和厚朴酚通过抑制cyclin D1影响Hep3B细胞增殖和周期

Honokiol Inhibits the Proliferation and Cell Cycle of Hep3B Cells via Regulating Cyclin D1

目的研究和厚朴酚(honokiol,HNK)对肝癌Hep3B细胞增殖能力的影响并探讨可能机制。方法在不同浓度梯度或时间的药物刺激下,培养Hep3B细胞及永生化人肝细胞LO2,观察HNK对两种细胞生长能力的影响。采用平板克隆形成实验观察HNK对Hep3B生长能力的影响,并采用流式细胞技术探究HNK对Hep3B细胞周期所产生影响。构建用裸鼠皮下成瘤模型,明确在体内水平下HNK抑制肝癌细胞增殖能力。最后采用Western blot法检测不同浓度HNK刺激下细胞周期相关蛋白水平,从而探索HNK抗肝癌细胞增殖的可能机制。结果HNK在体外可显著抑制肝癌细胞Hep3B增殖能力,有统计学意义(P<0.05),并呈现明显的浓度和时间依赖性;但相同浓度或者时间刺激下,HNK对正常肝细胞(LO2)毒性较小。HNK可能是通过诱导细胞周期G1/S阻滞,有统计学意义(P<0.05)从而发挥抑制肝癌细胞的增殖能力。裸鼠皮下成瘤模型结果显示,HNK亦可抑制皮下肿瘤生长,没有统计学意义(P<0.05)。进一步的机制研究发现HNK刺激后,Hep3B细胞周期蛋白cyclin D1表达降低,没有统计学意义(P<0.05),而细胞周期负性调控蛋白P53、P27、P21表达升高,没有统计学意义(P<0.05)。结论HNK通过抑制cyclin D1影响Hep3B细胞增殖和周期进程。

Objective To study the effects of honokiol(HNK)on the proliferation of hepatocellular carcinoma(HCC)cell Hep3B and explore the possible molecularmechanisms.Methods Hep3B cells and immortalized human hepatocytes LO2 was cultured under different concentrations or timeof HNK and the cell survival rate was observed.The effect of HNK on the growth capability of Hep3B wasevaluated by clone formation assay,and the effect of HNK on the cell cycle of Hep3B was investigated by Flow cytometric analysis.To clarify the ability of HNK inhibiting the proliferation of HCC cells in vivo,a subcutaneous tumorigenic model was builded in nude mice.Finally,Western blot was used to detect the levels of cell cycle-related proteins stimulated by different concentrations of HNK,so as to explore the possible mechanism of HNK in inhibiting the proliferation of HCC cells.Results HNK significantly inhibited(P<0.05)the proliferation of HCC cell Hep3B in vitro in a concentration-and time-dependence manner.However,under the same concentration or time stimulation,HNK had less toxicity to normal hepatocytes(LO2).HNK inhibited the proliferation of HCC cells by inducing G1/S cell cycle arrest(P<0.05).Furthermore,the results of subcutaneous tumorigenesis model in nude mice showed that HNK could inhibit the growth of subcutaneous tumors(P<0.05).Further mechanism analysis revealed that HNK inhibited the expression of cyclin D1(P<0.05)and upregulated the the expression of proteins P21,P27,P53(P<0.05)that negativelyregulate the cell cycle.Conclusion HNK inhibits the proliferation and cell cycle of Hep3B cells viaregulatingcyclin D1.