目的:研究哺乳动物雷帕霉素(mammalian target of rapamycin,mTOR)新型小分子抑制剂Torin-2对人小细胞肺癌NCI-H446细胞增殖及凋亡的影响并探讨其机制。方法:体外培养并取对数生长期人小细胞肺癌NCI-H446细胞,分别给予不同浓度Torin-2或...目的:研究哺乳动物雷帕霉素(mammalian target of rapamycin,mTOR)新型小分子抑制剂Torin-2对人小细胞肺癌NCI-H446细胞增殖及凋亡的影响并探讨其机制。方法:体外培养并取对数生长期人小细胞肺癌NCI-H446细胞,分别给予不同浓度Torin-2或1μmol/L处理不同时间,然后于镜下观察细胞离巢凋亡,通过克隆形成实验检验细胞生长抑制,采用MTS比色法测定吸光度值并计算相对细胞活性,流式细胞术检测细胞周期,免疫印迹法检测mTOR、p-mTOR(Ser2448)、p-Akt(Ser473)、Cyclin-B1、Cyclin-D1、Cyclin-E1、Cleaved Caspase-3等相关蛋白表达。结果:与对照组相比,Torin-2可有效抑制小细胞肺癌NCI-H446细胞增殖及相对细胞活性(P<0.05);诱导细胞周期阻滞(P<0.05);抑制p-mTOR、p-Akt磷酸化激活,降低Cyclin-D1、Cyclin-E1等细胞周期蛋白表达并诱导Cleaved Caspase-3蛋白激酶切割激活。结论:Torin-2可能通过抑制Akt-mTOR相关信号通路和激活凋亡相关蛋白诱导人小细胞肺癌NCI-H446细胞增殖抑制并促进其凋亡。展开更多
Background:Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma(HCC).However,resistance to sorafenib...Background:Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma(HCC).However,resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib.As the activation of AKT/mTOR played a pivotal role in sorafenib resistance,we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells.Methods:The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines.The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method.Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting.Results:Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells,which underscores the significance of AKT activation.Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction.Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis,leading to the dephosphorylation of Ser136 in BAD protein,and increased the expression of total BAD,which contributed to the apoptosis in sorafenibresistant HCC cells.Conclusions:In this study,Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenibresistant HCC cells,via the suppression of mTORC2-AKT-BAD pathway.Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.展开更多
Torin 1 is an ATP-competitive TOR inhibitor which inhibits the signaling of TOR and S6K kinase in mammals and plants.The objective of this research is to determine the effect of Torin 1 in a relatively simple and homo...Torin 1 is an ATP-competitive TOR inhibitor which inhibits the signaling of TOR and S6K kinase in mammals and plants.The objective of this research is to determine the effect of Torin 1 in a relatively simple and homogeneous plant system such as the NT-1 tobacco suspension cell cultures.Cultures of NT-1 cells were tested with 5,50,150 and 250 nM of Torin 1.During kinetics growth of NT-1 tobacco suspension cell cultures,150 and 250 nM Torin 1 inhibits the early growth and later enhanced the cellular proliferation during exponential growth by means of an increased expression of E2F1 and cyclin B.Furthermore,Torin 1 stimulates the growth of NT-1 cells during log phase with small shaped cell,characteristic of tobacco suspension cell cultures with high mitotic activity.展开更多
In this study, we reported the effect of the ATP binding site competitive inhibitor Torin1 on activated α2-macroglobulin (α2M*)-induced cell proliferation and activation of mTORC1 and mTORC2 signaling in prostate ca...In this study, we reported the effect of the ATP binding site competitive inhibitor Torin1 on activated α2-macroglobulin (α2M*)-induced cell proliferation and activation of mTORC1 and mTORC2 signaling in prostate cancer cells. Torin1 significantly inhibited α2M*-induced cellproliferation as measured by protein and DNA synthesis. Translational activity, a major cellular response in malignant cells,is coordinately regulated by the mTORC1-S6-kinaseand mTORC1-4EBP1 axes. Torin1 significantly inhibited α2M*- and insulin-induced activation of mTORC1 as determined by phosphorylation of S6-kinaseat Thr389 and 4EBP1 at Thr37/46 compared to untreated cells employing Raptor immunoprecipitates. Torin1 also significantly inhibited α2M*- and insulin-induced upregulation of p-AktT308 and p-AktS473 in prostate cancer cells. The effect was comparable to that of insulin employed as a positive control. Finally, Torin1 inhibited α2M*- and insulin-induced activation of mTORC2 kinase assayas measured by phosphorylation of Akt at Ser473 inRictor immunoprecipitates of prostate cancer cells.展开更多
