冬凌草甲素抑制结肠癌细胞HT-29的作用及其机制

Study on the oridonin- mediated inhibition of colon cancer and its mechanism

目的探讨冬凌草甲素抑制结肠癌细胞HT-29的作用及其机制。方法浓度为1—50μmol／L的冬凌草甲素分别作用结肠癌细胞或转染小干扰RNA（siRNA）的结肠癌细胞，噻唑蓝（MTY）法观察冬凌草甲素对结肠癌细胞活力的影响，Western blot检测冬凌草甲素作用的HT-29细胞中腺苷酸活化蛋白激酶α（AMPKα）、磷酸化AMPKα（p-AMPKα）、乙酰辅酶A羧化酶（ACC）、磷酸化ACC（p-ACC）、磷酸化p53（p-p53）、磷酸化S6K（p-s6K）及磷酸化S6（p-S6）的表达。结果冬凌草甲素具有抑制结肠癌细胞活力的作用，其抑制作用呈时间剂量曲线，50μmol／L的冬凌草甲素作用HT-29细胞72h后的细胞活力最低，为21．6％。随着作用时间和剂量的增加，HT-29细胞P—AMPKot、p-ACC和p-p53蛋白表达逐渐增加，p-S6K及P—S6的蛋白表达减弱。转染AMPKotsiRNA的结肠癌细胞活力为（73．3±2．8）％，明显高于转染错义siRNA组的细胞活力[（19．5±1．2）％]，两组间差异有统计学意义（P〈0．05），p-AMPKα、p-ACC、AMPKα和p—p53蛋白表达强于转染错义siRNA组，而p—S6的蛋白表达弱于转染错义siRNA组。结论冬凌草甲索通过AMPKα活化通路抑制HT-29细胞活力。冬凌草甲素抑制结肠癌细胞活力与p-AMPKot、p-p53蛋白激活及p-S6K与p-S6的蛋白抑制相关，p53、S6可能是冬凌草甲素抑制结肠癌细胞p-AMPKα活化通路的下游蛋白。

Objective To investigate the Oridonin - mediated inhibition of colon cancer and its mechanism. Methods After administration of 1 -50 μmol/L Oridonin, the methyl thiazol tetrazolium （MTT） method was used to investigate the cell viability rate by Oridonin on HT- 29 cells and small inter- feting RNA （siRNA） transfected cells. The caspase -3 activity was investigated by Spectrophotometric as- say. The expressions of adenosine monophosphate （AMP） -activated protein kinase α（ AMPKα）, phos- phorylated AMPKα （ p - AMPKα）, acetyl-coA carboxylase-ser 79 （ ACC ） , phosphorylated ACC （p -ACC）, phosphorylated p53 （ p - p53 ）, phosphorylated S6K （ p - S6K） and phosphorylated S6 （ p - S6） proteins were determined by Western blotting. Results Oridonin inhibited the colon cancer cell viability associated with the increased the protein expressions of p - AMPKα, p - ACC and p - 1953 （ P 〈 0.05）. The cell viability inhibited by Oridonin on the AMPKα siRNA transfected colon cancer cells was （73.3 ± 2. 8 ） % , which was more than the expression on the Scramble siRNA transfected colon cancer cells [ （ 19. 5 ± 1.2 ） % , P 〈 0. 05 ]. And the protein expressions for p - AMPKα, p - ACC, AMPKα and p - p53 were increased with the increased concentrations of Oridonin except for p - S6. Conclusion Our study has confirmed the inhibition effects of oridonin on colon cancer. These results indicate that the inhibi- tion of celt viability may be associated with the protein expressions for p - AMPKα and p - p53. The p - p53 and S6 may be the downstream protein of the p - AMPKα protein in the AMPK pathway for the Oridonin - mediated inhibition of colon cancer.