NPRL2调控去势抵抗型前列腺癌对奥拉帕尼的敏感性
|
摘要
目的探讨NPRL2在改善去势抵抗型前列腺癌(castration-resistant prostate cancer,CRPC)对Olaparib敏感性的作用。方法收集良性前列腺增生(benign prostatic hyperplasia,BPH)、普通前列腺癌(prostate cancer,PCa)和CRPC患者手术标本进行NPRL2的免疫组化染色,观察NPRL2在上述组织中的表达差异。Western blot检测正常前列腺上皮细胞RWPE-1、前列腺癌细胞LNCaP和耐恩杂鲁胺的去势抵抗型前列腺癌细胞LNPER中NPRL2蛋白的表达情况。通过慢病毒转染到CRPC细胞建立NPRL2的过表达和干扰,实时定量荧光PCR(qPCR)和Western blot验证NPRL2过表达和干扰效率,蛋白免疫印迹检测ATM蛋白表达。CCK-8法检测细胞增殖和细胞毒性,流式细胞术检测细胞凋亡,Transwell小室侵袭和迁移实验检测细胞侵袭转移。结果免疫组化结果显示NPRL2在CRPC组织中呈现高表达。Western blot结果显示NPRL2在RWPE-1细胞中低表达,在LNCaP和LNPER细胞中高表达,并成功构建过表达和干扰NPRL2的稳定株,q PCR及Western blot结果显示慢病毒转染能明显增高和降低NPRL2的mRNA和蛋白水平(P <0. 01)。CCK-8、流式细胞术和Transwell实验结果显示,与阴性对照比较,沉默NPRL2联合Olaparib能明显抑制细胞生长、阻止细胞侵袭转移和促进细胞凋亡(P <0. 01)。NPRL2与ATM表达呈正相关,NPRL2过表达联合Olaparib显著增加ATM表达。结论沉默NPRL2可增加CRPC细胞对Olaparib的敏感性。
Objective To investigate the effect of nitrogen permease regulator like-2( NPRL2) on the sensitivity of castration-resistant prostate cancer( CRPC) to olaparib. Methods Immunohistochemical staining of NPRL2 was performed in surgical specimens from the patients with benign prostatic hyperplasia( BPH),common prostate cancer( PCa) and CRPC. The expression of NPRL2 in the above tissues was observed and compared. Western blot assay was used to detect the protein level of NPRL2 in normal prostate epithelial cell line RWPE-1,prostate cancer cell line LNCaP and enzaliline-resistant castrated prostate cancer cell line LNPER. Overexpression and interference of NPRL2 were established by transfection of lentivirus into CRPC cells. The overexpression and interference efficiencies of NPRL2 were verified by real-time fluorescent PCR and Western blotting. ATM protein expression was detected by Western blotting. Cell proliferation and cytotoxicity were detected by CCK-8 assay,cell apoptosis was detected by flow cytometry,and invasion and migration were used with Transwell chamber test. Results Immunohistochemical assay showed that NPRL2 was highly expressed in the CRPC tissues. Western blotting showed that NPRL2 was down-regulated in RWPE-1cells but highly expressed in LNCaP and LNPER cells. After successfully constructed a stable strain that overexpressed and interfered with NPRL2,q PCR and Western blotting showed that lentiviral transfection significantly increased and decreased the expression of NPRL2 at mRNA and protein levels,respectively( P <0. 01). And the results of CCK8 assay,flow cytometry and Transwell test indicated that silencing NPRL2 combined with olaparib treatment significantly inhibited cell growth,prevented cell invasion and metastasis,and promoted cell apoptosis when compared with the negative control group( P < 0. 01). NPRL2 was positively correlated with ATM expression,and NPRL2 overexpression combined with olaparib treatment significantly enhanced ATM expression. Conclusion NPRL2 interference increases the sensitivity of CRPC cells to olaparib.
