NKX3.1, which is a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as a tumour suppressor gene. In this study, we investigated the inhibitory effect of NKX3,1 on insul...NKX3.1, which is a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as a tumour suppressor gene. In this study, we investigated the inhibitory effect of NKX3,1 on insulin-like growth factor （IGF）- 1R expression and its downstream signalling pathway in PC3 cells, PC3 cells were stably transfected with NKX3.1 expression plasmid （pcDNA3.1-NKX3.1） or vector plasmid （pcDNA3.1＋）. The IGF-IR mRNA and protein expression levels were assessed in PC3-NKX3.1 transfectants by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. The expression and activation of IGF-1/IGF-1R downstream signalling targets were examined by Western blotting and luciferase reporter assay. The cells were subsequently treated with relevant concentrations of IGF-1. The effect of IGF-1 on cell growth was examined by 3-（4,5）-dimethylthiahiazo（-z-yl）-3, 5-diphenytetrazoliumromide （MTT） assay and flow cytometry analysis. A significant suppression of IGF-1R mRNA and protein expression was observed after forced expression of NKX3.1 in PC3 cells. Correspondingly, the forced expression of NKX3.1 decreased IGF-l-induced phosphorylation of extracellular signal-regulated kinase 1/2 （ERK1/2） and protein kinase B （AKT） and activation of the Elk-1 transcription factor and downregulated the expression of the downstream target genes c-fos and cyclin D1. Furthermore, the forced expression of NKX3.1 inhibited IGF-l-induced cell growth. In conclusion, NKX3.1 could downregulate IGF-1R expression and could inhibit IGF-1R-mediated mitogen-activated protein kinase （MAPK）/ERK and AKT signalling pathways, which might partially leads to the inhibition of IGF-l-induced cell growth. This study provides new insights into the molecular mechanisms that NKX3,1 exerts against prostate cancer and ultimately expands the scope of alternative approaches in advanced prostate cancer therapy.展开更多
Aim： To explore whether the anti-tumor action of 17β-estradiol is enhanced by re-expression of the homeodomain transcription factor Nkx3.1 in PC3 human prostate cancer cells. Methods： PC3 cells were stably transfec...Aim： To explore whether the anti-tumor action of 17β-estradiol is enhanced by re-expression of the homeodomain transcription factor Nkx3.1 in PC3 human prostate cancer cells. Methods： PC3 cells were stably transfected with pcDNA3.1-Nkx3.1-His vector, which carries a full-length cDNA of human Nkx3.1. The PC3 cells stably transfected with vector pcDNA3.1 were set as a control. The expression of Nkx3.1 protein in the cells was confirmed by Western blot analysis. The effect of Nkx3.1 on cell proliferation of PC3 cells was examined with MTT assay. The antiproliferative and apoptotic effects of 17β-estradiol alone or in combination with Nkx3.1 were estimated on PC3 cells by using MTT growth tests and flow cytometric analyses. The expression of apoptosis-related proteins was analyzed using Western blotting. Results： The plasmid carrying Nkx3.1 gene induced high expression of Nkx3.1 protein in PC3 cells. The re-expression of exogenous Nkx3.1 did not cause a significant reduction in cellular proliferation, whereas the expression of Nkx3.1 enhanced the 17β-estradiol anti-proliferative effect in PC3 cells. Nkx3.1 expres- sion promoted 17β-estradiol-induced apoptosis of PC3 cells, as shown by analysis of Bcl-2, Bax, Caspase-3 and poly （ADP-ribose） polymerase expression. Conclusion： The present study demonstrates that re-expression of Nkx3.1 enhances 17β-estradiol anti-tumor action in PC3 human prostate cancer cells. The in vitro study suggests that re- expression of Nkx3.1 is worthy of further consideration as an adjuvant treatment of androgen independent prostate cancer with estrogen anti-tumor therapies.展开更多
Aim:To study the regulatory effects of 9-cis retinoic acid(RA)on the expression of human homeobox gene NKX3.1 in prostate cancer cell line LNCaP.Methods:Flow cytometry,reverse transcriptase polymerase chain reaction a...Aim:To study the regulatory effects of 9-cis retinoic acid(RA)on the expression of human homeobox gene NKX3.1 in prostate cancer cell line LNCaP.Methods:Flow cytometry,reverse transcriptase polymerase chain reaction and Western blotting were performed to evaluate the effects of 9-cis RA on NKX3.1 expression and cell cycle of LNCaP cells.To identify a regulatory region within the NKX3.1 promoter contributing to the regulation induced by 9-cis RA, we have constructed an NKX3.1 promoter-reporter plasmid,pGL_3-1040bp,and its 5′-deletion mutants,which were transfected into LNCaP cells with treatment of 9-cis RA in indicated concentrations.Results:With the treatment of 9-cis RA,the NKX3.1 promoter activity was increased in reporter gene assay and NKX3.1 expression was enhanced at both mRNA and protein levels in LNCaP cells.We found that the region between -936 and -921 in the upstream of NKX3.1 gene involved the inducible regulation by 9-cis RA treatment.In flow cytometry,9-cis RA treatment caused accumulation of cells in the G_1 phase of the cell cycle and a fewer cells pass through to G_2/M.Conclusion:Our results demonstrated that 9-cis RA as a differentiating agent can arrest prostate cancer cells in G_1 phase and reduce cell mitosis,and upregulate the expression of human homeobox gene NKX3.1,which is thought to play an important role in prostate differentiation and to act as a tumor suppressor gene in the prostate.(Asian J Androl 2006 Jul;8:435-441)展开更多
