白头翁皂苷PSA对SW480人结直肠癌细胞糖酵解途径关键蛋白及调节因子HIF-1α的影响

Effects of Pulsatilla Saponin A on key proteins and regulatory factor HIF-1α of glycolytic pathway in SW480 human colorectal cancer cells

目的探讨白头翁皂苷PSA对人结直肠癌SW480细胞糖酵解的影响。方法MTT法检测细胞存活率;分光光度试剂盒检测葡萄糖消耗及糖酵解产物;荧光定量PCR法检测己糖激酶II(HKII)、丙酮酸激酶M2(PKM2)和乳酸脱氢酶A(LDHA)以及葡萄糖转运体1(GLUT1)、单羧酸转运体4(MCT4)mRNA表达;流式细胞术检测凋亡;Westernblot法检测低氧诱导因子(HIF 1α)蛋白表达。结果在无血清条件下,白头翁皂苷PSA(1~9μg/mL)能够显著抑制细胞的增殖(P<0.01),降低葡萄糖的消耗、乳酸和ATP的生成(P<0.05,P<0.01),下调HK II、PKM2以及GLUT1 mRNA的表达(P<0.05,P<0.01),诱导凋亡(P<0.01),降低HIF 1α蛋白表达水平(P<0.05)。结论白头翁皂苷PSA能够有效干扰SW480细胞糖酵解途径,其机制可能与抑制HIF 1α蛋白表达,下调HK II、PKM2及GLUT1 mRNA有关。

AIM To investigate the effects of Pulsatilla Saponin A(PSA)on the glycolysis of human colorectal cancer cells SW480.METHODS The following procedures were performed:detection of cell viability by MTT assay,the glucose consumption and glycolysis products by spectrophotometric kit,and the mRNA expres sions of hexokinase II(HKII),pyruvate kinase M2(PKM2),lactate dehydrogenase A(LDHA),glucose trans porter 1(GLUT1)and monocarboxylate transporter 4(MCT4)by fluorescence quantitative PCR.The determina tions of apoptosis by flow cytometry and the expression of hypoxia inducible factor(HIF 1α)by Western blot were practicedaswell.RESULTS Underserum freeconditions,PSA(1μg/mLto9μg/mL)significantlyinhibited cellular proliferation,decreased glucose consumption and the production of lactic acid and ATP(P<0.05,P<0.01),down regulatedtheexpressionofHK II,PKM2andGLUT1(P<0.05,P<0.01),inducedapoptosis(P<0.01),anddecreasedHIF 1αexpression(P<0.05).CONCLUSION PSAcaneffectivelyinterferewith the glycolysis pathway of SW480 cells,induce apoptosis and decrease cell viability.The mechanism may contribute to PSA’s efficacy in the inhibition of HIF 1αprotein expression and the down regulation of HK II,PKM2 and GLUT1.