Objective To investigate the effect of nitrogen permease regulator like-2( NPRL2) on the sensitivity of castration-resistant prostate cancer( CRPC) to olaparib. Methods Immunohistochemical staining of NPRL2 was performed in surgical specimens from the patients with benign prostatic hyperplasia( BPH),common prostate cancer( PCa) and CRPC. The expression of NPRL2 in the above tissues was observed and compared. Western blot assay was used to detect the protein level of NPRL2 in normal prostate epithelial cell line RWPE-1,prostate cancer cell line LNCaP and enzaliline-resistant castrated prostate cancer cell line LNPER. Overexpression and interference of NPRL2 were established by transfection of lentivirus into CRPC cells. The overexpression and interference efficiencies of NPRL2 were verified by real-time fluorescent PCR and Western blotting. ATM protein expression was detected by Western blotting. Cell proliferation and cytotoxicity were detected by CCK-8 assay,cell apoptosis was detected by flow cytometry,and invasion and migration were used with Transwell chamber test. Results Immunohistochemical assay showed that NPRL2 was highly expressed in the CRPC tissues. Western blotting showed that NPRL2 was down-regulated in RWPE-1cells but highly expressed in LNCaP and LNPER cells. After successfully constructed a stable strain that overexpressed and interfered with NPRL2,q PCR and Western blotting showed that lentiviral transfection significantly increased and decreased the expression of NPRL2 at mRNA and protein levels,respectively( P <0. 01). And the results of CCK8 assay,flow cytometry and Transwell test indicated that silencing NPRL2 combined with olaparib treatment significantly inhibited cell growth,prevented cell invasion and metastasis,and promoted cell apoptosis when compared with the negative control group( P < 0. 01). NPRL2 was positively correlated with ATM expression,and NPRL2 overexpression combined with olaparib treatment significantly enhanced ATM expression. Conclusion NPRL2 interference increases the sensitivity of CRPC cells to olaparib.
引文
[1] ABESHOUSE A,AHN J,AKBANI R,et al. The molecular taxonomy of primary prostate cancer[J]. Cell,2015,163(4):1011-1025. DOI:10. 1016/j. cell. 2015. 10. 025.
[2] NA R,YE D,QI J,et al. Prostate health index significantly reduced unnecessary prostate biopsies in patients with PSA 2-10 ng/m L and PSA> 10 ng/m L:Results from a multicenter study in China[J]. Prostate,2017,77(11):1221-1229.DOI:10. 1002/pros. 23382.
[3] ROSS R W,XIE W,REGAN M M,et al. Efficacy of androgen deprivation therapy(ADT)in patients with advanced prostate cancer:association between Gleason score,prostatespecific antigen level,and prior ADT exposure with duration of ADT effect[J]. Cancer,2008,112(6):1247-1253.DOI:10. 1002/cncr. 23304.
[4] ITO Y,SADAR M D. Enzalutamide and blocking androgen receptor in advanced prostate cancer:lessons learnt from the history of drug development of antiandrogens[J]. Res Rep Urol,2018,10:23-32. DOI:10. 2147/RRU. S157116.
[5] MOTTET N,BELLMUNT J,BOLLA M,et al. EAU guidelines on prostate cancer. PartⅡ:Treatment of advanced,relapsing,and castration-resistant prostate cancer[J]. Eur Urol,2011,59(4):572-583. DOI:10. 1016/j. eururo.2011. 01. 025.
[6] LERMAN M I,MINNA J D. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21. 3:identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome3p21. 3 Tumor Suppressor Gene Consortium[J]. Cancer Res,2000,60(21):6116-6133.
[7] LI J,WANG F,HARALDSON K,et al. Functional characterization of the candidate tumor suppressor gene NPRL2/G21located in 3p21. 3C[J]. Cancer Res,2004,64(18):6438-6443. DOI:10. 1158/0008-5472. can-03-3869.
[8] FARMER H,MCCABE N,LORD C J,et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy[J]. Nature,2005,434(7035):917-921. DOI:10. 1038/nature03445.
[9] ROBINSON D,VAN ALLEN E M,WU Y M,et al. Integrative clinical genomics of advanced prostate cancer[J]. Cell,2015,161(5):1215-1228. DOI:10. 1016/j. cell. 2015.05. 001.
[10] JAYACHANDRAN G,UEDA K,WANG B,et al. NPRL2sensitizes human non-small cell lung cancer(NSCLC)cells to cisplatin treatment by regulating key components in the DNA repair pathway[J]. PLo S ONE,2010,5(8):e11994.DOI:10. 1371/journal. pone. 0011994.
[11] LIU S,LIU B. Overexpression of nitrogen permease regulator like-2(NPRL2)enhances sensitivity to irinotecan(CPT-11)in colon cancer cells by activating the DNA damage checkpoint pathway[J]. Med Sci Monit,2018,24:1424-1433. DOI:10. 12659/msm. 909186.
