SIRT1抑制高良姜素诱导的HepG2细胞凋亡的研究
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摘要
目的高良姜素是一种具有抗肿瘤活性的天然黄酮类化合物,SIRT1是参与许多生理过程的Sirtuin家族蛋白重要成员之一。文中旨在探讨SIRT1在高良姜素诱导Hep G2细胞凋亡过程中的作用。方法实验设置DMSO溶剂对照组、EX-527组(50μmol/L EX-527处理细胞2h)、高良姜素组(高良姜素处理细胞24h)和EX-527+高良姜素组(先用50μmol/L EX-527处理细胞2 h再用高良姜素处理细胞24 h)。用Hochest33342染色法和流式细胞术、Western blot分析细胞凋亡的变化。RNA干扰和转染外源基因调控SIRT1的表达后,130μmol/L高良姜素处理细胞24 h,并设置未感染腺病毒组、载体对照组(感染空载体腺病毒)、SIRT1敲降组(感染敲降SIRT1腺病毒)、未转染质粒组、空载体对照组(转染空载体质粒组)、SIRT1上调组(转染过表达SIRT1质粒),用流式细胞术和Western blot检测细胞凋亡。结果与DMSO溶剂对照组细胞凋亡率、PARP1剪切带与GAPDH灰度比值[(2.49±0.22)%,0.06±0.00]比较,高良姜素组[(11.62±0.55)%,0.89±0.01]、EX-527+高良姜素组[(25.75±0.61)%,1.15±0.06]均升高(P<0.01);其中EX-527+高良姜素组较高良姜素组亦明显升高(P<0.01)。Western blot结果表明,SIRT1敲降组中SIRT1与GAPDH灰度比值(0.06±0.01)较载体对照组(1.11±0.05)和未感染腺病毒组(1.10±0.04)明显减小(P<0.01)。与载体对照组和未感染腺病毒组比较,SIRT1敲降组细胞凋亡率、PARP1剪切带与GAPDH灰度比值均增加(P<0.01)。SIRT1上调组中SIRT1与GAPDH灰度比值(1.63±0.04)较载体对照组(0.89±0.02)和未转染质粒组(0.87±0.03)相比明显增大(P<0.01)。与未转染质粒组和空载体对照组比较,SIRT1上调组细胞凋亡率和SIRT1剪切带与GAPDH灰度比值降低(P<0.01)。结论 SIRT1抑制高良姜素诱导的Hep G2细胞凋亡。
Objective Galangin is a natural flavonoid with antineoplastic activity.SIRT1 is an important member of Sirtuin family which parcitipate in many physiological process.The aim of this study was to investigate the effect of SIRT1 on HepG2 cell apoptosis induced by galangin.Methods HepG2 cells were pre-treated with SIRT1 inhibitor EX-527 for 2 hours,and then galangin for24 hours.DMSO solvent control group,EX-527 treatment group,galangin treatment group and EX-527 and galangin co-treatment group were established.Hoechst 33342 staining,flow cytometry and western blot were performed to detect the apoptosis of HepG2 cells.After regulating the expression of SIRT1 in HepG2 cells with RNA interference and transfection of exogenous genes,these cells were treated with galangin for 24 hours.Negative control group,vector control group,SIRT1 knock down group,blank control group,blank vector group,and SIRT1 upregulation group were established.Western blot and Flow cytometry were performed to detect the apoptosis of HepG2 cells.Results The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of galangin group [(11.62 ±0.55) %,0.89±0.01]and EX-527+ galangin group[(25.75± 0.61) %,1.15± 0.06] were all increased(P < 0.01),when these were compared with DMSO solvent control group[(2.49±0.22) %,0.06±0.00]; and those in EX-527+ galangin group were also markedly increased compared with galangin group(P<0.01).The result of western bolt was that the gray level ratio of PARP1 and GAPDH of SIRT1 knocked down group(0.06±0.01) was markedly decreased compared with vector control group(1.11±0.05) and without adenovirus infection group(1.10±0.04)(P<0.01).The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of SIRT1 knocked down group were markedly increased compared with vector control group and without adenovirus infection group(P <0.01).The gray level ratio of PARP1 and GAPDH of SIRT1 up-regulated group(1.63±0.04) was markedly increased compared with blank control group(0.89±0.02) and without plasmid transfection group(0.87±0.03)(P<0.01).The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of SIRT1 up-regulated group were markedly decreased compared with blank control group and without plasmid transfection group(P<0.01).Conclusion SIRT1 inhibited galangin-induced apoptosis in HepG2 cells.
