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
References
Hsing AW, Sakoda LC, Rashid A, Chen J, Shen MC, Han TQ, Wang BS, Gao YT (2008) Body size and the risk of biliary tract cancer: a population-based study in China. Br J Cancer 99(5):811–815
Hsing AW, Bai Y, Andreotti G, Rashid A, Deng J, Chen JB, Goldstein AM, Han TQ, Shen MC, Fraumeni JF et al (2007) Family history of gallstones and the risk of biliary tract cancer and gallstones: a population-based study in Shanghai, China. Int J Cancer 121(4):832–838
Wernberg JA, Lucarelli DD (2014) Gallbladder cancer. Surg Clin N Am 94(2):343–360
Zheng YH, Xu DY, Bu ZD (2016) Chinese version of NCCN clinical practice guidelines in oncology officially authorized by NCCN. Chin J Cancer Res 28(1):144–145
Pedersen KS, Kim GP, Foster NR, Wang-Gillam A, Erlichman C, McWilliams RR (2015) Phase II trial of gemcitabine and tanespimycin (17AAG) in metastatic pancreatic cancer: a Mayo Clinic Phase II Consortium study. Investig New Drugs 33(4):963–968
Chen YM, Perng RP, Tsai CM, Whang-Peng J (2006) A phase II trial of gemcitabine plus UFUR combination chemotherapy in non-small-cell lung cancer patients failing previous chemotherapy. Lung Cancer 52(3):333–338
Sun L, Lu J, Niu Z, Ding K, Bi D, Liu S, Li J, Wu F, Zhang H, Zhao Z, Ding S (2015) A potent chemotherapeutic strategy with Eg5 inhibitor against gemcitabine resistant bladder cancer. PLoS One 10(12):e0144484
Hidalgo M (2010) Pancreatic cancer. N Engl J Med 362(17):1605–1617
Valle J (2010) Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer (vol 362, pg 1273, 2010). New Engl J Med 363(2):198
Qu K, Liu SN, Chang HL, Liu C, Xu XS, Wang RT, Zhou L, Tian F, Wei JC, Tai MH et al (2012) Gallbladder cancer: a subtype of biliary tract cancer which is a current challenge in China. Asian Pac J Cancer Prev 13(4):1317–1320
Hirooka Y, Ishikawa T, Kawashima H, Ohno E, Nonogaki K, Kanamori A, Hirai T, Uchida H, Shirai O, Ishikawa H et al (2017) Prospective multicenter phase II study of gemcitabine plus cisplatin in patients with unresectable gallbladder cancer. Cancer Chemother Pharmacol 80(1):119–125
Iyer Renuka V, Pokuri Venkata K, Adrienne Groman et al (2018) A multicenter phase II study of gemcitabine, capecitabine, and bevacizumab for locally advanced or metastatic biliary tract cancer. Am J Clin Oncol 41:649–655
Moehler M, Maderer A, Schimanski C et al (2014) Gemcitabine plus sorafenib versus gemcitabine alone in advanced biliary tract cancer: a double-blind placebo-controlled multicentre phase II AIO study with biomarker and serum programme. Eur J Cancer 50:3125–3135
Siebenhüner Alexander R, Heike Seifert, Helga Bachmann et al (2018) Adjuvant treatment of resectable biliary tract cancer with cisplatin plus gemcitabine: a prospective single center phase II study. BMC Cancer 18:72
White E (2015) The role for autophagy in cancer. J Clin Investig 125(1):42–46
Zhang XF, Zhao P, Wang CH, Xin BR (2019) SNHG14 enhances gemcitabine resistance by sponging miR-101 to stimulate cell autophagy in pancreatic cancer. Biochem Biophys Res Commun 510(4):508–514
Xiong JJ, Wang D, Wei AL, Ke NW, Wang YC, Tang J, He SR, Hu WM, Liu XB (2017) MicroRNA-410-3p attenuates gemcitabine resistance in pancreatic ductal adenocarcinoma by inhibiting HMGB1-mediated autophagy. Oncotarget 8(64):107500–107512
Li CG, Zhao ZM, Zhou ZP, Liu R (2016) Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis. Cancer Chemother Pharmacol 78(6):1199–1207
Song B, Bian Q, Zhang YJ, Shao CH, Li G, Liu AA, Jing W, Liu R, Zhou YQ, Jin G et al (2015) Downregulation of ASPP2 in pancreatic cancer cells contributes to increased resistance to gemcitabine through autophagy activation. Mol Cancer 14:177
Ma T, Chen W, Zhi X, Liu H, Zhou Y, Chen BW, Hu LQ, Shen J, Zheng XX, Zhang SF et al (2018) USP9X inhibition improves gemcitabine sensitivity in pancreatic cancer by inhibiting autophagy. Cancer Lett 436:129–138
