Akt抑制剂MK2206对膀胱癌细胞增殖凋亡的影响
|
摘要
目的探讨Akt抑制剂MK2206对膀胱癌细胞(T24)增殖、凋亡的影响及其作用机制。方法采用CCK-8法检测不同时间、不同浓度(0,1,5,10,20μmol·L-1) MK2206对T24细胞增殖的影响,流式细胞术检测不同浓度MK2206对T24细胞凋亡的影响; Western blotting检测Akt磷酸化蛋白表达情况及GSK-3β/β-catenin通路蛋白的表达水平。结果 CCK-8法检测显示,MK2206对T24细胞增殖活力具有显著的抑制效果(P<0.05),且在0～20μmol·L-1内呈浓度-时间依赖性。流式细胞术检测结果表明,MK2206在作用T24细胞24 h后,能够促进T24细胞凋亡。Western blotting检测显示,p-Akt(ser347)蛋白表达量显著降低(P<0.01);同时,MK2206在对GSK-3β总蛋白表达无明显的影响,P-GSK-3β、β-catenin蛋白表达水平显著降低(P<0.05)。结论 MK2206能够抑制T24细胞增殖,促进其发生凋亡,从而缓解膀胱癌发展进程,其作用机制可能是抑制GSK-3β/β-catenin信号通路。
Objective To explore the effect and mechanism of Akt inhibitor MK2206 on proliferation and apoptosis of T24 cells. Methods The CCK-8 method was used to detect the cell proliferation. Then the effects of different concentrations( 0,1,5,10,20 μmol·L-1) MK2206 on T24 cell apoptosis were tested by flow cytometry. Mealwhile,Western blotting was used to detect the phosphorylation of Akt protein and GSK-3β/β-catenin pathway proteins. Results The result of CCK-8 assay showed that MK2206 had a significant inhibitory effect on the proliferation activity of T24 cells( P<0.05),and the effect was in time-dose dependence manner arranged 0-20 μmol· L-1 concentration. Flow cytometry results showed that the MK2206 could promote apoptosis of T24 cells in 24 h. Comparing to control group,the level of p-Akt( ser347) protein significantly decreased( P<0.01). There was a significantly inhibitory effect on the GSK-3β/β-catenin signaling pathway,which showed the expression of p-GSK-3β and β-catenin proteins was significantly reduced( P<0.05) in the premise of no influence on the expression of GSK-3β. Conclusion MK2206 has inhibited T24 cell proliferation and promoted cell apoptosis via restraining Akt phosphorylation( ser473) and inhibiting the GSK-3β/β-catenin signaling pathway. MK2206 may relieve the tumor development process.
Objective To explore the effect and mechanism of Akt inhibitor MK2206 on proliferation and apoptosis of T24 cells. Methods The CCK-8 method was used to detect the cell proliferation. Then the effects of different concentrations( 0,1,5,10,20 μmol·L-1) MK2206 on T24 cell apoptosis were tested by flow cytometry. Mealwhile,Western blotting was used to detect the phosphorylation of Akt protein and GSK-3β/β-catenin pathway proteins. Results The result of CCK-8 assay showed that MK2206 had a significant inhibitory effect on the proliferation activity of T24 cells( P<0.05),and the effect was in time-dose dependence manner arranged 0-20 μmol· L-1 concentration. Flow cytometry results showed that the MK2206 could promote apoptosis of T24 cells in 24 h. Comparing to control group,the level of p-Akt( ser347) protein significantly decreased( P<0.01). There was a significantly inhibitory effect on the GSK-3β/β-catenin signaling pathway,which showed the expression of p-GSK-3β and β-catenin proteins was significantly reduced( P<0.05) in the premise of no influence on the expression of GSK-3β. Conclusion MK2206 has inhibited T24 cell proliferation and promoted cell apoptosis via restraining Akt phosphorylation( ser473) and inhibiting the GSK-3β/β-catenin signaling pathway. MK2206 may relieve the tumor development process.
