| Introdution and objectiveCarcinoma of the prostate is the most commonly diagnosed malignancy in older men and the second leading cause of cancer death in the western world. Although hormonal ablation is extremely effective, androgen-independent disease will ultimately intervene in most patients. Treatment options for androgen-independent prostate cancer are extremely limiting, with no regimen showing a survival advantage.The presence of estrogen receptor (ER)-αand ER-βin prostatic tissues has been reported. Some studies showed that ER-Pexpressed in normal prostate epithelium but it significantly decreased in prostatic carcinoma. Recent reviews renewed interest in the presence of ER in prostatic tissues and the potential benefits of estrogen therapy in prostate cancer.Estrogen administration has long been recognized as an effective hormonal ablative therapy to treat disseminated prostate carcinoma. Selective estrogen receptor modulators (SERMs) are compounds that antagonize the growth promoting effects of estradiol by inhibiting the binding of the hormone to its receptor. Tamoxifen and raloxifene, belonging to SERM, have been shown to prevent breast cancer and osteoporosis, respectively. Recent studies showed that they both could induce apoptosis in prostate cancer.Intracellular transmission of extracellular signals is mediated by the mitogen-activated protein kinase (MAPK), which were classified into three subfamilies: extra cellular regulated protein kinases (ERK1/2); c-Jun N-terminal protein kinase (JNK); and p38 kinase (p38). Some studies showed that estrogen, tamoxifen and raloxifene could activate MAPK cascades through ER.So we examine whether estrogen, tamoxifen and raloxifene have an antiproliferative effect on and stimulates the ERK1/2, JNK and p38 cascades in androgen independent prostate cancer lines PC3 cells through ER.Materials and Methods1,The effect of 17β-estradiol, tamoxifen and raloxifene on the growth of PC3 cellsPC3 cells were treated with different concentrations of 17β-estradiol, tamoxifen and raloxifene, and then method of MTT was used to detect the inhibition rate. Inhibition rate=(1-A490 of treated group/A490 of control group)×100%.2,Detection of morphology changes of apoptosisPC3 cells were treated with 17beta-estradiol, tamoxifen and raloxifene for 48 hours, and morphology changes of apoptosis were observed by hoechst staining.3,Detection of the expressions of Bcl-2 and caspase-3 by immuneohisto-chemical methodsPC3 cells were treated with 17β-estradiol, tamoxifen and raloxifene for 48 hours, and the expressions of Bcl-2 and caspase-3 were measured by immunohistochemical methods.4,The activation of ERK1/2, JNK and p38 was determined by western blotPC3 cells were exposed to different treatment for different time, then cells were harvested and the concentration of protein was determined using the Bradford assay. The samples were boiled for 10 min, and electrophoresis was carried out. And phosphorylated ERK1/2, phosphorylated JNK, phosphorylated p38, ERK1/2, JNK and p38 were detected. MAPKs activity was expressed as the ratio of the density of phosphorylated MAPKs band to that of the total-MAPKs band.5,Flow cytometry analysis PC3 cells were exposed to different treatment for 48 hours. Cells were harvested and stained with propidium iodide for DNA staining. Ten thousand cells were counted by a fluorescence-activated cell sorting calibur cytometer; red fluorescence due to PI staining of DNA was expressed on a logarithmic scale simultaneously to the