siRNA靶向沉默B7-H4基因对人前列腺癌DU145细胞增值和凋亡的影响

目的研究siRNA沉默B7-H4基因对人前列腺癌DUl45细胞增殖和凋亡的影响。方法以脂质体Lipofeclami-ne“2000（Lipo）为载体转染siRNA-B7-H4至DUl45细胞,应用RT-PCR和WesternBlot检测B7-H4表达水平,CCK-8法检测细胞增殖的变化,Annexinv／PI双染流式细胞术检测细胞凋亡情况。结果与空白组和阴性对照组（NC）相比,转染B7-H4-siRNA的细胞B7-H4mRNA和蛋白表达明显减少（P〈0．05）,DUl45转染B7-H4siRNA后,增殖能力减弱,凋亡显著。结论通过B7-H4-siRNA抑制DUl45细胞B7-H4的表达,可以抑制细胞增殖,促进其凋亡,表明B7-4在前列腺癌的发生发展中发挥重要作用。     

薄荷醇抑制前列腺癌DU145细胞的增殖和迁移

目的探讨薄荷醇对雄激素非依赖性前列腺癌DU145细胞增殖和迁移能力的影响。方法通过RT-PCR、免疫组织化学和Western blot方法检测TRPM8和TRPA1的表达;MTT和划痕试验检测薄荷醇对DU145细胞的增殖和迁移能力的影响;流式细胞术检测TRPM8对DU145细胞周期和凋亡的影响。结果 RT-PCR、免疫组织化学和Western blot提示TRPM8在DU145细胞中高表达而TRPA1在DU145细胞中不表达;薄荷醇能诱导细胞周期阻滞于G0/G1期,与未经薄荷醇处理的细胞相比,经100μmol/L薄荷醇处理的细胞培养24、48和72h后,G0/G1期细胞显著增加(49.12%±1.92%vs61.71%±2.70%、77.65%±1.63%、71.81%±2.46%,P<0.05,P<0.01),进而抑制细胞增殖(P<0.05),并抑制细胞迁移(P<0.05),流式细胞术检测显示薄荷醇并不引起细胞凋亡。结论 TRPM8可能成为前列腺癌治疗的一个新靶点,对于高表达TRPM8的雄激素非依赖性前列腺癌针对TRPM8通道的药物治疗可能比TRPM8基因治疗更为实用,因此,薄荷醇作为一个潜在的抗肿瘤药物也拥有很大的发展前景。     

Rock蛋白抑制剂法舒地尔对前列腺癌PC3、DU145细胞侵袭、迁移和凋亡的影响

目的:探讨Rho A/Rock信号传导通路在前列腺癌形成过程中的可能作用;研究Rock蛋白抑制剂法舒地尔潜在抗肿瘤侵袭、迁移和促凋亡的作用。方法:分别用5、10、20、40、80、160μmol/L浓度的法舒地尔作用于前列腺癌PC3、DU145细胞24 h,MTT法检测细胞增殖抑制率,并计算其IC50,取法舒地尔IC50的1/4作为给药剂量用于进一步研究。免疫细胞化学法检测法舒地尔对PC3、DU145细胞Rho A、RockⅠ、RockⅡ蛋白表达的影响;Western印迹检测法舒地尔对PC3、DU145细胞Rho A总蛋白、Rho A膜蛋白、RockⅠ、RockⅡ蛋白表达的影响;通过Transwell、划痕实验、流式细胞术评估细胞的侵袭、迁移、凋亡情况。结果:随着法舒地尔作用浓度的增加,对PC3、DU145的抑制率分别从(9.29±1.23)%、(7.59±1.54)%增加到(81.37±3.97)%、(76.53±2.67)%,呈一定剂量依赖性(P均0.05);细胞迁移实验中实验组穿入下室的PC3、DU145细胞为(39.2±8.4)个和(34.2±6.7)个,阴性对照组分别为(116.8±9.3)和(112.5±10.8)个,差异有统计学意义(P均0.05);划痕实验结果显示实验组PC3、DU145细胞24 h划痕愈合率为(37.26±1.17)%和(32.38±2.73)%,阴性对照组愈合率为(78.12±4.16)%和(69.47±6.71)%(P均0.05);Annexin V-FITC/PI双染色法的结果显示,法舒地尔作用PC3、DU145细胞后凋亡率分别为(31.88±2.49)%、(28.65±2.99)%,显著高于阴性对照组[(7.51±2.28)%、(7.13±1.61)%,P均0.05]。免疫细胞化学检测显示,法舒地尔能明显降低RockⅠ和RockⅡ的表达(与阴性对照组比较P均0.05),而Rho A的表达与阴性对照组比较差异无统计学意义(P0.05);Western印迹也显示,法舒地尔也明显降低Rho A膜蛋白、RockⅠ、RockⅡ蛋白的表达(与阴性对照组比较P均0.05),而Rho A总蛋白表达与阴性对照组差异无统计学意义(P0.05)。结论:Rho A/Rock信号传导途径可能参与了前列腺癌的形成;法舒地尔能够有效地抑制前列腺癌细胞的增殖、侵袭、迁移,并促进其凋亡,其机制可能与法舒地尔下调Rho A/Rock信号通路有关。     

