Objective To investigate the effect of nutritional deficiency on autophagic flux in human hepatoblastoma HepG2 cells and the autophagy-dependent molecular pathway mediated by ATG5 against nutritional stress.Methods Lentiviral siRNA knockdown of ATG5 was performed to knock down the expression in HepG2 cells,and the transfection was verified by Western blotting and qRT-PCR.HBSS was used to treat HepG2 cells for 0,1,2,3,4,5,6 and 7 h for starvation-induced autophagy.Monodansylcadaverine(MDC)fluorescence staining was employed to detect the formation of intracellular autophagic vesicles.Western blotting was performed to measure the expression changes in microtubule-associated protein 1A/1B-light chain 3(LC3),autophagy receptor protein(sequestosome 1,P62),autophagy-related gene 5(ATG5)and nutrient stress pathway(AMPK/mTOR pathway)related proteins.CCK-8 assay was utilized to test cell proliferation.Results Compared with the HepG2 cells under conventional culture medium,starvation induction resulted in more autophagic vacuoles(accumulation of autofluorescent marker,MDC),which peaked at 6 h of starvation(P<0.000 1),up-regulated protein levels of LC3B-Ⅱ and ATG5(P<0.05),decreased protein expression of P62(P<0.05),gradual activation of the AMPK/mTOR pathway with elapse of starvation time,and increase in the intensity of autophagic flux.However,in the HepG2 cells with ATG5 knockdown,starvation treatment led to decreased ATG5 expression,no significant difference in LC3B-II and P62 changes,inhibited autophagic flux,and suppressed cell proliferation(P<0.000 1).Conclusion Human hepatoblastoma HepG2 cells can regulate autophagic flux to against nutritional stress by inducing ATG5 expression.

Objective To investigate the combined effect and mechanism of metformin (Met) and 2-deoxy-D-glucose (2DG) on cell proliferation and apoptosis in liver cancer cells HepG2 and Hep3B.Methods Wst-1 reagent was used to determine the anti-proliferation effects after treatments with Met and 2DG alone or combined in HepG2 and Hep3B cells.Microscopy was used to observe cell morphological changes after treatments with Met and 2DG alone or combined in HepG2 and Hep3B cells.Cell apoptosis was observed by flow cytometry after treatment of different kinds of drugs.Western blotting was used to analyze the protein expressions of Caspase-3,PARP,Mcl-1 of HepG2.Results The survival rate of HepG2 cells in the combination group was (22.48 ± 0.51)％,and compared with the control group (100.00 ± 5.05)％,Met group (80.68 ±5.10)％ and 2DG group (72.56 ±4.34)％,the differences were statistically significant (P ＜ 0.001;P ＜ 0.001;P =0.001).The survival rate of Hep3B cells in the combination group was (29.16 ± 1.34) ％,and compared with the control group (100.00 ± 1.23) ％,Met group (59.58 ± 1.92) ％ and 2DG group (33.87 ± 1.95) ％,the differences were statistically significant (P ＜ 0.001;P ＜ 0.001;P =0.001).Microscopy observation showed that combined treatment of Met and 2DG caused less viable adherent cells of HepG2,but more floating dead cells.While the combination group also caused a decrease in the density of Hep3B cells,but did not significantly increase the shedding of cells.The apoptosis of HepG2 cells in the combination group was (39.63 ± 0.21) ％,and compared with the control group (7.12 ± 0.14) ％,Met group (12.56 ± 0.35) ％ and 2DG group (15.16 ± 1.93) ％,the differences were statistically significant (P ＜0.001;P ＜ 0.001;P =0.001).The apoptosis of Hep3B cells in the combination group was (12.58 ± 1.03) ％,and compared with the control group (2.82 ± 0.51) ％ and Met group (8.98 ± 0.86) ％,the differences were statistically significant (P ＜ 0.001;P =0.007),but compared with the 2DG group (12.40 ± 1.78) ％,the difference was not statistically significant (P =1.000).Furthermore,Western blotting demonstrated that the combined treatment induced evident Caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavages,and decreased expression of Mcl-1.Conclusion The combination of Met and 2DG can effectively inhibit cell proliferation of HepG2 and Hep3B,and induce apoptosis of HepG2 cells.The mechanism may be involved with Caspase-3 activation,cutting PARP substrate and decreasing Mcl-1 protein.

Primary hepatic carcinoma is one of the high incidence malignant tumors and the mortality ranked the second in the death caused by malignant tumors in China. The research progress on the epidemiology of primary hepatic carcinoma, the related environmental factors(virus, parasite, bacterium infected, aflatoxin ingested, drinking water pollution, smoking and drinking alcoholic beverages) and genes were reviewed and the mechanism of primary hepatic carcinoma was discussed in the present paper.