佛手柑内酯抑制p62/NF-κB信号轴增强顺铂对肝癌杀伤的影响

Bergapten strengthens the anti-tumor activity of cisplatin by suppressing p62/NF-κB pathway in hepatocellular carcinoma

目的观察佛手柑内酯与顺铂联用对肝癌细胞的凋亡及p62/NF-κB信号轴的影响。方法利用低浓度(1 mol/L)佛手柑内酯和1 mol/L顺铂联用或单用,处理体外培养的肝癌细胞Hep G2和Bel-7402,观察两肝癌细胞的凋亡核形态和Annexin V阳性的凋亡细胞群比例。进一步利用荧光定量聚合酶链反应检测两肝癌细胞的抗凋亡相关基因表达变化,利用荧光素酶报告基因检测NF-κB转录因子的转录活性,利用Western blot法检测细胞内p65及p62的表达水平。结果低浓度佛手柑内酯与顺铂联用能显著诱导Hep G2和Bel-7402的凋亡核形态和凋亡细胞群比例,两肝癌细胞的凋亡核形态比单用顺铂组分别增加1.99倍和2.00倍(P<0.05),凋亡细胞群比例比单用顺铂组分别增加1.86倍和2.00倍(P<0.05)。低浓度佛手柑内酯与顺铂联用能不同程度降低抗凋亡基因BCL2、BCL2L1、XIAP和BIRC5的表达,并抑制NF-κB转录因子的转录活性和细胞内p65及p62的表达水平。结论低浓度佛手柑内酯能增强顺铂对肝癌细胞凋亡的诱导,其可能原因与抑制p62/NF-κB信号轴相关。

Objective To investigate the effect of bergapten strengthening the anti-tumor activity of cisplatin in hepatocellular carcinoma in vitro. Methods The hepatocellular carcinoma cells( Hep G2 and Bel-7402) were treated with 1 mol/L bergapten and/or 1 mol/L cisplatin,and the apoptosis ratio was detected by Hoechst33258 staining and Annexin V-FITC/PI analysis. The expression of anti-apoptosis related genes were detected by real-time quantitive polymerase chain reaction. The activity of NF-κB transcription factors were detected by luciferase reporter gene analysis. The expression of p65 and p62 were detected by Western blot. Results Compared with cisplatin only treatment,the ratio of apoptosis nucleus and cells was remarkable increased by combination of bergapten and cisplatin both in Hep G2 and Bel-7402( P < 0. 05 for each). The m RNA level of BCL2,BCL2 L1,XIAP and BIRC5 was decreased by combination treatment with bergapten and cisplatin. The transcript ability of NF-κB andthe protein level of p65 and p62 was decreased by combination treatment with bergapten and cisplatin. Conclusion Bergapten could strengthen the anti-tumor activity of cisplatin by suppressing p62/NF-κB pathway in hepatocellular carcinoma in vitro.