Influence of Autophagy inhibition on 5-Fluorouracil-Induced Apoptosis of Hepatocellular Carcinoma and Its Mechanism_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

DAI Jingyou , YANG Shumeng ,  ZHANG Xinchen , WU Dequan , YANG Weiliang

Department of General Surgery，The Second Affiliated Hospital of Harbin Medical University， Harbin 150086，Heilongjiang Province，China;

Corresponding author：

ZHANG Xinchen, Email: marschenxin@hotmail.com

Keywords：

Hepatocellular carcinoma; Autophagy; 5-fluorouracil; 3-methyladenine

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Abstract Full text Figures/Tables Video References Cited by

Objective 　To investigate the effects and mechanism of 3-methyladenine （3-MA， an autophagy inhib-itor） on the apoptosis of hepatocellular carcinoma cell line SMMC7721 induced by 5-fluorouracil （5-FU）.
Methods 　The autophagy was observed using fluorescent microscope by monodansyicadaverin （MDC） staining. The viability of SMMC-7721 cell induced by 5-FU was measured using CCK8 assay before and after autophagy inhibited by 3-MA， meanwhile the apoptosis of SMMC7721 cell was determined via AnnexinⅤ/PI assay. The light chain 3 protein （LC3， the autophagy
specific protein） and caspase-3， PARP protein were detected by Western blot.
Results 　The autophagy of SMMC7721 cell could be induced by 5-FU after treatment for 48 h， the cell survival rate was （60.73±2.65）%， and the apoptosis rate was （40.42±2.34）%. Compared with the group of 5-FU treatment， the survival rate of SMMC7721 cell in the combination of 5-FU and 3-MA after treatment for 48 h decreased to （42.31±1.32）% （P＜0.01）， and the apoptosis rate increased to （60.92±2.99）% （P＜0.01）， meanwhile the expressions of LC3-Ⅱ， activation fragment of caspase-3， and cleavage frag-ment of PARP significantly increased （P＜0.01）.
Conclusions 　Autophagy is a protective phenomenon during the SMMC7721 cell line apoptosis induced by 5-FU， and autophagy inhibition may enhance the sensitivity of SMMC7721 cell line to 5-FU treatment， which is probably associated with the activation of caspase-3 and cleavage of PARP. Therefore， autophagy inhibition could be a promising strategy for adjuvant chemotherapy in hepatocellular carcinoma.

Citation： DAI Jingyou,YANG Shumeng,ZHANG Xinchen,WU Dequan,YANG Weiliang,.. Influence of Autophagy inhibition on 5-Fluorouracil-Induced Apoptosis of Hepatocellular Carcinoma and Its Mechanism. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2013, 20(8): 872-877. doi: Copy

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1. Nishikawa T， Tsuno NH， Okaji Y， et al. Inhibition of autophagy potentiates sulforaphane-induced apoptosis in human colon cancer cells[J]. Ann Surg Oncol， 2010， 17(2)：592-602.
2. Li J， Hou N， Faried A， et al. Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model[J]. Eur J Cancer， 2010， 46(10)：1900-1909.
3. Kanematsu S， Uehara N， Miki H， et al. Autophagy inhibition enhances sulforaphane-induced apoptosis in human breast cancer cells[J]. Anticancer Res， 2010， 30(9)：3381-3390.
4. Liu D， Yang Y， Liu Q， et al. Inhibition of autophagy by 3-MA potentiates cisplatin-induced apoptosis in esophageal squamous cell carcinoma cells[J]. Med Oncol， 2011， 28(1)：105-111.
5. Shimizu S， Takehara T， Hikita H， et al. Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma[J]. Int J Cancer， 2012， 131(3)：548-557.
6. Hsieh MJ， Yang SF， Hsieh YS， et al. Autophagy inhibition enhances apoptosis induced by dioscin in huh7 cells[J]. Evid Based Complement Alternat Med， 2012， 2012：134512.
7. Clark HP， Carson WF， Kavanagh PV， et al. Staging and current treatment of hepatocellular carcinoma[J]. Radiographics， 2005， 25 Suppl 1：S3-S23.
8. Chen S， Rehman SK， Zhang W， et al. Autophagy is a therapeutictarget in anticancer drug resistance[J]. Biochim Biophys Acta， 2010， 1806(2)：220-229.
9. Hu YL， Jahangiri A， Delay M， et al. Tumor cell autophagy as an adaptive response mediating resistance to treatments such as antiangiogenic therapy[J]. Cancer Res， 2012， 72(17)：4294-4299.
10. Lin JF， Tsai TF， Liao PC， et al. Benzyl isothiocyanate induces protective autophagy in human prostate cancer cells via inhibition of mTOR signaling[J]. Carcinogenesis， 2013， 34(2)：406-414.
11. Tu YJ， Fan X， Yang X， et al. Evodiamine activates autophagy as a cytoprotective response in murine Lewis lung carcinoma cells[J]. Oncol Rep， 2013， 29(2)：481-490.
12. Bareford MD， Hamed HA， Tang Y， et al. Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells[J]. Autophagy， 2011， 7(10)：1261-1262.
13. Chiu HW， Chen YA， Ho SY， et al. Arsenic trioxide enhances the radiation sensitivity of androgen-dependent and -independent humanprostate cancer cells[J]. PLoS One， 2012， 7(2)：e31579.
14. Singh BN， Kumar D， Shankar S， et al. Rottlerin induces autop-hagy which leads to apoptotic cell death through inhibition of PI3K/Akt/mTOR pathway in human pancreatic cancer stem cells[J]. Biochem Pharmacol， 2012， 84(9)：1154-1163.
15. Mizushima N. Methods for monitoring autophagy[J]. Int J Biochem Cell Biol， 2004， 36(12)：2491-2502.
16. Kabeya Y， Mizushima N， Ueno T， et al. LC3， a mammalian homologue of yeast Apg8p， is localized in autophagosome membranes after processing[J]. EMBO J， 2000， 19(21)：5720-5728.
17. Klionsky DJ， Abeliovich H， Agostinis P， et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes[J]. Autophagy， 2008， 4(2)：151-175.
18. Kumar S. Caspase function in programmed cell death[J]. Cell Death Differ， 2007， 14(1)：32-43.
19. Murcia JM， Niedergang C， Trucco C， et al. Requirement of poly (ADP-ribose) polymerase in recovery from DNA damage in mice and in cells[J]. Proc Natl Acad Sci USA， 1997， 94(14)：7303-7307.
20. Javle M， Curtin NJ. The role of PARP in DNA repair and its thera-peutic exploitation[J]. Br J Cancer， 2011， 105(8)：1114-1122.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Influence of Autophagy inhibition on 5-Fluorouracil-Induced Apoptosis of Hepatocellular Carcinoma and Its Mechanism

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