GLUT1和 PKM2在二甲双胍抑制人乳头状甲状腺癌细胞增殖中的作用

摘要

目的：观察不同浓度二甲双胍对人乳头状甲状腺癌（papillary thyroid cancer，PTC）细胞增殖、凋亡的影响，检测细胞中葡萄糖转运关键蛋白—葡萄糖转运体1（ GLUT1）和糖酵解关键酶—M2型丙酮酸激酶（PKM2）表达水平的变化。方法：体外培养人 PTC 细胞 BCPAP 和 K1，分为对照组和不同浓度二甲双胍（1、5和10mmol/ L）处理组，并分别培养24h、48h 和72h。四甲基偶氮唑蓝（MTT）法检测细胞增殖，Annexin V -FITC/ PI 流式细胞术检测细胞凋亡，实时定量 PCR 检测 GLUT1和 PKM2 mRNA 的表达，Western blot 检测GLUT1和 PKM2蛋白的表达。结果：与对照组相比，二甲双胍抑制 PTC 细胞增殖，随浓度及处理时间的增加，抑制效应加大，呈剂量-时间依赖性（P ﹤0.05）。二甲双胍诱导 PTC 细胞凋亡，随浓度及处理时间的增加，其中晚期凋亡率明显增加，呈剂量-时间依赖性（P ﹤0.05）。二甲双胍可抑制 PTC 细胞中 GLUT1和 PKM2 mRNA 的表达水平，呈剂量-时间依赖性（P ﹤0.05）。二甲双胍可抑制 PTC 细胞中 GLUT1和 PKM2蛋白的表达水平，呈时间依赖性。结论：二甲双胍抑制人 PTC 细胞增殖并诱导凋亡，其机制之一可能是抑制葡萄糖转运关键蛋白和糖酵解关键酶的表达，负面影响癌细胞生长的能量供应。%Objective:To investigate the effects of different concentration of metformin on the proliferation and ap-optosis of papillary thyroid cancer(PTC),and to determine whether metformin causes alterations of both GLUT1 and PKM2 levels. Methods:After treating with metformin(0,1,5,10 mmol/ L),the viability of PTC cell lines(BCPAP, K1)was analyzed by 3 -(4,5 - dimethyl - thiazol - 2 - yl)- 2,5 - diphenyltetrazolium bromide(MTT). Cell cycle analysis was performed by Annexin V - FITC/ PI flow cytometry,real - time polymerase chain reaction and Western blot were performed to determine the expression of GLUT1 and PKM2. Results:Compared with control group,MTT as-says showed that the proliferation of PTC cell lines was inhibited in a dose - time dependent manner after treating with metformin(P ﹤ 0. 05). Moreover,metformin induced the late apoptosis of the PTC cell lines(P ﹤ 0. 05). The relative mRNA levels of GLUT1 and PKM2 were inhibited in a dose - time dependent manner by metformin(P ﹤ 0. 05). The protein expression of GLUT1 and PKM2 in PTC cells was decreased by metformin in a time dependent manner. Con-clusion:Metformin provides an antitumoral effect in PTC cells. This effect may be mediated through a decrease of GLUT1 and PKM2 expression which leading to an energy crisis in cancer cells.