Aim： To characterize the matrix metalloproteinases （MMP）-2 promoter and to identify androgen response elements （AREs） involved in androgen-induced MMP-2 expression. Methods： MMP-2 mRNA levels was determined by r...Aim： To characterize the matrix metalloproteinases （MMP）-2 promoter and to identify androgen response elements （AREs） involved in androgen-induced MMP-2 expression. Methods： MMP-2 mRNA levels was determined by reverse transcription-polymerase chain reaction （RT-PCR）. MMP-2 promoter-driven luciferase assays were used to determine the fragments responsible for androgen-induced activity. Chromatin-immunoprecipitation assay and electrophoretic mobility shift assays （EMSA） were used to verify the identified AREs in the MMP-2 promoter. Results： Androgen significantly induced MMP-2 expression at the mRNA level, which was blocked by the androgen antagonist bicalutamide. Deletion of a region encompassing base pairs -1591 to -1259 （relative to the start codon） of the MMP-2 promoter led to a significant loss of androgen-induced reporter activity. Additional deletion of the 5＇-region up to -562 bp further reduced the androgen-induced MMP-2 promoter activity. Sequence analysis of these two regions revealed two putative ARE motifs. Introducing mutations in the putative ARE motifs by site-directed mutagenesis approach resulted in a dramatic loss of androgen-induced MMP-2 promoter activity, indicating that the putative ARE motifs are required for androgen-stimulated MMP-2 expression. Most importantly, the androgen receptor （AR） interacted with both motif-containing promoter regions in vivo in a chromatin immunoprecipitation assay after androgen treatment. Furthermore, the AR specifically bound to the wild-type but not mutated ARE motifs-containing probes in an in vitro EMSA assay. Conclusion： Two ARE motifs were identified to be responsible for androgen-induced MMP-2 expression in prostate cancer cells.展开更多
Background There is increasing interest in the role of dietary factors in both the development and behaviour of prostate cancer.This study was carried out to evaluate the impact of the dietary factor lycopene on DNA s...Background There is increasing interest in the role of dietary factors in both the development and behaviour of prostate cancer.This study was carried out to evaluate the impact of the dietary factor lycopene on DNA synthesis,activity and expression of the androgen receptor gene element in prostate LnCaP cells and to report our pilot phase Ⅱ study investigating its effect on prostate-specific antigen velocity over one year.Methods LnCaP cells were grown with or without different concentrations of lycopene or tetrahydrofuran （THF solvent）added to the culture medium for 48 hours.DNA synthesis was measured by the incorporation of bromodeoxyuridine （Brdu） into DNA during a 4-hour pulse, followed by immunostaining and visualization of stained cells using fluorescence microscopy.A transient transfection of a plasmid DNA recombinant containing an androgen receptor element-luciferase （ARE-Luc） report gene into LnCaP cells was developed and the impact of different concentrations of lycopene on the androgen receptor element was reflected by quantitative analysis of the luciferase enzyme function.Expression of the androgen gene was also studied by Western blotting.The phase Ⅱ pilot study patients （n=41） previously diagnosed with prostate cancer were enrolled and given lycopene supplement, 10 mg per