新型双核铂(Ⅱ)配合物通过p53-Bak途径诱导人乳腺癌MCF-7细胞凋亡

New Polynuclear Platinum(Ⅱ) Complex Induces Apoptosis of Human Breast Cancer MCF-7 Cells by p53-Bak Pathway

新型双核铂(Ⅱ)配合物{[cis-Pt(NH_3)_2Cl]2L}(NO_3)_2(L=4,4'-methylenedianiline)对多种肿瘤细胞有一定的抑制作用,但作用机制不明。本研究以顺铂为对照,探讨了该双核铂(Ⅱ)配合物对MCF-7细胞增殖抑制、细胞周期、细胞凋亡及凋亡相关因子p53、P-p53(ser15)、p21、Bcl-2、Bak、Cleaved-caspase-3、c PARP(cleavage of poly(ADP-ribose)polymerase)的影响。MTT法检测配合物或顺铂在不同浓度或不同作用时间后对MCF-7增殖的影响,其中作用48 h后配合物对MCF-7的IC50为1.59μmol/L,顺铂为7.95μmol/L。原位移植瘤实验显示,配合物组肿瘤抑制率为54.1%,高于顺铂组(36.2%)。同等条件下,配合物处理的MCF-7细胞,经Hoechst33342染色后出现明显细胞体积缩小和染色质固缩现象。流式细胞检测技术分析显示,经该配合物处理后,大部分MCF-7细胞停滞在S期,并出现了细胞膜外表面的磷脂酰丝氨酸外翻与线粒体内膜急剧下降等典型的细胞凋亡现象。Western印迹结果显示,随着配合物浓度增加,Cleaved-caspase-3、p53、P-p53(ser15)、c PARP、Bak蛋白表达增强,而p21、Bcl-2表达水平下调。上述结果表明,该配合物可能通过p53-Bak途径诱导DNA损伤,进而导致MCF-7细胞发生凋亡。

Dinuelear platinum （ Ⅱ ） complex { [ eis-Pt （NH3） 2C1] 2L} （NO3） 2 （L = 4, 4＇- methylenedianiline） can inhibit growth of tumor cells. However, the mechanisms remained unclear. In the present study, we investigated the anti-tumor aetivity of the dinuclear platinum （ Ⅱ ）eomplex with eisplatin as a control, whieh ineludes eell eyele, apoptosis, and related faetors such as p53, p21, Bcl-2, Bak, Cleaved-caspase-3 and cPARP}. MTT was used to detect the inhibition of MCF-7 eells proliferation by using dinuelear platinum （ Ⅱ ） eomplex at different times or different concentrations. The IC50 values of the complex or cisplatin against MCF-7 cells proliferation were 1.59 μmol/L or 7.95μmol/L after incubation for 48 hours. Results showed that the tumor growth inhibition rate of the complex was 54.1% , higher than that of cisplatin group （36.2%） in mouse xenografls. Under the same condition, MCF-7 cells treated with the eomplex showed more cell shrinkage and ehromatin pyknosis by Hoechst33342 staining. The complex arrested the cell cycle of MCF7 cells in S phase by flow cytometry. The complex induced MCF-7 cells apoptosis which was characterized by exposure of phosphatidyl serine and dissipation of mitochondrial membrane potential. The results of Western blot also demonstrated that the complex caused a dose-dependent increase of cleaved-caspase-3, p53, as a reduction of p21 and Bcl-2. The results indicated that p53-Bak pathway. p-p53 （serl5） , cPARP and Bak, as well the complex induced apoptosis through the