mTOR通路增强宫颈癌紫杉醇耐药细胞株HeLa/PTX对紫杉醇的敏感性
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摘要
目的探讨mTOR在宫颈癌紫杉醇耐药中的作用及mTORC1/2双重抑制剂OSI-027联用紫杉醇后HeLa/PTX细胞对紫杉醇(paclitaxel,PTX)敏感性的变化并对相关机制进行研究。方法用sh-RNA-Raptor及sh-RNA-Rictor质粒转染HeLa/PTX细胞以下调Rictor及Raptor的表达,采用western blot法检测mTOR信号通路相关蛋白(Raptor、Rictor、p-Akt(ser473)、Akt和p-p70S6K)和凋亡相关蛋白(Bcl-2和Bax)的表达情况;采用平板克隆形成实验、CCK-8法及Western blot法检测单用mTORC1/2双重抑制剂(OSI-027)、PTX及联用OSI-027和PTX后细胞克隆形成能力、增殖活力的变化并计算药物联用指数(combination index,CI)及mTOR信号通路相关蛋白和凋亡相关蛋白的表达情况。结果与对照组相比,在HeLa/PTX细胞中下调Raptor后p-p70S6K表达显著降低(P<0.001),p-Akt(ser473)/Akt比率显著升高(P<0.001),Bcl-2/Bax比值无显著差异(P>0.05)。下调Rictor后p-p70S6K蛋白表达、p-Akt(ser473)/Akt及Bcl-2/Bax比值均显著降低(P<0.001);与单独使用OSI-027或PTX相比,联合用药组细胞的克隆形成能力及增殖活力均明显下降且两药联用具有协同作用;与空白对照组相比,单用PTX处理HeLa/PTX细胞后p-Akt(ser473)/Akt比率略降低(P<0.05),p-p70S6K表达无明显变化(P>0.05),而单用OSI-027处理HeLa/PTX细胞后p-Akt(ser473)/Akt比率及p-p70S6K表达均显著降低(P<0.001);与单用OSI-027及PTX相比,联合用药处理HeLa/PTX细胞后p-Akt(ser473)/Akt比值、Bcl-2/Bax比值及p-p70S6K蛋白表达均显著降低(P<0.001)。结论抑制mTORC2信号通路的活性可减弱mTORC1抑制引起的p-Akt(ser473)反馈激活可成为克服紫杉醇耐药的有效靶点;mTORC1/2双重抑制剂OSI-027与紫杉醇联用可产生协同抗肿瘤作用。
Objective To explore the role of mTOR in paclitaxel resistance of cervical cancer and the sensitivity change of HeLa/PTX cells treated with the combination of paclitaxel and the dual inhibitor of mTORC1 and mTORC2 pathway(OSI-027) to paclitaxel.Methods HeLa/PTX cells were transfected with sh-RNA-Raptor and sh-RNA-Rictor plasmid in order to knock down the expression level of Raptor and Rictor,respectively.And western blot assay was used to detect the expression level of mTOR pathway associated proteins(Raptor,Rictor,p-Akt(ser473),Akt,and p-p70 S6 K) and apoptosis related proteins(Bcl-2 and Bax).The colony forming ability and proliferation activity of the HeLa/PTX cells treated with OSI-027 alone,paclitaxel alone,and OSI-027 combined with paclitaxel were tested by using plate colony formation and CCK-8 assays,and then drug combination index(CI)was calculated in the case of the combination of OSI-027 and paclitaxel.The expression level of mTOR pathway associated proteins and apoptosis related proteins were tested by using western blot assay.Results As compared with control group,after downregulating Raptor expression in HeLa/PTX cells,the expression level of p-p70 S6 K remarkably decreased(P<0.001) and the p-Akt(ser473)/Akt ratio increased obviously(P<0.001).Moreover,Bcl-2/Bax ratio did not differ apparently(P>0.05).The expression level of p-p70 S6 K,p-Akt(ser473)/Akt ratio,and Bcl-2/Bax ratio reduced entirely after knocking down Rictor in HeLa/PTX cells(P<0.001);As compared with OSI-027 or paclitaxel treatment alone,the combination group was attenuated significantly in aspect of the colony formation and proliferation activity and synergistic effects were seen with the combination of two drugs(P<0.001);As compared with blank group,after HeLa/PTX cells being treated with PTX alone,p-Akt(ser473)/Akt ratio was decreased slightly(P<0.05) and p-p70 S6 K expression did not differ apparently(P>0.05).However,p-Akt(ser473)/Akt ratio and p-p70 S6 K expression were significantly decreased after cells being treated with OSI-027 alone;As compared with OSI-027 or paclitaxel treatment alone,the decline of p-Akt(ser473)/Akt ratio,Bcl-2/Bax ratio,and p-p70 S6 K expression was obviously in HeLa/PTX cells treated the combination of the two drugs.Conclusion Inhibition of mTORC2 pathway attenuates the activation of p-Akt(ser473) induced by mTORC2 feedback loop,which can be an effective target to overcome paclitaxel resistance.The combination between the dual mTORC1/2 inhibitor OSI-027 and paclitaxel has a synergistically antitumous effect.
