogether, the data from in vitro and in vivo assays confirmed that inhibition of autophagy by CQ could significantly increase chemosensitivity to GEM for human GBC.

Discussion

Although the diagnosis and treatment technology of GBCs has made great progress in recent years, the overall survival rate and prognosis of the patients have not improved significantly. Chemotherapy is an alternative treatment for those patients who have lost their surgical opportunity or patients who have a recurrence after surgery. However, CQ synergistically enhanced GEM-induced apoptosis and cycle arrest in GBC cells. a, b GBC-SD, SGC-996, and NOZ cells treated by GEM (20 μM) implement with or without CQ (10 µM) for 48 h, the percentage of apoptotic cells was investigated using Annexin V-FITC and PI. (AnV +) (PI −) cells were considered early apoptotic and (AnV +) (PI +) cells were considered late apoptotic. The columns represent the mean ± SD of the three independent experiments. c, d GBC-SD, SGC-996, and NOZ cells were treated by GEM (20 μM) implement with or without CQ (10 µM) for 48 h, respectively. The PARP, Bcl-2, BAX, and β-actin expressions were detected by western blotting. Densitometry represents the expression of the proteins relative to β-actin. e, f Flow cytometric analysis of cell cycle progression in GBC cells treated by GEM (20 μM) with or without CQ (10 μM) for 48 h. The results were representative of three independent experiments. The percentages of cells in G1, S, and G2-M are shown as histograms. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. control group traditional chemotherapy cannot significantly prolong the survival time of GBC patients due to the insensitivity or resistance of chemotherapy. GEM is an anti-metabolic nucleoside analog that has been shown to have anticancer effects in various tumors [37–40]. GEM or combined with other anticancer drugs has achieved certain effects in clinical trials of GBC treatment [9]. However, the increased chemotherapy resistance attenuates the chemotherapy effect of GEM on GBCs. Therefore, an in-depth understanding of the effects and molecular mechanisms in vitro and in vivo that reduce the efficacy of GEM chemotherapy may play an important role in improving the prognosis of GBC patients.
Autophagy is a dynamic process that involves extensive degradation of cytoplasm, organelles, and proteins. Based on the function of cell recycling, autophagy plays a key role in the quality control of cell components and provides nutrients and materials for newly constructed structures in cells under metabolic stress [41]. Recent studies have shown that autophagy not only plays an important regulatory role in the occurrence and development of malignant tumors but also plays an important role in the response of tumor cells to anticancer therapies [42, 43], which may be related to the increased demand for tumor tissue metabolism and biosynthesis during chemotherapy. Studies indicate that autophagy is upregulated in hypoxic tumor areas, which is important for the survival of tumor cells in them [44]. Although the genetic background to autophagy dependence in cancer is still poorly understood, autophagy has been shown to protect tumor cells during the antitumor process of various drugs because it can reduce cell death caused by DNA damage [45, 46]. In pancreatic cancer, GEM induces protective autophagy in pancreatic cancer cells through the AMPK/ mTOR signaling pathway, which affects its chemotherapy effect [22]. In addition, in human non-small cell lung cancer cells, triple-negative MDA-MB-231 breast cancer cells, and urothelial cancer cells, GEM can induce autophagy to reduce cancer cell apoptosis [23, 24, 47]. In this study, we found that after GEM treatment of GBC cells, the expression of autophagy marker proteins LC3-II/LC3-I increased significantly, and p62/SQSTM-1 expression decreased, indicating that GEM can induce autophagy in GBC cells, which is consistent with reports that GEM can induce autophagy in tumor cells such as pancreatic cancer [22–24, 47].
