Induction Of Autophagic Flux In Oxaliplatin Treated Human Hepatocarcinoma HepG2Cell Line Arid Its Role In Cell Survival

Backgroud: Hepatocellular carcinoma （hepatocellular carcinoma, HCC） is the one ofthe most common and malignant tumor in the world. The incidence and mortality ofHCC rank the third and the second separately in China. Live resection is the most usefuland effective method in the treatment of HCC. Unfortunately, nearly80%of patients areat advanced stage and have lost the best opportunity of surgery when they arediagnosed.Chemotherapy is still the important treatment of HCC, especially inadvanced stage.HCC treatment of cytotoxic drugs, oxaliplatin （Oxa） is one of the few drugs that have apotential advantage admired. Now, there are many oxaliplatin combination therapies areused for the treatment of primary liver cancer, and achieved good results. But whetheroxaliplatin can introduce autophagy and the process is through what pathway inadvanced liver cancer treatment are rarely reported.The endoplasmic reticulum （ER） is an important eukaryotic organelle, the conditionwhich is caused by the aggregation of misfolded or unfolded protein and the state of Ca²⁺balance disorders is called ER Stress（ERS）. When the stimulation intensity is largeenough, ERS will cause cell death, recent studies have shown that the ERS can mediateanother programmed cell death way which is named autophagy except apoptosis.Autophagy is the process which can clear cytoplasm substances including long-livedproteins and damaged organelles by lysosomal. Autophagy which plays an extremelyimportant and complex role in tumor development has become the research focus oftargeted cancer therapy. In present research, it is explored whether oxa can induce autophagy through ERS pathway and affect the chemosensitivity of liver cancer cells invitro. Hoping to find a new strategy in the chemotherapy of HCC.Objectives: To investigate the autophagy which is caused by oxa and the effect onvitality of human hepatoma cell line HepG2and investigate the possible mechanisms invitro.Methods:1. Cell viability was detected by cell counting kit-8（CCK-8） assay;2. Thedistribution and abundance of autophagic vacuoles were detected by FITC-LC3-Ⅱ;3.Autophagosomes were observed directly under transmission electron microscopy（TEM）.4. The expression of the marker protein of ERS, apoptin caspase3andautophagic LC3conversion （from LC3-Ⅰ t o LC3-Ⅱ） were detected by Western-blotassay.Results:1. CCK-8demonstrated that Oxa could dose-dependently induce HepG2cellsdeath, and when was given in combination with3-methyladenine（3-MA） orchloroquine（CQ）, Oxa could significantly inhibit the growth of HepG2cells;2. Theresults of FITC-LC3-Ⅱa ntibody which was used to label LC3-Ⅱ clearly shown thatthere were a lot of green fluorescence dots namely autophagic vacuoles whichscattered in nearby the cell membrane after treated by Oxa, and the number issignificantly more than the control group;3. Formation of autophagosomes in HepG2cells was demonstrated under TEM after Oxa treatment;4. The results of western-blotshown the expression of LC3-Ⅱi ncreased markedly after inhibiting autophagicdegradation pathway by CQ, which reflected an enhancement of autophagic fluxinduced by Oxa and3-MA could inhibit the formation of autophagosomes while theexpression of proteins of GRP78and caspase3. Conclusion: Oxa can enhance autophagic function in HepG2cells maybe through theway of ERS while an increase of autophagosome formation; different targets ofautophagy inhibitor3-MA or CQ can facilitate proliferation inhibition of Oxa, so it canincrease the antitumor effect of Oxa.