敲低富含亮氨酸的三角状五肽重复结构蛋白(LRPPRC)促进激素抵抗性前列腺癌细胞凋亡
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摘要
目的研究富含亮氨酸的三角状五肽重复结构蛋白(LRPPRC)对激素抵抗性前列腺癌细胞凋亡的影响。方法Western blot法检测前列腺癌DU145细胞和LNCaP细胞LRPPRC和雄激素受体(AR)蛋白水平;在DU145细胞中瞬转LRPPRC小干涉RNA(siLRPPRC);反转录PCR检测转染细胞LRPPRC mRNA水平; Western blot法检测LRPPRC蛋白水平;噻唑蓝(MTT)法检测细胞存活率; Hoechst33258法和流式细胞术检测细胞凋亡率;荧光素酶法检测细胞ATP水平;Western blot法检测Bcl2、 BAX和胱天蛋白酶3(caspase-3)蛋白水平。结果 LRPPRC在DU145细胞和LNCaP细胞均高表达,但DU145细胞不表达AR。敲低DU145细胞LRPPRC水平后,细胞存活率显著降低、细胞凋亡增加、 ATP水平下降、活化的caspase-3蛋白水平增高、 Bcl2蛋白水平降低。结论敲低激素抵抗性前列腺癌DU145细胞LRPPRC促进细胞凋亡。
Objective To investigate the effects of leucine-rich pentatricopeptide repeat containing(LRPPRC) on proliferation and apoptosis in hormone resistant prostate cancer cells. Methods LRPPRC and androgen receptor(AR) expressions in DU145 and LNCaP cells were detected by Western blot analysis. The siRNA targeting LRPPRC gene was transiently transfected into DU145 cells. LRPPRC expression was detected by reverse transcription PCR and Western blot analysis. The cell survival rate was analyzed by MTT assay. ATP was measured by luciferin-luciferase assay. The cell apoptosis was investigated by flow cytometry and Hoechst 33258 staining. Caspase-3, Bcl2 and BAX protein expressions were detected by Western blot analysis. Results DU145 and LNCaP cells highly expressed LRPPRC. LNCaP cells highly expressed AR, while no expression of AR was observed in DU145 cells. Down-regulation of LRPPRC decreased cell survival rate and ATP level, increased apoptosis rate and caspase-3 activation, and decreased Bcl2 expression in DU145 cells. Conclusion Knock-down of LRPPRC promotes apoptosis of hormone resistant prostate cancer cells.
Objective To investigate the effects of leucine-rich pentatricopeptide repeat containing(LRPPRC) on proliferation and apoptosis in hormone resistant prostate cancer cells. Methods LRPPRC and androgen receptor(AR) expressions in DU145 and LNCaP cells were detected by Western blot analysis. The siRNA targeting LRPPRC gene was transiently transfected into DU145 cells. LRPPRC expression was detected by reverse transcription PCR and Western blot analysis. The cell survival rate was analyzed by MTT assay. ATP was measured by luciferin-luciferase assay. The cell apoptosis was investigated by flow cytometry and Hoechst 33258 staining. Caspase-3, Bcl2 and BAX protein expressions were detected by Western blot analysis. Results DU145 and LNCaP cells highly expressed LRPPRC. LNCaP cells highly expressed AR, while no expression of AR was observed in DU145 cells. Down-regulation of LRPPRC decreased cell survival rate and ATP level, increased apoptosis rate and caspase-3 activation, and decreased Bcl2 expression in DU145 cells. Conclusion Knock-down of LRPPRC promotes apoptosis of hormone resistant prostate cancer cells.
引文
[1] Filson C P,Marks L S,Litwin M S.Expectant management for men with early stage prostate cancer[J].CA Cancer J Clin,2015,65(4):265-282.
[2] Wu Y S,Wu X B,Zhang N,et al.Evaluation of PSA-age volume score in predicting prostate cancer in Chinese population[J].Asian J Androl,2018,20(4):324-329.
