TRPM8对前列腺癌细胞PC-3细胞增殖和迁移影响的研究

目的 探讨TRPM8对雄激素非依赖性前列腺癌PC-3细胞增殖和迁移能力的影响. 方法 PC-3细胞分别转染空载体pcDNA3和目的 基因TRPM8,筛选稳定表达细胞株;分别通过免疫荧光和钙成像检测TRPM8蛋白表达、分布以及功能;通过流式细胞术和划痕实验检测TRPM8对PC-3细胞周期和细胞迁移率的影响. 结果 免疫细胞化学和钙成像提示PC-3-TRPM8细胞表达有功能的TRPM8通道;TRPM8能诱导细胞周期阻滞于G0/G1期,与PC-3和PC-3-vector细胞相比,处于G0/G1期的PC-3-TRPM8细胞显著增加(71.89%±3.43% vs 54.67%±4.59%、51.48%±3.54%,P＜0.05),易化饥饿诱导的细胞凋亡(12.56%±1.78% vs 4.67%±1.49%、5.28±1.35%,P＜0.05),并抑制细胞迁移(P＜0.01). 结论 TRPM8并不为PC-3细胞存活所必需,相反,高表达的TRPM8能抑制PC-3细胞的增殖和迁移,可能为前列腺癌治疗的一个新靶点.     

Effects of gametogenetin-binding protein 2 on proliferation,invasion and migration of prostate cancer PC-3 cells

We aimed to assess the effects of gametogenetin-binding protein 2 (GGNBP2) on the proliferation, invasion and migration of prostate cancer PC-3 cells. PcDNA3-HisC-GGNBP2 was transfected to overexpress GGNBP2. Proliferation was tested by MTT assay, and migration and invasion were detected by Transwell assay. Cell cycle was detected by flow cytometry. The protein expressions of COX-2, cyclin D1, PI3K, Akt and p-Akt were detected by Western blot. A subcutaneous xenograft model of prostate cancer was established. Mice were randomly divided into three groups (n = 9) and intratumorally injected with pcDNA3-HisC-GGNBP2, pcDNA3-HisC and normal saline respectively. The xenograft tumour volume was measured every 3 days, and weight was measured after 2 weeks. After GGNBP2 overexpression, the proliferation, migration and invasion capacities of PC-3 cells decreased, and cell cycle was arrested in the G1 phase. The protein expressions of COX-2, cyclin D1, PI3K, Akt and p-Akt all reduced. The tumour volume and weight of pcDNA3-HisC-GGNBP2 group were significantly lower than those of pcDNA3-HisC group (p < .05). The proliferation capacity of GGNBP2-overexpressing prostate cancer cells is significantly attenuated, tumour growth is significantly inhibited, and cell cycle is arrested in the G1 phase. GGNBP2 overexpression affects the growth of castration-resistant prostate cancer via the PI3K/Akt signalling pathway.     

星形孢菌素对前列腺癌细胞株PC-3增殖和凋亡的影响

目的:探讨蛋白激酶C抑制剂星形孢菌素(ST)对前列腺癌细胞株PC-3增殖及凋亡的影响。方法:以ST(10-8mol/L)处理体外培养的PC-3细胞株。采用Western印迹检测细胞周期蛋白cyclin A、cyclin D1表达的变化。通过MTT实验、平板克隆实验检测ST对PC-3细胞增殖能力的影响。流式细胞仪检测各组细胞凋亡的情况。光镜观察细胞形态学的改变。结果:PC-3细胞经ST处理后cyclin A、cyclin D1蛋白表达明显降低。MTT实验结果显示ST对PC-3细胞的抑制作用自48 h后(19.35%)有显著性(F=31.06,P<0.01)。平板克隆实验结果显示组细胞克隆形成率ST组[(37.10±3.43)%]明显低于对照组((64.80±4.34)%,χ2=14.59,P<0.05]及DMSO组[(62.80±4.36)%,χ2=12.50,P<0.05]。流式细胞术结果显示ST组凋亡率(19.6±2.20)%与对照组(5.33±1.40)%和DMSO组(5.50±0.96)%比较显著增加(F=104.36,P<0.01)。光镜下可见:与对照组及DMSO组比较ST组细胞生长状态明显变差,细胞变圆,边缘模糊。结论:ST可以抑制前列腺癌细胞的生长和增殖,诱导细胞凋亡。     

