高糖状态下Nrf2在人甲状腺乳头状癌K1细胞凋亡中的作用及其机制
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摘要
目的研究高糖状态下核转录因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)对人甲状腺乳头状癌K1细胞凋亡的影响,并探讨氧化应激在Nrf2影响K1细胞凋亡中的作用。方法 25 mmol/L高糖条件下,将甲状腺乳头状癌K1细胞分为对照组、Nrf2siRNA组(转染siRNA抑制Nrf2的表达)、NcsiRNA组(阴性转染),培养48 h后,流式细胞仪检测细胞凋亡率,western blot检测Nrf2(核和浆蛋白)及凋亡相关蛋白B淋巴细胞瘤基因2(B-cell lymphoma-2,Bcl-2)和半胱氨酸蛋白酶3(Caspase-3)表达水平,逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测Nrf2mRNA表达水平,相关试剂盒检测活性氧(reactive oxygen species,ROS)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)水平。结果与对照组比较,Nrf2siRNA组K1细胞早期凋亡率和晚期凋亡率明显增加(P均<0.01),K1细胞Nrf2mRNA、核蛋白及浆蛋白表达水平明显下降(P均<0.01),K1细胞抗凋亡相关蛋白Bcl-2水平下降、促凋亡相关蛋白Caspase-3水平增加(P均<0.01),K1细胞ROS水平增加、GSH及SOD水平下降(P均<0.01);NcsiRNA组上述指标与对照组比较均无统计学差异(P均>0.05)。结论高糖状态下,沉默Nrf2的表达可以增强K1细胞氧化应激水平、下调抗凋亡相关蛋白的表达、上调促凋亡相关蛋白的表达,促进K1细胞凋亡,提示Nrf2可能通过抗氧化应激损伤抑制高糖状态下K1细胞的凋亡。
Objective To study the effect of nuclear factor erythroid 2-related factor 2(Nrf2) on the apoptosis of human papillary thyroid carcinoma K1 cells under high glucose environmental conditions, and investigate the role of oxidative stress in the apoptosis of K1 cells regulated by Nrf2. Methods K1 cells were cultured with 25 mmol/L glucose and then divided into three groups:the control group, Nrf2 siRNA group, and NcsiRNA group.After 48 hours of culturing,the apoptosis rate was detected by flow cytometry.The expression levels of Nrf2(nuclear and cytoplasmic protein),the apoptosis-related proteins B-cell lymphoma-2(Bcl-2) and Caspase-3 were detected by western blot. The expression of Nrf2 mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR).The levels of reactive oxygen species(ROS),superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were detected by related kits. Results Compared with control group, the early apoptosis and late apoptosis of K1 cells in Nrf2 siRNA group were significantly increased(all P<0.01),the expression levels of Nrf2 mRNA,nuclear protein and cytoplasmic protein of K1 cells in Nrf2 siRNA group were significantly decreased(all P<0.01),the protein level of Bcl-2 was decreased and Caspase-3 was increased in Nrf2 siRNA group(all P<0.01), the level of ROS was increased and the levels of GSH-Px and SOD were decreased in Nrf2 siRNA group(all P<0.01).All above test indicators had no significant difference between control group and NcsiRNA group(all P>0.05). Conclusion In high glucose state,silencing Nrf2 can enhance K1 cells oxidative stress, down-regulate the expression of anti-apoptosis related proteins and up-regulate the expression of pro-apoptosis related proteins,and can promote the apoptosis of K1 cells.It suggests that Nrf2 may inhibit the apoptosis of K1 cells in the high glucose state through antioxidant stress damage.
Objective To study the effect of nuclear factor erythroid 2-related factor 2(Nrf2) on the apoptosis of human papillary thyroid carcinoma K1 cells under high glucose environmental conditions, and investigate the role of oxidative stress in the apoptosis of K1 cells regulated by Nrf2. Methods K1 cells were cultured with 25 mmol/L glucose and then divided into three groups:the control group, Nrf2 siRNA group, and NcsiRNA group.After 48 hours of culturing,the apoptosis rate was detected by flow cytometry.The expression levels of Nrf2(nuclear and cytoplasmic protein),the apoptosis-related proteins B-cell lymphoma-2(Bcl-2) and Caspase-3 were detected by western blot. The expression of Nrf2 mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR).The levels of reactive oxygen species(ROS),superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were detected by related kits. Results Compared with control group, the early apoptosis and late apoptosis of K1 cells in Nrf2 siRNA group were significantly increased(all P<0.01),the expression levels of Nrf2 mRNA,nuclear protein and cytoplasmic protein of K1 cells in Nrf2 siRNA group were significantly decreased(all P<0.01),the protein level of Bcl-2 was decreased and Caspase-3 was increased in Nrf2 siRNA group(all P<0.01), the level of ROS was increased and the levels of GSH-Px and SOD were decreased in Nrf2 siRNA group(all P<0.01).All above test indicators had no significant difference between control group and NcsiRNA group(all P>0.05). Conclusion In high glucose state,silencing Nrf2 can enhance K1 cells oxidative stress, down-regulate the expression of anti-apoptosis related proteins and up-regulate the expression of pro-apoptosis related proteins,and can promote the apoptosis of K1 cells.It suggests that Nrf2 may inhibit the apoptosis of K1 cells in the high glucose state through antioxidant stress damage.
