拓扑替康通过调控缺氧诱导因子-1α影响U87MG细胞的增殖和迁移
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摘要
目的:探讨拓扑替康(TPT)对神经胶质瘤细胞中的缺氧诱导因子-1(HIF-1α)及其相关信号通路的调控作用。方法:应用Western Blot技术分别检测不同浓度TPT作用下的U87MG细胞内HIF-1α、去乙酰化酶sirtuin1(SIRT1)、上皮转化因子Twist和涉及Warburg效应的下游蛋白己糖激酶Ⅱ(HK-Ⅱ)、乳酸脱氢酶A(LDHA)及丙酮酸脱氢酶激酶1(PDK1)的表达,使用同位素标记法,测定TPT对U87MG细胞葡萄糖摄取、乳酸堆积、脂肪酸β氧化的影响,并测定低浓度的TPT作用下拓扑异构酶Ⅰ活性的变化。最后通过台盼蓝染色实验和细胞划痕实验分别检测拓扑替康对U87MG细胞增殖和迁移能力的影响。结果:以TPT处理U87MG细胞后,SIRT1的表达量增加,HIF-1α表达量减少。当U87MG以TPT处理时,涉及Warburg效应的下游蛋白HK-Ⅱ、LDHA及PDK1的表达量均减少。而且,在U87MG细胞中TPT也减缓了细胞增殖和TWIST蛋白的表达量。结论:拓扑替康可能通过抑制HIF-1α及其下游蛋白HK-Ⅱ、LDHA、PDK1的表达从而减少肿瘤的能量及脂肪酸来源,影响癌细胞的Warburg效应进而抑制癌细胞的增殖。
Objective: To investigate the regulation of Topotecan on hypoxia-inducible factor 1α(HIF-1α)and its related signaling pathway in glioma cells.Methods: Western Blot technique was used to detect the expression level of HIF-1α, sirtuin1(SIRT1), TWIST and the Warburg effect related proteins HK-Ⅱ, LDHA and PDK1 in the U87 MG cells treated with topotecan. The isotope labeling method was used to detect glucose uptake, lactic acid accumulation and fatty acid β-oxidation of U87 MG cells treated with topotecan. And the activity change of topoisomerase Ⅰ treated with low-dose topotecan was determined. Finally, the effects of topotecan on the proliferation and migration of U87 MG cells were detected by trypan blue staining and cell scratching experiments, respectively.Results: After treatment of U87 MG human glioma cells with topotecan, the expression of SIRT1 was increased and the expression of HIF-1α decreased. When U87 MG was treated with topotecan, the expression levels of Warburg effect related proteins HK-Ⅱ, LDHA and PDK1 were reduced. SIRT1 increased and the HIF-1α expression decreased after U87 MG human glioma cells were treated with topotecan. Moreover, topotecan also slowed cell proliferation and TWIST protein expression in U87 MG cells.Conclusion: Topotecan may reduce the energy and fatty acid sources of tumor by inhibiting the expression of HIF-1α and its downstream proteins HK-Ⅱ, LDHA and PDK1, which may affect the Warburg effect and inhibit the proliferation of cancer cells.
Objective: To investigate the regulation of Topotecan on hypoxia-inducible factor 1α(HIF-1α)and its related signaling pathway in glioma cells.Methods: Western Blot technique was used to detect the expression level of HIF-1α, sirtuin1(SIRT1), TWIST and the Warburg effect related proteins HK-Ⅱ, LDHA and PDK1 in the U87 MG cells treated with topotecan. The isotope labeling method was used to detect glucose uptake, lactic acid accumulation and fatty acid β-oxidation of U87 MG cells treated with topotecan. And the activity change of topoisomerase Ⅰ treated with low-dose topotecan was determined. Finally, the effects of topotecan on the proliferation and migration of U87 MG cells were detected by trypan blue staining and cell scratching experiments, respectively.Results: After treatment of U87 MG human glioma cells with topotecan, the expression of SIRT1 was increased and the expression of HIF-1α decreased. When U87 MG was treated with topotecan, the expression levels of Warburg effect related proteins HK-Ⅱ, LDHA and PDK1 were reduced. SIRT1 increased and the HIF-1α expression decreased after U87 MG human glioma cells were treated with topotecan. Moreover, topotecan also slowed cell proliferation and TWIST protein expression in U87 MG cells.Conclusion: Topotecan may reduce the energy and fatty acid sources of tumor by inhibiting the expression of HIF-1α and its downstream proteins HK-Ⅱ, LDHA and PDK1, which may affect the Warburg effect and inhibit the proliferation of cancer cells.
引文
[1] Allemani C, Matsuda T, Di Carlo V, et al. Global surveillance of trends in cancer survival 2000-14(CONCORD-3):analysis of individual records for 37 513 025patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries[J]. Lancet, 2018,391(10 125):1 023-1 075.
