Bcl-2和Bax在前列腺癌不同周期时相的表达

目的 探讨凋亡相关基因Bel-2和Bax在前列腺癌不同周期时相的差异表达及意义.方法 应用改良的胸腺嘧啶核苷和高压笑气双阻断法把前列腺癌细胞系PC-3同步在不同的周期时相,应用流式细胞术检测同步化的效率.进而应用RT-PCR及Western blot方法检测不同周期时相Bcl-2和Bax在mRNA和蛋白水平的表达.结果 前列腺癌细胞系PC-3的M、G1、S和G2期细胞同步化效率分别为92.1%、87.0%、80.2%和75.9%;Bcl-2在不同周期时相均有表达且存在差异,G1期表达水平最高,S、M和G2期表达水平显著降低,mRNA和蛋白水平表达具有一致性;Bax在各周期的mRNA水平及蛋自水平表达没有明显差别.结论 细胞周期对凋亡相关基因Bel-2的表达有显著影响,对Bax无影响,对于采用Bcl-2途径治疗前列腺癌具有指导意义.     

米非司酮对前列腺癌PC-3细胞周期及其蛋白的影响

目的 探讨米非司酮（MIF）对前列腺癌PC-3细胞周期及其调控蛋白的影响及其作用机制。方法 MTT法检测1、10、50、100μmol／LMIF作用于PC-3细胞24～120h的吸光度（A）值，流式细胞仪检测10、50μmol／LMIF作用PC-3细胞48h后细胞周期的变化，免疫组化法和Western blot法检测10、50μmol／LMIF处理48h后PC-3细胞cyclinD1、bax、bcl-2蛋白表达的变化情况。结果 1μmol／LMIF作用24～120h的A值与对照组相比差异无统计学意义（P〉0．05）；10、50、100μmol／L组A值与对照组比较差异有统计学意义（P〈0．01）；MIF对PC-3细胞的抑制作用呈时间-剂量依赖性。MIF作用48h后使PC-3细胞停滞于G1／G0期，并使此期细胞比例从对照组的27．4％增加到10μmol／L组的50．4％和50μmol／L组的59．2％，差异有统计学意义（P〈0．05）。处理后PC-3细胞中bcl-2蛋白和cyclinD1蛋白表达量，与对照组比较差异有统计学意义（P〈0．05）；而bax表达量显著增加。结论 MIF以时间．剂量依赖性方式抑制前列腺癌PC-3细胞的增殖，可能通过下调cyclinD1蛋白表达，阻止PC-3细胞G1期向S期的转换，使其停留于G1／G0期；同时降低bcl-2蛋白的表达及激活bax蛋白的表达等抑制前列腺癌PC-3细胞增殖。     

靛玉红对前列腺癌PC-3细胞增殖的抑制作用

目的:研究中药成分靛玉红对雄激素非依赖性前列腺癌PC-3细胞的杀伤作用及可能的机制。方法:采用MTT方法研究靛玉红对前列腺癌PC-3细胞增殖抑制作用,采用Western印迹检测细胞周期蛋白cyclin D1及c-myc表达,采用流式细胞术检测细胞周期。结果:靛玉红降低前列腺癌细胞的存活率呈浓度依赖性,当浓度为5μmol/L时,可降低存活率至52.2%,当浓度为10μmol/L时,PC-3细胞存活率降至13.6%。靛玉红浓度为5μmol/L时可明显抑制PC-3细胞的细胞周期,结果显示G0/G1期PC-3细胞增多,而与此同时S期和G2/M期细胞减少。此外,靛玉红可以抑制细胞周期进展关键调控蛋白cyclin D1,以及与之相关Wnt信号通路的下游基因c-myc的表达。结论:靛玉红可以抑制雄激素非依赖性前列腺癌PC-3细胞的增殖,其机制可能与其抑制细胞周期以及抑制Wnt信号通路有关。     

