HSP90α抑制剂17-AAG抑制甲状腺癌细胞增殖和迁移机制探讨
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摘要
目的在体内,热休克蛋白90α(heat shock protein 90α,HSP90α)对肿瘤的发生发展起着重要的作用。HSP90α抑制剂在体内可阻断肿瘤赖以生存的信号通路网络。本研究探讨HSP90α特异性抑制剂烯丙胺基-17-去甲氧基格尔德(17-allyl-amino-17-demethoxygeldanamycin,17-AAG)对甲状腺癌细胞增殖和迁移的影响及相关信号分子的调控机制。方法体外培养甲状腺乳头状癌细胞TPC-1和甲状腺未分化癌细胞TAK,分别给予0.001～10.000μmol/L浓度的17-AAG作用24和48h。MTT实验、划痕实验观察细胞生长和迁移情况。蛋白质印迹法检测细胞HSP90α及信号传导及转录激活因子(signal transducers and activators of transcription,STAT3)、丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase,AKT)、细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)相关信号蛋白的表达及磷酸化水平变化。结果与未处理的细胞相比,经0.001～10.000μmol/L浓度的17-AAG作用24和48h的TPC-1和TAK细胞增殖能力降低,且有明显的时间和剂量依赖性。TPC-1(F=7.326,P=0.005)和TAK细胞(F=22.770,P<0.001)浓度组间比较差异有统计学意义,时间组间比较差异有统计学意义,F值分别为1 048.000和663.800,均P<0.001。与未处理的细胞相比,经0.1和1.0μmol/L浓度的17-AAG作用24h的TPC-1和TAK细胞迁移能力降低,各浓度组间比较差异有统计学意义,F=1.228,P<0.005;两细胞株间比较差异无统计学意义,F=0.365,P=0.578。经0.1和1.0μmol/L浓度的17-AAG作用24h的TPC-1和TAK细胞以及STAT3、AKT、ERK蛋白磷酸化水平降低,却对STAT3和ERK总蛋白表达影响差异无统计学意义,P>0.05。结论 17-AAG通过下调甲状腺癌细胞HSP90α相关信号蛋白STAT3/AKT/ERK磷酸化水平,从而抑制甲状腺癌细胞的增殖和迁移能力。
OBJECTIVE Heat shock protein 90 alpha plays an important role in tumor development,HSP90αinhibitor can block tumor survival signaling pathway in the body.This study was designed to explore the effects of HSP90αinhibitor 17-AAG on the proliferation and migration of thyroid cancer cell and the underlying signaling mechanisms.METHODS Papillary thyroid cancer cell TPC-1 and undifferentiated thyroid cancer cell TAK were treated with 17-AAG,which was given the specific dose of 0.001-10.000μmol/L for 24 hand 48 hrespectively,MTT experiment and scratching assay were used to detect cell growth and migration.Western blot was used to detected HSP90αand STAT3/AKT/ERK signaling protein expression and phosphorylation level changes.RESULTS Compared with untreated cells,cell proliferation capacity was reduced after 24 hand 48 htreated with the concentration of 0.001-10.000μmol/L of 17-AAG,showing a significant time-and dose-dependent manner,comparison between more concentration groups(F=7.326,P=0.005;F=22.770,P<0.001),and the comparison between two-time groups(F=1 048.000,P<0.001;F=663.800,P<0.001)was statistically significant.TPC-1 and TAK cells migration capacities were reduced in the dose of 0.1μmol/L and1.0μmol/L of 17-AAG after 24 hcompared with untreated cells.Comparison between more concentration groups(F=1.228,P<0.005)was statistically significant,and comparison between two cells(F=0.365,P=0.578)was not statistically significant.Meanwhile,the phosphorylation levels of STAT3/AKT/ERK protein were reduced treated with 0.1 and1.0μmol/L of 17-AAG after 24 hin TPC-1 and TAK cells,but there was no statistical significance of general protein of STAT3 and ERK.CONCLUSION 17-AAG inhibits the proliferation and migration of thyroid cancer cell by reducing the phosphorylation of STAT3/AKT/ERK signaling proteins.