[3] Crumbaker M,Khoja L,Joshua A M.AR signaling and the PI3K pathway in prostate cancer[J/OL].Cancers (Basel),2017,9(4):E34.DOI:10.3390/cancers9040034.
[4] Schweizer M T,Yu E Y.AR-signaling in human malignancies:Prostate cancer and beyond[J].Cancers (Basel),2017,9 (1):E7.DOI:10.3390/cancers9010007.
[5] Joshi J B,Patel D,Morton D J,et al.Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52[J].Mol Oncol,2017,11(4):337-357.
[6] Pal S K,Patel J,He M,et al.Identification of mechanisms of resistance to treatment with abiraterone acetate or enzalutamide in patients with castration-resistant prostate cancer (CRPC) [J].Cancer,2018,124(6):1216-1224.
[7] Zhao H,Freedland S.Enzalutamide treatment for the whole spectrum of CRPC[J].Nat Rev Urol,2018,15(11):663-665.
[8] Gross M E,Dorff T B,Quinn D I,et al.Safety and efficacy of docetaxel,bevacizumab,and everolimus for castration-resistant prostate cancer (CRPC)[J].Clin Genitourin Cancer,2018,16(1):e11-e21.
[9] Twum-Ampofo J,Fu D X,Passaniti A,et al.Metabolic targets for potential prostate cancer therapeutics[J].Curr Opin Oncol,2016,28(3):241-247.
[10] Zhao Y,Zeng X,Tang H,et al.Combination of metformin and paclitaxel suppresses proliferation and induces apoptosis of human prostate cancer cells via oxidative stress and targeting the mitochondria-dependent pathway[J].Oncol Lett,2019,17(5):4277-4284.
[11] Siira S J,Spahr H,Shearwood A J,et al.LRPPRC-mediated folding of the mitochondrial transcriptome[J/OL].Nat Commun,2017,8(1):1532.DOI:10.1038/s41467-017-01221-z.
[12] Zou J,Yue F,Li W,et al.Autophagy inhibitor LRPPRC suppresses mitophagy through interaction with mitophagy initiator Parkin[J/OL].PLoS One,2014,9(4):e94903.DOI:10.1371/journal.pone.0094903.eCollection 2014.
[13] Zou J,Li W,Misra A,et al.The viral restriction factor tetherin prevents leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) from association with beclin 1 and B-cell CLL/lymphoma 2 (Bcl2) and enhances autophagy and mitophagy[J].J Biol Chem,2015,290(11):7269-7279.
[14] Jiang X,Zhong W,Huang H,et al.Autophagy defects suggested by low levels of autophagy activator MAP1S and high levels of autophagy inhibitor LRPPRC predict poor prognosis of prostate cancer patients[J].Mol Carcinog,2015,54(10):1194-1204.
[15] Zhang H Y,Ma Y D,Zhang Y,et al.Elevated levels of autophagy-related marker ULK1 and mitochondrion-associated autophagy inhibitor LRPPRC are associated with biochemical progression and overall survival after androgen deprivation therapy in patients with metastatic prostate cancer[J].J Clin Pathol,2017,70(5):383-389.
[16] Jiang X,Li X,Huang H,et al.Elevated levels of mitochondrion-associated autophagy inhibitor LRPPRC are associated with poor prognosis in patients with prostate cancer[J].Cancer,2014,120(8):1228-1236.
[17] Nishio T,Kurabe N,Goto-Inoue N,et al.Immunohistochemical expression analysis of leucine-rich PPR-motif-containing protein (LRPPRC),a candidate colorectal cancer biomarker identified by shotgun proteomics using iTRAQ.Clin Chim Acta[J].2017,471:276-282.
[18] Gao W,Xua J,Wang F,et al.Mitochondrial proteomics approach reveals voltage-dependent anion channel 1 (VDAC1) as a potential biomarker of gastric cancer[J].Cell Physiol Biochem,2015,37(6):2339-2354.