day, and response was measured by observing changes in the plasma prostate-specific antigen （PSA） levels.Results The addition of 0.5 μmol/L, 5 μmol/L, 10 μmol/L and 15 μmol/L of lycopene was shown to inhibit cell growth by 2.66%, 4.29%, 3.73% and 13.66%, respectively, compared with the THF solvent control samples （P=0.015）.As compared with the RPMI1640 medium group, cell proliferation in the presence of 5 μmol/L, 10 μmol/L, and 15 μmol/L lycopene was inhibited by 8.12%, 6.33% and 12.00%, respectively （P=0.024）.We showed for the first time that lycopene inhibited the activity of the androgen receptor gene element in a dose-related manner.Inhibition was seen in the transcription of the luciferase construct and confirmed by androgen receptor element expression assayed by Western blotting.Regression slopes of （log） PSA vs.time decreased in 26/37 （70%, 95% CI 53%-84%） of the patients after supplementation and in eight cases （21%） the post-treatment slope was negative.For these eight patients, the average fall in PSA was equivalent to 2% over 28 days （i.e.an average slope/d of -0.000 713）.The Wilcoxon rank-sum test showed an overall statistically significant decrease in slope （P=0.0007）.Analysis of the PSA doubling time （pretreatment vs.posttreatment） showed a median increase after supplementation for 174 days; however, this was not statistically significant （P=0.18）.Conclusions Lycopene as an antioxidant dietary factor could significantly inhibit DNA synthesis in a dose-dependent pattern; the result revealed lycopene might inhibit androgen receptor gene element activity and expression.Dietary lycopene may play an important role in prostate cancer cell proliferation and further supports a large randomized study into the role of lycopene supplementation in malignant prostate disease.展开更多
We cloned the three androgen response elements（AREs, including AREI, AREII, and AREIII ） with a core transactivation TATA element of the prostate-specific antigen（PSA） promoter into pGL2 basic vector to create an ...We cloned the three androgen response elements（AREs, including AREI, AREII, and AREIII ） with a core transactivation TATA element of the prostate-specific antigen（PSA） promoter into pGL2 basic vector to create an artificial pGL2/AREs-TATA reporter system, which was applied to evaluating the effects of different xeno- oestrogens[bisphenol A（BPA）, 4-nonylphenol（4-NP）, dichlorodiphenyl trichloroethane（DDT） or diethylstilbestrol （DES）] on androgen receptor（AR） abnormal activation to regulate PSA expression and cell proliferation. In all the three AREs, AREIII-TATA displayed as a major element responsive to AR-mediated DHT stimulation of PSA promoter. Therefore, pGL2/AREIII-TATA reporter was adopted to analyze the activation capacity of AR activated by four different xeno-oestrogens. The activation of pGL2/AREIII-TATA reporter by each xeno-oestrogen was analyzed in two different cell lines, one was HEK293T（Human Embryonic Kidney 293T） cell line, and the other was AR stably expressed DU145 cell line, which was produced by infecting AR with pLenti-puro-AR into the prostate cancer DU145 cells and that were scanned with puromycin and tested by AR antibody. In both the two cell lines, BPA or DES significantly induced AR-mediated transcriptional activity of AREIII-TATA reporter, whereas DDT or 4-nonylphenol did not. Moreover, AR-mediated cell proliferation in response to each of four xeno-oestrogens was measured in MTT assays in