PLCε沉默后通过Wnt/β-catenin信号通路抑制比卡鲁胺耐药的前列腺癌细胞增殖
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摘要
目的:探讨磷脂酰肌醇特异性磷脂酶Cε(phosphoinositide-speci c phospholipase Cε,PLCε)对前列腺癌细胞增殖和比卡鲁胺敏感性的影响及其对Wnt/β-catenin信号通路的调控作用。方法:采用比卡鲁胺浓度递增及间歇诱导法建立对比卡鲁胺耐药的前列腺癌B-LNCAP细胞。应用实时荧光定量PCR和蛋白质印迹法检测前列腺癌LNCAP和B-LNCAP细胞中雄激素受体(androgen receptor,AR)和PLCεmRNA和蛋白的表达,CCK-8法检测LNCAP和B-LNCAP细胞对比卡鲁胺的敏感性。将干扰PLCε表达的重组慢病毒LV-shPLCε或阴性对照(negative control,NC)LV-NC感染B-LNCAP细胞(称为LVshPLCε/B-LNCAP或LV-NC/B-LNCAP),Wnt/β-catenin信号通路激活剂AZD2858处理LV-shPLCε/B-LNCAP细胞,以未进行任何干预的B-LNCAP细胞作为空白组。应用CCK-8法检测各组细胞的增殖情况及对比卡鲁胺的敏感性,应用克隆形成实验检测各组细胞的克隆形成能力,蛋白质印迹法检测各组细胞的细胞质和细胞核中β-catenin和AR蛋白表达水平,实时荧光定量PCR和蛋白质印迹法检测各组细胞中c-myc、cyclin D1mRNA和蛋白表达水平。结果:B-LNCAP细胞中PLCε、AR mRNA和蛋白的表达水平均高于LNCAP细胞(P值均<0.05),B-LNCAP细胞对比卡鲁胺的耐药系数为132.87,成功构建前列腺癌耐药细胞B-LNCAP。LV-shPLCε/B-LNCAP细胞增殖和克隆形成能力均弱于空白组和LV-NC/B-LNCAP组(P值均<0.01),比卡鲁胺对LV-shPLCε/B-LNCAP细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)值均低于空白组和LV-NC/B-LNCAP组(P值均<0.05),LV-shPLCε/B-LNCAP细胞核中β-catenin和AR蛋白表达水平明显低于空白组和LV-NC/B-LNCAP组(P值均<0.01),LVshPLCε/B-LNCAP细胞中c-myc、cyclin D1 mRNA和蛋白的表达水平均低于空白组和LV-NC/B-LNCAP组(P值均<0.01);AZD2858处理的LVshPLCε/B-LNCAP细胞增殖和克隆形成能力强于LV-shPLCε/B-LNCAP细胞(P值均<0.05),比卡鲁胺对AZD2858处理的LV-shPLCε/B-LNCAP细胞IC50值高于未处理的LV-shPLCε/B-LNCAP细胞(P<0.05),AZD2858处理的LV-shPLCε/B-LNCAP细胞核中β-catenin和AR蛋白表达水平均高于LV-shPLCε/B-LNCAP细胞(P值均<0.05),AZD2858处理的LV-shPLCε/B-LNCAP细胞中c-myc、cyclin D1 mRNA和蛋白表达水平均高于LV-shPLCε/B-LNCAP细胞(P值均<0.05)。结论:PLCε下调后通过抑制Wnt/β-catenin信号通路,增强前列腺癌B-LNCAP细胞对比卡鲁胺的敏感性,抑制前列腺癌B-LNCAP细胞的增殖。
Objective: To investigate the effects of phosphoinositide-specific phospholipase Cε(PLCε) on the proliferation and sensitive of prostate cancer cells to bicalutamide as well as the regulation of Wnt/β-catenin signaling pathway.Methods: Bicalutamide-resistant prostate cancer B-LNCAP cells were constructed by increasing concentration of bicalutamide in a stepwise and intermittent manner. The expressions of androgen receptor(AR) and PLCε mRNA and protein in LNCAP and B-LNCAP cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The sensitivity of LNCAP and B-LNCAP cells to bicalutamide was detected by CCK-8 assay. The recombination lentivirus interfering PLCε expression(LV-shPLCε) and its negative control lentivirus(LV-NC) were used to infect B-LNCAP cells(named as LV-shPLCε/B-LNCAP or LV-NC/B-LNCAP), respectively.LV-shPLCε/B-LNCAP cells were treated with Wnt/β-catenin signaling pathway activator AZD2858,while B-LNCAP cells without any intervention was used as a blank group. Then the sensitivity of cells to bicalutamide and the cell proliferation in each group were tested by CCK-8 assay.The cell clonality in each group was tested by clonal formation assay. The expressions ofβ-catenin and AR proteins in cell cytoplasm and nucleus were detected by Western blotting. The expressions of c-myc and cyclin D1 mRNA and protein in each group were detected by real-time fluorescent quantitative PCR and Western blotting, respectively.Results: The expression levels PLCε and AR mRNA and protein in B-LNCAP cells were statistically higher than those in LNCAP cells(all P < 0.05). The drug resistance