Objective Galangin is a natural flavonoid with antineoplastic activity.SIRT1 is an important member of Sirtuin family which parcitipate in many physiological process.The aim of this study was to investigate the effect of SIRT1 on HepG2 cell apoptosis induced by galangin.Methods HepG2 cells were pre-treated with SIRT1 inhibitor EX-527 for 2 hours,and then galangin for24 hours.DMSO solvent control group,EX-527 treatment group,galangin treatment group and EX-527 and galangin co-treatment group were established.Hoechst 33342 staining,flow cytometry and western blot were performed to detect the apoptosis of HepG2 cells.After regulating the expression of SIRT1 in HepG2 cells with RNA interference and transfection of exogenous genes,these cells were treated with galangin for 24 hours.Negative control group,vector control group,SIRT1 knock down group,blank control group,blank vector group,and SIRT1 upregulation group were established.Western blot and Flow cytometry were performed to detect the apoptosis of HepG2 cells.Results The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of galangin group [(11.62 ±0.55) %,0.89±0.01]and EX-527+ galangin group[(25.75± 0.61) %,1.15± 0.06] were all increased(P < 0.01),when these were compared with DMSO solvent control group[(2.49±0.22) %,0.06±0.00]; and those in EX-527+ galangin group were also markedly increased compared with galangin group(P<0.01).The result of western bolt was that the gray level ratio of PARP1 and GAPDH of SIRT1 knocked down group(0.06±0.01) was markedly decreased compared with vector control group(1.11±0.05) and without adenovirus infection group(1.10±0.04)(P<0.01).The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of SIRT1 knocked down group were markedly increased compared with vector control group and without adenovirus infection group(P <0.01).The gray level ratio of PARP1 and GAPDH of SIRT1 up-regulated group(1.63±0.04) was markedly increased compared with blank control group(0.89±0.02) and without plasmid transfection group(0.87±0.03)(P<0.01).The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of SIRT1 up-regulated group were markedly decreased compared with blank control group and without plasmid transfection group(P<0.01).Conclusion SIRT1 inhibited galangin-induced apoptosis in HepG2 cells.
引文
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[7]Zhang HT,Wu J,Wen M,et al.Galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-Bid mitochondrial pathway[J].J Asian Nat Prod Res,2012,14(7):626-633.
[8]Zhang HT,Luo H,Wu J,et al.Galangin induces apoptosis of hepatocellular carcinoma cells via the mitochondrial pathway[J].World J Gastroenterol,2010,16(27):3377-3384.
[9]Wen M,Wu J,Luo H,et al.Galangin induces autophagy through upregulation of p53 in Hep G2 cells[J].Pharmacology,2012,89(5-6):247-255.
[10]Su L,Chen X,Wu J,et al.Galangin inhibits proliferation of hepatocellular carcinoma cells by inducing endoplasmic reticulum stress[J].Food Chem Toxicol,2013,62:810-816.
[11]Zhang H,Li N,Wu J,et al.Galangin inhibits proliferation of Hep G2 cells by activating AMPK via increasing the AMP/TAN ratio in a LKB1-independent manner[J].Eur J Pharmacol,2013,718(1-3):235-244.
[12]Wang Y,Wu J,Lin B,et al.Galangin suppresses Hep G2 cell proliferation by activating the TGF-beta receptor/Smad pathway[J].Toxicology,2014,326:9-17.
[13]Li X,Wang Y,Xiong Y,et al.Galangin Induces Autophagy via Deacetylation of LC3 by SIRT1 in Hep G2 Cells[J].Sci Rep,2016,6:30496.
[14]Huang R,Xu Y,Wan W,et al.Deacetylation of nuclear LC3drives autophagy initiation under starvation[J].Mol Cell,2015,57(3):456-466.
[15]Lee IH,Cao L,Mostoslavsky R,et al.A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy[J].Proc Natl Acad Sci USA,2008,105(9):3374-3379.
[16]Haigis MC,Sinclair DA.Mammalian sirtuins:biological insights and disease relevance[J].Annu Rev Pathol,2010,5:253-295.
[17]Ou X,Chae HD,Wang RH,et al.SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation,and embryonic and adult hematopoiesis in the mouse[J].Blood,2011,117(2):440-450.