膀胱移行细胞癌组织中聚集素、Ki-67蛋白表达和细胞凋亡的测定

目的 探讨聚集素在膀胱移行细胞癌组织中的表达及其与细胞增殖和凋亡的关系。方法制作87例膀胱移行细胞癌组织的组织芯片,分别应用免疫组织化学和末端脱氧核苷酸转移酶介导缺口末端标记方法,检测膀胱移行细胞癌组织中聚集素和Ki-67蛋白表达及细胞凋亡情况,分析聚集素蛋白表达与肿瘤分化、临床分期等病理特征及肿瘤细胞凋亡、增殖及Ki-67蛋白表达的关系。结果43％（37／87）患者为聚集素蛋白过度表达,且聚集素蛋白表达与肿瘤细胞的分化程度、肿瘤的浸润深度均有显著的相关性（r分别为14．1,10．2,P均〈0．01）;71％（20／28）低分化（G3）和62％（23／37）浸润型（T2-4期）患者的肿瘤组织聚集素蛋白过度表达,过度表达率明显高于分化较好（G1-2,29％,17／59）和浅表型（Ta-1期,28％,14／50）者。聚集素蛋白表达与肿瘤的凋亡指数（AI）呈负相关（r=7．31,P〈0．01）,但与Ki-67的表达水平无关;聚集素过度表达的肿瘤,57％（21／37）表现为低AI值（≤1．2％）,而聚集素正常表达的肿瘤,则72％（36／50）为高AI值。结论膀胱移行细胞癌组织中聚集素蛋白过度表达与肿瘤的分化程度和浸润深度呈正相关,与肿瘤细胞的AI呈负相关。     

GM-CSF引起的MAPK途径磷酸化在激素非依赖性前列腺癌增殖中的作用

目的:研究丝裂原活化蛋白激酶(MAPK)信号途径中Erk1/2分子磷酸化在粒细胞-单核细胞集落刺激因子(GM-CSF)促进前列腺癌增殖中的作用。方法:前列腺癌PC-3M细胞经过GM-CSF刺激前后,流式细胞术和半定量RT-PCR分别检测细胞周期和细胞周期蛋白K i-67表达的变化;W estern印迹法对比GM-CSF作用前后Erk1/2分子磷酸化程度的变化。结果:GM-CSF可以显著增加PC-3M细胞S期和G2/M期的百分率以及K i-67蛋白的表达,同时Erk1/2分子磷酸化程度显著增强。结论:Erk1/2分子磷酸化可能是GM-CSF促进前列腺癌增殖的重要分子机制。     

维生素E琥珀酸酯诱导前列腺癌PC-3细胞凋亡的实验研究

目的：研究维生素E琥珀酸酯（VES）对非雄激素敏感的前列腺癌PC-3细胞株的凋亡诱导作用及分子机制。方法：以体外培养的PC-3细胞为研究对象,采用含不同浓度的VES培养液作用于细胞。采用噻唑兰（MTT）比色法检测细胞增殖活性;采用Wright—Giemsa染色、流式细胞术（FCM）检测细胞凋亡并测定Fas蛋白表达水平;采用酶联免疫法检测转化生长因子口（TGF-β）的表达水平的变化。结果：VES可显著抑制PC-3细胞的生长及增殖,光镜下可见到PC-3细胞呈典型凋亡的形态学改变。FCM细胞周期分析显示G2／M期细胞增加而s期细胞明显减少,Fas蛋白和TGF-β表达明显上调。结论：VES可诱导前列腺癌PC3细胞凋亡,其作用机制可能与其上调Fas、TGF-β的表达有关。     

