中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2008年
4期
271-275
,共5页
前列腺肿瘤%雷洛昔芬%丝裂原活化蛋白激酶%细胞周期%凋亡
前列腺腫瘤%雷洛昔芬%絲裂原活化蛋白激酶%細胞週期%凋亡
전렬선종류%뢰락석분%사렬원활화단백격매%세포주기%조망
Prostate carcinoma%Raloxifene%Mitogen-activated protein kinase%Cell cycle%Apoptosis
目的 探讨丝裂原活化蛋白激酶(MAPK)信号转导通路在雷洛昔芬(RAL)引起雄激素非依赖性前列腺癌PC3细胞凋亡和细胞周期阻滞中的作用.方法 应用四甲基偶氮唑蓝(MTT)法检测不同浓度RAL作用48、72、96、120 h后对PC3细胞生长抑制率.不同处理因素作用后流式细胞仪分析细胞周期分布和亚二倍体峰,TUNEL染色检测细胞凋亡.Western印迹检测细胞外信号调节蛋白激酶(ERK1/2)、c-Jun N端应力激活的蛋白激酶(JNK)和p38丝裂原活化蛋白激酶(p38)活性以及Bel-2、磷酸化Bcl-2(p-Bel-2)、Bax和半胱氨酸蛋白水解酶3(caspase-3)的表达.逆转录-聚合酶链反应(RT-PCR)法检测雌激素受体(ER)α、ERβ、细胞周期蛋白依赖激酶抑制蛋白(P21WAF1)、细胞周期蛋白D1(cyclinD1)基因的表达.结果 RAL呈浓度依赖性抑制PC3细胞增殖.10-6mol/L RAL可以快速持续激活ERK1/2和p38,不激活JNK.处理因素作用48 h后,对照组、RAL、RAL+PD98059、RAL + SB203580组细胞凋亡率分别为0.9%±0.1%;22.9%±1.5%;15.2%±1.8%和9.7%±0.6%(P＜0.05).10-6 mol/L RAL使细胞阻滞在G1期,10 μm PD98059或SB203580预孵育1 h可减轻RAL此种作用.PC3细胞表达ERα和ERβ.RAL、RAL+PD98059、RAL+SB203580组中cyclinD1和P21WAF1基因表达分别为对照组的0.50±0.02、4.48±0.12倍;0.49±0.02、1.77±0.06倍;2.36±0.08、4.50±0.03倍(P＜0.05).Bcl-2、Bax和caspase-3(对照组、RAL、RAL+PD98059、RAL+SB203580)的表达分别是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.RAL作用1.5 h后p-Bcl-2增加,SB203580可以抑制RAL引起的p-Bcl-2增加.结论 RAL激活ERK1/2增加P21WAF1.基因表达,并激活p38抑制cyclinD1表达使细胞阻滞在G1期.RAL激活ERK1/2促进Bax表达,同时激活p38磷酸化Bcl-2使其表达减少,引起PC3细胞凋亡.