茶多酚对前列腺癌细胞DU145生长的影响

目的:探讨茶多酚对前列腺癌细胞生长的影响及其作用机制。方法:选取激素非依赖性前列腺癌细胞系DU145为研究对象,在药物组的培养基中加入茶多酚使其终浓度分别为50、100、250、500μg/ml,对照组加入正常细胞培养基。各组细胞培养48 h后,采用MTT比色法检测细胞生存率,然后通过Western印迹分析和荧光定量RT-PCR法检测各组细胞survivin基因表达情况。结果:加入茶多酚溶液48 h后,各药物组细胞存活率分别为0.97±0.12、0.71±0.07、0.20±0.03和0.08±0.01,与对照组相比,除50μg/ml组(P=0.42)外,其余3个药物组细胞存活率均明显低于对照组(P﹤0.01)。随茶多酚作用时间增加,各组细胞存活率呈现下降趋势,到96 h时,除50μg/ml组,其余3组细胞存活率均在5%以下。加药48 h后,对照组、100、250、500μg/ml药物组的sur-vivin表达条带灰度值分别为15 075±48、13 425±31、2 017±24、1 274±22,与对照组相比,3个药物组survivin蛋白表达均明显减少(P均﹤0.01)。另外,50、100、250、500μg/ml药物组给药48 h时的survivin mRNA量分别为0.74±0.03、0.64±0.02、0.52±0.01、0.21±0.02,均明显少于对照组(P均﹤0.01)。结论:茶多酚可以抑制前列腺癌DU145细胞的生长,且这种作用可能与survivin基因表达减少有关。     

表皮生长因子信号通路对前列腺癌细胞DU145黏附和迁移能力的影响

目的 探讨表皮生长因子（EGF）信号通路对雄激素非依赖型前列腺癌细胞株DU145黏附和迁移能力的影响。方法 应用细胞黏附能力测定和划痕实验测定EGF对DU145黏附和迁移能力的作用，应用流式细胞术测定EGF对DU145细胞表面整联蛋白α5和β1表达的影响，应用逆转录-聚合酶链反应（RT-PCR）和Western blot测定EGF对DUl45细胞整联蛋白α5和β1亚基总蛋白和mRNA表达的影响。结果 EGF可促进DUl45对纤连蛋白（Fn）的黏附和迁移作用，同时，Fn的受体a5β1的表达发生了变化，EGF上调细胞表面β1亚基的表达，而丝裂源激活蛋白激酶（MAPK）信号通路抑制剂PD98059则具有相反的作用，这种调节作用主要发生在mRNA水平上。结论 EGF可能通过MAPK信号通路，上调β1的表达，从而促进DU145细胞的侵袭能力。     

隐丹参酮对前列腺癌DU145细胞中异黏蛋白表达的影响

目的:探讨隐丹参酮对前列腺癌DU145细胞增殖及细胞凋亡的作用,并初步探讨隐丹参酮对DU145细胞中异黏蛋白(MTDH)表达及下游PI3K/AKT信号通路的影响。方法:四氮唑蓝(MTT)比色法检测不同浓度隐丹参酮分别作用DU145细胞24、48、72 h后对细胞的生长抑制作用;原位末端转移酶标记(TUNEL)法检测细胞凋亡情况;Western印迹检测不同浓度隐丹参酮及作用不同时间对DU145细胞中MTDH蛋白的表达影响;RT-PCR技术检测隐丹参酮分别作用DU145细胞12、24、48 h后细胞中MTDH mRNA的表达情况;Western印迹检测隐丹参酮作用DU145细胞48 h后细胞中MTDH、AKT、p-AKT、Bcl-2蛋白的表达情况。结果:隐丹参酮能够明显抑制DU145细胞增殖,且抑制效应呈剂量和时间依赖性(P0.05);以10μmol/L隐丹参酮作用DU145细胞24、48、72 h后细胞凋亡率分别为(29.42±4.51)%、(55.07±5.67)%和(70.84±4.66)%,明显高于对照组(3.1±2.48)%(P0.05)。Western印迹和RT-PCR结果显示,隐丹参酮可在转录和翻译水平下调MTDH的表达(P0.05),抑制AKT信号通路和抗凋亡蛋白Bcl-2的表达(P0.05)。结论:隐丹参酮可抑制前列腺癌DU145细胞的增殖,促进细胞凋亡,其机制可能是通过下调MTDH表达,抑制其下游PI3K/AKT信号通路。     