Kaur S, Bafna S, Rachagani S, Ganti AK, Batra SK (2012) Mucin MUC4 resists gemcitabine-induced pancreatic cancer cell death by promoting autophagy. Pancreas 41(8):1374–1375
Zhu JH, Chen Y, Ji Y, Yu YQ, Jin Y, Zhang XX, Zhou JL (2018) Gemcitabine induces apoptosis and autophagy via the AMPK/mTOR signaling pathway in pancreatic cancer cells. Biotechnol Appl Biochem 65(5):665–671
Wu HM, Shao LJ, Jiang ZF, Liu RY (2016) Gemcitabine-induced autophagy protects human lung cancer cells from apoptotic death. Lung 194(6):959–966
Chen M, He MY, Song YJ, Chen LQ, Xiao P, Wan XP, Dai F, Shen P (2014) The cytoprotective role of gemcitabine-induced autophagy associated with apoptosis inhibition in triple-negative MDA-MB-231 breast cancer cells. Int J Mol Med 34(1):276–282
Sui X, Chen R, Wang Z et al (2013) Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis 4:e828
Donohue E, Thomas A, Maurer N, Manisali I, Zeisser-Labouebe M, Zisman N, Anderson HJ, Ng SSW, Webb M, Bally M et al (2013) The autophagy inhibitor verteporfin moderately enhances the antitumor activity of gemcitabine in a pancreatic ductal adenocarcinoma model. J Cancer 4(7):585–596
Yin L, Liu S, Li CS, Ding ST, Bi DB, Niu ZH, Han LP, Li WJ, Gao DX, Liu Z et al (2016) CYLD downregulates Livin and synergistically improves gemcitabine chemosensitivity and decreases migratory/invasive potential in bladder cancer: the effect is autophagy-associated. Tumor Biol 37(9):12731–12742
Fu Z, Cheng X, Kuang J, Feng H, Chen L, Liang J, Shen X, Yuen S, Peng C, Shen B et al (2018) CQ sensitizes human pancreatic cancer cells to gemcitabine through the lysosomal apoptotic pathway via reactive oxygen species. Mol Oncol 12(4):529–544
Papademetrio DL, Cavaliere V, Simunovich T, Costantino S, Campos MD, Lombardo T, Kaiser CMF, Alvarez E (2014) Interplay between autophagy and apoptosis in pancreatic tumors in response to gemcitabine. Target Oncol 9(2):123–134
Li XS, Yan J, Wang LS, Xiao FJ, Yang YF, Guo XZ, Wang H (2013) Beclin1 inhibition promotes autophagy and decreases gemcitabine-induced apoptosis in Miapaca2 pancreatic cancer cells. Cancer Cell Int 13:26
Bjorkoy G, Lamark T, Pankiv S, Overvatn A, Brech A, Johansen T (2009) Monitoring autophagic degradation of P62/Sqstm1. Methods Enzymol Autophagy Mamm Syst 452(Pt B):181–197
Hasanain M, Bhattacharjee A, Pandey P, Ashraf R, Singh N, Sharma S, Vishwakarma AL, Datta D, Mitra K, Sarkar J (2015) Alpha-solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway. Cell Death Dis 6:e1860
Tanida I (2011) Autophagosome formation and molecular mechanism of autophagy. Antioxid Redox Signal 14(11):2201–2214
Saxton RA, Sabatini DM (2017) mTOR signaling in growth, metabolism, and disease (vol 168, pg 960, 2017). Cell 169(2):362
Nicholson KM, Anderson NG (2012) The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal 14:381–395
Majumder PK, Sellers WR (2005) Akt-regulated pathways in prostate cancer. Oncogene 24:7465–7474
Toyota Y, Iwama H, Kato K, Tani J, Katsura A, Miyata M, Fujiwara S, Fujita K, Sakamoto T, Fujimori T et al (2015) Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth. Int J Oncol 47(4):1293–1302
Kilani R, Tamimi Y, Karmali S, Mackey J, Hanel E, Wong KK, Moore RB (2002) Selective cytotoxicity of gemcitabine in bladder cancer cell lines. Anticancer Drugs 13(6):557–566
Kang YW, Lee JE, Jung KH, Son MK, Shin SM, Kim SJ, Fang ZH, Yan HH, Park JH, Han B et al (2018) KRAS targeting antibody synergizes anti-cancer activity of gemcitabine against pancreatic cancer. Cancer Lett 438:174–186
Teng JP, Yang ZY, Zhu YM, Ni D, Zhu ZJ, Li XQ (2018) Gemcitabine and cisplatin for treatment of lung cancer in vitro and vivo. Eur Rev Med Pharmacol Sci 22(12):3819–3825
Tsuchihara K, Fujii S, Esumi H (2009) Autophagy and cancer: dynamism of the metabolism of tumor cells and tissues. Cancer Lett 278(2):130–138
Du HL, Yang WP, Chen L, Shi MM, Seewoo V, Wang JY, Lin AD, Liu ZR, Qiu WH (2012) Role of autophagy in resistance to oxaliplatin in hepatocellular carcinoma cells. Oncol Rep 27(1):143–150