引文
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[13] Pizzino G,Irrera N,Cucinotta M,et al.Oxidative stress:harms and benefits for human health[J].Oxid Med Cell Longev,2017:8416763
[14] Saha SK,Lee SB,Won J,et al.Correlation between oxidative stress,nutrition,and cancer initiation[J].Int J Mol Sci,2017,18(7):E1544
[15] Chakraborty S,Balan M,Flynn E.Activation of c-Met in cancer cells mediates growth-promoting signals against oxidative stress through Nrf2-HO-1[J].Oncogenesis,2019,8(2):7
[16] Panneer Selvam S,Roth BM,Nganga R,et al.Balance between senescence and apoptosis is regulated by telomere damage-induced association between p16 and caspase-3[J].J Biol Chem,2018,293(25):9784-9800
[17] Warren CFA,Wong-Brown MW,Bowden NA.BCL-2 family isoforms in apoptosis and cancer[J].Cell Death Dis,2019,10(3):177
[18] Khalifeh S,Oryan S,Digaleh H,et al.Involvement of Nrf2 in development of anxiety-like behavior by linking Bcl2 to oxidative phosphorylation:estimation in rat hippocampus,amygdala,and prefrontal cortex[J].J Mol Neurosci,2015,55(2):492-499
[2] Rapp K,Schroeder J,Klenk J,et al.Fasting blood glucose and cancer risk in a cohort of more than 140,000 adults in Austria[J].Diabetologia,2006,49(5):945-952
[3] 倪静,乐岭,向光大,等.甲状腺恶性结节与代谢综合征组分及尿碘的相关性研究[J].华南国防医学杂志,2016,30(11):714-717
[4] Menegon S,Columbano A,Giordano S.The dual roles of NRF2 in cancer[J].Trends Mol Med,2016,22(7):578-593
[5] 倪静,乐岭,王永,等.高糖对甲状腺乳头状癌K1细胞增殖及Nrf2表达和转位影响[J].中华肿瘤防治杂志,2017,24(19):1335-1341
[6] Gao AM,Ke ZP,Wang JN,et al.Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway[J].Carcinogenesis,2013,34(8):1806-1814
[7] Suzuki T,Yamamoto M.Molecular basis of the Keap1-Nrf2 system[J].Free Radic Biol Med,2015,88(PtB):93-100
[8] Leinonen HM,Kansanen E,P?l?nen P,et al.Role of the Keap1-Nrf2 pathway in cancer[J].Adv Cancer Res,2014,122:281-320
[9] Ziros PG,Manolakou SD,Habeos IG,et al.Nrf2 is commonly activated in papillary thyroid carcinoma,and it controls antioxidant transcriptional responses and viability of cancer cells[J].J Clin Endocrinol Metab,2013,98(8):E1422-E1427
[10] Du ZX,Yan Y,Zhang HY,et al.Proteasome inhibition induces a p38 MAPK pathway-dependent antiapoptotic program via Nrf2 in thyroid cancer cells[J].J Clin Endocrinol Metab,2011,96(5):E763-E771
[11] Cha HY,Lee BS,Chang JW,et al.Downregulation of Nrf2 by the combination of TRAIL and Valproic acid induces apoptotic cell death of TRAIL-resistant papillary thyroid cancer cells via suppression of Bcl-xL[J].Cancer Lett,2016,372(1):65-74
[12] Li C,Miao X,Li F,et al.Oxidative stress-related mechanisms and antioxidant therapy in diabetic retinopathy[J].Oxid Med Cell Longev,2017:9702820
[13] Pizzino G,Irrera N,Cucinotta M,et al.Oxidative stress:harms and benefits for human health[J].Oxid Med Cell Longev,2017:8416763
[14] Saha SK,Lee SB,Won J,et al.Correlation between oxidative stress,nutrition,and cancer initiation[J].Int J Mol Sci,2017,18(7):E1544
[15] Chakraborty S,Balan M,Flynn E.Activation of c-Met in cancer cells mediates growth-promoting signals against oxidative stress through Nrf2-HO-1[J].Oncogenesis,2019,8(2):7
[16] Panneer Selvam S,Roth BM,Nganga R,et al.Balance between senescence and apoptosis is regulated by telomere damage-induced association between p16 and caspase-3[J].J Biol Chem,2018,293(25):9784-9800
[17] Warren CFA,Wong-Brown MW,Bowden NA.BCL-2 family isoforms in apoptosis and cancer[J].Cell Death Dis,2019,10(3):177
[18] Khalifeh S,Oryan S,Digaleh H,et al.Involvement of Nrf2 in development of anxiety-like behavior by linking Bcl2 to oxidative phosphorylation:estimation in rat hippocampus,amygdala,and prefrontal cortex[J].J Mol Neurosci,2015,55(2):492-499