OBJECTIVE Heat shock protein 90 alpha plays an important role in tumor development,HSP90αinhibitor can block tumor survival signaling pathway in the body.This study was designed to explore the effects of HSP90αinhibitor 17-AAG on the proliferation and migration of thyroid cancer cell and the underlying signaling mechanisms.METHODS Papillary thyroid cancer cell TPC-1 and undifferentiated thyroid cancer cell TAK were treated with 17-AAG,which was given the specific dose of 0.001-10.000μmol/L for 24 hand 48 hrespectively,MTT experiment and scratching assay were used to detect cell growth and migration.Western blot was used to detected HSP90αand STAT3/AKT/ERK signaling protein expression and phosphorylation level changes.RESULTS Compared with untreated cells,cell proliferation capacity was reduced after 24 hand 48 htreated with the concentration of 0.001-10.000μmol/L of 17-AAG,showing a significant time-and dose-dependent manner,comparison between more concentration groups(F=7.326,P=0.005;F=22.770,P<0.001),and the comparison between two-time groups(F=1 048.000,P<0.001;F=663.800,P<0.001)was statistically significant.TPC-1 and TAK cells migration capacities were reduced in the dose of 0.1μmol/L and1.0μmol/L of 17-AAG after 24 hcompared with untreated cells.Comparison between more concentration groups(F=1.228,P<0.005)was statistically significant,and comparison between two cells(F=0.365,P=0.578)was not statistically significant.Meanwhile,the phosphorylation levels of STAT3/AKT/ERK protein were reduced treated with 0.1 and1.0μmol/L of 17-AAG after 24 hin TPC-1 and TAK cells,but there was no statistical significance of general protein of STAT3 and ERK.CONCLUSION 17-AAG inhibits the proliferation and migration of thyroid cancer cell by reducing the phosphorylation of STAT3/AKT/ERK signaling proteins.
引文
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[11]Armstrong HK,Gillis JL,Johnson IRD,et al.Dysregulated fibronectin trafficking by hsp90inhibition restricts prostate cancer cell invasion[J].Sci Rep,2018,8(1):2090.
[12]Kosova F,Kasar Z,Tuglu I,et al.Apoptosis of colon cancer cells under the effect of geldanamycin derivate[J].Bratisl Lek Listy,2017,118(5):288-291.
[13]Weber H,Valbuena JR,Barbhuiya MA,et al.Small molecule inhibitor screening identifified hsp90inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer[J].Oncotarget,2017,8(16):26169-26184.
[14]Abu-Elsaad NM,Serrya MS,El-Karef AM,et al.The heat shock protein 90inhibitor,17-aag,attenuates thioacetamide induced liver fibrosis in mice[J].Pharmacol Rep,2016,68(2):275-282.
[15]Woo JK,Jang JE,Kang JH,et al.Lectin,galactoside-binding soluble 3binding protein promotes 17-n-allylamino-17-demethoxygeldanamycin resistance through pi3k/akt pathway in lung cancer cell line[J].Mol Cancer Ther,2017,16(7):1355-1365.
[16]Zhang Z,Zhao J,Mi Z,et al.Effects of salinomycin and 17-AAGon proliferation of human gastric cancer cells in vitro[J].Mol Med Rep,2017,16(2):1063-1070.
[17]王娜娜,李之珩,陶燕芳,等.Hsp90选择性抑制剂17-AAG诱导白血病细胞株凋亡的基因谱研究[J].中国实验血液学杂志,2016,24(3):672-680.
[18]Zhao X,Wang J,Xiao L,et al.Effects of 17-allylamino-17-demethoxygeldanamycin on the induction of apoptosis and cell cycle arrest in hct-116cells[J].Oncol Lett,2017,14(2):2177-2185.
[19]Massimini M,Palmieri C,De MR,et al.17-AAG and apoptosis,autophagy,and mitophagy in canine osteosarcoma cell lines[J].Vet Pathol,2017,54(3):405-412.
[2]刘杨,吴高松,王文斌,等.17-AAG联合紫杉醇对人未分化甲状腺癌FRO细胞增殖和凋亡影响的研究[J].中国普外基础与临床杂志,2016,23(3):292-296.
