白藜芦醇通过调控雷帕霉素靶蛋白/M2型丙酮酸激酶轴抑制口腔鳞状细胞癌细胞的糖酵解作用
|
摘要
目的:通过雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)/M2型丙酮酸激酶(M2 isoform pyruvate kinase,PKM2)轴探讨白藜芦醇对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞的糖酵解的影响。方法:培养CAL-27和SCC-15细胞,采用不同浓度白藜芦醇(0、50、100、200、400、800μmol/L)分别处理细胞24 h,噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测其对细胞增殖的影响并计算半数抑制浓度(half maximal inhibitory concentration,IC_(50))值。选用250μmol/L白藜芦醇或10 nmol/L mTOR抑制剂Rapamycin干预细胞24 h,采用2-脱氧葡萄糖类似物[(2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose,2-NBDG]法检测细胞葡萄糖摄取率;比色法检测细胞上清中乳酸含量、细胞内糖酵解关键酶己糖激酶(hexokinase,HK)和丙酮酸激酶(pyruvate kinase,PK)活性;Western blot检测细胞中己糖激酶2(hexokinase2,HK2)、PKM2、mTOR和磷酸化mTOR(phosphorylation mTOR,p-mTOR)等蛋白表达情况。结果:白藜芦醇对两株OSCC细胞的增殖有明显的抑制作用(P<0.05),并呈现浓度依赖性。白藜芦醇可显著抑制两株OSCC细胞的葡萄糖摄取能力以及降低乳酸的产生量(P<0.05),并可抑制两株细胞PK活性(P<0.05)、下调PKM2蛋白的表达(P<0.05),但对HK活性及HK2蛋白的表达无显著影响(P>0.05)。同时,白藜芦醇干预还可显著降低细胞内p-mTOR水平(P<0.05)。另外,Rapamycin干预也可抑制两株细胞的葡萄糖摄取能力、降低细胞上清中乳酸的产生量和细胞内p-mTOR水平,同时也可下调PKM2蛋白的表达。结论:白藜芦醇可抑制OSCC细胞的糖酵解作用,其机制可能与抑制mTOR/PKM2轴通路相关。
Objective: To investigate the effect resveratrol on glycolysis of oral squamous cell carcinoma(OSCC) by regulating mTOR/PKM2 axis. Methods: OSCC cell lines CAL-27 and SCC-15 were cultured and treated with different concentrations of resveratrol(0 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L, and 800 μmol/L) for 24 h. Cell proliferation was evaluated by MTT assay, and the value of IC_(50) was calculated. OSCC cells were treated with 250 μmol/L resveratrol or 10 nmol/L rapamycin(mTOR inhibitor) for 24 h, and then the cellular glucose uptake was assessed by 2-NBDG staining. Lactate production in the cell supernatant and the activity of hexokinase and pyruvate kinase in the cells were detected using colorimetric method. The proteins expression of hexokinase-2(HK2), M2 isoform pyruvate kinase(PKM2), mTOR, and p-mTOR in the cells were analyzed by Western blotting. Results: Resveratrol had an obvious inhibitory effect on the proliferation of CAL-27 and SCC-15 cells in a concentration dependent manner(P<0.05). Resveratrol intervention significantly inhibited the glucose uptake and decreased the production of lactate in both OSCC cells(P<0.05). It also significantly inhibited the activity of pyruvate kinase and decreased the protein expression of PKM2 in CAL-27 and SCC-15 cells(P<0.05). However, it had no significant effect on the activity of hexokinase and the protein expression of HK-2(P>0.05). At the same time,resveratrol intervention also significantly decreased the phosphorylation level of mTOR(P <0.05).In addition,rapamycin intervention could also inhibit glucose uptake,decrease the production of lactate and the phosphorylation level of mTOR,and down-regulate the protein expression of PKM2 in CAL-27 and SCC-15 cells.Conclusion:Resveratrol inhibits the glycolysis of OSCC cells,and its mechanism might relate to the inhibition of the mTOR/PKM2 axis pathway.
Objective: To investigate the effect resveratrol on glycolysis of oral squamous cell carcinoma(OSCC) by regulating mTOR/PKM2 axis. Methods: OSCC cell lines CAL-27 and SCC-15 were cultured and treated with different concentrations of resveratrol(0 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L, and 800 μmol/L) for 24 h. Cell proliferation was evaluated by MTT assay, and the value of IC_(50) was calculated. OSCC cells were treated with 250 μmol/L resveratrol or 10 nmol/L rapamycin(mTOR inhibitor) for 24 h, and then the cellular glucose uptake was assessed by 2-NBDG staining. Lactate production in the cell supernatant and the activity of hexokinase and pyruvate kinase in the cells were detected using colorimetric method. The proteins expression of hexokinase-2(HK2), M2 isoform pyruvate kinase(PKM2), mTOR, and p-mTOR in the cells were analyzed by Western blotting. Results: Resveratrol had an obvious inhibitory effect on the proliferation of CAL-27 and SCC-15 cells in a concentration dependent manner(P<0.05). Resveratrol intervention significantly inhibited the glucose uptake and decreased the production of lactate in both OSCC cells(P<0.05). It also significantly inhibited the activity of pyruvate kinase and decreased the protein expression of PKM2 in CAL-27 and SCC-15 cells(P<0.05). However, it had no significant effect on the activity of hexokinase and the protein expression of HK-2(P>0.05). At the same time,resveratrol intervention also significantly decreased the phosphorylation level of mTOR(P <0.05).In addition,rapamycin intervention could also inhibit glucose uptake,decrease the production of lactate and the phosphorylation level of mTOR,and down-regulate the protein expression of PKM2 in CAL-27 and SCC-15 cells.Conclusion:Resveratrol inhibits the glycolysis of OSCC cells,and its mechanism might relate to the inhibition of the mTOR/PKM2 axis pathway.
