汉黄岑素通过调节P53信号通路增强宫颈癌对顺铂的化疗敏感性

Enhancement on chemosensitivity of cervical carcinoma to cisplatin by Wogonin regulating P53 signaling pathway

目的:探究汉黄岑素增强宫颈癌对顺铂的化疗敏感性的作用机制。方法:体外培养CC细胞株,建立顺铂敏感性的CC细胞株CaSki/DDP并检测CaSki和CaSki/DDP的IC50值,分为:空白对照组(Control)、贝伐单抗组(BVZ 2.5μg/ml)、顺铂组(DDP 10 mg/L)、低剂量汉黄岑素组(DDP 10 mg/L+WG 5μmol/L)、中剂量汉黄岑素组(DDP10 mg/L+WG 10μmol/L)、高剂量汉黄岑素组(DDP 10 mg/L+WG 20μmol/L)、P53抑制剂组(DDP+PFT-α)和汉黄岑素+P53抑制剂组(DDP+WG 20μmol/L+PFT-α)。使用CCK8检测细胞生长;使用蛋白质印迹法检测Ki67、PCNA Bcl-2、Bax、cytC、Caspase3、LC3Ⅱ、LC3Ⅰ、Beclin1、P62的表达水平;流式细胞术分析检测细胞凋亡;使用免疫荧光检测自噬LC3核转位;使用蛋白印迹法检测P53水平;使用P53抑制剂Pifithrin-α处理后,使用蛋白印迹法、CCK8、流式和免疫荧光检测P53水平和细胞生长、凋亡、自噬情况。结果:宫颈癌细胞株CaSki/DDP的IC50值为50 mg/L;相比空白对照组,贝伐单抗组宫颈癌细胞Ki67、PCNA、Bcl-2、P62表达水平和细胞存活率显著降低(P<0.05),Bax、cytC、Caspase3表达水平和LC3Ⅱ/LC3Ⅰ值、细胞凋亡率、细胞内荧光强度显著升高(P<0.05);相比顺铂组,使用汉黄岑素处理各组宫颈癌细胞Ki67、PCNA、Bcl-2、P62表达水平、细胞存活率显著降低,Bax、cytC、Caspase3表达水平、LC3Ⅱ/LC3Ⅰ值、细胞凋亡率、细胞内荧光强度显著升高(P<0.05),且呈剂量依赖性;相比P53抑制剂组,汉黄岑素+P53抑制剂组P53宫颈癌细胞凋亡率、单个细胞内LC3+荧光点数显著升高(P<0.05),宫颈癌细胞存活率显著降低(P<0.05)。结论:汉黄岑素可以通过调节P53信号通路增强宫颈癌细胞的自噬和凋亡,抑制其增殖,实现增强宫颈癌对顺铂的化疗敏感性,且在一定剂量范围内呈剂量依赖性。

Objective:To investigate the mechanism of Wogonin(WG)enhances the sensitivity of cervical carcinoma(CC)to cisplatin(DDP)chemotherapy.Methods:CC cell lines were cultured in vitro.The DDP-sensitive CC cell line CaSki/DDP was established.IC50 values of CaSki and CaSki/DDP were detected.Grouping was performed:blank control group(Control),bevacizumab(BVZ)group(BVZ 2.5μg/ml),DDP group(DDP 10 mg/L),low-dose Wogonin group(DDP 10 mg/L and WG 5μmol/L),medium-dose Wogonin group(DDP 10 mg/L and WG 10μmol/L),high-dose Wogonin group(DDP 10 mg/L and WG 20μmol/L),P53 inhibitor group(DDP and PFT-α)and Wogonin with P53 inhibitor group(DDP with WG 20μmol/L and PFT-α).Cell growth was detected by CCK8.The expression levels of Ki67,PCNA Bcl-2,Bax,cytC,Caspase3,LC3Ⅱ,LC3Ⅰ,Beclin1 and P62 were detected by Western blot.Apoptosis was analyzed and detected by flow cytometry.The immunofluorescence assay was performed to detect autophagy LC3 nuclear translocation.P53 levels were detected by Western blot.After treatment with P53 inhibitor Pifithrin-α,P53 levels,cell growth,apoptosis and autophagy were detected by Western blot,CCK8,flow cytometry and immunofluorescence.Results:The IC50 value of CC cell line CaSki/DDP was 50 mg/L.Compared with blank control group,expression levels of Ki67,PCNA,Bcl-2 and P62,and cell survival rate of cervical cancer cells in bevacizumab group were significantly decreased(P<0.05),while expression levels of Bax,cytC and Caspase3,LC3Ⅱ,LC3Ⅰvalue,apoptosis rate and intracellular fluorescence intensity were significantly increased(P<0.05).Compared with DDP group,expression levels of Ki67,PCNA,Bcl-2 and p62,and cell survival rate of CC cells in all groups treated with Wogonin were significantly decreased,while expression levels of Bax,cytC and Caspase3,LC3Ⅱ,LC3Ⅰvalue,apoptosis rate and intracellular fluorescence intensity were significantly increased(P<0.05),showing dose-dependence.Compared with P53 inhibitor group,apoptosis rate of P53 CC cells and number of LC3+fluorescent dots in per cell were significantly increased in Wogonin with P53 inhibitor group(P<0.05),while survival rate of cervical cancer cells was significantly decreased(P<0.05).Conclusion:Wogonin can enhance autophagy and apoptosis of cervical cancer cells by regulating P53 signaling pathway,inhibit their proliferation,and enhance the chemotherapy sensitivity of cervical cancer to cisplatin,and it is dose-dependent in a certain dose range.