Objective To explore the role of mTOR in paclitaxel resistance of cervical cancer and the sensitivity change of HeLa/PTX cells treated with the combination of paclitaxel and the dual inhibitor of mTORC1 and mTORC2 pathway(OSI-027) to paclitaxel.Methods HeLa/PTX cells were transfected with sh-RNA-Raptor and sh-RNA-Rictor plasmid in order to knock down the expression level of Raptor and Rictor,respectively.And western blot assay was used to detect the expression level of mTOR pathway associated proteins(Raptor,Rictor,p-Akt(ser473),Akt,and p-p70 S6 K) and apoptosis related proteins(Bcl-2 and Bax).The colony forming ability and proliferation activity of the HeLa/PTX cells treated with OSI-027 alone,paclitaxel alone,and OSI-027 combined with paclitaxel were tested by using plate colony formation and CCK-8 assays,and then drug combination index(CI)was calculated in the case of the combination of OSI-027 and paclitaxel.The expression level of mTOR pathway associated proteins and apoptosis related proteins were tested by using western blot assay.Results As compared with control group,after downregulating Raptor expression in HeLa/PTX cells,the expression level of p-p70 S6 K remarkably decreased(P<0.001) and the p-Akt(ser473)/Akt ratio increased obviously(P<0.001).Moreover,Bcl-2/Bax ratio did not differ apparently(P>0.05).The expression level of p-p70 S6 K,p-Akt(ser473)/Akt ratio,and Bcl-2/Bax ratio reduced entirely after knocking down Rictor in HeLa/PTX cells(P<0.001);As compared with OSI-027 or paclitaxel treatment alone,the combination group was attenuated significantly in aspect of the colony formation and proliferation activity and synergistic effects were seen with the combination of two drugs(P<0.001);As compared with blank group,after HeLa/PTX cells being treated with PTX alone,p-Akt(ser473)/Akt ratio was decreased slightly(P<0.05) and p-p70 S6 K expression did not differ apparently(P>0.05).However,p-Akt(ser473)/Akt ratio and p-p70 S6 K expression were significantly decreased after cells being treated with OSI-027 alone;As compared with OSI-027 or paclitaxel treatment alone,the decline of p-Akt(ser473)/Akt ratio,Bcl-2/Bax ratio,and p-p70 S6 K expression was obviously in HeLa/PTX cells treated the combination of the two drugs.Conclusion Inhibition of mTORC2 pathway attenuates the activation of p-Akt(ser473) induced by mTORC2 feedback loop,which can be an effective target to overcome paclitaxel resistance.The combination between the dual mTORC1/2 inhibitor OSI-027 and paclitaxel has a synergistically antitumous effect.
引文
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[6] PENG D J,WANG J,ZHOU JY,et al.Role of the Akt/mTOR survival pathway in cisplatin resistance in ovarian cancer cells[J].Biochem Biophys Res Commun,2010,394(3):600-605.
[7] YANG Z,LIU Y,SHI C,et al.Suppression of PTEN/AKT signaling decreases the expression of TUBB3 and TOP2A with subsequent inhibition of cell growth and induction of apoptosis in human breast cancer MCF-7 cells via ATP and caspase-3 signaling pathways[J].Oncol Rep,2017,37(2):1011-1019.
[8] JULIEN L A,CARRIERE A,MOREAU J,et al.mTORC1-activated S6K1 phosphorylates rictor on threonine 1135 and regulates mTORC2 signaling[J].Mol Cell Biol,2010,30(4):908-921.
[9] EFEYAN A,SABATINI D M.mTOR and cancer:many loops in one pathway[J].Curr Opin Cell Biol,2010,22(2):169-176.
[10] GOHR K,HAMACHER A,ENGELKE L H,et al.Inhibition of PI3K/Akt/mTOR overcomes cisplatin resistance in the triple negative breast cancer cell line HCC38[J].Bmc Cancer,2017,17(1):647-656.
[11] LAPLANTE M,SABATINI D M.mTOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.
[12] ZHOU F,XUE M,QIN D,et al.HIV-1 tat promotes kaposi′s sarcoma-associated herpesvirus (KSHV) vIL-6-induced angiogenesis and tumorigenesis by regulating PI3K/PTEN/AKT/GSK-3beta signaling pathway[J].PLoS One,2013,8(1):e53145.
[13] GURI Y,HALL M N.mTOR signaling confers resistance to targeted cancer drugs[J].Trends in Cancer,2016,2(11):688-697.
[14] WU S H,BI J F,CLOUGHESY T,et al.Emerging function of mTORC2 as a core regulator in glioblastoma:metabolic reprogramming and drug resistance[J].Cancer Biol Med,2014,11(4):255-263.