[12] PENG Y,DAI H,WANG E,et al. TUSC4 functions as a tumor suppressor by regulating BRCA1 stability[J]. Cancer Res,2015,75(2):378-386. DOI:10. 1158/0008-5472.can-14-2315.
[13] LIU M N,LIU A Y,PEI F H,et al. Functional mechanism of the enhancement of 5-fluorouracil sensitivity by TUSC4 in colon cancer cells[J]. Oncol Lett,2015,10(6):3682-3688. DOI:10. 3892/ol. 2015. 3801.
[14] HUANG N,CHENG S,MI X,et al. Downregulation of nitrogen permease regulator like-2 activates PDK1-AKT1 and contributes to the malignant growth of glioma cells[J]. Mol Carcinog,2016,55(11):1613-1626. DOI:10. 1002/mc. 22413.
[15] TANG Y,JIANG L,TANG W. Decreased expression of NPRL2 in renal cancer cells is associated with unfavourable pathological,proliferation and apoptotic features[J]. Pathol Oncol Res,2014,20(4):829-837. DOI:10. 1007/s12253-014-9761-2.
[16] BAR-PELED L,CHANTRANUPONG L,CHERNIACK A D,et al. A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to m TORC1[J]. Science,2013,340(6136):1100-1106. DOI:10.1126/science. 1232044.
[17] CHEN Z,LUO S,CHEN Y,et al. High expression of NPRL2 is linked to poor prognosis in patients with prostate cancer[J]. Hum Pathol,2018,76:141-148. DOI:10.1016/j. humpath. 2018. 02. 011.
[18] MATEO J,CARREIRA S,SANDHU S,et al. DNA-Repair defects and olaparib in metastatic prostate cancer[J]. N Engl J Med,2015,373(18):1697-1708. DOI:10. 1056/NEJMoa1506859.
[19] ZHANG D,TANG B,XIE X,et al. The interplay between DNA repair and autophagy in cancer therapy[J]. Cancer Biol Ther,2015,16(7):1005-1013. DOI:10. 1080/15384047. 2015. 1046022.
[20] LI Y,YANG D Q. The ATM inhibitor KU-55933 suppresses cell proliferation and induces apoptosis by blocking Akt in cancer cells with overactivated Akt[J]. Mol Cancer Ther,2010,9(1):113-125. DOI:10. 1158/1535-7163. mct-08-1189.
[21] REDDY V,WU M,CIAVATTONE N,et al. ATM inhibition potentiates death of androgen receptor-inactivated prostate cancer cells with telomere dysfunction[J]. J Biol Chem,2015,290(42):25522-25533. DOI:10. 1074/jbc. M115. 671404.
[22] WESTON V J,OLDREIVE C E,SKOWRONSKA A,et al.The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo[J].Blood,2010,116(22):4578-4587. DOI:10. 1182/blood-2010-01-265769.
[2] NA R,YE D,QI J,et al. Prostate health index significantly reduced unnecessary prostate biopsies in patients with PSA 2-10 ng/m L and PSA> 10 ng/m L:Results from a multicenter study in China[J]. Prostate,2017,77(11):1221-1229.DOI:10. 1002/pros. 23382.
[3] ROSS R W,XIE W,REGAN M M,et al. Efficacy of androgen deprivation therapy(ADT)in patients with advanced prostate cancer:association between Gleason score,prostatespecific antigen level,and prior ADT exposure with duration of ADT effect[J]. Cancer,2008,112(6):1247-1253.DOI:10. 1002/cncr. 23304.
[4] ITO Y,SADAR M D. Enzalutamide and blocking androgen receptor in advanced prostate cancer:lessons learnt from the history of drug development of antiandrogens[J]. Res Rep Urol,2018,10:23-32. DOI:10. 2147/RRU. S157116.
[5] MOTTET N,BELLMUNT J,BOLLA M,et al. EAU guidelines on prostate cancer. PartⅡ:Treatment of advanced,relapsing,and castration-resistant prostate cancer[J]. Eur Urol,2011,59(4):572-583. DOI:10. 1016/j. eururo.2011. 01. 025.
