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Abstract：
OBJECTIVE: The aim of the present study was to examine whether GLUT1 was involved in the antiproliferative activity of curcumin and doxorubicin by understanding mechanistically how curcumin regulated GLUT1.
METHODS: Expression level of GLUT1 in MCF-7 and MDA-MB-231 cells were quantitated using quantitative real-time PCR and western blot. GLUT1 activity was inhibited in MDA-MB-231 cells with the pharmacological inhibitor WZB117 to assess the anti-proliferative effects of doxorubicin using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). To examine cell proliferation, trypan blue assay was used in cells transfected with GLUT1 siRNA or plasmid overexpressing GLUT1 with doxorubicin and/or commercially available curcumin. The role of PPARδ and Akt on the regulation of GLUT1 by curcumin was examined by overexpressing these proteins and western blot was employed to examine their protein expression.
RESULTS: The data revealed that there was a 1.5 fold increase in GLUT1 mRNA and protein levels in MDA-MB-231 compared to MCF-7. By inhibiting GLUT1 in triple negative breast cancer cell line, MDA-MB-231 with either the pharmacological inhibitor WZB117 or with GLUT1 siRNA, we observed the enhanced antiproliferative effects of doxorubicin. Additional observations indicated these effects can be reversed by the overexpression of GLUT1. Treatment of MDA-MB-231 with curcumin also revealed downregulation of GLUT1, with further growth suppressive effects when combined with doxorubicin. Overexpression of GLUT1 blocked the growth suppressive role of curcumin and doxorubicin (p< 0.05). Mechanistically, we also observed that the regulation of GLUT1 by curcumin was mediated by the Peroxisome proliferator-activated receptor (PPAR) δ/Akt pathway.
CONCLUSIONS: Our study demonstrates that regulation of GLUT1 by curcumin via the PPARδ/Akt signaling improves the efficacy of doxorubicin by promoting its growth inhibitory effects in MDA-MB-231 cells.
METHODS: Expression level of GLUT1 in MCF-7 and MDA-MB-231 cells were quantitated using quantitative real-time PCR and western blot. GLUT1 activity was inhibited in MDA-MB-231 cells with the pharmacological inhibitor WZB117 to assess the anti-proliferative effects of doxorubicin using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). To examine cell proliferation, trypan blue assay was used in cells transfected with GLUT1 siRNA or plasmid overexpressing GLUT1 with doxorubicin and/or commercially available curcumin. The role of PPARδ and Akt on the regulation of GLUT1 by curcumin was examined by overexpressing these proteins and western blot was employed to examine their protein expression.
RESULTS: The data revealed that there was a 1.5 fold increase in GLUT1 mRNA and protein levels in MDA-MB-231 compared to MCF-7. By inhibiting GLUT1 in triple negative breast cancer cell line, MDA-MB-231 with either the pharmacological inhibitor WZB117 or with GLUT1 siRNA, we observed the enhanced antiproliferative effects of doxorubicin. Additional observations indicated these effects can be reversed by the overexpression of GLUT1. Treatment of MDA-MB-231 with curcumin also revealed downregulation of GLUT1, with further growth suppressive effects when combined with doxorubicin. Overexpression of GLUT1 blocked the growth suppressive role of curcumin and doxorubicin (p< 0.05). Mechanistically, we also observed that the regulation of GLUT1 by curcumin was mediated by the Peroxisome proliferator-activated receptor (PPAR) δ/Akt pathway.
CONCLUSIONS: Our study demonstrates that regulation of GLUT1 by curcumin via the PPARδ/Akt signaling improves the efficacy of doxorubicin by promoting its growth inhibitory effects in MDA-MB-231 cells.
摘要：
目的：本研究的目的是通过从机制上了解姜黄素如何调节GLUT1，来检查GLUT1是否参与姜黄素和多柔比星的抗增殖活性。
方法：使用定量实时PCR和蛋白质印迹定量MCF-7和MDA-MB-231细胞中GLUT1的表达水平。用药理学抑制剂WZB117在MDA-MB-231细胞中抑制GLUT1活性，以使用MTT3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）评估阿霉素的抗增殖作用。为了检查细胞增殖，锥虫蓝测定法用于用GLUT1siRNA或使用阿霉素和/或市售姜黄素过表达GLUT1的质粒转染的细胞。通过过表达这些蛋白质来检查PPARδ和Akt对姜黄素调节GLUT1的作用，并采用蛋白质印迹来检查它们的蛋白质表达。
结果：数据显示，与MCF-7相比，MDA-MB-231中的GLUTlmRNA和蛋白质水平增加了1.5倍。通过抑制三阴性乳腺癌细胞系中的GLUT1，MDA-MB-231与药理学抑制剂WZB117或GLUT1siRNA，我们观察到阿霉素的抗增殖作用增强。另外的观察表明这些作用可以通过GLUT1的过表达而逆转。用姜黄素处理MDA-MB-231也显示GLUT1的下调,当与多柔比星组合时具有进一步的生长抑制作用。GLUT1的过表达阻断了姜黄素和阿霉素的生长抑制作用(p<0.05)。机械上,我们还观察到姜黄素对GLUT1的调节是通过过氧化物酶体增殖物激活受体(PPAR)δ/Akt途径介导的。
结论：我们的研究表明，姜黄素通过PPARδ/Akt信号传导对GLUT1的调节通过促进阿霉素在MDA-MB-231细胞中的生长抑制作用来提高其功效。
方法：使用定量实时PCR和蛋白质印迹定量MCF-7和MDA-MB-231细胞中GLUT1的表达水平。用药理学抑制剂WZB117在MDA-MB-231细胞中抑制GLUT1活性，以使用MTT3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）评估阿霉素的抗增殖作用。为了检查细胞增殖，锥虫蓝测定法用于用GLUT1siRNA或使用阿霉素和/或市售姜黄素过表达GLUT1的质粒转染的细胞。通过过表达这些蛋白质来检查PPARδ和Akt对姜黄素调节GLUT1的作用，并采用蛋白质印迹来检查它们的蛋白质表达。
结果：数据显示，与MCF-7相比，MDA-MB-231中的GLUTlmRNA和蛋白质水平增加了1.5倍。通过抑制三阴性乳腺癌细胞系中的GLUT1，MDA-MB-231与药理学抑制剂WZB117或GLUT1siRNA，我们观察到阿霉素的抗增殖作用增强。另外的观察表明这些作用可以通过GLUT1的过表达而逆转。用姜黄素处理MDA-MB-231也显示GLUT1的下调,当与多柔比星组合时具有进一步的生长抑制作用。GLUT1的过表达阻断了姜黄素和阿霉素的生长抑制作用(p<0.05)。机械上,我们还观察到姜黄素对GLUT1的调节是通过过氧化物酶体增殖物激活受体(PPAR)δ/Akt途径介导的。
结论：我们的研究表明，姜黄素通过PPARδ/Akt信号传导对GLUT1的调节通过促进阿霉素在MDA-MB-231细胞中的生长抑制作用来提高其功效。