细胞周期素D1反义寡核苷酸对激素非依赖性前列腺癌细胞增殖的影响

目的：观察细胞周期素D1(Cyclin D1)反义寡核苷酸(ASODN)对激素非依赖性前列腺癌(AIPC)细胞生长的影响。方法：将ASODN转染AIPC细胞系PC-3m(ASODN组);逆转录一聚合酶链式反应(RT—PCR)和免疫组化法(SP)检测PC-3m细胞Cyclin D1 mRNA和蛋白的表达,锥虫蓝活细胞拒染法和流式细胞术(FCM)检测PC-3m细胞生长和细胞周期分布。并与Cyclin D1正义寡核苷酸转染者(SODN组)对比。结果：ASODN组PC-3m细胞Cyclin D1 mRNA和蛋白表达明显降低;ASODN浓度在0．05～0．3umol／L时,可对PC-3m细胞生长发挥抑制作用,且呈明显的剂量依赖性关系(P＜0．05);PC3m细胞G1期细胞比例明显上升而S期细胞比例下降(P＜0．01);SODN组s期细胞百分比稍有增加,但与空白对照组差异无统计学意义(P＞0．05)。结论：Cyclin D1 ASODN可通过有效逆转AIPC细胞内源性Cyclin D1过表达而导致的细胞G1～s期限制点失控,使细胞停滞于G1期,实现对AIPC细胞的生长抑制作用,提示用Cyclin D1 ASODN对AIPC的治疗有望成为可能。     

聚集素在前列腺癌中的表达及意义

目的　探讨抗细胞凋亡因子聚集素 (clusterin)在前列腺癌中的表达及意义。　方法　RT PCR方法检测聚集素在前列腺癌组织 (3例 )、癌细胞系 (1株 )及正常前列腺组织 (3例 )中的表达水平。　结果　 3例前列腺癌组织及 1例前列腺癌细胞株中聚集素与内参基因 β actin相比较的相对表达量明显高于 3例正常前列腺组织。　结论　聚集素在前列腺癌中高表达 ,提示聚集素可能通过抗凋亡机制在前列腺癌的生物特性中发挥作用。     

滇重楼皂苷对前列腺癌细胞周期与凋亡的影响

目的探讨滇重楼皂苷对前列腺癌细胞增殖、周期和促凋亡的作用,以及对磷酸化CDK2表达的影响。方法使用不同浓度(0.1、0.3、1.0、3.0、10.0μmol/L)的滇重楼皂苷处理前列腺癌细胞株PC-3和Du145,应用MTT和流式细胞技术检测细胞增殖、周期分布和凋亡的改变,进一步运用免疫印迹法检测细胞周期及凋亡相关蛋白表达的改变。结果滇重楼皂苷明显抑制前列腺癌细胞株(PC-3及Du145)的增殖,使细胞在G0/G1期发生阻滞,并诱导细胞显著凋亡,抑制效果呈时间剂量梯度依赖关系;p21蛋白与Cleaved-Caspase-3表达水平明显上调,磷酸化CDK2、磷酸化RB蛋白下调。结论滇重楼皂苷增加p21蛋白水平,抑制细胞周期关键调控因子CDK2及RB的磷酸化,并且激活Caspase-3参与的细胞凋亡信号通路,从而抑制前列腺癌细胞增殖、阻断细胞周期,并诱导细胞凋亡,具有抗肿瘤活性。     

ID3蛋白在前列腺癌中的表达及临床病理学意义

目的:研究ID3蛋白在前列腺癌中的表达及临床病理学意义。方法:应用间接免疫荧光技术检测ID3在PC-3M细胞中的表达情况;应用免疫组化技术检测29例前列腺癌和15例前列腺增生标本中ID3的表达情况,分析ID3与临床病理参数之间的关系。结果:ID3蛋白在PC-3M细胞中主要表达在细胞核;29例前列腺癌中ID3阳性表达率为82.7%(24/29),15例前列腺增生标本中ID3阳性表达率为6.6%(1/15),前者显著高于后者(P<0.05);ID3蛋白表达程度与前列腺癌的G leason评分显著相关(P<0.05)。结论:ID3蛋白在前列腺癌中表达,其表达量随着G leason评分的升高而升高。     