Recent studies have shown that autophagy and apoptotic signaling pathways can interact with each other [48–51]. Inhibition of autophagy can enhance apoptosis in pancreatic cancer cells [29, 30], and autophagy promotes apoptosis of mesenchymal stem cells in the inflammatory microenvironment [52]. Therefore, understanding the interaction between apoptosis and autophagy may help to explore new treatment strategies for GBC. Studies on the relationship between autophagy and apoptosis of pancreatic cancer cells induced by GEM have shown that inhibition of autophagy can significantly enhance the pro-apoptotic effect of GEM on pancreatic cancer [29, 30]. Based on these basic studies, a clinical study showed that the autophagy inhibitor hydroxy chloroquine (HCQ) combined with GEM in the treatment of patients with pancreatic cancer is biosafe and tolerated, and the results are encouraging. The tumor marker CA19-9 has been reduced to a certain extent, and the PFS and OS of patients have improved to varying degrees. These results support a larger number of more randomized clinical trials to verify the efficacy of GEM combined with autophagy inhibitors in the treatment of pancreatic cancer [53]. In addition, in non-small cell lung cancer cells and bladder cancer cells, inhibition of autophagy can increase the sensitivity of cancer cells to GEM [27, 54]. In this study, GEM induced autophagy and apoptosis in GBC cells. As an advanced autophagy inhibitor, CQ not only effectively eliminates GEM-induced autophagy in GBC cells but also enhances the anticancer effects of GEM on GBC by inhibiting autophagy in vitro and in vivo, including inhibition of proliferation, induction of apoptosis and cell cycle arrest. These results indicate that the interaction between cell autophagy and apoptosis plays an important role in the resistance of GBC treatment, and this new regulatory mechanism is conducive to the antitumor effect of GEM in the treatment of GBC.
In recent years, many studies have pointed out that autophagy and apoptosis affect the chemotherapy effect of malignant tumors through various molecular pathways. It has been reported that GEM can induce autophagy and apoptosis in pancreatic cancer cells through the AMPK/ mTOR signaling pathway, and blocking this pathway can inhibit autophagy to enhance the apoptosis-inducing effect of GEM on pancreatic cancer cells [22]. Endoplasmic reticulum-stress-mediated autophagy is also thought to be associated with chemotherapy resistance of GEM for pancreatic cancer [28]. In bladder cancer, CYLD-Livin is also a target for improving the therapeutic effect of GEM by inhibiting autophagy. Another study reported that non-coding RNAs play an important role in the sensitivity of breast cancer cells to chemotherapy by regulating autophagy and apoptosis induced by GEM [55]. In addition, the AKT signaling pathway is one of the main signaling pathways that regulate tumor cells [56]; and there is increasing evidence that the overactive AKT/mTOR pathway can promote tumor cell proliferation and survival [57–59]. As a downstream of VEGFR, the AKT/mTOR signaling pathway is involved in apoptosis of VEGFR-targeted therapy [60]. mTOR is also mainly related to apoptosis and autophagy. GEM has been reported to have VEGF-C inhibitory effects [61], which can inhibit the migration and invasion of GBC. Therefore, the AKT/mTOR signaling pathway may be closely related to the growth inhibitory effect of GEM in GBC. Our results show that GEM upregulates cell autophagy marker proteins LC3-II/LC3-I and pro-apoptotic protein BAX, downregulates p62/SQSTM-1, anti-apoptotic proteins Bcl-2, PARP, and inhibits the expression of p-AKT and p-mTOR protein, indicating that GEM may induce autophagy and apoptosis of GBC cells by inhibiting the AKT/mTOR signaling pathway. Therefore, cell autophagy may become a new potential therapeutic target for adjuvant therapy of GBC and autophagy inhibitors such as CQ as an adjuvant or potential synergist of GEM can increase the anticancer effect and efficacy of GEM, which could be a new strategy for adjuvant treatment of GBC.
In conclusion, our results confirm that GEM induces apoptosis and autophagy in GBC cell lines, which may be related to the AKT/mTOR pathway; GEM in combination with CQ, a late autophagy inhibitor, can enhance the antitumor effect on GBC cell lines in vitro and in vivo. These findings suggest that GEM in combination with autophagy inhibitors may be a potential new strategy for adjuvant treatment of GBC.

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