目的 探討絲裂原活化蛋白激酶(MAPK)信號轉導通路在雷洛昔芬(RAL)引起雄激素非依賴性前列腺癌PC3細胞凋亡和細胞週期阻滯中的作用.方法 應用四甲基偶氮唑藍(MTT)法檢測不同濃度RAL作用48、72、96、120 h後對PC3細胞生長抑製率.不同處理因素作用後流式細胞儀分析細胞週期分佈和亞二倍體峰,TUNEL染色檢測細胞凋亡.Western印跡檢測細胞外信號調節蛋白激酶(ERK1/2)、c-Jun N耑應力激活的蛋白激酶(JNK)和p38絲裂原活化蛋白激酶(p38)活性以及Bel-2、燐痠化Bcl-2(p-Bel-2)、Bax和半胱氨痠蛋白水解酶3(caspase-3)的錶達.逆轉錄-聚閤酶鏈反應(RT-PCR)法檢測雌激素受體(ER)α、ERβ、細胞週期蛋白依賴激酶抑製蛋白(P21WAF1)、細胞週期蛋白D1(cyclinD1)基因的錶達.結果 RAL呈濃度依賴性抑製PC3細胞增殖.10-6mol/L RAL可以快速持續激活ERK1/2和p38,不激活JNK.處理因素作用48 h後,對照組、RAL、RAL+PD98059、RAL + SB203580組細胞凋亡率分彆為0.9%±0.1%;22.9%±1.5%;15.2%±1.8%和9.7%±0.6%(P＜0.05).10-6 mol/L RAL使細胞阻滯在G1期,10 μm PD98059或SB203580預孵育1 h可減輕RAL此種作用.PC3細胞錶達ERα和ERβ.RAL、RAL+PD98059、RAL+SB203580組中cyclinD1和P21WAF1基因錶達分彆為對照組的0.50±0.02、4.48±0.12倍;0.49±0.02、1.77±0.06倍;2.36±0.08、4.50±0.03倍(P＜0.05).Bcl-2、Bax和caspase-3(對照組、RAL、RAL+PD98059、RAL+SB203580)的錶達分彆是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.RAL作用1.5 h後p-Bcl-2增加,SB203580可以抑製RAL引起的p-Bcl-2增加.結論 RAL激活ERK1/2增加P21WAF1.基因錶達,併激活p38抑製cyclinD1錶達使細胞阻滯在G1期.RAL激活ERK1/2促進Bax錶達,同時激活p38燐痠化Bcl-2使其錶達減少,引起PC3細胞凋亡.
목적 탐토사렬원활화단백격매(MAPK)신호전도통로재뢰락석분(RAL)인기웅격소비의뢰성전렬선암PC3세포조망화세포주기조체중적작용.방법 응용사갑기우담서람(MTT)법검측불동농도RAL작용48、72、96、120 h후대PC3세포생장억제솔.불동처리인소작용후류식세포의분석세포주기분포화아이배체봉,TUNEL염색검측세포조망.Western인적검측세포외신호조절단백격매(ERK1/2)、c-Jun N단응력격활적단백격매(JNK)화p38사렬원활화단백격매(p38)활성이급Bel-2、린산화Bcl-2(p-Bel-2)、Bax화반광안산단백수해매3(caspase-3)적표체.역전록-취합매련반응(RT-PCR)법검측자격소수체(ER)α、ERβ、세포주기단백의뢰격매억제단백(P21WAF1)、세포주기단백D1(cyclinD1)기인적표체.결과 RAL정농도의뢰성억제PC3세포증식.10-6mol/L RAL가이쾌속지속격활ERK1/2화p38,불격활JNK.처리인소작용48 h후,대조조、RAL、RAL+PD98059、RAL + SB203580조세포조망솔분별위0.9%±0.1%;22.9%±1.5%;15.2%±1.8%화9.7%±0.6%(P＜0.05).10-6 mol/L RAL사세포조체재G1기,10 μm PD98059혹SB203580예부육1 h가감경RAL차충작용.PC3세포표체ERα화ERβ.RAL、RAL+PD98059、RAL+SB203580조중cyclinD1화P21WAF1기인표체분별위대조조적0.50±0.02、4.48±0.12배;0.49±0.02、1.77±0.06배;2.36±0.08、4.50±0.03배(P＜0.05).Bcl-2、Bax화caspase-3(대조조、RAL、RAL+PD98059、RAL+SB203580)적표체분별시1、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.RAL작용1.5 h후p-Bcl-2증가,SB203580가이억제RAL인기적p-Bcl-2증가.결론 RAL격활ERK1/2증가P21WAF1.기인표체,병격활p38억제cyclinD1표체사세포조체재G1기.RAL격활ERK1/2촉진Bax표체,동시격활p38린산화Bcl-2사기표체감소,인기PC3세포조망.