人工合成小分子RNA对前列腺癌细胞周期及增殖的影响

目的 研究人工合成的dsP21-555对前列腺癌细胞株PC-3和LNCaP细胞周期和增殖的影响。方法 合成dsP21-555（实验组）和dsControl（阴性对照组）,分别转染至PC-3和LNCaP。使用Real-time PCR及Western blotting分别检测分析前列腺癌细胞转染后的p21mRNA及p21蛋白的表达情况。流式细胞术检测细胞周期分布,使用MTT实验及集落形成实验检测细胞的活力及增殖能力。结果 转染dsP21-555后PC-3和LNCaP细胞中的p21 mRNA水平分别上调至2.90倍(P<0.01)和2.05倍(P<0.01)。Western blotting实验结果符合这一趋势。流式细胞术检测显示,转染dsP21-555后,在S期和G2/M期的细胞比例下降,在G0/G1期的细胞比例则增加。MTT实验显示,与dsControl组相比,转染dsP21-555后,PC-3和LNCaP细胞的活力明显降低。集落形成实验显示,dsP21-555组的集落的数量较少,细胞增殖能力降低。结论 人工合成的dsP21-555能明显激活前列腺癌细胞中p21基因的表达,并显著抑制前列腺癌细胞周期的进展和增殖。     

Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model

OBJECTIVE: To evaluate the effect of clusterin overexpression on radiation-induced tumour growth rates and apoptosis in human prostate LNCaP cells, as prostate cancer cells are relatively resistant to radiation-induced apoptosis and local recurrences are common, but overexpression of the anti-apoptotic protein clusterin can accelerate progression to androgen-independence and to confer a chemoresistant phenotype in various prostate cancer models. MATERIALS AND METHODS: Western blot analysis and immunohistochemistry were used to compare clusterin expression levels in parental (P) and clusterin-transfected (T) LNCaP cells in vitro and in vivo. The effects of radiation on clusterin-expression in both parental LNCaP/P and clusterin-transfected LNCaP/T tumours were analysed by Northern blot analysis. The cellular response to radiation was determined up to 3 weeks after irradiation using tetrazolium and re-growth assays, and cell-cycle analysis by flow cytometry. RESULTS: Clusterin mRNA expression increased from undetectable to low levels in LNCaP/P tumours after radiation and more than three-fold in LNCaP/T tumours. Clusterin overexpression decreased the radiosensitivity in a time-dependent manner, reducing the extent of growth arrest and apoptosis by up to 54%. Re-growth assays showed that the improved survival rates of LNCaP/T cells after radiation did not change after 3 days, remaining constant over 3 weeks. CONCLUSIONS: These results identify clusterin as a promoter of cell survival that may help mediate resistance to radiation-induced apoptosis. Furthermore, clusterin overexpression seems to provide an extended protection against radiation-induced cell cycle arrest and apoptosis.     

黏附分子T-cadherin表达诱导C6胶质母细胞瘤细胞G2期阻滞

目的 研究T-cadherin分子表达抑制胶质母细胞瘤C6细胞增殖的机制。方法 利用脂质体转染pcDNA3和pcDNA3-T-cadherin质粒后获得的表达和不表达T-cadherin的C6细胞克隆，以流式细胞仪检测两种克隆细胞的细胞周期的变化，同时分析其细胞周期与细胞分裂指数的关系。结果 转染后表达T-cadherin的C6细胞在去血清培养48h使细胞周期同步后，分别用血清刺激培养12h后行流式细胞仪检测，与转染空载体的1、2克隆相比，转染T-cadherin基因后表达T-cadherin的3、4克隆于G2／M期的细胞比例显著增加（3、4克隆为52．60％和51．84％，1、2克隆为16．17％和13．47％），而G1期的细胞比例显著下降（3、4克隆为0．66％和0．39％，1、2克隆为50．6％和57．9％）。在血清刺激0、4、8、12、24和48h时，空载体克隆1在各时间点的分裂指数与其G2／M期的细胞比例一致，而表达T．cadherin的克隆3在各时间点的分裂指数显著低于其G2／M期的细胞比例数。结论 T．cadherin分子表达能诱导C6细胞G2期细胞阻滞，该机制可能是T-cadherin抑制C6细胞增殖的主要机制。     

Differential expression of cell cycle regulatory molecules and evidence for a "cyclin switch" during progression of prostate cancer