both HEK293T cell or AR stably expressed DUI45 cell lines. BPA or DES, as an AR inducer, exhibited an enhanced effect in cell proliferation, rather than the effect of DDT or 4-NP, in both cell lines. Finally, we demonstrated that BPA or DES stimulated PSA expression and enhanced the recruitment of AR onto the PSA promoter, resulting in stronger binding to AREIII sites. Taken together, four xeno-oestrogens were identified to have different activities on AR. BPA and DES are demonstrated to be androgenic effectors in the regulation of PSA activation or cell proliferation.展开更多
目的我们前期研究中进行全基因组表达谱检测发现在雄激素受体（androgen receptor，Ar）基因睾丸支持细胞特异性敲除小鼠（S-Ar^-/y）睾丸组织中泛素特异性蛋白酶25（ubiquitin specific peptidase 25，usp25）基因表达较低。本研究的...目的我们前期研究中进行全基因组表达谱检测发现在雄激素受体（androgen receptor，Ar）基因睾丸支持细胞特异性敲除小鼠（S-Ar^-/y）睾丸组织中泛素特异性蛋白酶25（ubiquitin specific peptidase 25，usp25）基因表达较低。本研究的目的是了解雄激素及其受体是否可以作用于Usp25基因，并测定其雄激素反应元件（androgen-responsive element，ARE）。方法采用RT-qPCR方法检测Usp25基因表达量。通过生物信息学预测脚25基因上游可能的ARE，构建Usp25基因ARE报告质粒pGLP／Usp25。在TM4细胞中，采用荧光素酶报告系统分析雄激素及其受体对Usp25基因ARE活性的调控作用。结果在S-Ar^-/y小鼠睾丸组织中矾p25基因表达量比在野生型小鼠中显著降低。在TM4细胞中睾酮可以显著提高Usp25基因表达量。在TM4细胞中，雄激素可以显著提高pGLP／Usp25的荧光素酶活性。结论Usp25基因表达可以被雄激素睾酮激活，Usp25基因第一内含子519至1102bp区域含有ARE，可以调控启动子的转录水平。展开更多
基金Acknowledgment We thank Dr Etty N. Benveniste （University of Alabama at Birmingham, Birmingham, AL, USA） for the truncated MMP-2 promoter-driven luciferase constructs and Ms Donna Barnes for excellent secretarial assistance. This study was supported by KU William L.Valk Endowment and Kansas Mason＇s Foundation, and a grant from KUMC Lied Foundation to Dr Ben-Yi Li. This study was also partially supported by grants from the National Natural Science Foundation of China （No. 30370509 and No. 30370645） to Dr Ping-Yi Xu.
文摘Aim： To characterize the matrix metalloproteinases （MMP）-2 promoter and to identify androgen response elements （AREs） involved in androgen-induced MMP-2 expression. Methods： MMP-2 mRNA levels was determined by reverse transcription-polymerase chain reaction （RT-PCR）. MMP-2 promoter-driven luciferase assays were used to determine the fragments responsible for androgen-induced activity. Chromatin-immunoprecipitation assay and electrophoretic mobility shift assays （EMSA） were used to verify the identified AREs in the MMP-2 promoter. Results： Androgen significantly induced MMP-2 expression at the mRNA level, which was blocked by the androgen antagonist bicalutamide. Deletion of a region encompassing base pairs -1591 to -1259 （relative to the start codon） of the MMP-2 promoter led to a significant loss of androgen-induced reporter activity. Additional deletion of the 5＇-region up to -562 bp further reduced the androgen-induced MMP-2 promoter activity. Sequence analysis of these two regions revealed two putative ARE motifs. Introducing mutations in the putative ARE motifs by site-directed mutagenesis approach resulted in a dramatic loss of androgen-induced MMP-2 promoter activity, indicating that the putative ARE motifs are required for androgen-stimulated MMP-2 expression. Most importantly, the androgen receptor （AR） interacted with both motif-containing promoter regions in vivo in a chromatin immunoprecipitation assay after androgen treatment. Furthermore, the AR specifically bound to the wild-type but not mutated ARE motifs-containing probes in an in vitro EMSA assay. Conclusion： Two ARE motifs were identified to be responsible for androgen-induced MMP-2 expression in prostate cancer cells.