腺病毒介导的PTEN基因对前列腺癌细胞株PC-3增殖及cyclinD1、p21表达的影响

目的:构建携带抑癌基因PTEN的腺病毒载体,探讨PTEN基因对前列腺癌细胞株PC-3增殖及cyclin D1、p21表达的影响。方法:采用RT-PCR方法从大鼠海马神经元扩增目的基因PTEN,连接入pENTR2A载体,在293A细胞中进行重组腺病毒的包装及扩增。以携带PTEN基因的腺病毒载体感染体外培养的前列腺癌细胞株PC-3。采用间接免疫荧光法检测细胞中PTEN的过表达情况。Western印迹检测PTEN、cyclin D1和p21表达的变化,通过细胞生长实验、平板克隆实验检测PTEN对PC-3细胞增殖能力的影响。结果:成功构建重组腺病毒载体Ad-PTEN;PC-3细胞经Ad-PTEN感染后PTEN蛋白表达水平明显增加。Western印迹实验所得条带进行灰度值分析后与β-actin求比值,PTEN蛋白在Ad-PTEN感染组细胞中表达(0.215±0.065)明显高于对照组[(0.052±0.009),t=4.30,P0.05]和Ad-LacZ组[(0.056±0.008),t=4.21,P0.05]。cyclin D1蛋白在Ad-PTEN感染组细胞中表达(0.256±0.072)明显低于对照组[(0.502±0.087,t=3.77,P0.05)]和Ad-LacZ组[(0.498±0.081,t=3.87,P0.05)]。p21蛋白在Ad-PTEN感染组细胞中表达(0.589±0.076)明显高于对照组[(0.146±0.026,t=9.55,P0.01)]和Ad-LacZ组[(0.163±0.024,t=9.26,P0.01)]。MTT实验显示Ad-PTEN对PC-3细胞的抑制作用自48h后(21.98%)有显著性(t=6.80,P0.01)。平板克隆实验显示Ad-PTEN组克隆形成率[(37.4±4.18)%]明显低于对照组[(54.9±4.81)%,t=4.76,P0.01]及Ad-LacZ组[(56.5±5.42)%,t=4.83,P0.01]。结论:通过腺病毒载体Ad-PTEN使PC-3细胞表达PTEN基因,可以明显抑制细胞的增殖,并可降低cyclin D1表达,同时增加p21的表达。     

3-溴丙酮酸对前列腺癌PC-3细胞增殖、迁移及侵袭的影响

目的:观察3-溴丙酮酸(3-BrPA)对前列腺癌细胞PC-3增殖、迁移和侵袭能力的影响,并初步探讨其内在机制。方法:体外培养PC-3细胞,倒置显微镜下观察不同浓度3-BrPA对PC-3细胞形态学的影响;应用MTT法、细胞划痕和Transwell法检测不同浓度3-BrPA在不同时间段(24、48、72 h)对前列腺癌PC-3细胞增殖、迁移和侵袭的影响;应用Western印迹检测3-BrPA作用后各组PC-3细胞葡萄糖转运蛋白1(GLUT1)和基质金属蛋白酶14、9、2(MMP-14、MMP-9、MMP-2)蛋白表达的变化。结果:在一定浓度范围内,随着3-BrPA浓度的升高,细胞形态的改变愈明显。MTT结果表明,随着3-BrPA浓度的增加、作用时间延长PC-3细胞抑制率增大(P<0.01)。细胞划痕和Transwell细胞侵袭实验显示:与对照组相比,培养24 h后25、50、100μmol/L 3-BrPA浓度组细胞划痕迁移愈合率降低,细胞侵袭数目减少,且培养48 h与24 h相比,各组细胞迁移愈合率和侵袭细胞数目均降低,表明细胞体外迁移率和侵袭细胞数目与3-BrPA作用浓度、作用时间有关,具有剂量和时间依赖性(P<0.01)。Western印迹检测结果表明:与对照组相比,25、50、100μmol/L 3-BrPA浓度组中GLUT1、MMP-14、MMP-9、MMP-2蛋白表达量明显降低(P<0.01)。结论:糖酵解抑制剂3-BrPA降低PC-3增殖、迁移和侵袭能力,可能与下调GLUT1、MMP-14、MMP-9及MMP-2蛋白表达有关。     

