斑蝥提取物通过细胞周期G2/M期阻滞诱导人胆管细胞癌QBC939细胞凋亡

Cantharis extract induces G2/M phase arrest and apoptosis in human cholangiocarcinoma QBC939 cells

目的:研究斑蝥提取物(cantharis extract,CTE)体外诱导人胆管癌QBC939细胞凋亡作用及其对细胞周期分布的影响。方法:体外培养人胆管细胞癌QBC939细胞,CTE作用于QBC939细胞48小时,利用CCK-8法检测QBC939细胞增殖率并计算半数抑制浓度(IC50);光学显微镜下观察细胞形态的改变;AnnexinⅤ/PI双染法检测细胞凋亡情况;用PI染色检测对QBC939细胞周期分布的影响;Western blot法检测细胞凋亡相关蛋白及细胞周期相关蛋白表达变化。结果:CTE显著抑制QBC939细胞增殖,呈浓度及时间依赖性,处理48小时的IC50为4.16μg/ml。经光学显微镜下观察可见随着药物浓度增加,细胞密度减少,游离变圆的细胞明显增多。AnnexinⅤ/PI双染流式凋亡检测示,随着药物浓度的增加,凋亡细胞数量逐渐增加,药物处理组(1.25μg/ml、2.5μg/ml、5.0μg/ml)的细胞凋亡率分别为(9.52±1.01)%、(15.62±1.88)%、(46.03±4.88)%,与对照组(4.59±0.43)%比较,差异有统计学意义(P<0.05)。PI检测细胞周期示,CTE能将QBC939细胞周期阻滞在G2/M期,随药物浓度增加而增加,G2/M期细胞比例逐渐增加,与对照组比较差异有统计学意义,而G1期细胞比例逐渐减少。Western blot检测示,CTE处理组细胞增殖相关蛋白增殖细胞核抗原(PCNA)表达降低,相对蛋白表达量与对照组比较,差异有统计学意义(P<0.05);Bcl-2家族蛋白Bax和Bid表达增加,而Bcl-2、Mcl-1表达降低,抑制凋亡蛋白家族蛋白Survivin表达显著下降,凋亡相关蛋白半胱氨酸蛋白水解酶3(Caspase-3)表达下降,与对照组比较CTE处理组上述相对蛋白表达量差异有统计学意义(P<0.05);CTE处理组细胞周期相关蛋白Chk1表达无显著改变,相对蛋白表达量与对照组比较差异无统计学意义(P<0.05),磷酸化的Chk1蛋白及p53蛋白表达逐渐增加,相对蛋白表达量与对照组比较差异有统计学意义(P<0.05)。结论:CTE显著抑制QBC939细胞增殖,诱导QBC939细胞凋亡,其可能机制与CTE调节凋亡相关蛋白表达,同时调节肿瘤细胞周期相关。

Objective:To investigate the influence of cantharis extract(CTE)on human cholangiocarcinoma cell line QBC939 apoptosis and cell cycle distribution.Methods:Human cholangiocarcinoma cells QBC939 were cultured in vitro.QBC939 cells were treated with CTE for 48 h.CCK-8 assay was used to assess QBC939 cells viability and calculate half inhibitory concentration(IC50).Cell morphology change was observed under optical microscope.Flow cytometery based on Annexin V/PI double staining was performed to evaluate the ratio of apoptosis.Flow cytometery based on PI staining was performed to evaluate cell cycle distribution.Western blot assay was used to detect apoptosis and cell cycle related protein expression.Results:CTE significantly inhibited the proliferation of QBC939 cells in a dose and time dependent manner.And the IC50 of CTE was 4.16μg/ml.Under optical microscope,apparent anoikis and apoptosis occurred in tumor cells and increased with the increase of CTE concentration.Annexin V/PI double staining flow cytometry also showed that CTE triggered a dose dependent apoptosis.The apoptosis rate of QBC939 cells treated with 1.25,2.5,5.0μg/ml CTE for 48 h was(9.52±1.01)%,(15.62±1.88)%,(46.03±4.88)%respectively,statistically different from those of control group(4.59±0.43)%(P<0.05).PI staining flow cytometery showed that CTE caused cell cycle arrest in G2/M phase.With the increase of the CTE concentration,the ratio of G2/M phase cells increased and G1 phase cells decreased gradually,significantly different from the control group.Western blot showed that compared with control group,the expression of proliferation related protein PCNA decreased(P<0.05),expression of Bax and Bid increased(P<0.05),expression of Bcl-2,Mcl-1 and Survivin,apoptotic protein Caspase-3 decreased(P<0.05),expression of cell cycle related protein Chk1 had no change(P>0.05),expression of cell cycle related protein p-Chk1 and p53 increased(P<0.05),in the CTE treated group.Conclusion:Our data suggest that CTE significantly exerts anti-cancer effects through inducing proliferation inhibition and apoptosis,which may be mediated by impairing the balance of anti-and pro-apoptotic proteins and cell cycle distribution.