[2] Kumar S, Visvanathan A, Arivazhagan A, et al. Assessment of radiation resistance and therapeutic targeting of cancer stem cells:a raman spectroscopic study of glioblastoma[J]. Anal Chem, 2018,90(20):12 067-12 074.
[3] Da Ros M, De Gregorio V, Iorio AL, et al. Glioblastoma chemoresistance:the double play by microenvironment and blood-brain barrier[J]. Int J Mol Sci, 2018,19(10):pii:E2879.
[4] Sasine JP, Savaraj N, Feun LG. Topoisomerase I inhibitors in the treatment of primary CNS malignancies:an update on recent trends[J]. Anticancer Agents Med Chem, 2010,10(9):683-696.
[5] Xue H, Yu Z, Liu Y, et al. Delivery of miR-375 and doxorubicin hydrochloride by lipid-coated hollow mesoporous silica nanoparticles to overcome multiple drug resistance in hepatocellular carcinoma[J]. Int J Nanomedicine, 2017,12:5 271-5 287.
[6] Yu M, Yongzhi H, Chen S, et al. The prognostic value of GLUT1 in cancers:a systematic review and meta-analysis[J]. Oncotarget, 2017,8(26):43 356-43 367.
[7] Tao L, Wei L, Liu Y, et al. Gen-27, a newly synthesized flavonoid, inhibits glycolysis and induces cell apoptosis via suppression of hexokinaseⅡin human breast cancer cells[J]. Biochem Pharmacol, 2017,125:12-25.
[8] Hirschhaeuser F, Sattler UG, Mueller-Klieser W. Lactate:a metabolic key player in cancer[J]. Cancer Res,2011,71(22):6 921-6 925.
[9] Warburg O. On the origin of cancer cells[J]. Science,1956,123(3 191):309-314.
[10] Weidenfeld K, Barkan D. EMT and stemness in tumor dormancy and outgrowth:are they intertwined processes[J]? Front Oncol, 2018,8:381.
[11] Zhang H, Gong J, Kong D, et al. Anti-proliferation effects of Twist gene silencing in gastric cancer SGC7901cells[J]. World J Gastroenterol, 2015, 21(10):2 926-2 936.
[12] Lim JH, Lee YM, Chun YS, et al. Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1alpha[J]. Mol Cell, 2010,38(6):864-878.
[2] Kumar S, Visvanathan A, Arivazhagan A, et al. Assessment of radiation resistance and therapeutic targeting of cancer stem cells:a raman spectroscopic study of glioblastoma[J]. Anal Chem, 2018,90(20):12 067-12 074.
[3] Da Ros M, De Gregorio V, Iorio AL, et al. Glioblastoma chemoresistance:the double play by microenvironment and blood-brain barrier[J]. Int J Mol Sci, 2018,19(10):pii:E2879.
[4] Sasine JP, Savaraj N, Feun LG. Topoisomerase I inhibitors in the treatment of primary CNS malignancies:an update on recent trends[J]. Anticancer Agents Med Chem, 2010,10(9):683-696.
[5] Xue H, Yu Z, Liu Y, et al. Delivery of miR-375 and doxorubicin hydrochloride by lipid-coated hollow mesoporous silica nanoparticles to overcome multiple drug resistance in hepatocellular carcinoma[J]. Int J Nanomedicine, 2017,12:5 271-5 287.
[6] Yu M, Yongzhi H, Chen S, et al. The prognostic value of GLUT1 in cancers:a systematic review and meta-analysis[J]. Oncotarget, 2017,8(26):43 356-43 367.
[7] Tao L, Wei L, Liu Y, et al. Gen-27, a newly synthesized flavonoid, inhibits glycolysis and induces cell apoptosis via suppression of hexokinaseⅡin human breast cancer cells[J]. Biochem Pharmacol, 2017,125:12-25.
[8] Hirschhaeuser F, Sattler UG, Mueller-Klieser W. Lactate:a metabolic key player in cancer[J]. Cancer Res,2011,71(22):6 921-6 925.
[9] Warburg O. On the origin of cancer cells[J]. Science,1956,123(3 191):309-314.
[10] Weidenfeld K, Barkan D. EMT and stemness in tumor dormancy and outgrowth:are they intertwined processes[J]? Front Oncol, 2018,8:381.
[11] Zhang H, Gong J, Kong D, et al. Anti-proliferation effects of Twist gene silencing in gastric cancer SGC7901cells[J]. World J Gastroenterol, 2015, 21(10):2 926-2 936.
[12] Lim JH, Lee YM, Chun YS, et al. Sirtuin 1 modulates cellular responses to hypoxia by deacetylating hypoxia-inducible factor 1alpha[J]. Mol Cell, 2010,38(6):864-878.