index of B-LNCAP cells to bicalutamide was 132.87, which indicated that the drug-resistant prostate cancer B-LNCAP cells were successfully constructed. The clone formation and cell proliferation abilities of LV-shPLCε/B-LNCAP cells were lower than those of the blank and LV-NC/B-LNCAP groups(all P < 0.01). The half maximal inhibitory concentration(IC50) value of bicalutamide in LV-shPLCε/B-LNCAP cells was lower than that of the blank or LV-NC/B-LNCAP groups(both P < 0.05). The expression levels of nuclear protein β-catenin and AR in LV-shPLCε/B-LNCAP cells were decreased as compared with the blank and LV-NC/B-LNCAP groups(all P < 0.01). The expression levels of c-myc and cyclin D1 mRNA and protein in LV-shPLCε/B-LNCAP cells were decreased as compared with the blank and LV-NC/B-LNCAP groups(all P < 0.01). The abilities of clone formation and proliferation of LV-shPLCε/B-LNCAP cells treated with AZD2858 were stronger than those of untreated LV-shPLCε/B-LNCAP cells(both P < 0.05). The IC50 value of AZD2858 in LV-shPLCε/B-LNCAP cells was increased as compared with untreated LV-shPLCε/B-LNCAP cells(P < 0.05). The expression levels of nuclear protein β-catenin and AR, as well as the expression levels of c-myc and cyclin D1 mRNA and protein in LV-shPLCε/B-LNCAP cells treated with AZD2858 were higher than those in untreated LV-shPLCε/B-LNCAP cells(all P < 0.05).Conclusion: Down-regulation of PLCε enhances the sensitivity of prostate cancer B-LNCAP cells to bicalutamide, and inhibits the cell proliferation through suppressing Wnt/β-catenin signaling pathway.
Objective: To investigate the effects of phosphoinositide-specific phospholipase Cε(PLCε) on the proliferation and sensitive of prostate cancer cells to bicalutamide as well as the regulation of Wnt/β-catenin signaling pathway.Methods: Bicalutamide-resistant prostate cancer B-LNCAP cells were constructed by increasing concentration of bicalutamide in a stepwise and intermittent manner. The expressions of androgen receptor(AR) and PLCε mRNA and protein in LNCAP and B-LNCAP cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The sensitivity of LNCAP and B-LNCAP cells to bicalutamide was detected by CCK-8 assay. The recombination lentivirus interfering PLCε expression(LV-shPLCε) and its negative control lentivirus(LV-NC) were used to infect B-LNCAP cells(named as LV-shPLCε/B-LNCAP or LV-NC/B-LNCAP), respectively.LV-shPLCε/B-LNCAP cells were treated with Wnt/β-catenin signaling pathway activator AZD2858,while B-LNCAP cells without any intervention was used as a blank group. Then the sensitivity of cells to bicalutamide and the cell proliferation in each group were tested by CCK-8 assay.The cell clonality in each group was tested by clonal formation assay. The expressions ofβ-catenin and AR proteins in cell cytoplasm and nucleus were detected by Western blotting. The expressions of c-myc and cyclin D1 mRNA and protein in each group were detected by real-time fluorescent quantitative PCR and Western blotting, respectively.Results: The expression levels PLCε and AR mRNA and protein in B-LNCAP cells were statistically