引文
[1] 王倩,侯大为.口腔鳞状细胞癌发病及转移机制研究进展[J].口腔医学研究,2018,34(11):1164-1167.
[2] Allemani C,Matsuda T,Di CV,et al.Global surveillance of trends in cancer survival 2000-14 (CONCORD-3):analysis of individual records for 37,513,025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries [J].Lancet,2018,391(10125):1023-1075.
[3] Shahruzaman SH,Fakurazi S,Maniam S.Targeting energy metabolism to eliminate cancer cells [J].Cancer Manag Res,2018,10:2325-2335.
[4] Diaz-Ruiz R,Rigoulet M,Devin A.The Warburg and Crabtree effects:On the origin of cancer cell energy metabolism and of yeast glucose repression [J].Biochim Biophys Acta,2011,1807(6):568-576.
[5] Alayev A,Berger SM,Holz MK.Resveratrol as a novel treatment for diseases with mTOR pathway hyperactivation [J].Ann N Y Acad Sci,2015,1348(1):116-123.
[6] Jiang S,Zou Z,Nie P,et al.Synergistic effects between mTOR complex 1/2 and glycolysis inhibitors in non-small-cell lung carcinoma cells [J].PLoS One,2015,10 (7):e0132880.
[7] Yu L,Chen X,Sun X,et al.The glycolytic switch in tumors:how many players are involved [J].J Cancer,2017,8(17):3430-3440.
[8] Abd El-Hafez YG,Moustafa HM,Khalil HF,et al.Total lesion glycolysis:a possible new prognostic parameter in oral cavity squamous cell carcinoma [J].Oral Oncol,2013,49(3):261-268.
[9] Dong G,Mao Q,Xia W,et al.PKM2 and cancer:The function of PKM2 beyond glycolysis [J].Oncol Lett,2016,11(3):1980-1986.
[10] He XY,Yin YR,Shi SS,et al.The mTOR pathway regulates PKM2 to affect glycolysis in esophageal squamous cell carcinoma [J].Technol Cancer Res Treat,2018,17:1-10.
[11] Cheng KY,Hao M.Mammalian target of rapamycin (mTOR) regulates transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition via decreased pyruvate kinase M2 (PKM2) expression in cervical cancer cells [J].Med Sci Moni,2017,23:2017-2028.
[2] Allemani C,Matsuda T,Di CV,et al.Global surveillance of trends in cancer survival 2000-14 (CONCORD-3):analysis of individual records for 37,513,025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries [J].Lancet,2018,391(10125):1023-1075.
[3] Shahruzaman SH,Fakurazi S,Maniam S.Targeting energy metabolism to eliminate cancer cells [J].Cancer Manag Res,2018,10:2325-2335.
[4] Diaz-Ruiz R,Rigoulet M,Devin A.The Warburg and Crabtree effects:On the origin of cancer cell energy metabolism and of yeast glucose repression [J].Biochim Biophys Acta,2011,1807(6):568-576.
[5] Alayev A,Berger SM,Holz MK.Resveratrol as a novel treatment for diseases with mTOR pathway hyperactivation [J].Ann N Y Acad Sci,2015,1348(1):116-123.
[6] Jiang S,Zou Z,Nie P,et al.Synergistic effects between mTOR complex 1/2 and glycolysis inhibitors in non-small-cell lung carcinoma cells [J].PLoS One,2015,10 (7):e0132880.
[7] Yu L,Chen X,Sun X,et al.The glycolytic switch in tumors:how many players are involved [J].J Cancer,2017,8(17):3430-3440.
[8] Abd El-Hafez YG,Moustafa HM,Khalil HF,et al.Total lesion glycolysis:a possible new prognostic parameter in oral cavity squamous cell carcinoma [J].Oral Oncol,2013,49(3):261-268.
[9] Dong G,Mao Q,Xia W,et al.PKM2 and cancer:The function of PKM2 beyond glycolysis [J].Oncol Lett,2016,11(3):1980-1986.
[10] He XY,Yin YR,Shi SS,et al.The mTOR pathway regulates PKM2 to affect glycolysis in esophageal squamous cell carcinoma [J].Technol Cancer Res Treat,2018,17:1-10.
[11] Cheng KY,Hao M.Mammalian target of rapamycin (mTOR) regulates transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition via decreased pyruvate kinase M2 (PKM2) expression in cervical cancer cells [J].Med Sci Moni,2017,23:2017-2028.