文摘Background There is increasing interest in the role of dietary factors in both the development and behaviour of prostate cancer.This study was carried out to evaluate the impact of the dietary factor lycopene on DNA synthesis,activity and expression of the androgen receptor gene element in prostate LnCaP cells and to report our pilot phase Ⅱ study investigating its effect on prostate-specific antigen velocity over one year.Methods LnCaP cells were grown with or without different concentrations of lycopene or tetrahydrofuran （THF solvent）added to the culture medium for 48 hours.DNA synthesis was measured by the incorporation of bromodeoxyuridine （Brdu） into DNA during a 4-hour pulse, followed by immunostaining and visualization of stained cells using fluorescence microscopy.A transient transfection of a plasmid DNA recombinant containing an androgen receptor element-luciferase （ARE-Luc） report gene into LnCaP cells was developed and the impact of different concentrations of lycopene on the androgen receptor element was reflected by quantitative analysis of the luciferase enzyme function.Expression of the androgen gene was also studied by Western blotting.The phase Ⅱ pilot study patients （n=41） previously diagnosed with prostate cancer were enrolled and given lycopene supplement, 10 mg per day, and response was measured by observing changes in the plasma prostate-specific antigen （PSA） levels.Results The addition of 0.5 μmol/L, 5 μmol/L, 10 μmol/L and 15 μmol/L of lycopene was shown to inhibit cell growth by 2.66%, 4.29%, 3.73% and 13.66%, respectively, compared with the THF solvent control samples （P=0.015）.As compared with the RPMI1640 medium group, cell proliferation in the presence of 5 μmol/L, 10 μmol/L, and 15 μmol/L lycopene was inhibited by 8.12%, 6.33% and 12.00%, respectively （P=0.024）.We showed for the first time that lycopene inhibited the activity of the androgen receptor gene element in a dose-related manner.Inhibition was seen in the transcription of the luciferase construct and confirmed by androgen receptor element expression assayed by Western blotting.Regression slopes of （log） PSA vs.time decreased in 26/37 （70%, 95% CI 53%-84%） of the patients after supplementation and in eight cases （21%） the post-treatment slope was negative.For these eight patients, the average fall in PSA was equivalent to 2% over 28 days （i.e.an average slope/d of -0.000 713）.The Wilcoxon rank-sum test showed an overall statistically significant decrease in slope （P=0.0007）.Analysis of the PSA doubling time （pretreatment vs.posttreatment） showed a median increase after supplementation for 174 days; however, this was not statistically significant （P=0.18）.Conclusions Lycopene as an antioxidant dietary factor could significantly inhibit DNA synthesis in a dose-dependent pattern; the result revealed lycopene might inhibit androgen receptor gene element activity and expression.Dietary lycopene may play an important role in prostate cancer cell proliferation and further supports a large randomized study into the role of lycopene supplementation in malignant prostate disease.
文摘We cloned the three androgen response elements（AREs, including AREI, AREII, and AREIII ） with a core transactivation TATA element of the prostate-specific antigen（PSA） promoter into pGL2 basic vector to create an artificial pGL2/AREs-TATA reporter system, which was applied to evaluating the effects of different xeno- oestrogens[bisphenol A（BPA）, 4-nonylphenol（4-NP）, dichlorodiphenyl trichloroethane（DDT） or diethylstilbestrol （DES）] on androgen receptor（AR） abnormal activation to regulate PSA expression and cell proliferation. In all the three AREs, AREIII-TATA displayed as a major element responsive to AR-mediated DHT stimulation of PSA promoter. Therefore, pGL2/AREIII-TATA reporter was adopted to analyze the activation capacity of AR activated by four different xeno-oestrogens. The activation of pGL2/AREIII-TATA reporter by each xeno-oestrogen was analyzed in two different cell lines, one was HEK293T（Human Embryonic Kidney 293T） cell line, and the other was AR stably expressed DU145 cell line, which was produced by infecting AR with pLenti-puro-AR into the prostate cancer DU145 cells and that were scanned with puromycin and tested by AR antibody. In both the two cell lines, BPA or DES significantly induced AR-mediated transcriptional activity of AREIII-TATA reporter, whereas DDT or 4-nonylphenol did not. Moreover, AR-mediated cell proliferation in response to each of four xeno-oestrogens was measured in MTT assays in both HEK293T cell or AR stably expressed DUI45 cell lines. BPA or DES, as an AR inducer, exhibited an enhanced effect in cell proliferation, rather than the effect of DDT or 4-NP, in both cell lines. Finally, we demonstrated that BPA or DES stimulated PSA expression and enhanced the recruitment of AR onto the PSA promoter, resulting in stronger binding to AREIII sites. Taken together, four xeno-oestrogens were identified to have different activities on AR. BPA and DES are demonstrated to be androgenic effectors in the regulation of PSA activation or cell proliferation.