[6] LERMAN M I,MINNA J D. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21. 3:identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome3p21. 3 Tumor Suppressor Gene Consortium[J]. Cancer Res,2000,60(21):6116-6133.
[7] LI J,WANG F,HARALDSON K,et al. Functional characterization of the candidate tumor suppressor gene NPRL2/G21located in 3p21. 3C[J]. Cancer Res,2004,64(18):6438-6443. DOI:10. 1158/0008-5472. can-03-3869.
[8] FARMER H,MCCABE N,LORD C J,et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy[J]. Nature,2005,434(7035):917-921. DOI:10. 1038/nature03445.
[9] ROBINSON D,VAN ALLEN E M,WU Y M,et al. Integrative clinical genomics of advanced prostate cancer[J]. Cell,2015,161(5):1215-1228. DOI:10. 1016/j. cell. 2015.05. 001.
[10] JAYACHANDRAN G,UEDA K,WANG B,et al. NPRL2sensitizes human non-small cell lung cancer(NSCLC)cells to cisplatin treatment by regulating key components in the DNA repair pathway[J]. PLo S ONE,2010,5(8):e11994.DOI:10. 1371/journal. pone. 0011994.
[11] LIU S,LIU B. Overexpression of nitrogen permease regulator like-2(NPRL2)enhances sensitivity to irinotecan(CPT-11)in colon cancer cells by activating the DNA damage checkpoint pathway[J]. Med Sci Monit,2018,24:1424-1433. DOI:10. 12659/msm. 909186.
[12] PENG Y,DAI H,WANG E,et al. TUSC4 functions as a tumor suppressor by regulating BRCA1 stability[J]. Cancer Res,2015,75(2):378-386. DOI:10. 1158/0008-5472.can-14-2315.
[13] LIU M N,LIU A Y,PEI F H,et al. Functional mechanism of the enhancement of 5-fluorouracil sensitivity by TUSC4 in colon cancer cells[J]. Oncol Lett,2015,10(6):3682-3688. DOI:10. 3892/ol. 2015. 3801.
[14] HUANG N,CHENG S,MI X,et al. Downregulation of nitrogen permease regulator like-2 activates PDK1-AKT1 and contributes to the malignant growth of glioma cells[J]. Mol Carcinog,2016,55(11):1613-1626. DOI:10. 1002/mc. 22413.
[15] TANG Y,JIANG L,TANG W. Decreased expression of NPRL2 in renal cancer cells is associated with unfavourable pathological,proliferation and apoptotic features[J]. Pathol Oncol Res,2014,20(4):829-837. DOI:10. 1007/s12253-014-9761-2.
[16] BAR-PELED L,CHANTRANUPONG L,CHERNIACK A D,et al. A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to m TORC1[J]. Science,2013,340(6136):1100-1106. DOI:10.1126/science. 1232044.
[17] CHEN Z,LUO S,CHEN Y,et al. High expression of NPRL2 is linked to poor prognosis in patients with prostate cancer[J]. Hum Pathol,2018,76:141-148. DOI:10.1016/j. humpath. 2018. 02. 011.
[18] MATEO J,CARREIRA S,SANDHU S,et al. DNA-Repair defects and olaparib in metastatic prostate cancer[J]. N Engl J Med,2015,373(18):1697-1708. DOI:10. 1056/NEJMoa1506859.
[19] ZHANG D,TANG B,XIE X,et al. The interplay between DNA repair and autophagy in cancer therapy[J]. Cancer Biol Ther,2015,16(7):1005-1013. DOI:10. 1080/15384047. 2015. 1046022.
[20] LI Y,YANG D Q. The ATM inhibitor KU-55933 suppresses cell proliferation and induces apoptosis by blocking Akt in cancer cells with overactivated Akt[J]. Mol Cancer Ther,2010,9(1):113-125. DOI:10. 1158/1535-7163. mct-08-1189.
[21] REDDY V,WU M,CIAVATTONE N,et al. ATM inhibition potentiates death of androgen receptor-inactivated prostate cancer cells with telomere dysfunction[J]. J Biol Chem,2015,290(42):25522-25533. DOI:10. 1074/jbc. M115. 671404.
[22] WESTON V J,OLDREIVE C E,SKOWRONSKA A,et al.The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo[J].Blood,2010,116(22):4578-4587. DOI:10. 1182/blood-2010-01-265769.