天然二萜衍生物Jar-TTA双重抑制糖酵解及氧化磷酸化诱导食管癌细胞凋亡
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摘要
目的研究二萜衍生物Jar-TTA对糖酵解/氧化磷酸化(OXPHOS)的双重抑制作用,并探讨其抗食管癌机制。方法MTT法检测Jar-TTA对食管癌细胞EC109、KYSE~(-1)50的抗增殖作用;荧光显微镜观察线粒体膜电位(MMP);流式细胞术定量分析细胞凋亡、MMP及葡萄糖摄取;能量代谢分析仪实时检测细胞糖酵解及OXPHOS; Western blot检测蛋白表达。结果 Jar-TTA明显抑制食管癌细胞的增殖,且呈浓度依赖性; 2、4、8μmol·L~(-1)Jar-TTA诱导EC109早期凋亡率分别为(27. 9±6. 1)%、(71. 1±9. 3)%和(65. 0±9. 5)%,与对照组相比,差异均具有显著性(P <0. 01); Jar-TTA明显抑制细胞的糖酵解和OXPHOS;此外,Jar-TTA诱导MMP下降,抑制葡萄糖摄取,同时下调葡萄糖转运蛋白4(GLUT4)及乳酸脱氢酶A(LDHA)表达。结论 Jar-TTA通过双重抑制糖酵解/OXPHOS诱导食管癌细胞凋亡,其机制可能与Jar-TTA降低MMP而干扰线粒体OXPHOS功能,以及下调GLUT4和LDHA而抑制葡萄糖摄取和糖酵解进程有关。
Aim To investigate the effects of Jar-TTA in dual inhibition of glycolysis/oxidative phosphorylation in human esophageal cancer cells and explore the related molecular mechanism. Methods The effect of Jar-TTA on the esophageal cancer cell EC109 and KYSE~(-1)50 viability was examined using MTT assay.The effect of Jar-TTA in apoptosis morphology and mitochondrial membrane potential( MMP) was observed by a fluorescence microscopy. The apoptosis of cell lines treated with Jar-TTA,the quantitative analysis of MMP falling,as well as the glucose uptake was analyzed by flow cytometry. The mitochondrial OXPHOS and glycolysis of EC109 cells in response to Jar-TTA were analyzed using a Seahorse XFp extracellular flux analyzer by real-time measurements of the oxygen consumption rate( OCR,indicative of mitochondrial OXPHOS) and extracellular acidification rate( ECAR,indicative of glycolysis). The expression of the proteins related with glycolysis were detected by Western blot.Results Jar-TTA caused strong antiproliferation in EC109 and KYSE~(-1)50 cells in a concentration-dependent manner. 2,4 and 8 μmol · L~(-1) Jar-TTA treat-ments of EC109 cells for 24 h resulted in a significant increase of early apoptosis population up to( 27. 9 ±6. 1) %,( 71. 1 ± 9. 3) % and( 65. 0 ± 9. 5) %,respectively,compared to control treated cells( 5. 6 ±3. 2) %. The mitochondrial OXPHOS and glycolysis were significantly inhibited in EC109 incubated by 4 and 8 μmol · L~(-1) Jar-TTA for 2 h. In addition,JarTTA induced the drop of MMP. Furthermore,the glucose uptake and the expression of GLUT4 and LDHA were distinctly inhibited in EC109 treated by Jar-TTA.Conclusions Jar-TTA induces apoptosis of human esophageal cancer cells through dual inhibition of glycolysis and oxidative phosphorylation,which is related with the drop of MMP collapse,the decrease of glucose uptake and the down-regulation of GLUT4 and LDHA in EC109 treated by Jar-TTA.
Aim To investigate the effects of Jar-TTA in dual inhibition of glycolysis/oxidative phosphorylation in human esophageal cancer cells and explore the related molecular mechanism. Methods The effect of Jar-TTA on the esophageal cancer cell EC109 and KYSE~(-1)50 viability was examined using MTT assay.The effect of Jar-TTA in apoptosis morphology and mitochondrial membrane potential( MMP) was observed by a fluorescence microscopy. The apoptosis of cell lines treated with Jar-TTA,the quantitative analysis of MMP falling,as well as the glucose uptake was analyzed by flow cytometry. The mitochondrial OXPHOS and glycolysis of EC109 cells in response to Jar-TTA were analyzed using a Seahorse XFp extracellular flux analyzer by real-time measurements of the oxygen consumption rate( OCR,indicative of mitochondrial OXPHOS) and extracellular acidification rate( ECAR,indicative of glycolysis). The expression of the proteins related with glycolysis were detected by Western blot.Results Jar-TTA caused strong antiproliferation in EC109 and KYSE~(-1)50 cells in a concentration-dependent manner. 2,4 and 8 μmol · L~(-1) Jar-TTA treat-ments of EC109 cells for 24 h resulted in a significant increase of early apoptosis population up to( 27. 9 ±6. 1) %,( 71. 1 ± 9. 3) % and( 65. 0 ± 9. 5) %,respectively,compared to control treated cells( 5. 6 ±3. 2) %. The mitochondrial OXPHOS and glycolysis were significantly inhibited in EC109 incubated by 4 and 8 μmol · L~(-1) Jar-TTA for 2 h. In addition,JarTTA induced the drop of MMP. Furthermore,the glucose uptake and the expression of GLUT4 and LDHA were distinctly inhibited in EC109 treated by Jar-TTA.Conclusions Jar-TTA induces apoptosis of human esophageal cancer cells through dual inhibition of glycolysis and oxidative phosphorylation,which is related with the drop of MMP collapse,the decrease of glucose uptake and the down-regulation of GLUT4 and LDHA in EC109 treated by Jar-TTA.