[18]Chae HD,Broxmeyer HE.SIRT1 deficiency downregulates PTEN/JNK/FOXO1 pathway to block reactive oxygen species-induced apoptosis in mouse embryonic stem cells[J].Stem Cells Dev,2011,20(7):1277-1285.
[19]Liu B,Lei M,Hu T,et al.Inhibitory effects of SRT1720 on the apoptosis of rabbit chondrocytes by activating SIRT1[J].J Huazhong Univ Sci Technolog Med Sci,2016,36(3):350-355.
[20]Gu X,Han D,Chen W,et al.SIRT1-mediated Fox Os pathways protect against apoptosis by promoting autophagy in osteoblast-like MC3T3-E1 cells exposed to sodium fluoride[J].Oncotarget,2016,7(40):65218-65230.
[21]Chung S,Yao H,Caito S,et al.Regulation of SIRT1 in cellular functions role of polyphenols[J].Arch Biochem Biophys,2010,501(1):79-90.
[22]Tian Z,Jiang H,Liu Y,et al.MicroRNA-133b inhibits hepatocellular carcinoma cell progression by targeting Sirt1[J].Exp Cell Res,2016,343(2):135-147.
[23]Feng S,Li M,Zhang J,et al.Regulation of Hep G2 cell apoptosis by hepatitis C virus(HCV)core protein via the sirt1-p53-bax pathway[J].Virus Genes,2015,51(3):338-346.
[24]Chen WY,Wang DH,Yen RC,et al.Tumor suppressor HIC1directly regulates SIRT1 to modulate p53-dependent DNA-damage responses[J].Cell,2005,123(3):437-448.
[25]Ikenoue T,Inoki K,Zhao B,et al.PTEN acetylation modulates its interaction with PDZ domain[J].Cancer Res,2008,68(17):6908-6912.
[26]Sundaresan NR,Pillai VB,Wolfgeher D,et al.The deacetylase SIRT1 promotes membrane localization and activation of Akt and PDK1 during tumorigenesis and cardiac hypertrophy[J].Sci Signal,2011,4(182):ra46.
[27]Orecchia A,Scarponi C,Di Felice F,et al.Sirtinol treatment reduces inflammation in human dermal microvascular endothelial cells[J].PLo S One,2011,6(9):e24307.
[28]Eades G,Yao Y,Yang M,et al.miR-200a regulates SIRT1 expression and epithelial to mesenchymal transition(EMT)-like transformation in mammary epithelial cells[J].J Biol Chem,2011,286(29):25992-26002.
[29]Zhang Y,Zhang M,Dong H,et al.Deacetylation of cortactin by SIRT1 promotes cell migration[J].Oncogene,2009,28(3):445-460.
[30]Chen HC,Jeng YM,Yuan RH,et al.SIRT1 promotes tumorigenesis and resistance to chemotherapy in hepatocellular carcinoma and its expression predicts poor prognosis[J].Ann Surg Oncol,2012,19(6):2011-2019.
[31]Yeung F,Hoberg JE,Ramsey CS,et al.Modulation of NF-kappa B-dependent transcription and cell survival by the SIRT1deacetylase[J].EMBO J,2004,23(12):2369-2380.
[32]Nie Y,Erion DM,Yuan Z,et al.STAT3 inhibition of gluconeogenesis is downregulated by Sir T1[J].Nat Cell Biol,2009,11(4):492-500.
[33]Gao Z,Ye J.Inhibition of transcriptional activity of c-JUN by SIRT1[J].Biochem Biophys Res Commun,2008,376(4):793-796.
[34]Firestein R,Blander G,Michan S,et al.The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth[J].PLo S One,2008,3(4):e2020.
[35]Oberdoerffer P,Michan S,Mc Vay M,et al.SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging[J].Cell,2008,135(5):907-918.
[36]Palacios JA,Herranz D,De Bonis ML,et al.SIRT1 contributes to telomere maintenance and augments global homologous recombination[J].J Cell Biol,2010,191(7):1299-1313.
[37]孙鹏,于红,张文卿,等.血管内皮生长因子小干扰RNA诱导胃癌细胞凋亡机制的研究[J].医学研究生学报,2011,24(4):350-353.
[38]冯安宁,樊祥山,黄勤,等,沉默信息调节因子1在肿瘤形成中的作用机制[J].医学研究生学报,2009,22(7):767-769.
[39]Howitz KT,Bitterman KJ,Cohen HY,et al.Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[J].Nature,2003,425(6954):191-196.
