Mechanisms of Resveratrol Inhibiting Glycolysis in Glioblastoma Cells through G6PC
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摘要:
目的 探讨白藜芦醇（resveratrol，RES）是否通过调控G6PC抑制胶质母细胞瘤（glioblastoma，GBM）细胞的糖酵解过程，并阐明其具体分子机制。 方法 分别用RES处理GBM细胞A172、U87 MG和U118 MG并在细胞中转染sh-NC、sh-G6PC、pcDNA-NC和pcNA-G6PC。通过CCK-8、试剂盒和Annexin V-FITC/PI细胞凋亡检测试剂盒探讨白藜芦醇和G6PC对A172、U87 MG和U118 MG细胞糖酵解的作用；通过Western blot、试剂盒和RT-qPCR检测RES对G6PC表达的调控作用。 结果 在A172、U87 MG和U118 MG胶质母细胞瘤细胞中，RES（20 μM、50 μM和100 μM）处理显著抑制糖酵解（P < 0.05）、降低细胞活力（P < 0.05）并诱导凋亡（P < 0.05）。这些细胞中G6PC表达水平高于人星形胶质细胞（P < 0.001），且RES可下调G6PC蛋白表达（P < 0.01）。敲低G6PC可模拟RES的抗肿瘤效应（抑制糖酵解、降低活力、促进凋亡，P < 0.05），而过表达G6PC则相反。此外，RES能增强敲低G6PC的抑制作用，并逆转过表达G6PC对糖酵解的促进。 结论 RES通过下调G6PC表达，抑制GBM细胞的糖酵解过程，从而抑制细胞增殖并促进细胞凋亡。 Abstract:Objective To investigate whether resveratrol （RES） inhibits glycolysis in glioblastoma （GBM） cells by regulating G6PC and to elucidate the underlying molecular mechanisms. Methods GBM cell lines A172, U87 MG, and U118 MG were treated with RES and transfected with sh-NC, sh-G6PC, pcDNA-NC, and pcDNA-G6PC, respectively. The effects of RES and G6PC on glycolysis, cell viability, and apoptosis in these cells were assessed using CCK-8 assay, specific assay kits, and Annexin V-FITC/PI apoptosis detection kit. Western blot, assay kits, and RT-qPCR were employed to examine the regulatory role of RES on G6PC expression. Results RES treatment （20 μM, 50 μM, and 100 μM） significantly inhibited glycolysis （P < 0.05）, reduced cell viability （P < 0.05）, and induced apoptosis （P < 0.05） in A172, U87 MG, and U118 MG glioblastoma cells. G6PC expression levels in these cells were higher than in human astrocytes （P < 0.001）, and RES downregulated G6PC protein expression （P < 0.01）. G6PC knockdown mimicked the anti-tumor effects of RES （inhibiting glycolysis, reducing viability, and promoting apoptosis, P < 0.05）, while G6PC overexpression showed the opposite effects. Additionally, RES enhanced the inhibitory effects of G6PC knockdown and reversed the glycolysis-promoting effect induced by G6PC overexpression. Conclusion RES inhibits glycolysis in GBM cells by downregulating G6PC expression, thereby suppressing cell proliferation and promoting apoptosis. -
Key words:
- Glioblastoma /
- Resveratrol /
- Glycolysis /
- G6PC /
- Cell viability
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图 1 RES抑制GBM细胞糖酵解（$ \bar x \pm s $，n = 3）
A～C：CCK-8检测A172、U87 MG和U118 MG细胞活力；D～F：试剂盒检测细胞消耗葡萄糖的量；G～I：试剂盒检测细胞生成乳酸的量；J～L：ELISA试剂盒检测细胞中的HK2水平；M～O：ELISA试剂盒检测细胞中的LDHA水平；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 1. RES inhibits glycolysis in GBM cells （$ \bar x \pm s $，n = 3）
图 2 RES诱导GBM细胞凋亡（$ \bar x \pm s $，n = 3）
Annexin V-FITC/PI 检测A172 （A和D）、U87 MG （B和E）和U118 MG （C和F）细胞凋亡率；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 2. RES induces apoptosis in GBM cells （$ \bar x \pm s $，n = 3）
图 3 RES处理抑制GBM细胞中的G6PC表达（$ \bar x \pm s $，n = 3）
A～B：Western blot检测HA和人GBM细胞系A172、U87 MG和U118 MG中G6PC蛋白表达；C～F：Western blot检测RES对G6PC蛋白表达的影响；G～I：试剂盒检测RES对G6PC酶活性的影响；J～L：试剂盒检测RES对G6PC mRNA表达的影响；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 3. RES treatment suppresses G6PC expression in GBM cells （$ \bar x \pm s $，n = 3）
图 4 转染sh-G6PC和RES处理对G6PC表达的影响（$ \bar x \pm s $，n = 3）
A～D：Western blot检测转染sh-G6PC对A172、U87 MG和U118 MG细胞中G6PC表达的作用；E～H：Western blot检测转染sh-G6PC并经RES处理的癌细胞中G6PC表达；I～K：试剂盒检测细胞中的G6PC酶活性；L～N：RT-qPCR检测细胞中的G6PC mRNA相对表达；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 4. Effects of sh-G6PC transfection and RES treatment on G6PC expression （$ \bar x \pm s $，n = 3）
图 5 RES处理和敲低G6PC对GBM细胞糖酵解的影响（$ \bar x \pm s $，n = 3）
A～C：CCK-8试剂盒检测细胞活力；D～F：试剂盒检测细胞中葡萄糖的消耗量；G～I：试剂盒检测细胞中乳酸生成量；J～L：ELISA试剂盒检测细胞中的HK2水平；M～O：ELISA试剂盒检测细胞中的LDHA水平；P～U：Annexin V-FITC/PI凋亡检测试剂盒检测细胞的凋亡率；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 5. Effects of RES treatment and G6PC knockdown on glycolysis in GBM cells （$ \bar x \pm s $，n = 3）
图 6 RES处理和过表达G6PC对GBM细胞糖酵解的影响（$ \bar x \pm s $，n = 3）
A～D：Western blot检测转染pcDNA-G6PC和RES处理对细胞中G6PC表达的影响；E～G：试剂盒检测G6PC酶活性；H～J：RT-qPCR检测G6PC mRNA表达；K～M：CCK-8检测癌细胞活性；N～P：试剂盒检测细胞的葡萄糖消耗量；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 6. Effects of RES treatment and G6PC overexpression on glycolysis in GBM cells （$ \bar x \pm s $，n = 3）
图 7 RES处理和过表达G6PC对GBM细胞凋亡的影响（$ \bar x \pm s $，n = 3）
A～C：试剂盒检测细胞中的乳酸生成量；D～F：试剂盒检测细胞中的HK2水平；G～I：试剂盒检测细胞中的LDHA水平；J～O：Annexin V-FITC/PI试剂盒检测细胞凋亡率；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 7. Effects of RES treatment and G6PC overexpression on apoptosis in GBM cells （$ \bar x \pm s $，n = 3）
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