Pardo R, Lo Re A, Archange C, Ropolo A, Papademetrio DL, Gonzalez CD, Alvarez EM, Iovanna JL, Vaccaro MI (2010) Gemcitabine induces the VMP1-mediated autophagy pathway to promote apoptotic death in human pancreatic cancer cells. Pancreatology 10(1):19–26
Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen GH, Mukherjee C, Shi YF, Gelinas C, Fan YJ et al (2006) Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 10(1):51–64
Abedin MJ, Wang D, McDonnell MA, Lehmann U, Kelekar A (2007) Autophagy delays apoptotic death in breast cancer cells following DNA damage. Cell Death Differ 14(3):500–510
Mani J, Vallo S, Rakel S, Antonietti P, Gessler F, Blaheta R, Bartsch G, Michaelis M, Cinatl J, Haferkamp A et al (2015) Chemoresistance is associated with increased cytoprotective autophagy and diminished apoptosis in bladder cancer cells treated with the BH3 mimetic (−)-Gossypol (AT-101). BMC Cancer 15:224
Ojha R, Jha V, Singh SK (2016) Gemcitabine and mitomycin induced autophagy regulates cancer stem cell pool in urothelial carcinoma cells. Biochim Biophys Acta-Mol Cell Res 1863(2):347–359
Tavassoly I, Parmar J, Shajahan-Haq AN, Clarke R, Baumann WT, Tyson JJ (2015) Dynamic modeling of the interaction between autophagy and apoptosis in mammalian cells. Cpt-Pharmacomet Syst Pharmacol 4(4):263–272
Mukhopadhyay S, Panda PK, Sinha N, Das DN, Bhutia SK (2014) Autophagy and apoptosis: where do they meet? Apoptosis 19(4):555–566
Moretti L, Cha YI, Niermann KJ, Lu B (2007) Switch between apoptosis and autophagy: radiation-induced endoplasmic reticulum stress? Cell Cycle 6(7):793–798
Jiang Q, Li F, Shi K, Wu P, An J, Yang Y, Xu C (2014) Involvement of p38 in signal switching from autophagy to apoptosis via the PERK/eIF2 alpha/ATF4 axis in selenite-treated NB4 cells. Cell Death Dis 5:e1270
Dang SP, Yu ZM, Zhang CY, Zheng J, Li KL, Wu Y, Qian LL, Yang ZY, Li XR, Zhang YY et al (2015) Autophagy promotes apoptosis of mesenchymal stem cells under inflammatory microenvironment. Stem Cell Res Ther 6:247
Boone BA, Bahary N, Zureikat AH, Moser AJ, Normolle DP, Wu WC, Singhi AD, Bao P, Bartlett DL, Liotta LA et al (2015) Safety and biologic response of pre-operative autophagy inhibition in combination with gemcitabine in patients with pancreatic adenocarcinoma. Ann Surg Oncol 22(13):4402–4410
Jiang YF, Han ZD, Wang Y, Hao WB (2018) Depletion of SIRT7 sensitizes human non-small cell lung cancer cells to gemcitabine therapy by inhibiting autophagy. Biochem Biophys Res Commun 506(1):266–271
Chen Y-M, Liu Y, Wei H-Y, Lv K-Z, Fu P-F (2016) Large intergenic non-coding RNA-ROR reverses gemcitabine-induced autophagy and apoptosis in breast cancer cells. Oncotarget 7(37):59604–59617
Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411(6835):355–365
Pene F, Claessens YE, Muller O, Viguie F, Mayeux P, Dreyfus F, Lacombe C, Bouscary D (2002) Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma. Oncogene 21(43):6587–6597
LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA (2008) Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updat 11(1–2):32–50
Hu HZ, Yang YB, Xu XD, Shen HW, Shu YM, Ren Z, Li XM, Shen HM, Zeng HT (2007) Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma HeLa cell line. Acta Pharmacol Sin 28(11):1819–1826
Liu Y, Chen ZJ, Cheng HX, Chen J, Qian J (2017) Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling. Oncotarget 8(1):979–987
Yang MH, Lee KT, Yang S, Lee JK, Lee KH, Moon IH, Rhee JC (2012) Guggulsterone enhances antitumor activity of gemcitabine in gallbladder cancer cells through suppression of NF-kappa B. J Cancer Res Clin Oncol 138(10):1743–1751
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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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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DOI: https://doi.org/10.1007/s00280-020-04100-5