[40]Kume S,Haneda M,Kanasaki K,et al.Silent inform ation regulator 2(SIRT1)attenuates oxidative stress-induced m esangia1cell apoptosis via p53 deacetylation[J].Free Radic Biol Med,2006,40(12):2175-2182.
[41]Rodgers JT,Lerin C,Haas W,et al.Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1[J].Nature,2005,434(7029):113-118.
[42]Hughes KJ,Meares GP,Hansen PA,et al.FOXO1 and SIRT1regulate beta-cell responses to nitric oxide[J].J Biol Chem,2011,286(10):8338-8348.
[2]黎旭,熊禹真,汪亚君,等.数字全息技术检测高良姜素诱导的Hep G2细胞凋亡[J].医学研究生学报,2016,29(8):790-795.
[3]Phang JM,Poore CM,Lopaczynska J,et al.Flavonol-stimulated efflux of 7,12-dimethylbenz(a)anthracene in multidrug-resistant breast cancer cells[J].Cancer Res,1993,53(24):5977-5981.
[4]Bestwick CS,Milne L.Influence of galangin on HL-60 cell proliferation and survival[J].Cancer Lett,2006,243(1):80-89.
[5]王竹君,张强,赵新淮.高良姜素对人食管鳞癌KYSE-510细胞的抑制作用[J].中国生物化学与分子生物学报,2009,25(6):563-569.
[6]Otake Y,Walle T.Oxidation of the flavonoids galangin and kaempferide by human liver microsomes and CYP1A1,CYP1A2,and CYP2C9[J].Drug Metab Dispos,2002,30(2):103-105.
[7]Zhang HT,Wu J,Wen M,et al.Galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-Bid mitochondrial pathway[J].J Asian Nat Prod Res,2012,14(7):626-633.
[8]Zhang HT,Luo H,Wu J,et al.Galangin induces apoptosis of hepatocellular carcinoma cells via the mitochondrial pathway[J].World J Gastroenterol,2010,16(27):3377-3384.
[9]Wen M,Wu J,Luo H,et al.Galangin induces autophagy through upregulation of p53 in Hep G2 cells[J].Pharmacology,2012,89(5-6):247-255.
[10]Su L,Chen X,Wu J,et al.Galangin inhibits proliferation of hepatocellular carcinoma cells by inducing endoplasmic reticulum stress[J].Food Chem Toxicol,2013,62:810-816.
[11]Zhang H,Li N,Wu J,et al.Galangin inhibits proliferation of Hep G2 cells by activating AMPK via increasing the AMP/TAN ratio in a LKB1-independent manner[J].Eur J Pharmacol,2013,718(1-3):235-244.
[12]Wang Y,Wu J,Lin B,et al.Galangin suppresses Hep G2 cell proliferation by activating the TGF-beta receptor/Smad pathway[J].Toxicology,2014,326:9-17.
[13]Li X,Wang Y,Xiong Y,et al.Galangin Induces Autophagy via Deacetylation of LC3 by SIRT1 in Hep G2 Cells[J].Sci Rep,2016,6:30496.
[14]Huang R,Xu Y,Wan W,et al.Deacetylation of nuclear LC3drives autophagy initiation under starvation[J].Mol Cell,2015,57(3):456-466.
[15]Lee IH,Cao L,Mostoslavsky R,et al.A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy[J].Proc Natl Acad Sci USA,2008,105(9):3374-3379.
[16]Haigis MC,Sinclair DA.Mammalian sirtuins:biological insights and disease relevance[J].Annu Rev Pathol,2010,5:253-295.
[17]Ou X,Chae HD,Wang RH,et al.SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation,and embryonic and adult hematopoiesis in the mouse[J].Blood,2011,117(2):440-450.
[18]Chae HD,Broxmeyer HE.SIRT1 deficiency downregulates PTEN/JNK/FOXO1 pathway to block reactive oxygen species-induced apoptosis in mouse embryonic stem cells[J].Stem Cells Dev,2011,20(7):1277-1285.
[19]Liu B,Lei M,Hu T,et al.Inhibitory effects of SRT1720 on the apoptosis of rabbit chondrocytes by activating SIRT1[J].J Huazhong Univ Sci Technolog Med Sci,2016,36(3):350-355.
[20]Gu X,Han D,Chen W,et al.SIRT1-mediated Fox Os pathways protect against apoptosis by promoting autophagy in osteoblast-like MC3T3-E1 cells exposed to sodium fluoride[J].Oncotarget,2016,7(40):65218-65230.