PPARγ对前列腺癌细胞增殖及糖酵解的影响

目的:研究PPAR-γ基因表达对前列腺癌细胞增殖和肿瘤特殊糖酵解代谢的影响。方法:采用RNAi技术构建PPARγ低表达的腺病毒载体并转染到前列腺癌细胞株,并以空白载体为对照组,进行细胞增殖检测、细胞凋亡检测、糖酵解代谢基因及产物乳酸检测,并比较两组之间的结果差异。结果:经RNAi抑制的PPAR-γ前列腺癌细胞株与对照组相比,蛋白表达量降低至(26.00±4.06)%,增殖抑制率最大为第2天达(39.50±4.92)%,细胞凋亡率升高至(21.03±3.08)%,糖酵解代谢基因产物(Myc和Glut-1)下降,第4天培养液中乳酸浓度为对照组的69.71%,上述结果具有统计学差异(P<0.01)。结论:抑制PPAR-γ基因的表达有望成为治疗前列腺癌新方法。     

靶向ADAM17基因siRNA对前列腺癌PC-3细胞增殖能力的影响

目的:探讨靶向抑制ADAM17基因对雄激素非信赖性前列腺癌PC-3细胞增殖的影响。方法:应用ADAM17 siRNA转染PC-3细胞后,通过RT-PCR、Western印迹方法分别检测ADAM17 mRNA和蛋白表达变化;MTT、BrdU掺入法检测下调ADAM17对PC-3细胞的增殖和DNA合成能力的影响;流式细胞术检测ADAM17 siR-NA对PC-3细胞细胞周期的影响;Western印迹检测下调ADAM17对PC-3细胞增殖相关基因表达的影响。结果:两对ADAM17 siRNAs均可有效地降低PC-3细胞ADAM17 mRNA和蛋白的表达;MTT结果显示与对照组(0.80±0.51)相比,两对ADAM17 siRNAs均可显著抑制细胞的生长(0.43±0.57、0.44±0.64,P均<0.05);Br-dU掺入实验显示与对照组(0.79±0.72)相比,ADAM17 siRNAs均能显著下调DNA的合成能力(0.48±0.43、0.54±0.59,P<0.05);流式细胞术结果显示,与对照组(41.38±1.53)%相比,ADAM17 siRNAs可显著增加G1期细胞数量[(61.83±2.41)%、(59.78±1.92)%,P均<0.05]、降低S期细胞数量[从(33.51±1.47)%减少到(23.64±2.56)%、(25.24±1.86)%,P均<0.05],同时伴随着cyclin D1蛋白的表达下降而p21蛋白的表达升高。结论:ADAM17 siRNA可以通过下调cyclin D1、上调p21的表达而抑制前列腺癌PC-3细胞增殖,ADAM17可能成为前列腺癌基因治疗的靶点。     

Tetrandrine suppresses proliferation,induces apoptosis,and inhibits migration and invasion in human prostate cancer cells