引文
[1]FREDULSPERGER C,HORN D,WEISSFUSS S,et al.Phosphorylation of AKT(Ser473)serves as an independent prognostic marker for radiosensitivity in advanced head and neck squamous cell carcinoma[J].Int J Cancer,2015,136(12):2775-2785.
[2]BERGES C,BEDKE T,STUEHLER C,et al.Combined PI3K/Akt and Hsp90 targeting synergistically suppresses essential functions of alloreactive T cells and increases tregs[J].J Leukoc Biol,2015,98(6):1091-1105.
[3]CHEN J M,LIAN X L,DU J,et al.Inhibition of phosphorylated Ser473-Akt from translocating into the nucleus contributes to 2-cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis[J].Dev Growth Differ,2016,58(3):280-292.
[4]HIRAI H,SOOTOME H,NAKATSURU Y,et al.MK2206,an allosteric Akt inhibitor,enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo[J].Mol Cancer Ther,2010,9(7):1956-1967.
[5]NARAYAN R S,FEDRIGO C A,BRANDS E,et al.The allosteric AKT inhibitor MK2206 shows a synergistic interaction with chemotherapy and radiotherapy in glioblastoma spheroid cultures[J].BMC Cancer,2017,17(1):204.
[6]KNOLLMAN H,GODWIN J L,JAIN R,et al.Muscleinvasive urothelial bladder cancer:an update on systemic therapy[J].Ther Adv Urol,2015,7(6):312-330.
[7]JIA Y L,XIAO Y W,GUAN N W,et al.FOXQ1 promotes cancer metastasis by PI3K/Akt signaling regulation in colorectal carcinoma[J].Am J Transl Res,2017,9(5):2207-2218.
[8]TESTA J R,BELLACOSA A.Comentary-AKT plays a central role in tumorigenesis[J].Proc Natl Acad Sci USA.2001,98(20):10983-10985.
[9]WANG Q,YU W N,CHEN X,et al.Spontaneous hepatocellular carcinoma after the combined deletion of Akt isoforms[J].Cancer Cell,2016,29(4):523-525.
[10]THOMAS C C,DEAK M,ALESSI D R.High-resolution strcture of the pleckstrin homology domain of protein kinase B/Akt bound to phosphatidylinositol(3,4,5)-trisphosphate[J].Curr Biol,2002,12(14):1256-1262.
[11]HEN J M,LIAN X L,DU J,et al.Inhibition of phosphorylated Ser473-Akt from translocating into the nucleus contributes to 2-cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis[J].Dev Growth Differ,2016,58(3):280-292.
[12]XUE G,ROMAO E,MASSI D,et al.Wnt/β-catenin signaling in melanoma:preclinical rationale and novel therapeutic insights[J].Cancer Treat Rev,2016,49(1):1-12.
[13]ALMHANNA K,CUBITT C L,ZHANG S,et al.MK2206,an Akt inhibitor,enhances carboplatinum/paclitaxel efficacy in gastric cancer cell lines[J].Cancer Bilo Ther,2013,14(10):14932-14936.
[14]YAP T A,YAN L,PATNAIK A,et al.First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors[J].J Clin Oncol,2011,29(35):4688-4695.
[15]HIRAI H,SOOTOME H,NAKATSURU Y,et al.MK2206,an allosteric Akt inhibitor,enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo[J].Mol Cancer Ther,2010,9(7):1956-1957.
[16]MATSUZAKI S,POULY J L,CANIS M.In vitro and in vivo effects of MK2206 and chloroquine combination therapy on endometriosis antophagy may be required for regrowth of endometriosis[J].Br J Pharmacol,2018,175(10):1637-1653.
[17]MATSUZAKI S,POULY J L,CANIS M.Effects of U0126and MK2206 on cell growth and regrowth of endometriotic stromal cels grown on substates of varying stiffness[J].Sci Rep,2017,7:42939.
[18]WU K,FAN J,ZHANG L,et al.PI3K/Akt to GSK-3β/β-catenin signaling cascade coordinatescell colonization for bladder cancer bone metastasis through regulating ZEB1transcription[J].Cell Signal,2012,24(12):2273-2282.