forward scatter of the particles, and the percentages of cells in the sub-G1 (DNA content＜2N), G1, S, and G2/M phases of the cell cycle were determined using ModFit LT software.6,Expression of ER-α, ER-β, P21WAF1, cyclinD1 andβ-actinCells were harvested, and total RNA was isolated using Trizol reagent. Once isolated, 1μg of total RNA was reverse transcribed using superscript and 2μl of the resulting cDNA were used in each PCR. And To visualize the PCR products, the samples were subjected to electrophoresis in 1% agarose gel followed by staining with ethidium bromide.β-actin cDNA levels served as a loading control.7,Detection of apoptosis rate by TUNELPC3 cells were exposed to different treatment for 48 hours. Cells were incubated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using an In Situ Cell Apoptosis Detection Kit. The incidence of apoptosis was determined by counting 100 cells and calculating the percentage of apoptotic cells.8,The expression of Bcl-2, caspase-3, p-Bcl-2 and BaxPC3 cells were exposed to different treatment for different time, then cells were harvested and the concentration of protein was determined using the Bradford assay. The expression of Bcl-2, caspase-3, p-Bcl-2 and Bax was measured by western blot.9,Statistical analysisStatistical analysis was performed using one-way analysis of variance (ANOVA), and P＜0.05 was considered significant. Data are expressed as the mean±SE.ResultsExpressions of mRNA of ER-a and ER-beta were detected in PC3 cells. There was exon 2 deletion variant of ER-α(ER-αΔ2) in PC3 cells. A dose-dependent proliferation inhibition of 17β-estradiol, tamoxifen and raloxifene was demonstrated in PC3. Morphology changes of apoptosis were detected in PC3 cells after they were treated with 17β-estradiol, tamoxifen and raloxifene for 48 hours. The expression of Bcl-2 decreased and the expression of caspase-3 increased in PC3 cells.10-4mol/l 17β-estradiol and 10-5mol/l tamoxifen induced the activation of ERK1/2, JNK and p38 in PC3 cells with different time courses. 10-6mol/l raloxifene could only induced the activation of ERK1/2 and p38. Inhibitor of MAPK at 10μmol/l completely prevented 17beta-estradiol, tamoxifen and raloxifene-induced MAPK activation.A G1 cell cycle arrest was induced in PC3 exposed to 17β-estradiol, tamoxifen and raloxifene. Suppression p38 activation by treatment with SB203580 blocked the cell-cycle arrest at the G1 phase by 17beta-estradiol. Suppression JNK activation by treatment with SP600125 attenuated the cell-cycle arrest at the G1 phase by 17β-estradiol. But suppression ERK1/2 by PD98059 enforced the cell-cycle arrest at the G1 phase by 17β-estradiol. Suppression ERK1/2 activation by treatment with PD98059 and p38 activation by treatment with SB203580 attenuated the cell-cycle arrest at the G1 phase by tamoxifen. Suppression ERK1/2 activation by treatment with PD98059 or p38 activation by treatment with SB203580 attenuated the cell-cycle arrest at the G1 phase by raloxifene, respectively.The expression of cyclinD1 and P21WAF1 mRNA in PC3 (control,10-4mol/l 17β-estradiol, 10-4mol/l 17β-estradiol+10μmol/l PD98059, 10-4mol/l 17β-estradiol+10μmol/l SP600125 and 10-4mol/l 17β-estradiol+10μmol/l SB203580) was 1,1; 0.42±0.03, 3.13±0.02; 0.21±0.03, 3.14±0.02; 0.43±0.01, 1.03±0.06; 2.81±0.02, 3.08±0.05, respectively. The expression of cyclinD1 and P21WAF1 mRNA in PC3 (control, 10-5mol/l tamoxifen, 10-5mol/l tamoxifen+10-5mol/l PD98059,10-5mol/l tamoxifen+10μmol/l SP600125 and 10-5mol/l tamoxifen+10μmol/l