| Hepatocellular carcinoma(HCC)is the third cause of death in patients with malignant tumors in the world,is the major diseases which cause serious damage to human health.Surgery is the most effective and exact treatment,but as a result of liver cancer onset hidden and malignant degree is high,the majority of patients that have been found in the late andloss surgery opportunity,therefore,chemotherapy is still the principal treatmentmeans of advanced liver cancer,but the occurrence of multidrug resistance of liver cancer often lead to treatment failure.Some reports showed that malignant tumors,including hepatocellular carcinoma,express the high level of Bcl-2,Bcl-XL and other anti-apoptotic proteins,promotes apoptosis escape of tumor.Therefore,the inhibition of Bcl-2 will be an effective method of tumor therapy.At present,ABT-737 is a B-cell lymphoma 2 homology 3mimetic,which specifically inhibits the binding of Bcl-2 / Bcl-XL and the BH3 domain of Bax / Bak and induces cells apoptosis.The latest research showed that autophagy promotes cell repair and survival through degradation of damaged organelles and intracellular proteins.Thus,autophagy protectstumor cell from apoptosis and necrosis,and becomes an important mechanism of cancer resistance.At present,Bcl-2 inhibitor ABT-737 was used for the treatment of hepatocellular carcinoma in the clinic.However,ABT-737 can induceautophagy,and leads to ABT-737 resistance in several tumors.This is an important bottleneck treatment of advanced hematopoietic cancers.Therefore,in this study,human hepatoma cell line Hep G-2 and adriamycin-resistant cell line Hep G-2 / ADM were used to investigate the effect of Bcl-2 inhibitor ABT-737 induced-autophagy in the apoptosis of Adriamycin-resistant humanhepatocellular carcinomacell line Hep G-2 /ADMcells.Hoping to provide a new treatment target and strategy for the treatment of hematopoietic cancers.Method:(1)Western blot was used to detect the expression of Bcl-2 and Bax proteins in human hepatoma cell line Hep G-2 and adriamycinresistant cell line Hep G-2 / ADM;(2)MTT assay was used to detect the effect of different doses and time of ABT-737 on the survival rate ofadriamycin-resistant cell line Hep G-2 / ADM;(3)Western blot was used to detect the expression of Bcl-2,Bax,Caspase-3 and Cleaved Caspase-3 proteins in adriamycin-resistant cell line Hep G-2 / ADM treated with different doses and time of ABT-737;Western blot was used to detect the expression of autophagy-related proteins Beclin1 and LC3 proteins in adriamycin-resistant cell line Hep G-2 / ADM treated with different doses and time of ABT-737;(4)After knockdown of Beclin1,detecting the expression of the autophagy-associated protein LC3 by Western blotting(5)Laser confocal microscopy was used to observe dot aggregation of the autophagy-related protein LC3;(6)The changes of intracellular ROS were observed by fluorescence microscopy using DCFH-DA fluorescent probe;(7)The apoptotic rate was detected by flow cytometry using Annexin V / PI staining;(8)Apoptosis was detected by TUNEL assay.Results:(1)Compared with Hep G-2 cells,Hep G-2 / ADM cells showed higher level of Bcl-2 protein,indicating that the high expression of Bcl-2protein may be the cause of drug resistance of Hep G-2cells.(2)After Hep G-2 / ADM cells were treated with different doses of ABT-737 for 24 h,gradually increasing ABT-737 obviously decreased the survival rate of Hep G-2 / ADM cells,augmented the apoptosis rate,meanwhile,decreased the expression of anti-apoptotic protein Bcl-2,and increased the expression of pro-apoptotic protein Bax.The ratio of Bax / Bcl-2and the expression of Cleaved Caspase-3were significantly increased.Thus indicated that ABT-737 could induce the apoptosis of adriamycin-resistant cell line Hep G-2 / ADM.(3)As the increasing dose and time of ABT-737,the expression of Beclin1 and the ratio of LC3-II / LC3-I were significantly increased,and the aggregation of LC3 II obviously increased,indicating that ABT-737 could induced time and dose-dependent autophagy of adriamycin-resistant cell line Hep G-2 / ADM.After knockdown of Beclin1,the ratio of LC3-II / LC3 I in ABT737 group was significantly decreased,indicating that ABT737 induced Beclin1-dependent.autophagyin Hep G-2 / ADM.(4)With the increasing dose of ABT737 in Hep G-2 / ADM cells,the level of ROS was obviously increased,accompanied by autophagy.NAC,ROS inhibitor,significantly decreased the ratio of LC3-II / LC3-I and dot aggregation of LC3 II,indicating thatABT-737 induced-autophagy in Hep G-2 / ADM cells was also dependent on ROS.(5)Further experiments showed that NAC obviously decreased the survival rate,increased the apoptosis rate of Hep G-2 / ADM cells,and markedly increased the expression of Cleaved Caspase-3,indicating that NAC could enhance ABT 737-induced apoptosis.(6)After 3-MA inhibiting autophagy,cell survival rate of Hep G-2 /ADM more was significantly decreased.And the expression of Cleaved Caspase3 was more significantly increased.Meanwhile,the ratio of LC3-II / LC3-I was significantly decreased,and the expression of Beclin1 was significantly reduced,indicating that inhibition of autophagy can increase the sensitivity of Hep G-2 /ADM cellsto apoptosis induced by ABT-737.Conclusion:Compared with Hep G-2 cells,Hep G-2 / ADM cells showed higher level of Bcl-2,indicating that Bcl-2 protein may be a cause of adriamycin resistance of Hep G-2 cells.ABT-737 can induce autophagy in adriamycinresistant cell line Hep G-2 / ADM through time and dose-dependent manner.While inhibiting the expression of Beclin1 or the production of ROS,it can enhance the sensitivity of Hep G-2 / ADM cellsto apoptosis induced by ABT-737 through inhibiting autophagy. |
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