[2]BERGES C,BEDKE T,STUEHLER C,et al.Combined PI3K/Akt and Hsp90 targeting synergistically suppresses essential functions of alloreactive T cells and increases tregs[J].J Leukoc Biol,2015,98(6):1091-1105.
[3]CHEN J M,LIAN X L,DU J,et al.Inhibition of phosphorylated Ser473-Akt from translocating into the nucleus contributes to 2-cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis[J].Dev Growth Differ,2016,58(3):280-292.
[4]HIRAI H,SOOTOME H,NAKATSURU Y,et al.MK2206,an allosteric Akt inhibitor,enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo[J].Mol Cancer Ther,2010,9(7):1956-1967.
[5]NARAYAN R S,FEDRIGO C A,BRANDS E,et al.The allosteric AKT inhibitor MK2206 shows a synergistic interaction with chemotherapy and radiotherapy in glioblastoma spheroid cultures[J].BMC Cancer,2017,17(1):204.
[6]KNOLLMAN H,GODWIN J L,JAIN R,et al.Muscleinvasive urothelial bladder cancer:an update on systemic therapy[J].Ther Adv Urol,2015,7(6):312-330.
[7]JIA Y L,XIAO Y W,GUAN N W,et al.FOXQ1 promotes cancer metastasis by PI3K/Akt signaling regulation in colorectal carcinoma[J].Am J Transl Res,2017,9(5):2207-2218.
[8]TESTA J R,BELLACOSA A.Comentary-AKT plays a central role in tumorigenesis[J].Proc Natl Acad Sci USA.2001,98(20):10983-10985.
[9]WANG Q,YU W N,CHEN X,et al.Spontaneous hepatocellular carcinoma after the combined deletion of Akt isoforms[J].Cancer Cell,2016,29(4):523-525.
[10]THOMAS C C,DEAK M,ALESSI D R.High-resolution strcture of the pleckstrin homology domain of protein kinase B/Akt bound to phosphatidylinositol(3,4,5)-trisphosphate[J].Curr Biol,2002,12(14):1256-1262.
[11]HEN J M,LIAN X L,DU J,et al.Inhibition of phosphorylated Ser473-Akt from translocating into the nucleus contributes to 2-cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis[J].Dev Growth Differ,2016,58(3):280-292.
[12]XUE G,ROMAO E,MASSI D,et al.Wnt/β-catenin signaling in melanoma:preclinical rationale and novel therapeutic insights[J].Cancer Treat Rev,2016,49(1):1-12.
[13]ALMHANNA K,CUBITT C L,ZHANG S,et al.MK2206,an Akt inhibitor,enhances carboplatinum/paclitaxel efficacy in gastric cancer cell lines[J].Cancer Bilo Ther,2013,14(10):14932-14936.
[14]YAP T A,YAN L,PATNAIK A,et al.First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors[J].J Clin Oncol,2011,29(35):4688-4695.
[15]HIRAI H,SOOTOME H,NAKATSURU Y,et al.MK2206,an allosteric Akt inhibitor,enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo[J].Mol Cancer Ther,2010,9(7):1956-1957.
[16]MATSUZAKI S,POULY J L,CANIS M.In vitro and in vivo effects of MK2206 and chloroquine combination therapy on endometriosis antophagy may be required for regrowth of endometriosis[J].Br J Pharmacol,2018,175(10):1637-1653.
[17]MATSUZAKI S,POULY J L,CANIS M.Effects of U0126and MK2206 on cell growth and regrowth of endometriotic stromal cels grown on substates of varying stiffness[J].Sci Rep,2017,7:42939.
[18]WU K,FAN J,ZHANG L,et al.PI3K/Akt to GSK-3β/β-catenin signaling cascade coordinatescell colonization for bladder cancer bone metastasis through regulating ZEB1transcription[J].Cell Signal,2012,24(12):2273-2282.