[19] Fahrmann J F,Grapov D,Phinney B S,et al.Proteomic profiling of lung adenocarcinoma indicates heightened DNA repair,antioxidant mechanisms and identifies LASP1 as a potential negative predictor of survival[J/OL].Clin Proteomics,2016,13:31.DOI:10.1186/s12014-016-9132-y.eCollection 2016.
[20] Tian T,Ikeda J,Wang Y,et al.Role of leucine-rich pentatricopeptide repeat motif-containing protein (LRPPRC) for anti-apoptosis and tumourigenesis in cancers[J].Eur J Cancer,2012,48(15):2462-2473.
[21] Li X,Lv L,Zheng J,et al.The significance of LRPPRC overexpression in gastric cancer[J/OL].Med Oncol,2014,31(2):818.DOI:10.1007/s12032-013-0818-y.Epub 2013 Dec 28.
[22] Michaud M,Barakat S,Magnard S,et al.Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells[J].Int J Oncol,2011,38(1):169-178.
[23] Vyas S,Zaganjor E,Haigis M C.Mitochondria and Cancer[J].Cell,2016,166(3):555-566.
[24] Weinberg S E,Chandel N S.Targeting mitochondria metabolism for cancer therapy[J].Nat Chem Bio,2015,11(1):9-15.
[25] Lopez J,Tait S W.Mitochondrial apoptosis:killing cancer using the enemy within[J].Br J Cancer,2015,112(6):957-962.
[26] Mukaneza Y,Cohen A,Rivard M E,et al.mTORC1 is required for expression of LRPPRC and cytochrome c oxidase but not HIF-1α in Leigh syndrome French Canadian type patient fibroblasts[J].Am J Physiol Cell Physiol,2019 Apr 17.DOI:10.1152/ajpcell.00160.2017.[Epub ahead of print].
[2] Wu Y S,Wu X B,Zhang N,et al.Evaluation of PSA-age volume score in predicting prostate cancer in Chinese population[J].Asian J Androl,2018,20(4):324-329.
[3] Crumbaker M,Khoja L,Joshua A M.AR signaling and the PI3K pathway in prostate cancer[J/OL].Cancers (Basel),2017,9(4):E34.DOI:10.3390/cancers9040034.
[4] Schweizer M T,Yu E Y.AR-signaling in human malignancies:Prostate cancer and beyond[J].Cancers (Basel),2017,9 (1):E7.DOI:10.3390/cancers9010007.
[5] Joshi J B,Patel D,Morton D J,et al.Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52[J].Mol Oncol,2017,11(4):337-357.
[6] Pal S K,Patel J,He M,et al.Identification of mechanisms of resistance to treatment with abiraterone acetate or enzalutamide in patients with castration-resistant prostate cancer (CRPC) [J].Cancer,2018,124(6):1216-1224.
[7] Zhao H,Freedland S.Enzalutamide treatment for the whole spectrum of CRPC[J].Nat Rev Urol,2018,15(11):663-665.
[8] Gross M E,Dorff T B,Quinn D I,et al.Safety and efficacy of docetaxel,bevacizumab,and everolimus for castration-resistant prostate cancer (CRPC)[J].Clin Genitourin Cancer,2018,16(1):e11-e21.
[9] Twum-Ampofo J,Fu D X,Passaniti A,et al.Metabolic targets for potential prostate cancer therapeutics[J].Curr Opin Oncol,2016,28(3):241-247.
[10] Zhao Y,Zeng X,Tang H,et al.Combination of metformin and paclitaxel suppresses proliferation and induces apoptosis of human prostate cancer cells via oxidative stress and targeting the mitochondria-dependent pathway[J].Oncol Lett,2019,17(5):4277-4284.
[11] Siira S J,Spahr H,Shearwood A J,et al.LRPPRC-mediated folding of the mitochondrial transcriptome[J/OL].Nat Commun,2017,8(1):1532.DOI:10.1038/s41467-017-01221-z.