higher than those in LNCAP cells(all P < 0.05). The drug resistance index of B-LNCAP cells to bicalutamide was 132.87, which indicated that the drug-resistant prostate cancer B-LNCAP cells were successfully constructed. The clone formation and cell proliferation abilities of LV-shPLCε/B-LNCAP cells were lower than those of the blank and LV-NC/B-LNCAP groups(all P < 0.01). The half maximal inhibitory concentration(IC50) value of bicalutamide in LV-shPLCε/B-LNCAP cells was lower than that of the blank or LV-NC/B-LNCAP groups(both P < 0.05). The expression levels of nuclear protein β-catenin and AR in LV-shPLCε/B-LNCAP cells were decreased as compared with the blank and LV-NC/B-LNCAP groups(all P < 0.01). The expression levels of c-myc and cyclin D1 mRNA and protein in LV-shPLCε/B-LNCAP cells were decreased as compared with the blank and LV-NC/B-LNCAP groups(all P < 0.01). The abilities of clone formation and proliferation of LV-shPLCε/B-LNCAP cells treated with AZD2858 were stronger than those of untreated LV-shPLCε/B-LNCAP cells(both P < 0.05). The IC50 value of AZD2858 in LV-shPLCε/B-LNCAP cells was increased as compared with untreated LV-shPLCε/B-LNCAP cells(P < 0.05). The expression levels of nuclear protein β-catenin and AR, as well as the expression levels of c-myc and cyclin D1 mRNA and protein in LV-shPLCε/B-LNCAP cells treated with AZD2858 were higher than those in untreated LV-shPLCε/B-LNCAP cells(all P < 0.05).Conclusion: Down-regulation of PLCε enhances the sensitivity of prostate cancer B-LNCAP cells to bicalutamide, and inhibits the cell proliferation through suppressing Wnt/β-catenin signaling pathway.
引文
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[14]XUE Y,OU L,MIN T,et al.Knockdown of PLCεinhibits inflammatory cytokine release via STAT3 phosphorylation in human bladder cancer cells[J].Tumour Biol,2015,36(12):9723-9732.
[15]ABNET CC,FREEDMAN ND,HU N,et al.Ashared susceptibility locus in PLCE1 at 10q23for gastric adenocarcinoma and esophageal squamous cell carcinoma[J].Nat Genet,2010,42(9):764-767.
[16]LASSI K,DAWSON N.Emerging therapies in castrate-resistant prostate cancer[J].Curr Opin Oncol,2009,21(3):260-265.
[17]GAO WQ,GUO Y,ZHANG K,et al.Numb-/low enriches a castration resistant prostate cancer cell subpopulation associated with enhanced Notch and Hedgehog signaling[J].Clin Cancer Res,2017,23(21):6744-6756.
[18]王希,鹿占鹏.去势抵抗性前列腺癌的药物治疗新进展[J].临床医药文献电子杂志,2017,4(55):10866-10867.
[19]ASEM MS,BUECHLER S,WATES RB,et al.Wnt5a signaling in cancer[J].Cancers(Basel),2016,8(9):79-96.
[20]CHEN G,SHUKEIR N,POTTI A,et al.Up-regulation of Wnt-1 and beta-catenin production in patients with advanced metastatic prostate carcinoma:potential pathogenetic and prognostic implications[J].Cancer,2004,101(6):1345-1356.
[21]移志刚,蒲彦川,王兴文,等.RIPK4、β-catenin和P-gp在骨肉瘤中的表达及临床意义[J].肿瘤,2016,36(9):1006-1012.
[22]TRUICACI,BYERSS,GELMAN NEP.β-Catenin affects androgen receptor transcriptional activity and lig and specificity[J].Cancer Res,2000,60(17):4709-4713.
[2]CHEN W,ZHENG R,BAADE PD,et al.Cancer statistics in China,2015[J].CACancer J Clin,2016,66(2):115-132.