[21]Chung S,Yao H,Caito S,et al.Regulation of SIRT1 in cellular functions role of polyphenols[J].Arch Biochem Biophys,2010,501(1):79-90.
[22]Tian Z,Jiang H,Liu Y,et al.MicroRNA-133b inhibits hepatocellular carcinoma cell progression by targeting Sirt1[J].Exp Cell Res,2016,343(2):135-147.
[23]Feng S,Li M,Zhang J,et al.Regulation of Hep G2 cell apoptosis by hepatitis C virus(HCV)core protein via the sirt1-p53-bax pathway[J].Virus Genes,2015,51(3):338-346.
[24]Chen WY,Wang DH,Yen RC,et al.Tumor suppressor HIC1directly regulates SIRT1 to modulate p53-dependent DNA-damage responses[J].Cell,2005,123(3):437-448.
[25]Ikenoue T,Inoki K,Zhao B,et al.PTEN acetylation modulates its interaction with PDZ domain[J].Cancer Res,2008,68(17):6908-6912.
[26]Sundaresan NR,Pillai VB,Wolfgeher D,et al.The deacetylase SIRT1 promotes membrane localization and activation of Akt and PDK1 during tumorigenesis and cardiac hypertrophy[J].Sci Signal,2011,4(182):ra46.
[27]Orecchia A,Scarponi C,Di Felice F,et al.Sirtinol treatment reduces inflammation in human dermal microvascular endothelial cells[J].PLo S One,2011,6(9):e24307.
[28]Eades G,Yao Y,Yang M,et al.miR-200a regulates SIRT1 expression and epithelial to mesenchymal transition(EMT)-like transformation in mammary epithelial cells[J].J Biol Chem,2011,286(29):25992-26002.
[29]Zhang Y,Zhang M,Dong H,et al.Deacetylation of cortactin by SIRT1 promotes cell migration[J].Oncogene,2009,28(3):445-460.
[30]Chen HC,Jeng YM,Yuan RH,et al.SIRT1 promotes tumorigenesis and resistance to chemotherapy in hepatocellular carcinoma and its expression predicts poor prognosis[J].Ann Surg Oncol,2012,19(6):2011-2019.
[31]Yeung F,Hoberg JE,Ramsey CS,et al.Modulation of NF-kappa B-dependent transcription and cell survival by the SIRT1deacetylase[J].EMBO J,2004,23(12):2369-2380.
[32]Nie Y,Erion DM,Yuan Z,et al.STAT3 inhibition of gluconeogenesis is downregulated by Sir T1[J].Nat Cell Biol,2009,11(4):492-500.
[33]Gao Z,Ye J.Inhibition of transcriptional activity of c-JUN by SIRT1[J].Biochem Biophys Res Commun,2008,376(4):793-796.
[34]Firestein R,Blander G,Michan S,et al.The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth[J].PLo S One,2008,3(4):e2020.
[35]Oberdoerffer P,Michan S,Mc Vay M,et al.SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging[J].Cell,2008,135(5):907-918.
[36]Palacios JA,Herranz D,De Bonis ML,et al.SIRT1 contributes to telomere maintenance and augments global homologous recombination[J].J Cell Biol,2010,191(7):1299-1313.
[37]孙鹏,于红,张文卿,等.血管内皮生长因子小干扰RNA诱导胃癌细胞凋亡机制的研究[J].医学研究生学报,2011,24(4):350-353.
[38]冯安宁,樊祥山,黄勤,等,沉默信息调节因子1在肿瘤形成中的作用机制[J].医学研究生学报,2009,22(7):767-769.
[39]Howitz KT,Bitterman KJ,Cohen HY,et al.Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[J].Nature,2003,425(6954):191-196.
[40]Kume S,Haneda M,Kanasaki K,et al.Silent inform ation regulator 2(SIRT1)attenuates oxidative stress-induced m esangia1cell apoptosis via p53 deacetylation[J].Free Radic Biol Med,2006,40(12):2175-2182.
[41]Rodgers JT,Lerin C,Haas W,et al.Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1[J].Nature,2005,434(7029):113-118.
[42]Hughes KJ,Meares GP,Hansen PA,et al.FOXO1 and SIRT1regulate beta-cell responses to nitric oxide[J].J Biol Chem,2011,286(10):8338-8348.