Calphostin C对乳腺癌细胞增殖的抑制作用及相关机制

Inhibition of Calphostin C on the proliferation of breast carcinoma cells and its mechanism

目的研究Calphostin C对乳腺癌细胞MDA-MB-435S增殖的影响及初步机制。方法通过观察Calphostin C处理MDA-MB-435S细胞前后细胞的生长速度、倍增时间、克隆形成率和细胞周期分布等变化,探讨Calphostin C对MDA-MB-435S细胞增殖的影响;通过Western blotting、免疫细胞化学染色和RT-PCR等方法探讨Calphostin C影响MDA-MB-435S细胞增殖的初步机制。结果与MDA-MB-435S亲本细胞(对照组)相比,Calphostin C处理的MDA-MB-435S细胞(实验组)生长速度明显减慢,倍增时间明显延长(P<0.01),并且呈现浓度和时间依赖性;对照组和实验组细胞克隆形成率分别为(82.33±6.81)%和(22.00±1.73)%,差异有统计学意义(P<0.01);实验组细胞周期出现明显的G1期阻滞(P<0.01)。Western blotting和免疫细胞化学染色结果显示,与对照组细胞相比,实验组细胞中蛋白激酶Cα(protein kinase Cα,PKCα)表达没有明显变化,但活性状态(胞质转位至胞膜)的PKCα显著减少。West-ern blotting和RT-PCR结果显示,与对照组细胞相比,实验组细胞中p53、Cyclin A和Cyclin B表达没有明显变化,但p21表达上调,Cyclin E表达下调。结论Calphostin C可显著抑制乳腺癌细胞MDA-MB-435S的增殖,该作用可能与其抑制PKCα活性、上调p21表达和下调Cyclin E表达有关。

Objective To explore the effects of Calphostin C on the proliferation of breast carcinoma cells , 〈 （MDA-MB-435S） and its preliminary mechanism. Methods The effects of Calphostin C on the proliferation of MDA-MB-435S cells were studied by observing changes of the growth rate, doubling time, clone-forming efficiency, and distribution of cell cycle of MDA-MB-435S cells before and after Calphostin C treatment. Furthermore, the mechanism involved was analyzed by Western blotting, immunocytochemistry and RT-PCR assay. Results Compared with those of the parental MDA-MB-435S ceils （control group）, the growth rate was suppressed markedly and the doubling time was prolonged significantly （P〈0.01） in the MDA-MB-435S cells treated with Calphostin C （experimental group）, and the changes presented concentration- and time-dependence patterns. Furthermore, the clone-forming efficiency in control group and experimental group was （82.33 ± 6.81） % and （22.00 ± 1.73） %, respectively, with a significant difference （P〈 0.01）. Moreover, for the experimental group cells, the cell cycle arrested in G1 phase （P〈0.01）. The results from Western blotting and immunocytochemical staining indicated that compared with that of the parental MDA-MB-435S cells, the expression of protein kinase Ca （PKCa） did not change significantly, but PKCa in active status （transposition from cytoplasm to cytomembrane） was decreased dramatically in the MDA-MB-435S cells treated with Calphostin C. Western blotting and RT-PCR results showed that compared with that of the parental MDA-MB-435S cells, the expression of p53, Cyclin A and Cyelin B had no significant changes, while the expression of p21 was increased and the expression of Cyclin E was decreased in the MDA-MB- 435S cells treated with Calphostin C. Conclusion Calphostin C could suppress markedly the proliferation of MDA- MB-435S cells, which may be correlated to the abilities of inhibiting PKCa activity, up-regulating the expression of p21 and down-regulating the expression of Cyclin E.