芹菜素与丹参酮Ⅱ_A的协同抗肿瘤作用及机制

Synergistic antitumor effects and mechanism of apigenin and tanshinone Ⅱ_A

目的研究芹菜素(Api)和丹参酮ⅡA(TanⅡA)联用对多种肿瘤细胞的协同抗肿瘤作用,并揭示其发生协同作用的机制。方法以人胃癌BGC823细胞、人乳腺癌MCF7细胞、人肝癌SMMC7721细胞为研究对象,采用MTT法检测Api和TanⅡA联用对细胞的增殖抑制作用;采用AV-PI双染法、PI染色法检测药物对BGC823细胞凋亡、细胞周期的影响;免疫印迹法检测BGC823细胞凋亡相关蛋白p53、BAX/BCL-2和周期蛋白B1、D1的表达;圆二色和DNA熔点法检测药物与DNA结合情况;S180荷瘤鼠模型检测药物的抑瘤效果。结果 Api与TanⅡA联用对BGC823等肿瘤细胞的增殖有协同抑制作用,联用指数CI在0.28左右。两药联用显著增加了细胞凋亡(P<0.01),上调了胞内BAX/BCL-2比值(P<0.01);周期蛋白水平发生变化,细胞周期阻滞在S期得到强化。两药与DNA以两种不同方式结合,DNA热变性曲线显著改变。体内抑瘤实验证明与单药相比,两药联用荷瘤鼠肿瘤体积和肿瘤质量均显著降低(P<0.01),抑瘤效果与环磷酰胺相当,但不良反应较小。结论 Api与TanⅡA联用具有协同抗肿瘤作用;药物与DNA以不同的方式结合,加剧了细胞周期的阻滞,是二者产生协同作用的核心机制。

Objective To evaluate the synergistic anticancer effects of the combination of apigenin(Api) and tanshinone ⅡA(TanⅡ_A), and investigate the mechanisms of pharmacological effects and their potential applications as an anticancer therapy in clinics. Methods MTT assay were used to determine anticancer effects of the combination of Api and Tan Ⅱ_A on BGC823, MCF7, and SMMC7721 cells. AV-PI dual stain and PI staining method were used for detecting the effect of the two drugs combination on BGC823 cell apoptosis and cell cycle. Expression of p53, BAX/BCL-2, cyclin B1 and D1 proteins were determined by Western blotting. Circular dichroism method and DNA melting point method were explored to detect interaction among the two drugs and DNA. S180 tumor xenograft mice model was used to evaluate the antitumor effects of the two drugs combination. Results Tan Ⅱ_A combined with Api exerted synergistic inhibitory effects on the proliferation of BGC823 and other tumor cells with the CI of 0.28. After tumor cell treated by combination of Tan Ⅱ_A and Api, the tumor cell apoptosis was significantly enhanced and the value of BAX/BCL-2 in cells was up-regulated(P < 0.01);The levels of cyclin B1, D1 protein were changed and cell cycle arrest was increased which mainly blocked in S phase. The interaction among the two drugs and DNA was in two different ways, leading to the curves of thermal denaturation of DNA changed significantly. Furthermore, the combination of Tan Ⅱ_A and Api showed a stronger inhibitory effect on tumor volume and weight in S180 mice model than monotherapy, which was similar to cyclophosphamide therapy but less side effects. Conclusion Tan Ⅱ_A combined with Api exerted synergistic antitumor effects. The two drugs interacted with DNA in different ways and aggravated the cell cycle arrest, which were the key mechanisms of their synergistic antitumor effects.