硼替佐米增强前列腺癌细胞对NK细胞介导细胞杀伤作用的敏感性

目的:探讨硼替佐米是否能够增强前列腺癌细胞对NK细胞介导杀伤作用的敏感性,以及是否在不同类型的人前列腺癌细胞系中有相似的作用。方法:以激素依赖性的前列腺癌细胞株LNCaP和激素非依赖性的前列腺癌细胞株DU145为模型,不同浓度(0、5、10、15、20、25nmol/L)硼替佐米处理细胞后,CCK-8法检测肿瘤细胞的增殖,Annexin V/PI法检测细胞凋亡率。结果:15、20、25nmol/L硼替佐米处理DU145细胞48、72h后,各处理组细胞的增殖率分别为(82.79±2.04)%、(73.59±2.95)%、(74.16±6.16)%和(71.24±5.30)%、(51.20±2.91)%、(38.02±2.67)%,同样处理LNCaP细胞后,各处理组细胞的增殖率分别为(77.04±7.74)%、(42.61±6.62)%、(23.85±6.04)%和(36.45±7.02)%、(14.94±5.76)%、(11.65±5.87)%。与对照组相比,硼替佐米强烈抑制两种细胞系的增殖(P0.05)。15、20、25nmol/L硼替佐米处理DU145细胞24h后,DU145细胞的凋亡率分别为(14.41±1.32)%、(16.13±1.55)%、(14.48±1.42)%,而在LNCaP细胞,20、25nmol/L硼替佐米处理24h后,凋亡率为(12.77±1.28)%和(14.84±1.65)%,与对照组相比有统计学差异(P0.05),DU145细胞对硼替佐米诱导的凋亡作用较LNCaP细胞更加敏感。但是,在短期分析中硼替佐米不能致敏两种细胞系对NK细胞介导的杀伤作用。在长效分析中,用硼替佐米处理肿瘤细胞后,20nmol/L硼替佐米+NK组诱导的DU145细胞和LNCaP细胞凋亡率分别为(41.83±5.06)%和(30.31±3.62)%,较单独应用硼替佐米或者NK细胞更高(P0.05)。结论:硼替佐米能够应用于致敏前列腺癌细胞对NK细胞介导的杀伤作用的敏感性,提高当前前列腺癌的治疗水平。而且此治疗策略对雄激素非依赖性的前列腺癌患者更有效。     

环巴胺对DU145细胞增殖和细胞周期的影响

目的:研究Hedgehog信号通路阻断剂(环巴胺)对DU145细胞增殖的抑制作用。方法:不同浓度(1、10、50、100μmol/L)环巴胺干预DU145细胞,分别在24、48、72h后采用噻唑蓝比色法检测其对细胞增殖的抑制作用;流式细胞术检测环巴胺对细胞周期的影响;RT-PCR检测50μmol/L环巴胺作用48h后实验组和对照组细胞周期蛋白E(cyclinE)mRNA表达水平的差异。结果:环巴胺对DU145细胞的抑制作用呈时效和量效依赖关系,当浓度>10μmol/L作用24h后会显著抑制细胞增殖,10、50、100μmol/L浓度组对细胞的抑制率分别为7.42%、12.70%和59.15%,与空白对照组相比,差异有统计学意义(P<0.05)。流式细胞术检测发现,当环巴胺浓度达10μmol/L以上,干预48h后G1期细胞比例明显升高。对照组、10、50μmol/L浓度组的G1期细胞百分比分别为:(52.17±2.21)%、(60.13±2.75)%和(74.30±3.52)%,差异有统计学意义(P<0.01);凋亡峰也随环巴胺浓度增加而逐渐增高。50μmol/L环巴胺作用48h后DU145细胞cyclinEmRNA表达显著降低,与空白对照组相比降低61.90%(P<0.01)。结论:环巴胺可以抑制DU145细胞的增殖能力,其机制可能与下调DU145细胞cyc-linEmRNA表达水平,从而将DU145细胞阻滞于G1期有关。环巴胺亦可以诱导DU145细胞凋亡。     

