二十二碳六烯酸增强MDA-MB-231细胞对5-FU敏感性的研究

Effects of Docosahexaenoic Acid on Chemo Sensitivity of Human Breast Cancer MDA-MB-231 Cells to 5-fluorouracil

目的:探讨二十二碳六烯酸(DHA)对5-FU诱导乳腺癌细胞MDA-MB-231增殖与凋亡的作用及对Wnt/β-Catenin信号传导通路的影响。方法:采用四甲基偶氮唑蓝(MTT),测定DHA对5-FU诱导乳腺癌细胞MDA-MB-231增殖活性的作用;应用流式细胞仪检测细胞周期和凋亡率;RT-PCR检测MDA-MB-231细胞β-catenin、GSK-3β基因的表达情况;Western blot检测MDA-MB-231细胞β-catenin、GSK-3β、磷酸化GSK-3β(Ser9)蛋白的表达情况;细胞免疫组化染色观察DHA及DHA联合Licl对β-Catenin蛋白细胞内表达及定位的影响。结果:20μg/ml、40μg/ml的DHA分别与5-FU联合应用,可增强5-FU对MDA-MB-231细胞增殖抑制作用。DHA(20μg/ml)能促进5-FU增强MDA-MB-231细胞G0/G1期阻滞作用、降低细胞增殖指数及诱导细胞的凋亡。DHA作用于MDA-MB-231细胞后,β-catenin基因及蛋白表达水平下降(P<0.05)、GSK-3β的基因及蛋白表达水平无明显变化(P>0.05),磷酸化GSK-3β(Ser9)蛋白表达水平下降(P<0.05)。结论:DHA能增强5-FU对MDA-MB-231细胞增殖抑制和诱导凋亡,起化疗增敏作用,可能与其通过抑制GSK-3β磷酸化来阻断Wnt/β-Catenin信号通路有关。

Objective： To study the effect ofdocosahexaenoic（DHA） acid on 5-fluorouracil-induced proliferation and apoptosis in human breast cancer MDA-MB-231 cells and the role of Wnt/13-catenin signaling transduction pathway. Methods： The proliferation of DHA on 5-fluorouracil-induced in human breast cancer MDA-MB-231 cells was examined by MTT assay. The cell cycle arrest and apoptosis of MDA-MB-231 cells were tested by PI staining flow cytometry. The mRNA expression of 13-catenin, GSK-3β in MDA-MB-231 cells were investigated by Reverse transcription polymerase chain reaction （RT-PCR）. The expression and their activity change of 13-catenin, GSK-3β and phospho-GSK-3β （Ser9）protein in MDA-MB-231 cells were investigated by Western blot. The sub- cellular location of 13-catenin protein was investigated by immunocytochemistry tests in DHA and DHA combined with Licl treated cells. Results： 20 ~g/ml, 40 i^g/ml concentrations of DHA combined with 5-fluorouracil significantly increased the cytotoxicity of 5-fluo- rouracil on MDA-MB-231 cells. DHA （20 ~g/ml） promoted MDA-MB-231 cells effect of G0/G 1 phase arrest and apoptosis is induced by 5-fluorouracil, while in combined group the proliferation index of cells was more significantly reduced. In MDA-MB-231 cells treated with DHA in a concentration dependent manner, the expression levels of mRNA3β -catenin, 13-catenin and phospho-GSK-3β （Ser9） pro- rein were decreased（P〈0.05）, mRNA GSK-3β and GSK-3β protein were not significantly changed（P〉0.05）. Conclusions： DHA enhances the effect of growth inhibition and apoptosis induction of 5-fluorouracil on MDA-MB-231 cells. The effect of to 5-fluorouracil of DHA on MDA-MB-231 cells may be associated with inhibition of Wnt/13-catenin signaling pathway through inducing GSK-3β dephosphorylation.