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

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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.

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Abbreviations

GBC:

Gallbladder cancer

GEM:

Gemcitabine

OS:

Overall survival

PFS:

Progression-free survival

CQ:

Chloroquine

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Funding

This study was supported by funds from the National Nature Science Foundation of China (Grant nos. 30672073, 81072004 and 81372614) and the Shanghai Science and Technology Commission Research Project (Grant nos. 19411966300 and 19140902302).

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FTW, HW, QWW, WS and YZF conceived and designed the experiments. FTW, HW and QWW performed the experiments. FTW analyzed the data. HW, QWW, MSP, XPL and WS contributed reagents/materials/analysis tools. FTW and YZF wrote the paper. All authors have read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is appropriately investigated and resolved.

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Correspondence to Wei Sun or Yue-Zu Fan.

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Supplementary Fig.

 1 GEM inhibited the proliferation of GBC cells. (a) GBC cells were cultured in a series of concentrations of GEM for 24, 48 and 72 h. Cell viability was assessed by CCK-8 assay. (b) The IC50 of GEM treated in GBC-SD, SGC-996 and NOZ for 72 h. (c, d) Representative images of GBC-SD, SGC-996, and NOZ contact-dependent clone formation. Each experiment is representative of three independent experiments. *P < 0.05, **P < 0.01, vs. GEM (0 µM). (TIFF 10537 kb)

Supplementary Fig.

 2 GEM induced apoptosis and cycle arrest in GBC cell lines. (a) GBC-SD, SGC-996, and NOZ cells were treated with the indicated concentrations of GEM for 48 h. The ratio of apoptotic cells was measured by Annexin V-FITC and propidium iodide (PI) staining. The results were representative of three independent experiments. (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. (b) GBC-SD, SGC-996, and NOZ cells were treated with a series of concentrations of GEM for 48 h. The PARP, Bcl-2, BAX, and β-actin expressions were detected by western blotting. (c) Quantification of the relative gray value of bands compared with β-actin, as detected by Fig. 2B. (d) Flow cytometric analysis of cell cycle progression in GBC cells treated by GEM. Detected cell cycle phase distribution in GBC-SD, SGC-996, and NOZ cells treated with GEM (20 μM) for 48 h by flow cytometry. 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 < 0.0001, vs. control group. (TIFF 16134 kb)

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Wang, FT., Wang, H., Wang, QW. et al. Inhibition of autophagy by chloroquine enhances the antitumor activity of gemcitabine for gallbladder cancer. Cancer Chemother Pharmacol 86, 221–232 (2020). https://doi.org/10.1007/s00280-020-04100-5

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