蔓荆子黄素对p53突变型人乳腺癌细胞增殖和凋亡的影响

Vitexicarpin effects proliferation and apoptosis in mutated p53 breast cancer cell

目的了解蔓荆子黄素（vitexicarpin）对p53突变型人乳腺癌细胞Hs578T增殖和凋亡的影响，并观察vitexicarpin对Hs578T和p53野生型人乳腺癌细胞MCF-7中c—Myc、p21、Bcl-2蛋白表达的影响及c—Myc蛋白高表达对vitexicarpin作用的影响。方法在不同时间点（24、48和72h）用MTT方法来检测不同浓度vitexicarpin（0、0．1、0．2、0．5、1．0umol/L）对细胞增殖的影响。用TUNEL方法在24h时间点检测不同浓度vitexicarpin对细胞凋亡的影响。在vitexicarpin不同浓度作用下，检测Hs578T细胞中c—Myc、Bcl-2和p21三种蛋白的表达情况。在Hs578T和MCF-7细胞中瞬时转染c—Myc基因，用M1Tr方法检测c—Myc蛋白高表达对vitexicarpin抑制细胞增殖作用的影响。结果在vitexicarpin浓度〉0．2umol／L情况下，Hs578T及MCF-7细胞的增殖能力均受到明显抑制（IC50为0．25umol／L和0．53umoL／L，72h）。TUNEL实验显示vitexicarpin在0．1、0．2、0．5umol／L作用浓度下TUNEL阳性细胞率分别为10．15％，27．33％和35．34％，而对照组为4．65％。Hs578T细胞中c．Myc和Bcl-2的表达与vitexicarpin的浓度呈反比，p21的表达与vitexicarpin的浓度呈正比。转染c—Myc基因及c—Myc蛋白高表达后，在Hs578T细胞中vitexicarpin对Bcl-2和p21的影响减弱。在vitexicarpin 0．5umol／L组72h时比0h时的吸光度（A）值增加了1．53倍，抑制细胞增殖的能力下降；相反地，但在MCF-7／c—Myc细胞中，同样条件下A值减少了48％。结论vitexicarpin抑制p53突变型Hs578T细胞增殖的机制可能是通过c—Myc蛋白，而对于MCF-7细胞则是通过其他机制。vitexicarpin对恶性肿瘤细胞的作用机制在不同细胞中是不同的。

Objective To elucidate the effect of proliferation and apoptosis induced by vitexicarpin in mutated p53 Hs578T cell line and study the expression of c-Myc, p21 and Bcl-2 protein in Hs578T and wild p53 MCF-7 cell pre-treated with vitexiearpin. Methods Cells were treated with various concentrations of vitexicarpin （0, 0. 1, 0. 2, 0. 5, 1.0 umol/L）. MTT assays were used to detect cell proliferation at different time points with different doses of vitexicarpin. TUNEL assays were performed to examine apoptosis in cells pretreated with vitexicarpin. The authors detected three main proteins involved in apoptosis： c-Myc, bcl-2 and p21 protein in various concentrations of vitexicarpin-treated cells. To understand the function of c- Myc protein in the effect of vitexicarpin, the authors transiently transfected c-Myc protein in Hs578T cell and detected the cellular effect of vitexicarpin. Results Proliferation of Hs578T and MCF-7 cells were inhibited markedly by vitexicarpiu at concentrations above 0. 2 umol/L （ IC50 = 0. 25 umol/L and 0. 53 umol/L at 72 h respectively）. TUNEL assays revealed that the rates of TUNEL positive cells were 10. 15% , 27.33% and 35.34% when exposing Hs578T cells to 0. 1, 0. 2 and 0. 5 umoL/L of vitexicarpin respectively. In control cells, the rates of TUNEL positive cells were 4. 65%. Cells pretreated with higher concentrations of vitexicarpin expressed less e-Myc and Bcl-2 in Hs578T cells. In contrast, p21 decreased when cells were treated with the same conditions. When c-Myc transient transfection was performed in vitexicarpin-treated cells, the effect of p21 and Bcl-2 disappeared. The proliferative function of vitexicarpiu declined in Hs578T/c-Myc cells. When treated with 0. 5umoL/L vitexicarpin, A value increased 1.53 times at 72 h. Conversely, A value decreased 48% at the same condition in MCF-7/c-Myc cells. Conclusion The suppressing mechanism of vitexicarpin for malignant tumors is through c-Myc in p53 mutated Hs578T cells. And it is multi-directional and varies in different cells.