Tetrandrine (TET), a traditional Chinese medicine, exerts remarkable anticancer activity on various cancer cells. However, little is known about the effect of TET on human prostate cancer cells, and the mechanism of function of TET on prostate cancer has not yet been elucidated. To investigate the effects of TET on the suppression of proliferation, induction of apoptosis, and inhibition of migration and invasion in human prostate cancer cell lines, DU145 and PC-3. Inhibition of growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and clone formation assay, and flow cytometry analysis was performed to detect the induction of apoptosis. Activation of poly (ADP-ribose) polymerase, caspase-3, Akt, phospho-Akt, Bcl-2, and Bax was analyzed by Western blotting. Wound healing assay and transwell migration assay were used to evaluate the effect of TET on migration and invasion of cancer cells. TET inhibited the growth of DU145 and PC–3 cells in a dose- and time-dependent manner. Cell cloning was inhibited in the presence of TET in DU145 and PC-3 cells. TET suppressed the migration of DU145 and PC-3 cells. Transwell invasion assay showed that TET significantly weakened invasion capacity of DU145 and PC-3 cells. TET exhibited strong inhibitory effect on proliferation, migration, and invasion of prostate cancer cells. In addition, TET induced apoptosis in a dose-dependent manner by activating the caspase cascade and inhibiting phosphoinositide 3-kinase-Akt signal pathway. The accumulating evidence suggests that TET could be a potential therapeutic candidate against prostate cancer in a clinical setting.     

培养液中胆固醇缺乏对前列腺癌PC-3细胞生长和凋亡的影响

目的:探讨培养液中胆固醇水平对人前列腺癌PC-3细胞生长抑制及凋亡调控的作用。方法:前列腺癌PC-3细胞分别培养于普通和胆固醇缺乏培养液中,再分别加入不同剂量血小板源性生长因子PDGF或EGF作用后,倒置显微镜观察细胞形态,MTT法检测细胞的增殖抑制情况,流式细胞仪进行细胞凋亡率及细胞周期时相分析。结果:与普通培养液组比较,胆固醇缺乏培养液组细胞明显变圆、体积缩小、脱壁细胞增多。MTT法检测显示,胆固醇缺乏培养液组细胞增殖显著抑制,并呈剂量依赖性。流式细胞分析显示胆固醇缺乏培养液组细胞凋亡率同普通培养液组相比无显著差异。当加入PDGF或EGF刺激细胞增殖时,普通培养液组细胞数目显著增加,而在胆固醇缺乏培养液组脱壁细胞增加,细胞凋亡增多,同普通培养液组相比存在显著差异。流式细胞术分析细胞周期显示,同普通培养液组相比,胆固醇缺乏培养液组停滞在G0/G1期细胞增加,而S、G2/M期细胞减少。结论:胆固醇缺乏对PC-3细胞增殖有显著抑制作用,其作用机制并不是简单增加细胞凋亡,而可能是在不利增殖条件下,PC-3细胞的一种自我调节机制。     

PSA"灰区"值时前列腺癌组织中Trp-p8的表达及意义

目的 研究Trp-p8蛋白在PSA"灰区"前列腺组织中的表达规律,探讨其在前列腺癌(PCa)早期诊断中的作用.方法 通过免疫组织化学的方法研究了28例PSA"灰区"前列腺组织中Trp-p8蛋白的表达情况,其中前列腺增生症(BPH)和PCa标本各14例,采用图文数据成像分析系统判定各组织中Trp-p8蛋白的表达强度,分析其差异性.结果 BPH中Trp-p8蛋白的表达强度较弱,呈不表达或微量表达.在PCa组织中Trp-p8蛋白均呈不同程度的阳性表达,这种表达差异具有统计学意义.结论 Trp-p8在PSA"灰区"前列腺组织中的表达存在差异,在PCa组织中Trp-p8蛋白的表达强度高于BPH组织,这种差异性表达对于早期PCa诊断具有重要意义.     

