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目的研究天然产物白杨素(chrysin)对损伤DNA的抗肿瘤药物顺铂和喜树碱诱导肝肿瘤细胞(Hep G2)死亡的作用及机制。方法白杨素以不同浓度单独/联合顺铂或喜树碱处理Hep G2,于倒置显微镜下观察肿瘤细胞形态变化,以四甲基偶氮唑蓝(MTT)法分析细胞活性,获得细胞死亡的定量资料;并以Western blot方法分析凋亡标志蛋白caspase-3和多聚(ADP-核糖)聚合酶(PARP)以及凋亡抑制蛋白Bcl-x L、x IAP、FLIP相应的变化情况。结果形态学观察可发现白杨素联合顺铂或喜树碱处理可明显增加Hep G2细胞的死亡,MTT法分析显示,白杨素联合抗肿瘤药物处理细胞,无论与未处理对照比较还是与单独白杨素、单独顺铂或单独喜树碱处理比较,均使细胞活性显著降低(P<0.05);Hochest 33342荧光染色表明,联合白杨素和抗肿瘤药物处理细胞可观察到明显的核固缩,而未处理对照及单独白杨素、单独顺铂或喜树碱处理则无此作用;Thermo高内涵筛选系统分析结果显示,联合白杨素和抗肿瘤药物处理可使凋亡细胞显著增加(P<0.05)。Western blot检测到凋亡标志蛋白caspase-3和PARP活化降解;全caspase酶抑制剂z-VAD-fmk可有效阻止凋亡标志蛋白caspase-3和PARP的活化降解。白杨素联合顺铂可抑制顺铂上调的凋亡抑制蛋白FLIP和x IAP表达,而白杨素联合喜树碱可降低喜树碱引起的凋亡抑制蛋白Bcl-x L的高表达。结论天然产物白杨素能有效促进DNA损伤类抗肿瘤药物诱导的Hep G2细胞凋亡,核因子(NF)-κB调节的凋亡抑制蛋白表达降低是其重要的机制。
Objective To investigate the effect and mechanism of chrysin, a natural product, on the DNA damage induced by cisplatin and camptothecin in hepatocarcinoma cells (Hep G2). Methods Poplar was treated with different concentration of cisplatin or camptothecin alone or in combination with Hep G2. The morphological changes of tumor cells were observed under an inverted microscope. The cell viability was analyzed by MTT assay and the cell death was quantified Data were analyzed by Western blot. The changes of apoptotic marker protein caspase-3, poly (ADP-ribose) polymerase (PARP) and apoptosis-inhibitory proteins Bcl-x L, x IAP and FLIP were analyzed by Western blot. Results Morphological observation showed that the combination of chrysin and cisplatin or camptothecin significantly increased the death of Hep G2 cells. The results of MTT assay showed that chrysin and anti-tumor drugs, compared with untreated control or chrysin (P <0.05). Hochest 33342 fluorescence staining showed that significant nuclear pyknosis was observed in cells treated with chrysin and anti-tumor drugs, but not The results of Thermo high content screening system analysis showed that the combination of chrysin and anti-tumor drugs can significantly increase the number of apoptotic cells (P <0.05). Western blot showed that caspase-3 and PARP were activated and degraded. Z-VAD-fmk, a caspase inhibitor, could effectively prevent the activation and degradation of apoptotic markers caspase-3 and PARP. Chrysin combined with cisplatin could inhibit cisplatin-induced upregulation of FLIP and x IAP expression, while chrysin and CPT decreased the high expression of apoptosis-inducing protein Bcl-x L induced by CPT. Conclusions Chimonin, a natural product, can effectively promote the apoptosis of Hep G2 cells induced by DNA-damaging antitumor drugs and decrease the expression of apoptosis-inhibiting protein that is regulated by nuclear factor (NF) -κB.