BACKGROUND: Deregulation of the cell cycle can be viewed as both cause and consequence of cancer. Cyclin expression regulates progression through the cell cycle and although some cyclins have been examined in prostate cancer, the spatial and temporal changes in expression of these molecules during progression of autochthonous disease has not been fully explored. METHODS: Expression patterns of cyclins and cyclin dependent kinases during the different stages of progression in the spontaneous autochthonous TRAMP model were examined by RNAse protection assay, Western blot analysis, and immunohistochemistry. RESULTS: Differential expression of cell cycle regulatory molecules was observed during prostate cancer progression. Levels of the D-type cyclins decreased during progression while expression of cyclin E increased both at the mRNA and protein levels. The level of cyclin A and cyclin B expression increased beginning in early stage tumors and continued to increase throughout progression. The levels of cyclin dependent kinases did not change substantially during progression of the TRAMP model. CONCLUSIONS: The spatial and temporal pattern of mitotic cyclin expression during prostate cancer progression suggests that these molecules represent potential therapeutic targets. The differential expression of D-type cyclins may have implications with respect to androgen receptor mediated gene expression.     

Small G-protein RhoE is underexpressed in prostate cancer and induces cell cycle arrest and apoptosis

BACKGROUND: RhoE/Rnd3, a recently described novel member of the Rho GTPases family, was discussed as a possible antagonist of the RhoA protein that stimulates cell cycle progression and is overexpressed and/or overactivated in prostate cancer. We investigated the expression of RhoE and its role in cell cycle regulation and apoptosis in the human prostate. METHODS: RhoE expression in cell lines and tissue specimens was assessed by immunoblot analysis, real-time PCR (RT-PCR), and immunohistochemistry. To elucidate RhoE effects on the prostate, RhoE was cloned and overexpressed in DU-145 prostate cancer. Cell cycle modulation and apoptosis was investigated by immunoblot and FACS analysis. RESULTS: Immunoblot analysis showed a strong RhoE signal in both, benign epithelial and stromal cells. In contrast, almost no protein was detected in various prostate cancer cells. On RT-PCR and microarray analysis, RhoE mRNA expression was significantly reduced in malignant tissue when compared to benign samples. RhoE immunostaining was strong in benign tissue, especially in prostate epithelial cells, whereas it was minimal or absent in malignant tissue. Forced RhoE overexpression in a prostate cancer cell line inhibits the expression of two proteins essential for G2/M transition, namely CDC2 and cyclin B1, and induces G2/M arrest. In addition, apoptotic cell death as measured by a cleavage product of caspase 3 is significantly increased in RhoE-overexpressing cells. CONCLUSION: In conclusion, our findings suggest RhoE as a tumor suppressor gene that is downregulated early in the development of prostate cancer.     

Prostate specific antigen expression does not necessarily correlate with prostate cancer cell growth

PURPOSE: The antiproliferative effects of pharmacological agents used for androgen ablative therapy in prostate cancer, including goserelin, bicalutamide and cyproterone acetate (Fluka Chemie, Buchs, Switzerland), were tested in vitro. It was determined whether they affected prostate specific antigen mRNA and protein expression independent of growth inhibition. MATERIALS AND METHODS: Goserelin, bicalutamide (AstraZeneca, Zug, Switzerland) and cyproterone acetate were added to prostate specific antigen expressing, androgen dependent LNCaP and androgen independent C4-2 cell line (Urocor, Oklahoma City, Oklahoma) cultures. Proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay (Roche, Mannheim, Germany). Prostate specific antigen mRNA expression was assessed by quantitative real-time polymerase chain reaction. Secreted prostate specific antigen protein levels were quantified by microparticle enzyme-immunoassay. RESULTS: Goserelin inhibited cell growth and prostate specific antigen protein secretion in LNCaP and C4-2 cells. Prostate specific antigen mRNA expression was not decreased. Bicalutamide did not affect cell growth or prostate specific antigen mRNA expression in LNCaP or C4-2 cells, although it significantly decreased prostate specific antigen protein secretion in LNCaP and to a lesser extent in C4-2 cells. Cyproterone acetate decreased the growth of C4-2 but not of LNCaP cells. It did not affect prostate specific antigen mRNA or protein expression in either cell line. CONCLUSIONS: Prostate specific antigen expression does not necessarily correlate with cell growth. Without a substantial effect on cell growth bicalutamide lowers prostate specific antigen synthesis, whereas cyproterone acetate decreases cell growth with no effect on prostate specific antigen secretion. Prostate specific antigen expression may be influenced by growth inhibition but also by altered mRNA and protein levels depending on the agent, its concentration and the cell line evaluated. For interpreting clinical trials prostate specific antigen is not necessarily a surrogate end point marker for a treatment effect on prostate cancer cell growth.     