引文
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[2]Ding Y,Ding C Y,Ye N,et al.Discovery and development of natural product oridonin-inspired anticancer agents[J].Eur J Med Chem,2016,122:102-17.
[3]马永成,苏楠,秦玉花,等.新天然化合物Jaridonin通过诱导氧化应激选择性杀伤肿瘤细胞[J].中国药理学通报,2015,31(2):198-203.[3]Ma Y C,Su N,Qin Y H,et al.Jaridonin:Selective killing of cancer cells through oxidative stress[J].Chin Pharmacol Bull,2015,31(2):198-203.
[4]Vander Heiden M G.Targeting cancer metabolism:a therapeutic window opens[J].Nat Rev Drug Discov,2011,10(9):671-84.
[5]Moreno-Sánchez R,Marín-Hernández A,Saavedra E,et al.Who controls the ATP supply in cancer cells?Biochemistry lessons to understand cancer energy metabolism[J].Int J Biochem Cell Biol,2014,50(5):10-23.
[6]Le Bleu V S,O'Connell J T,Gonzalez Herrera K N,et al.PGC-1αmediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis[J].Nat Cell Biol,2014,16(10):992-1003.
[7]马永成,苏楠,石晓静,等.新型对映-贝壳杉烷二萜化合物Jaridonin体外抗肿瘤活性及其作用机制探讨[J].中国药学杂志,2013,48(15):1266-70.[7]Ma Y C,Su N,Shi X J,et al.Bioactivity and mechanism of Jaridonin,a novel ent-kaurene diterpenoid compound,on tumor cells growth in vitro[J].Chin Pharm J,2013,48(15):1266-70.
[8]汪春龙,郭苗苗,吴艺琦,等.生物能量分析仪在肿瘤细胞生物能量代谢中的应用研究[J].中国细胞生物学学报,2016,38(9):1066-76.[8]Wang C L,Guo M M,Wu Y Q,et al.Application of extracellular flux analyzer in the studies of cancer cell metabolism[J].Chin JCell Biol,2016,38(9):1066-76.
[9]Ferreira L M R.Cancer metabolism:the Warburg effect today[J].Exp Mol Pathol,2010,89(3):372-80.
[10]Dwarakanath B S,Singh D,Banerji A K,et al.Clinical studies for improving radiotherapy with 2-deoxy-D-glucose:present status and future prospects[J].J Cancer Res Ther,2009,5(Suppl 1):S21-6.
[11]郑杰.肿瘤能量代谢:糖酵解还是氧化磷酸化[J]?生命科学,2012,24(4):310-5.[11]Zheng J.Energy metabolism of cancer:glycolysis or oxidative phosphorylation[J]?Chin Bull Life Sci,2012,24(4):310-5.
[12]Solaini G,Sgarbi G,Baracca A.Oxidative phosphorylation in cancer cells[J].Biochim Biophys Acta,2011,1807(6):534-42.
[13]Fan L X,Liu C M,Gao A H,et al.Berberine combined with 2-deoxy-D-glucose synergistically enhances cancer cell proliferation inhibition via energy depletion and unfolded protein response disruption[J].Biochim Biophys Acta,2013,1830(11):5175-83.
[14]Cheong J H,Park E S,Liang J Y,et al.Dual inhibition of tumor energy pathway by 2-deoxy glucose and metformin is effective against a broad spectrum of preclinical cancer models[J].Mol Cancer Ther,2011,10(12):2350-62.
[15]Marín-Hernández A,Gallardo-Pérez J C,López-Ramírez S Y,et al.Casiopeina II-gly and bromo-pyruvate inhibition of tumor hexokinase,glycolysis,and oxidative phosphorylation[J].Arch Toxicol,2012,86(5):753-66.
[16]Zhao L J,Zhao X Y,Zhao K,et al.Theα-tocopherol derivative ESeroS-GS induces cell death and inhibits cell motility of breast cancer cells through the regulation of energy metabolism[J].Eur J Pharmacol,2014,745:98-107.
[17]余苏云,刘兆国,贾琦,等.葡萄糖转运蛋白1与肿瘤能量代谢关系的研究进展[J].中国药理学通报,2016,32(7):906-9.[17]Yu S Y,Liu Z G,Jia Q,et al.Research progress on relationship between glucose transporter 1 and tumor energy metabolism[J].Chin Pharmacol Bull,2016,32(7):906-9.
[18]Kohlmann A,Zech S G,Li F,et al.Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors[J].JMed Chem,2013,56(3):1023-40.