[15] GUERRERO-ZOTANO A,MAYER I A,ARTEAGA C L.PI3K/AKT/mTOR:role in breast cancer progression,drug resistance,and treatment[J].Cancer Metast Rev,2016,35(4):515-524.
[16] SCHMIDT K M,HELLERBRAND C,RUEMMELE P.Inhibition of mTORC2 component RICTOR impairs tumor growth in pancreatic cancer models[J].Oncotarget,2017,8(15):24491-24505.
[17] REHAN M.An anti-cancer drug candidate OSI-027 and its analog as inhibitors of mTOR:computational insights into the inhibitory mechanisms[J].J Cell Biochem,2017,118(12):4558-4567.
[18] CHEN B W,CHEN W,LIANG H,et al.Inhibition of mTORC2 induces cell-cycle arrest and enhances the cytotoxicity of doxorubicin by suppressing MDR1 expression in HCC cells[J].Mol Cancer Ther,2015,14(8):1805-1815.
[2] WAGNER W,CISZEWSKI W M,KANIA K D.L-and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation[J].Cell Commun Signal,2015,13(1):13-36.
[3] CORREIA A L,BISSELL M J.The tumor microenvironment is a dominant force in multidrug resistance[J].Drug Resist Update,2012,15(1/2):39-49.
[4] CHEN L,WANG L,SHEN H,et al.Anthelminthic drug niclosamide sensitizes the responsiveness of cervical cancer cells to paclitaxel via oxidative stress-mediated mTOR inhibition[J].Biochem Bioph Res Co,2017,484(2):416-421.
[5] ZHAI B,HU F,JIANG X,et al.Inhibition of akt reverses the acquired resistance to sorafenib by switching protective autophagy to autophagic cell death in hepatocellular carcinoma[J].Mol Cancer Ther,2014,13(6):1589-1598.
[6] PENG D J,WANG J,ZHOU JY,et al.Role of the Akt/mTOR survival pathway in cisplatin resistance in ovarian cancer cells[J].Biochem Biophys Res Commun,2010,394(3):600-605.
[7] YANG Z,LIU Y,SHI C,et al.Suppression of PTEN/AKT signaling decreases the expression of TUBB3 and TOP2A with subsequent inhibition of cell growth and induction of apoptosis in human breast cancer MCF-7 cells via ATP and caspase-3 signaling pathways[J].Oncol Rep,2017,37(2):1011-1019.
[8] JULIEN L A,CARRIERE A,MOREAU J,et al.mTORC1-activated S6K1 phosphorylates rictor on threonine 1135 and regulates mTORC2 signaling[J].Mol Cell Biol,2010,30(4):908-921.
[9] EFEYAN A,SABATINI D M.mTOR and cancer:many loops in one pathway[J].Curr Opin Cell Biol,2010,22(2):169-176.
[10] GOHR K,HAMACHER A,ENGELKE L H,et al.Inhibition of PI3K/Akt/mTOR overcomes cisplatin resistance in the triple negative breast cancer cell line HCC38[J].Bmc Cancer,2017,17(1):647-656.
[11] LAPLANTE M,SABATINI D M.mTOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.
[12] ZHOU F,XUE M,QIN D,et al.HIV-1 tat promotes kaposi′s sarcoma-associated herpesvirus (KSHV) vIL-6-induced angiogenesis and tumorigenesis by regulating PI3K/PTEN/AKT/GSK-3beta signaling pathway[J].PLoS One,2013,8(1):e53145.
[13] GURI Y,HALL M N.mTOR signaling confers resistance to targeted cancer drugs[J].Trends in Cancer,2016,2(11):688-697.
[14] WU S H,BI J F,CLOUGHESY T,et al.Emerging function of mTORC2 as a core regulator in glioblastoma:metabolic reprogramming and drug resistance[J].Cancer Biol Med,2014,11(4):255-263.
[15] GUERRERO-ZOTANO A,MAYER I A,ARTEAGA C L.PI3K/AKT/mTOR:role in breast cancer progression,drug resistance,and treatment[J].Cancer Metast Rev,2016,35(4):515-524.
[16] SCHMIDT K M,HELLERBRAND C,RUEMMELE P.Inhibition of mTORC2 component RICTOR impairs tumor growth in pancreatic cancer models[J].Oncotarget,2017,8(15):24491-24505.
[17] REHAN M.An anti-cancer drug candidate OSI-027 and its analog as inhibitors of mTOR:computational insights into the inhibitory mechanisms[J].J Cell Biochem,2017,118(12):4558-4567.
[18] CHEN B W,CHEN W,LIANG H,et al.Inhibition of mTORC2 induces cell-cycle arrest and enhances the cytotoxicity of doxorubicin by suppressing MDR1 expression in HCC cells[J].Mol Cancer Ther,2015,14(8):1805-1815.