SB203580) was 1,1; 0.40±0.05, 4.37±0.12; 0.41±0.05, 1.74±0.10; 0.43±0.06, 4.36±0.23; 3.92±0.22, 4.46±0.09, respectively. The expression of cyclinD1 and P21WAF1 mRNA in PC3 (control, 10-6mol/l raloxifene, 10-6mol/l raloxifene+10μmol/lPD98059 and 10-6mol/l raloxifene+10μmol/l SB203580) was 1,1; 0.50±0.02, 4.48±0.12; 0.49±0.02,1.07±0.06; 2.36±0.08,4.50±0.03, respectively.48 hours after treatment with 10-4mol/l 17β-estradiol, 10-4mol/l 17β-estradiol+10μmol/l PD98059, 10-4mol/l 17β-estradiol+10μmol/l SP600125 and 10-4mol/l 17β-estradiol+10μmol/l SB203580, apoptosis rate were 23.0±1.4%, 30.0±1.2%, 10.6±0.8% and 14.6±0.7%, respectively. 48 hours after treatment with 10-5mol/l tamoxifen, 10-5mol/l tamoxifen+10μmol/1 PD98059, 10-5mol/l tamoxifen+10μmol/l SP600125 and 10-5mol/l tamoxifen+10μmol/l SB203580, apoptosis rate were 41.5±1.9%, 36.3±1.9%, 27.1±1.6% and 17.4±2.0%, respectively. 48 hours after treatment with 10-6mol/l raloxifene, 10-6mol/l raloxifene+10μmol/l PD98059 and 10-6mol/l raloxifene+10μmol/l SB203580, apoptosis rate were 22.9±1.5%, 15.2±1.8% and 9.7±0.6%, respectively.The expression of Bcl-2 and caspase-3 (control, 10-5mol/l tamoxifen, 10-5mol/l tamoxifen+10μmol/L PD98059, 10-5mol/l tamoxifen+10μmol/L SP600125 and 10-5mol/l tamoxifen+10μmol/l SB203580) was 1, 0.21±0.03, 0.25±0.02, 0.42±0.05, 0.57±0.05; 1, 4.18±0.15, 4.03±0.06, 3.07±0.09, 2.07±0.12, respectively. The expression of p-Bcl-2 increased after exposed to tamoxifen 6 hours. SP600125 or SB203580 could prevent the expression of p-Bcl-2 induced by tamoxifen partly. The expression of Bcl-2, Bax and caspase-3 (control, 10-6mol/l raloxifene, 10-6mol/l raloxifene+10μmol/l PD98059 and 10-6mol/l raloxifene+10μmol/l SB203580) was 1, 0.33±0.02, 0.34±0.01, 0.81±0.05; 1, 3.14±0.02, 1.67±0.11, 3.15±0.03; 1, 4.16±0.02, 2.66±0.03, 1.80±0.06, respectively. The expression of p-Bcl-2 increased after exposed to raloxifene 6 hours. SB203580 could prevent the expression of p-Bcl-2 induced by raloxifene.Conclusions17β-estradiol inhibited proliferation and induced apoptosis and G1 cell cycle arrest via MAPK cascades in human prostate carcinoma cell lines PC3 cells. Up-regulation of P21WAF1 mRNA expression by activating JNK and down-regulation of cyclinD1 by activating p38 induced G1 cell cycle arrest in PC3. The JNK or p38 cascade is involved in the induction of apoptosis by 17β-estradiol. But the ERK1/2 cascade attenuated the effect of JNK and p38 cascade.Tamoxifen inhibit proliferation and induce apoptosis and G1 cell cycle arrest via MAPK cascades in human prostate carcinoma cell lines PC3 cells. Up-regulation of P21WAF1 mRNA expression by activating ERK1/2 and down-regulation of cyclinD1 by activating p38 induced G1 cell cycle arrest in PC3. The JNK or p38 cascade is involved in the induction of apoptosis by tamoxifen. The activation of JNK or p38 phosphorylated Bcl-2 then inactivated Bcl-2. Raloxifene inhibit proliferation and induce apoptosis and G1 cell cycle arrest via MAPK cascades in human prostate carcinoma cell lines PC3 cells. Up-regulation of P21WAF1 mRNA expression by activating ERK1/2 and down-regulation of cyclinD1 by activating p38 induced G1 cell cycle arrest in PC3. The ERK1/2 or p38 cascade is involved in the induction of apoptosis by raloxifene.The activation of ERK1/2 increased the expression of Bax. The activation of p38 phosphorylated Bcl-2 then inactivated Bcl-2.The studies showed that we could treat androgen-independent prostate cancer by 17beta-estradiol, tamoxifen or raloxifene.
|
PDF Full Text Request