芪蓝方对人前列腺癌DU145细胞增殖和凋亡的作用机制研究

目的:观察芪蓝方对人前列腺癌DU145细胞增殖和凋亡的影响,并探讨其作用机制。方法:以DU145细胞为研究对象,通过观察不同浓度芪蓝方组(400、200、100、50、25、12.5、6.25、3.125、1.56、0μg/ml)细胞形态变化,筛选出高、中、低浓度应用于后续实验,并采用CCK-8法检测DU145细胞增殖,流式细胞术检测DU145细胞周期、凋亡情况,Western印迹检测DU145细胞中细胞周期、凋亡相关蛋白Cyclin D1、Bax、Bcl-2、Cleaved-Caspase 3的表达。结果:筛选结果显示100、200、400μg/ml浓度的芪蓝方均能显著抑制DU145细胞的生长、降低轮廓清晰度和贴壁能力,且200、400μg/ml浓度的芪蓝方可显著降低DU145细胞活力,故确定高中低浓度为400、200、100μg/ml,并用于后续实验研究。与空白组比较,G2期各浓度组DU145细胞数显著增加(P<0.01),而S期高、中浓度组细胞数显著下降(P<0.01、P<0.05);与空白对照组相比,芪蓝方各组DU145细胞总凋亡数均显著增高(P<0.0...     

重楼皂苷Ⅰ对去势抵抗性前列腺癌DU145细胞增殖及凋亡的影响

目的 探讨重楼皂苷Ⅰ(PPⅠ)对去势抵抗性前列腺癌(CRPC)细胞株DU145细胞增殖的影响及其诱导细胞凋亡的分子机制。方法采用MTT法检测PPⅠ对CRPC细胞株DU145细胞增殖能力的影响,使用流式细胞仪检测DU145细胞的凋亡率,同时应用Western blot检测PPⅠ对DU145细胞p-ERK1/2、ERK1/2、特异性蛋白1(SP1)及Zeste基因增强子同源物2(EZH2)蛋白表达的影响;通过加入ERK1/2抑制剂(PD98059)检测PPⅠ对SP1表达的影响,通过转染SP1、EZH2过表达质粒分别检测PPⅠ对SP1、EZH2表达的影响;采用双荧光素酶报告基因检测EZH2启动子活性,并探讨ERK1/2、SP1和EZH2之间的关系。设未加药组为空白组。结果MTT法检测结果显示,PPⅠ能抑制DU145细胞体外生长,与空白组相比,细胞存活率从给药浓度0.4μmol·L-1开始明显下降,且呈时间和剂量依赖性,差异有统计学意义(P<0.01)。流式细胞仪检测结果显示,PPⅠ能诱导DU145细胞早期凋亡,与空白组相比,细胞早期凋亡率从给药浓度0.4μmol·L-1开始明显增加,差...     

Inhibition of cortactin and SIRT1 expression attenuates migration and invasion of prostate cancer DU145 cells

Objectives: Cortactin is overexpressed in various types of cancer and enhances cell motility. It has been recently reported that silent mating type information regulation 2 homolog 1 interacts with cortactin and promotes cell migration. Here, we examined the role of cortactin and silent mating type information regulation 2 homolog 1 in migration and invasion of prostate cancer cells. Methods: The cortactin expression levels in DU145, LNCaP and PC3 prostate cancer cells, and in PrEC normal human prostate epithelial cells were evaluated by western blot analysis. In DU145 cells, the expression of cortactin or silent mating type information regulation 2 homolog 1 was inhibited by small interfering RNA, and the effects of their knockdown on migration and invasion were examined by cell migration and invasion assays. To determine the localization of cortactin and silent mating type information regulation 2 homolog 1, western blot and immunofluorescence microscopic analyses were carried out. The functional interaction between silent mating type information regulation 2 homolog 1 and cortactin was also studied by in vivo acetylation assay. Results: The protein expression of cortactin was significantly higher in DU145 cells than in other cell lines. Knockdown of cortactin or silent mating type information regulation 2 homolog 1 expression inhibited both migration and invasion of DU145 cells. Similarly to cortactin, silent mating type information regulation 2 homolog 1 was found to be predominantly expressed in the cytoplasm. Finally, the knockdown of silent mating type information regulation 2 homolog 1 expression increased the acetylation level of cortactin. Conclusions: Our findings suggest that inhibition of cortactin or silent mating type information regulation 2 homolog 1 expression attenuates migration and invasion of DU145 cells and this could represent a promising strategy to regulate metastasis of prostate cancer.     