参附注射液对前列腺癌PC-3细胞凋亡诱导的影响

目的:研究参附注射液(SF)对人前列腺癌PC-3细胞凋亡的影响及可能机制。方法:实验设立对照组和SF 50、100、200μl/ml组,Annexin V/PI染色流式细胞术检测细胞凋亡,RT-PCR检测p53 mRNA表达。结果:与对照组比较,作用24、48、72 h后,SF 50、100、200μl/ml组PC-3细胞存活率显著减少(P均0.05)。SF各组24 h存活率分别为(93.76±2.63)%、(81.21±1.80)%、(18.01±3.84)%;48 h存活率分别为(94.67±1.11)%、(78.33±2.89)%、(10.34±1.44)%;72 h存活率分别为(91.30±0.47)%、(36.67±1.56)%、(1.33±0.32)%,呈浓度和时间依赖性。作用48 h后,p53 mRNA表达明显升高(P0.05)。结论:SF可以诱导PC-3细胞凋亡,其作用机制可能与p53表达增高相关。     

The multikinase inhibitor sorafenib induces caspase-dependent apoptosis in PC-3 prostate cancer cells

The present study investigated the effects of the multikinase inhibitor sorafenib on androgen-independent can- cer cells viability and intracellular signaling. Human androgen-independent PC-3 prostate cancer cells were treated with sorafenib. At concentration that suppresses extracellular signal-regulated kinase phosphorylation, sorafenib treatment reduced the mitochondrial transmembrane potential. Sorafenib also down-modulated the levels of mye- loid cell leukemia 1, survivin and cellular inhibitor of apoptosis protein 2. Sorafenib induced caspase-3 cleavage and the mitochondrial release of cytochrome c. However, no nuclear translocation of apoptosis inducing factor was detected after treatment and the pan-caspase inhibitor Z-VAD-FMK had an obvious protective effect against the drug. In conclusion, sorafenib induces apoptosis through a caspase-dependent mechanism with down-regulated antiapoptotic proteins in androgen-independent prostate cancer cells in vitro.     

CK2抑制剂四溴肉桂酸对前列腺癌细胞增殖及周期的影响

目的探讨一种新的人工合成CK2选择性抑制剂四溴肉桂（TBCA）,对前列腺癌细胞增殖及周期的影响。方珐CK2选择性抑制剂TBCA应用到多种前列腺癌细胞系,Alamarblue法和克隆形成实验检测细胞生长和增殖能力,流式细胞技术检测细胞周期分布,治疗组与对照组间差异是否具有显著意义采用SPSS统计软件进行分析。站杲低剂量（〈25μmol）TBCA干预下未发现细胞聚集和分离,而在高剂量（〉50μmol）则可观察到细胞明显分离,剂量依赖性抑制细胞生长和增殖（P〈0．05）,半抑制浓度值（IC50）为25μmol。TBCA诱导前列腺癌细胞停滞在G2／M期细胞周期。结论TBCA呈剂量依赖性抑制前列腺癌细胞增殖和细胞周期停滞在G2／M期。     

Physiological normal levels of androgen inhibit proliferation of prostate cancer cells in vitro

For more than 70 years, it has been believed that a severe reduction of serum androgen levels caused regression of prostate cancer （PCa） and that increasing androgen levels enhanced growth of PCa. However, numerous recent studies have questioned this traditional belief. In our study, LNCaP and MDA PCa 2b PCa cells were treated with various levels of androgens for 10 or 20 days, and the cell growth was measured with crystal violet mitogenic assay. The results indicated that the effect of androgens on the proliferation of PCa cells occurs in a biphasic pattern, with the androgen levels promoting optimal cell growth at approximately 0.23 ng m1-1 for LNCaP cells and between I and 2 ng m1-1 for MDA PCa 2b cells. Both of the optimal androgen levels are within the adult men＇s physiological low range （〈2.4 ng ml-1）. At lower concentrations than the optimal androgen level, increasing androgen concentration promoted the proliferation of PCa cells. However, at the higher concentrations, increasing androgen concentration resulted in a dose-dependent proliferative inhibition. We conclude that physiologically normal levels of androgen inhibit the proliferation of PCa cells in vitro. However, at very low levels androgens are essential for initial growth of PCa cells.