目的研究哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路抑制剂对卵丘及卵母细胞发育潜能的影响。方法收集小鼠较大有腔卵泡中卵丘-卵母细胞复合体(cumulusoocyte complexes,COCs)作为体外培养的研究对象,在培养液中...目的研究哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路抑制剂对卵丘及卵母细胞发育潜能的影响。方法收集小鼠较大有腔卵泡中卵丘-卵母细胞复合体(cumulusoocyte complexes,COCs)作为体外培养的研究对象,在培养液中加入不同浓度的mTOR通路抑制剂(雷帕霉素、Torin1),分析mTOR通路抑制剂对卵丘及卵母细胞发育潜能的影响。结果雷帕霉素、Torin1可以有效抑制COCs中mTOR信号通路,Torin1有明显影响卵丘细胞扩展的作用,使卵母细胞受精及着床前胚胎发育潜能下降。结论 mTOR信号通路抑制剂有明显降低卵母细胞质量的效应,是维持COCs活性及促进卵丘和卵母细胞的发育潜能所必需。展开更多
基金a grant from Medical and Health Science and Technology Program of Zhejiang Province(2019RC076).
文摘Background:Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma(HCC).However,resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib.As the activation of AKT/mTOR played a pivotal role in sorafenib resistance,we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells.Methods:The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines.The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method.Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting.Results:Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells,which underscores the significance of AKT activation.Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction.Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis,leading to the dephosphorylation of Ser136 in BAD protein,and increased the expression of total BAD,which contributed to the apoptosis in sorafenibresistant HCC cells.Conclusions:In this study,Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenibresistant HCC cells,via the suppression of mTORC2-AKT-BAD pathway.Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.
文摘Torin 1 is an ATP-competitive TOR inhibitor which inhibits the signaling of TOR and S6K kinase in mammals and plants.The objective of this research is to determine the effect of Torin 1 in a relatively simple and homogeneous plant system such as the NT-1 tobacco suspension cell cultures.Cultures of NT-1 cells were tested with 5,50,150 and 250 nM of Torin 1.During kinetics growth of NT-1 tobacco suspension cell cultures,150 and 250 nM Torin 1 inhibits the early growth and later enhanced the cellular proliferation during exponential growth by means of an increased expression of E2F1 and cyclin B.Furthermore,Torin 1 stimulates the growth of NT-1 cells during log phase with small shaped cell,characteristic of tobacco suspension cell cultures with high mitotic activity.
文摘In this study, we reported the effect of the ATP binding site competitive inhibitor Torin1 on activated α2-macroglobulin (α2M*)-induced cell proliferation and activation of mTORC1 and mTORC2 signaling in prostate cancer cells. Torin1 significantly inhibited α2M*-induced cellproliferation as measured by protein and DNA synthesis. Translational activity, a major cellular response in malignant cells,is coordinately regulated by the mTORC1-S6-kinaseand mTORC1-4EBP1 axes. Torin1 significantly inhibited α2M*- and insulin-induced activation of mTORC1 as determined by phosphorylation of S6-kinaseat Thr389 and 4EBP1 at Thr37/46 compared to untreated cells employing Raptor immunoprecipitates. Torin1 also significantly inhibited α2M*- and insulin-induced upregulation of p-AktT308 and p-AktS473 in prostate cancer cells. The effect was comparable to that of insulin employed as a positive control. Finally, Torin1 inhibited α2M*- and insulin-induced activation of mTORC2 kinase assayas measured by phosphorylation of Akt at Ser473 inRictor immunoprecipitates of prostate cancer cells.