Objective To investigate the role of mitogen-activated protein kinase (MAPK) in the apoptosis and cell cycle arrest of human prostate carcinoma cells induced by raloxifene (RAL).Methods Human prostate carcinoma cells of the line PC3 were cultured.RAL of the concentrations of 10-4,10-5,10-6,and 10-7 mol/L were added into the culture fluid.MTT method was used to detect the inhibitory rate of the PC3 proliferation.RAL of the concentrations of 10-6 mol/L or 10-6 mol/L+ 10 μmol/L PD98059,a MEK1/2 inhibitor.and 10-6 mol/L RAL+10 μmol/L SB203580.JNK inhibitor,and RAL+SB203580,a p38 inhibitor were added respectively for 48 h,and the flow cytometry (FCM) was used to detect the cell cycle.The cell apoptosis percentage was measured by TUNEL staining.The activation of extra cellular regulated protein kinases (ERK1/2),c-Jun N-terminal protein kinase (JNK),and p38 mitogen-activated protein kinase (p38),and the expressions of Bcl-2,Bax,phospho-Bcl-2(p-Bcl-2),and caspase-3 were determined by Western blotting.The expressions of estrogen receptor (ER) α,Erβ,cyclin dependent kinase inhibitor (P21WAF1).and cyclinD1 mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR).Results A dose-dependent proliferation inhibition of RAL was demonstrated in the PC3 cells.A G1 cell cycle arrest and apoptosis were induced in the PC3 cells exposed to 10-6 mol/L RAL.10-6 mol/L RAL induced the activation of ERK1/2 and p38 with different time courses,but it did not induce the activation of JNK.Suppression ERK1/2 activation by treatment with PD98059 or p38 activation by treatment with SB203580 attenuated the cell-cycle arrest at the G1 phase respectively.48 h after the treatment of 10-6 mol/L RAL the PC3 cells was arrested at G1 stage,however,48 h after the treatment of 10-6 mol/L RAL +10 μmol/L PD98059 and 10-6 mol/L RAL +10 μmol/L SB203580 the degree of PC3 cell arrest at the G1 stage was lower.18 h after the treatment of 10-6 mol/L RAL,the expressions of cyrclinD1 and P21WAF1 gene were(0.50±0.02) and (4.48±0.12)times that of the control group,and the expressions of cyclinD1 and P21WAF1 gene of the SB203580 pretreatment group and SB203580 pretreatment groups were (2.36±0.08) and (4.50±0.03) times,and (0.49±0.02) and (1.77±0.06)times respectively.48 h after treatment with 10-6 mol/L RAL,,the apoptosis rates were 22.9%±1.5%,significantly higher than those of the 10-6 mol/L RAL +10 μmol/L PD98059 and 10-6 mol/L RAL +10 μmol/L SB203580 groups (15.2%±1.8% and 9.7%±0.6% respectively,both P＜0.05).The expression of Bcl-2,Bax and caspase-3 (control,10-6 mol/L raloxifene,10-6 mol/L raloxifene +10 μmol/L PD98059 and 10-6 mol/L raloxifene +10 μmol/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.Western blotting showed that 48 h after the treatment of 10-6 mol/L RAL the expression of Bcl-2 decreased,and the expression of caspace-3 increased.Pretreatment with PD98059 inhibited the increase of caspase-3 and Bax expression induced by RAL.1.5 h after the treatment with RAL the p-Bcl-2 expression increased remarkable,pretreatment with SB203580 inhibited the p-Bcl-2 expression induced by RAL,and however,PD98059 did not show such effect.Conclusion RAL inhibits the proliferation and induces the apoptosis and G1 cell cycle arrest via MAPK cascades in human prostate carcinoma cells. Up-regulation of P21WAF1 mRNA expression by activating ERK1/2 and down-regulation of cyclinD1 by activating p38 induces G1 cell cycle arrest in the human prostate carcinoma cells.The ERK1/2 or p38 cascade is involved in the induction of apoptosis by RAL.The activation of ERK1/2 increases the expression of Bax.The activation of p38 phosphorylates Bcl-2 then inactivates Bcl-2.