[3]SIEGEL RL,MILLER KD,JEMAL A.Cancer statistics,2016[J].CA Cancer JClin,2016,66(1):7-30.
[4]LAM JS,LEPPERT JT,VEMULAPALLI SN,et al.Secondary hormonal therapy for advanced prostate cancer[J].J Urol,2006,175(1):27-34.
[5]HONG FD,LI PO,XUE Y,et al.A new PKCα/β/TBX3/E-cadherin pathway is involved in PLCε-regulated invasion and migration in human bladder cancer cells[J].Cell Signal,2014,26(3):580-593.
[6]SONG C,HU CD,MASAGO M,et al.Regulation of a novel human phospholipase C,PLCε,through membrane targeting by Ras[J].J Biol Chem,2001,276(4):2752-2757.
[7]WA N G Y,W U X,O U L,et al.P L Cεknockdown inhibits prostate cancer cell proliferation via suppression of Notch signalling and nuclear translocation of the androgen receptor[J].Cancer Lett,2015,362(1):61-69.
[8]CHEN G,SHUKEIR N,POTTI A,et al.Up-regulation of Wnt-1 and beta-catenin production in patients with advanced metastatic prostate carcinoma:potential pathogenetic and prognostic implications[J].Cancer,2004,101(6):1345-1356.
[9]GLINSKY GV,GLINSKII AB,STEPHENSON AJ,et al.Gene expression profiling predicts clinical outcome of prostate cancer[J].J Clin Invest,2004,113(6):913-923.
[10]WISSMANN C,WILD PJ,KAISER S,et al.WIF1,a component of the Wnt pathway,is down-regulated in prostate,breast,lung,and bladder cancer[J].J Pathol,2003,201(2):204-212.
[11]NANDANA S,TRIPATHI M,DUAN P,et al.Bone metastasis of prostate cancer can be therapeutically targeted at the TBX2-WNTsignaling axis[J].Cancer Res,2017,77(6):1331-1344.
[12]HICKS SN,JEZYK MR,GERSHBURG S,et al.General and versatile autoinhibition of PLCisozymes[J].Mol Cell,2008,31(3):383-394.
[13]WING MR,SNYDER JT,SONDEK J,et al.Direct activation of phospholipase C-epsilon by Rho[J].J Biol Chem,2003,278(42):41253-41258.
[14]XUE Y,OU L,MIN T,et al.Knockdown of PLCεinhibits inflammatory cytokine release via STAT3 phosphorylation in human bladder cancer cells[J].Tumour Biol,2015,36(12):9723-9732.
[15]ABNET CC,FREEDMAN ND,HU N,et al.Ashared susceptibility locus in PLCE1 at 10q23for gastric adenocarcinoma and esophageal squamous cell carcinoma[J].Nat Genet,2010,42(9):764-767.
[16]LASSI K,DAWSON N.Emerging therapies in castrate-resistant prostate cancer[J].Curr Opin Oncol,2009,21(3):260-265.
[17]GAO WQ,GUO Y,ZHANG K,et al.Numb-/low enriches a castration resistant prostate cancer cell subpopulation associated with enhanced Notch and Hedgehog signaling[J].Clin Cancer Res,2017,23(21):6744-6756.
[18]王希,鹿占鹏.去势抵抗性前列腺癌的药物治疗新进展[J].临床医药文献电子杂志,2017,4(55):10866-10867.
[19]ASEM MS,BUECHLER S,WATES RB,et al.Wnt5a signaling in cancer[J].Cancers(Basel),2016,8(9):79-96.
[20]CHEN G,SHUKEIR N,POTTI A,et al.Up-regulation of Wnt-1 and beta-catenin production in patients with advanced metastatic prostate carcinoma:potential pathogenetic and prognostic implications[J].Cancer,2004,101(6):1345-1356.
[21]移志刚,蒲彦川,王兴文,等.RIPK4、β-catenin和P-gp在骨肉瘤中的表达及临床意义[J].肿瘤,2016,36(9):1006-1012.
[22]TRUICACI,BYERSS,GELMAN NEP.β-Catenin affects androgen receptor transcriptional activity and lig and specificity[J].Cancer Res,2000,60(17):4709-4713.
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