Effects of bortezomib in sensitizing human prostate cancer cell lines to NK-mediated cytotoxicity

The proteasome inhibitor, bortezomib, has been demonstrated to sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Natural killer (NK) cells represent potent antitumor effector cells. They also express TRAIL. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing, and have the same effect in human prostate cancer cell lines (LNCaP and DU145). We found that bortezomib strongly inhibits proliferation in both cell lines. Furthermore, compared with LNCaP cells, DU145 cells are more sensitive to bortezomib-induced apoptosis. However, bortezomib is unable to sensitize these two cell lines to NK cell-mediated killing in short-term assays. In long-term assays, we found that killing mediated by activated NK cells following bortezomib treatment leads to greater antitumor effects than either treatment alone. In addition, treatment with bortezomib causes these cells to upregulate apoptosis-related mRNA as well as death receptors and downregulate the major histocompatibility class (MHC)-I molecule on the cell surface of DU145 cells. In contrast, LNCaP cells are not sensitized by this treatment. Death receptors and the MHC-I molecule did not change in this cell line. These data suggest that bortezomib can be used to sensitize prostate cancer cells to NK cell-mediated killing and improve current cancer therapies. This therapeutic strategy may be more effective in patients with androgen-insensitive prostate cancer.     

Effects of hepatocyte growth factor on urokinase-type plasminogen activator (uPA) and uPA receptor in DU145 prostate cancer cells

Urokinase-type plasminogen activator (uPA) and the uPA receptor (uPAR) are involved in a proteolytic cascade resulting of extracellular matrix degradation. Upstream, uPA and uPAR are regulated by various factors including hepatocyte growth factor (HGF), which stimulates the uPA/uPAR proteolytic system and increases invasion of cancers. We recently demonstrated that HGF induces invasion of DU145 prostate cancer cells into collagen gel matrix. We therefore examined effects of HGF on uPA and uPAR expression in DU145 cells. Effects of HGF on uPA expression in culture medium were determined by Western blotting and fibrin zymography, effects on uPAR expression in cell-associated protein were examined by Western blotting. HGF increased uPA and uPAR production in a dose-dependent manner up to 10 ng/mL, while effects of 20 ng/mL were approximately equal to those of 10 ng/mL. HGF stimulated uPA production beyond that in control cultures from 8 h until 48 h after HGF addition. HGF stimulated a uPA/uPAR proteolytic network in DU145 cells, which may be important for acquisition invasive potential by prostate cancer.     

Stable lower PAR expression decreased DU145 prostate cancer cell growth in SCID mice.

BACKGROUND: PAR is a novel gene ubiquitously expressed in normal and malignant tissues with a trend towards higher expression in tumor cells. PAR biological function is unknown. Here we report the effect of lowering PAR expression on in vitro and in vivo proliferation of DU145 cells. METHODS: Decreased PAR expression was achieved by stable transfection of DU145 cells with antisense PAR cDNA cloned in pCMV-Script expression vector. The proliferative potential of DU145 transfectants was studied by cell counts, colony formation in soft agar, flow cytometry, and growth in severe combined immunodeficient (SCID) mice. RESULTS: DU145 transfectants exhibited a decreased cell proliferation in tissue culture and a low efficiency of colony formation in soft agar. Flow cytometry revealed an arrest of these cells in G2-M phase of mitotic cycle. A dramatic decrease of tumor growth was observed when DU145 transfectant cells were inoculated in SCID mice, compared with controls. Histological examination of these tumors showed a marked decrease in cell density and in number of mitoses while control tumors showed a high cell density and numerous mitoses. CONCLUSIONS: The data presented here provide the first evidence for PAR gene cellular function and its possible implication in malignant transformation.     

Comment on ＂Effect of transferred NK4 gene on proliferation,migration, invasion,and apoptosis of human prostate cancer DU145 cells＂ by Dan Yue et al. in Asian Journal of Androtogy

Hepatocyte growth factor/scatter factor （HGF/SF） interacting with its cell surface receptor tyrosine kinase （RTK） c-met proto-oncogene drives downstream signaling pathways which lead to cell proliferation, migration, invasion, apoptotic cell-death protection, angiogenesis during embryogenesis, repair and regeneration, and neoplastic growth and metastatic progression [1-6].