Effects of high mobility group box-1 protein-nuclear factor-κB signaling pathway on autophagy and chemosensitivity of human hepatocellular carcinoma cells / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the effect of high mobility group box-1 protein (HMGB1)-nuclear factor-κB (NF-κB) signaling pathway on autophagy and chemosensitivity of human hepatocellular carcinoma cells and its possible mechanism.Methods:Human hepatocellular carcinoma BEL-7402 cells were cultured in vitro and divided into control group (BEL-7402 cells without any treatment), doxorubicin group, recombinant human HMGB1+doxorubicin group, anti-HMGB1 neutralizing antibody+doxorubicin group, pyrrolidine dithiocarbamate+doxorubicin group and 3-methyladenine + doxorubicin group. The methyl thiazolyl tetrazolium (MTT) method was used to detect cell proliferation inhibition rate. Western blot method was used to detect the expressions of HMGB1 and NF-κB subunit p-p65 protein, the autophagy-related proteins Beclin-1, LC3Ⅰ, LC3Ⅱ and apoptosis-related protein bcl-2. Enzyme labeling method was used to detect Caspase-9 and Caspase-3 activity.Results:The cell proliferation inhibition rates in the control group, doxorubicin group, recombinant human HMGB1+doxorubicin group, anti-HMGB1 neutralizing antibody+doxorubicin group, pyrrolidine dithiocarbamate+doxorubicin group and 3-methyladenine+doxorubicin group were (1.31±0.16)%, (47.80±6.30)%, (31.60±5.68)%, (67.20±6.83)%, (66.60±6.27)%, and (68.60±11.19)%, respectively, and the difference was statistically significant ( F = 75.91, P < 0.01), suggesting that doxorubicin had a proliferation inhibitory effect on BEL-7402 cells; the expression levels of HMGB1 were 1.17±0.11, 1.37±0.15, 1.43±0.15, 0.70±0.09, 1.27±0.12, 1.29±0.18, and the difference was statistically significant ( F = 18.70, P < 0.01), suggesting that doxorubicin could increase the expression of HMGB1 in BEL-7402 cells, and the anti-HMGB1 neutralizing antibody could block or attenuate this effect; after pretreatment with recombinant human HMGB1, the proliferation inhibitory effect of doxorubicin on BEL-7402 cells was weakened; after pretreatment with anti-HMGB1 neutralizing antibody, pyrrolidine dithiocarbamate and 3-methyladenine, the proliferation inhibitory effect of doxorubicin on BEL-7402 cells was enhanced. Compared with the control group, the expression of p-p65 protein in the doxorubicin group, recombinant human HMGB1+doxorubicin group and 3-methyladenine+doxorubicin group increased (all P < 0.05). The expression of p-p65 protein in the recombinant human HMGB1+doxorubicin group, anti-HMGB1 neutralizing antibody+doxorubicin group and pyrrolidine dithiocarbamate+doxorubicin group was lower than that in the doxorubicin group (all P < 0.05). Compared with the control group, the expression of bcl-2 protein in doxorubicin group, anti-HMGB1 neutralizing antibody+doxorubicin group, pyrrolidine dithiocarbamate+doxorubicin group and 3-methyladenine+ doxorubicin group decreased (all P < 0.05), and the activity of Caspase-9 and Caspase-3 was enhanced (all P < 0.05); after adding recombinant human HMGB1 pretreatment, the expression of bcl-2 protein in the cells increased compared with doxorubicin alone, and the activity of Caspase-9 and Caspase-3 was weakened (all P < 0.05). The expression levels of autophagy-related protein Beclin-1 in the control group, doxorubicin group, recombinant human HMGB1+doxorubicin group, anti-HMGB1 neutralizing antibody+doxorubicin group, pyrrolidine dithiocarbamate+doxorubicin group, and 3-methyl adenine+doxorubicin group were 0.77±0.12, 0.92±0.07, 1.29±0.10, 0.51±0.03, 0.49±0.06, and 0.42±0.05, and the difference was statistically significant ( F = 97.01, P < 0.01). The expression levels of LC3Ⅱ were 0.24±0.04, 0.39±0.04, 0.49±0.07, 0.23±0.05, 0.20±0.06, and 0.20±0.05, and the difference was statistically significant ( F = 26.98, P < 0.01). Conclusion:The activation of HMGB1-NF-κB signaling pathway can reduce the chemosensitivity of hepatocellular carcinoma cells to doxorubicin, and its mechanism may be related to the regulation of autophagy and down-regulation of doxorubicin inducing apoptosis of hepatocellular carcinoma cells.