Antisense oligodeoxynucleotide therapy targeting clusterin gene for prostate cancer: Vancouver experience from discovery to clinic

BACKGROUND: The objective of this study was to review our experience in the development of antisense (AS) oligodeoxynucleotide (ODN) therapy for prostate cancer targeting antiapoptotic gene, clusterin. METHODS: We initially summarized our data demonstrating that clusterin could be an optimal therapeutic target for prostate cancer, then presented the process of developing AS ODN therapy using several preclinical animal models. Finally, the preliminary data of the recently completed phase I clinical trial using AS clusterin ODN as well as the future prospects of this therapy are discussed. RESULTS: Expression of clusterin was highly up-regulated after androgen withdrawal and during progression to androgen-independence, but low or absent in untreated tissues in both prostate cancer animal model systems and human clinical specimens. Introduction of the clusterin gene into human prostate cancer cells confers resistance to several therapeutic stimuli, including androgen ablation, chemotherapy and radiation. AS ODN targeting the translation initiation site of the clusterin gene markedly inhibited clusterin expression in prostate cancer cells in a dose-dependent and sequence-specific manner. Systemic treatment with AS clusterin ODN enhanced the effects of several conventional therapies through the effective induction of apoptosis in prostate cancer xenograft models. Based on these findings, a phase I clinical trial was completed using AS clusterin ODN incorporating 2'-O-(2-methoxy)ethyl-gapmer backbone (OGX-011), showing up to 90% suppression of clusterin in prostate cancer. CONCLUSIONS: The data described above identified clusterin as an antiapoptotic gene up-regulated in an adaptive cell survival manner following various cell death triggers that helps confer a phenotype resistant to therapeutic stimuli. Inhibition of clusterin expression using AS ODN technology enhances apoptosis induced by several conventional treatments, resulting in the delay of AI progression and improved survival. Clinical trials using AS ODN confirm potent suppression of clusterin expression and phase II studies will begin in early 2005.     

细胞周期对Fas/APO-1在前列腺癌细胞系PC-3M中表达的影响

目的研究细胞周期对ＰＣ３Ｍ细胞表面Ｆａｓ／ＡＰＯ１蛋白表达的影响。方法采用直接免疫荧光流式细胞术对前列腺癌细胞系ＰＣ３Ｍ未同步化的细胞,及经改良的笑气阻断及ＴｄＲ双阻断方法同步化的不同细胞周期细胞表面Ｆａｓ／ＡＰＯ１表达情况进行检测。结果未同步化的ＰＣ３Ｍ细胞Ｆａｓ／ＡＰＯ１表达阳性率为３４２０％;ＰＣ３Ｍ细胞系不同细胞周期的细胞表面Ｆａｓ／ＡＰＯ１表达明显不同,以Ｓ期最高达８５０５％,Ｇ１及Ｍ期较低,分别为３３２％和４９５％。结论细胞周期对细胞表面Ｆａｓ／ＡＰＯ１的表达情况有明显影响。深入研究细胞周期对肿瘤细胞Ｆａｓ／ＡＰＯ１表达情况的影响,对于采用Ｆａｓ／ＡＰＯ１系统途径治疗恶性肿瘤有重要意义。