[12] Zou J,Yue F,Li W,et al.Autophagy inhibitor LRPPRC suppresses mitophagy through interaction with mitophagy initiator Parkin[J/OL].PLoS One,2014,9(4):e94903.DOI:10.1371/journal.pone.0094903.eCollection 2014.
[13] Zou J,Li W,Misra A,et al.The viral restriction factor tetherin prevents leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) from association with beclin 1 and B-cell CLL/lymphoma 2 (Bcl2) and enhances autophagy and mitophagy[J].J Biol Chem,2015,290(11):7269-7279.
[14] Jiang X,Zhong W,Huang H,et al.Autophagy defects suggested by low levels of autophagy activator MAP1S and high levels of autophagy inhibitor LRPPRC predict poor prognosis of prostate cancer patients[J].Mol Carcinog,2015,54(10):1194-1204.
[15] Zhang H Y,Ma Y D,Zhang Y,et al.Elevated levels of autophagy-related marker ULK1 and mitochondrion-associated autophagy inhibitor LRPPRC are associated with biochemical progression and overall survival after androgen deprivation therapy in patients with metastatic prostate cancer[J].J Clin Pathol,2017,70(5):383-389.
[16] Jiang X,Li X,Huang H,et al.Elevated levels of mitochondrion-associated autophagy inhibitor LRPPRC are associated with poor prognosis in patients with prostate cancer[J].Cancer,2014,120(8):1228-1236.
[17] Nishio T,Kurabe N,Goto-Inoue N,et al.Immunohistochemical expression analysis of leucine-rich PPR-motif-containing protein (LRPPRC),a candidate colorectal cancer biomarker identified by shotgun proteomics using iTRAQ.Clin Chim Acta[J].2017,471:276-282.
[18] Gao W,Xua J,Wang F,et al.Mitochondrial proteomics approach reveals voltage-dependent anion channel 1 (VDAC1) as a potential biomarker of gastric cancer[J].Cell Physiol Biochem,2015,37(6):2339-2354.
[19] Fahrmann J F,Grapov D,Phinney B S,et al.Proteomic profiling of lung adenocarcinoma indicates heightened DNA repair,antioxidant mechanisms and identifies LASP1 as a potential negative predictor of survival[J/OL].Clin Proteomics,2016,13:31.DOI:10.1186/s12014-016-9132-y.eCollection 2016.
[20] Tian T,Ikeda J,Wang Y,et al.Role of leucine-rich pentatricopeptide repeat motif-containing protein (LRPPRC) for anti-apoptosis and tumourigenesis in cancers[J].Eur J Cancer,2012,48(15):2462-2473.
[21] Li X,Lv L,Zheng J,et al.The significance of LRPPRC overexpression in gastric cancer[J/OL].Med Oncol,2014,31(2):818.DOI:10.1007/s12032-013-0818-y.Epub 2013 Dec 28.
[22] Michaud M,Barakat S,Magnard S,et al.Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells[J].Int J Oncol,2011,38(1):169-178.
[23] Vyas S,Zaganjor E,Haigis M C.Mitochondria and Cancer[J].Cell,2016,166(3):555-566.
[24] Weinberg S E,Chandel N S.Targeting mitochondria metabolism for cancer therapy[J].Nat Chem Bio,2015,11(1):9-15.
[25] Lopez J,Tait S W.Mitochondrial apoptosis:killing cancer using the enemy within[J].Br J Cancer,2015,112(6):957-962.
[26] Mukaneza Y,Cohen A,Rivard M E,et al.mTORC1 is required for expression of LRPPRC and cytochrome c oxidase but not HIF-1α in Leigh syndrome French Canadian type patient fibroblasts[J].Am J Physiol Cell Physiol,2019 Apr 17.DOI:10.1152/ajpcell.00160.2017.[Epub ahead of print].