文摘NKX3.1, which is a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as a tumour suppressor gene. In this study, we investigated the inhibitory effect of NKX3,1 on insulin-like growth factor （IGF）- 1R expression and its downstream signalling pathway in PC3 cells, PC3 cells were stably transfected with NKX3.1 expression plasmid （pcDNA3.1-NKX3.1） or vector plasmid （pcDNA3.1＋）. The IGF-IR mRNA and protein expression levels were assessed in PC3-NKX3.1 transfectants by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. The expression and activation of IGF-1/IGF-1R downstream signalling targets were examined by Western blotting and luciferase reporter assay. The cells were subsequently treated with relevant concentrations of IGF-1. The effect of IGF-1 on cell growth was examined by 3-（4,5）-dimethylthiahiazo（-z-yl）-3, 5-diphenytetrazoliumromide （MTT） assay and flow cytometry analysis. A significant suppression of IGF-1R mRNA and protein expression was observed after forced expression of NKX3.1 in PC3 cells. Correspondingly, the forced expression of NKX3.1 decreased IGF-l-induced phosphorylation of extracellular signal-regulated kinase 1/2 （ERK1/2） and protein kinase B （AKT） and activation of the Elk-1 transcription factor and downregulated the expression of the downstream target genes c-fos and cyclin D1. Furthermore, the forced expression of NKX3.1 inhibited IGF-l-induced cell growth. In conclusion, NKX3.1 could downregulate IGF-1R expression and could inhibit IGF-1R-mediated mitogen-activated protein kinase （MAPK）/ERK and AKT signalling pathways, which might partially leads to the inhibition of IGF-l-induced cell growth. This study provides new insights into the molecular mechanisms that NKX3,1 exerts against prostate cancer and ultimately expands the scope of alternative approaches in advanced prostate cancer therapy.
文摘Aim： To explore whether the anti-tumor action of 17β-estradiol is enhanced by re-expression of the homeodomain transcription factor Nkx3.1 in PC3 human prostate cancer cells. Methods： PC3 cells were stably transfected with pcDNA3.1-Nkx3.1-His vector, which carries a full-length cDNA of human Nkx3.1. The PC3 cells stably transfected with vector pcDNA3.1 were set as a control. The expression of Nkx3.1 protein in the cells was confirmed by Western blot analysis. The effect of Nkx3.1 on cell proliferation of PC3 cells was examined with MTT assay. The antiproliferative and apoptotic effects of 17β-estradiol alone or in combination with Nkx3.1 were estimated on PC3 cells by using MTT growth tests and flow cytometric analyses. The expression of apoptosis-related proteins was analyzed using Western blotting. Results： The plasmid carrying Nkx3.1 gene induced high expression of Nkx3.1 protein in PC3 cells. The re-expression of exogenous Nkx3.1 did not cause a significant reduction in cellular proliferation, whereas the expression of Nkx3.1 enhanced the 17β-estradiol anti-proliferative effect in PC3 cells. Nkx3.1 expres- sion promoted 17β-estradiol-induced apoptosis of PC3 cells, as shown by analysis of Bcl-2, Bax, Caspase-3 and poly （ADP-ribose） polymerase expression. Conclusion： The present study demonstrates that re-expression of Nkx3.1 enhances 17β-estradiol anti-tumor action in PC3 human prostate cancer cells. The in vitro study suggests that re- expression of Nkx3.1 is worthy of further consideration as an adjuvant treatment of androgen independent prostate cancer with estrogen anti-tumor therapies.
文摘Aim:To study the regulatory effects of 9-cis retinoic acid(RA)on the expression of human homeobox gene NKX3.1 in prostate cancer cell line LNCaP.Methods:Flow cytometry,reverse transcriptase polymerase chain reaction and Western blotting were performed to evaluate the effects of 9-cis RA on NKX3.1 expression and cell cycle of LNCaP cells.To identify a regulatory region within the NKX3.1 promoter contributing to the regulation induced by 9-cis RA, we have constructed an NKX3.1 promoter-reporter plasmid,pGL_3-1040bp,and its 5′-deletion mutants,which were transfected into LNCaP cells with treatment of 9-cis RA in indicated concentrations.Results:With the treatment of 9-cis RA,the NKX3.1 promoter activity was increased in reporter gene assay and NKX3.1 expression was enhanced at both mRNA and protein levels in LNCaP cells.We found that the region between -936 and -921 in the upstream of NKX3.1 gene involved the inducible regulation by 9-cis RA treatment.In flow cytometry,9-cis RA treatment caused accumulation of cells in the G_1 phase of the cell cycle and a fewer cells pass through to G_2/M.Conclusion:Our results demonstrated that 9-cis RA as a differentiating agent can arrest prostate cancer cells in G_1 phase and reduce cell mitosis,and upregulate the expression of human homeobox gene NKX3.1,which is thought to play an important role in prostate differentiation and to act as a tumor suppressor gene in the prostate.(Asian J Androl 2006 Jul;8:435-441)