[3]Armstrong HK,Gillis JL,Ird J,et al.Dysregulated fibronectin trafficking by hsp90inhibition restricts prostate cancer cell invasion[J].Sci Rep,2018,8(1):2090.
[4]Woo JK,Jang JE,Kang JH,et al.Lectin,galactoside-binding soluble 3binding protein promotes 17-n-allylamino-17-demethoxygeldanamycin resistance through pi3k/akt pathway in lung cancer cell line[J].Mol Cancer Ther,2017,16(7):1355-1365.
[5]Joshi SS,Jiang S,Unni E,et al.17-AAG inhibits vemurafenib-associated map kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model[J/CD].PLoS One,2018,13(2):e0191264.
[6]Pontes FSC,De Souza LL,De Jesus AS,et al.Effect of 17-allylamino-17-demethoxygeldanamycin(17-AAG)on akt protein expression is more effective in head and neck cancer cell lineages that retain pten protein expression[J].J Oral Pathol Med,2018,47(3):253-259.
[7]Takashi U,Kazue M,Shin S,et al.The HSP90inhibitor 17-N-allylamino-17-demethoxy geldanamycin(17-AAG)synergizes with cisplatin and induces apoptosis in cisplatin-resistant esophageal squamous cell carcinoma cell lines via the Akt/XIAP pathway[J].Oncol Rep,2014,31(2):619-624.
[8]Lv C,Zeng HW,Wang JX,et al.The antitumor natural product tanshinoneⅡA inhibits protein kinase C and acts synergistically with 17-AAG[J].Cell Death Dis,2018,9(2):165.
[9]Ghadban T,Jessen A,Reeh M,et al.In vitro study comparing the efficacy of the water-soluble HSP90inhibitors,17-AEPGA and17-DMAG,with that of the non water-soluble HSP90inhibitor,17-AAG,in breast cancer cell lines[J].Int J Mol Med,2016,38(4):1296-302.
[10]Sain N,Krishnan B,Ormerod MG,et al.Potentiation of paclitaxel activity by the hsp90inhibitor 17-allylamino-17-demethoxygeldanamycin in human ovarian carcinoma cell lines with high levels of activated akt[J].Mol Cancer Ther,2006,5(5):1197-208.
[11]Armstrong HK,Gillis JL,Johnson IRD,et al.Dysregulated fibronectin trafficking by hsp90inhibition restricts prostate cancer cell invasion[J].Sci Rep,2018,8(1):2090.
[12]Kosova F,Kasar Z,Tuglu I,et al.Apoptosis of colon cancer cells under the effect of geldanamycin derivate[J].Bratisl Lek Listy,2017,118(5):288-291.
[13]Weber H,Valbuena JR,Barbhuiya MA,et al.Small molecule inhibitor screening identifified hsp90inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer[J].Oncotarget,2017,8(16):26169-26184.
[14]Abu-Elsaad NM,Serrya MS,El-Karef AM,et al.The heat shock protein 90inhibitor,17-aag,attenuates thioacetamide induced liver fibrosis in mice[J].Pharmacol Rep,2016,68(2):275-282.
[15]Woo JK,Jang JE,Kang JH,et al.Lectin,galactoside-binding soluble 3binding protein promotes 17-n-allylamino-17-demethoxygeldanamycin resistance through pi3k/akt pathway in lung cancer cell line[J].Mol Cancer Ther,2017,16(7):1355-1365.
[16]Zhang Z,Zhao J,Mi Z,et al.Effects of salinomycin and 17-AAGon proliferation of human gastric cancer cells in vitro[J].Mol Med Rep,2017,16(2):1063-1070.
[17]王娜娜,李之珩,陶燕芳,等.Hsp90选择性抑制剂17-AAG诱导白血病细胞株凋亡的基因谱研究[J].中国实验血液学杂志,2016,24(3):672-680.
[18]Zhao X,Wang J,Xiao L,et al.Effects of 17-allylamino-17-demethoxygeldanamycin on the induction of apoptosis and cell cycle arrest in hct-116cells[J].Oncol Lett,2017,14(2):2177-2185.
[19]Massimini M,Palmieri C,De MR,et al.17-AAG and apoptosis,autophagy,and mitophagy in canine osteosarcoma cell lines[J].Vet Pathol,2017,54(3):405-412.