FGFR2Ⅲc胞外段的原核表达及其对前列腺癌细胞增殖的抑制作用

将FGFR2Ⅲc胞外段基因转入pEt3c载体,并通过大肠杆菌使其以包涵体形式表达.经透析法复性和肝素亲和层析纯化,得到纯度95%以上的目的蛋白.通过免疫共沉淀方法证实复性后的FGFR2Ⅲc胞外段与FGF2之间可特异性结合,并对前列腺癌DU145细胞的增殖具有明显的抑制作用(MTT法).免疫印迹结果显示FGFR2Ⅲc胞外段可显著下调DU145细胞膜上FGFRs的磷酸化水平,提示由于有效阻断FGFs的细胞内信号通路,从而导致FGFR2Ⅲc胞外段对肿瘤细胞增殖的抑制.     

FGFR2IIIc重组慢病毒载体的构建及其在肌原细胞L6中的表达

目的:构建人FGFR2IIIc重组慢病毒表达系统,感染并筛选获得大鼠肌原细胞L6表达人FGFR2IIIc的重组细胞株,初步研究FGFR2IIIc对L6细胞的作用。方法:从人胎盘组织中获得FGFR2IIIc基因,并克隆到Gateway慢病毒系统的入门载体pENTR-11,采用LR重组酶将重组的pENTR-FGFR2IIIc和表达载体pLenti6/V5-DEST进行重组反应得到pLenti6/V5-DEST-FGFR2IIIc。将该重组表达载体和Viral Packaging Mix包装质粒通过阳离子脂质体共转染293FT细胞,待细胞完全裂解后收集重组慢病毒颗粒上清液;取适量上清液感染L6细胞,杀稻瘟毒素筛选2~4周,挑单克隆细胞进一步扩大培养并建立重组细胞株。RT-PCR、间接免疫荧光和Western blot鉴定重组细胞株中目的基因FGFR2IIIc的表达,bFGF和硫酸乙酰肝素共同诱导各重组L6细胞株后流式细胞术分析细胞周期,形态观察检测成肌分化水平,并通过相关信号通路抑制剂分析与各信号通路的关系。结果:构建了含人FGFR2IIIc基因的重组慢病毒表达载体,获得的重组慢病毒颗粒能高效感染L6细胞,RT-PCR和间接免疫荧光实验说明正确表达目的基因;流式细胞术结果表明L6中高表达FGFR2IIIc的重组细胞株的G1/G0期的细胞增多;各重组细胞株分别加入Erk1/2和p38通路抑制剂抑制诱导2天,发现重组细胞株发生不同的形态变化。结论:通过慢病毒表达系统,成功构建高表达FGFR2IIIc的重组L6细胞株,FGFR2IIIc通过Erk1/2和p38通路影响了L6细胞的形态发生,该结果为下一步在大鼠L6细胞中研究FGFR2IIIc基因与成肌分化功能相关性奠定基础。     

PIAS3α过表达对前列腺癌细胞增殖和凋亡的影响

目的 研究过表达PIAS3α蛋白对人前列腺癌细胞系DU145细胞增殖、细胞周期和凋亡的影响.方法 构建pcDNA3.1(+)-HA-PIAS3α真核表达载体,转染前列腺癌细胞系DU145,以蛋白免疫印迹实验检测外源PIAS3α的表达,通过MTT法检测细胞增殖,以流式细胞术分析细胞周期和细胞凋亡.结果 成功构建PIAS3α真核表达质粒,蛋白免疫印迹实验证实外源性PIAS3蛋白质DU145细胞中获得表达.分析目的 蛋白质过表达后的细胞生物学效应,结果显示在DU145细胞中过表达PIAS3α后,细胞增殖受到抑制;同时,细胞周期G0/G1期细胞显著增加,而S期细胞减少,细胞凋亡比率增加.结论 过表达PIAS3α抑制前列腺癌细胞的增殖并诱导其凋亡.     

sFGFR1Ⅲc的原核表达及其抑制NIH3T3增殖的作用

目的:克隆sFGFR1Ⅲc胞外段进行原核表达和纯化。方法:将sFGFR1Ⅲc胞外段基因转入pET3c载体后转化BL21工程菌,以包涵体形式表达胞外段。经凝胶层析法复性(复性率为20%)和肝素亲和层析纯化后得到纯度95%以上的目的蛋白。通过电泳鉴定其纯度以及MTT法检测其生物学活性。结果:成功获得大小为660bp的人sFGFR1Ⅲc基因片段,转化菌诱导表达了24kDa的sFGFR1Ⅲc胞外段并通过MTT证实它对小鼠胚胎成纤维细胞(NIH3T3)的增殖具有明显的抑制作用,其抑制FGF2引起的细胞增殖呈剂量依赖性,其中作用最为明显的是80ng/ml。结论:成功获得了高纯度的sFGFR1Ⅲc胞外段,该胞外段具备较好的生物学活性,为下一步的研究工作奠定了良好的物质基础。     

成纤维细胞生长因子受体3胞外配体结合区的重组表达及其与A型肉毒毒素重链C端的相互作用

目的:实现成纤维细胞生长因子受体3(FGFR3)胞外配体结合区FGFR3D23的表达与纯化,并鉴定其与A型肉毒毒素重链C端(Bo NT/AHc)的相互作用。方法:全基因合成的FGFR3D23基因片段经酶切连入p TIG(+)质粒,转化大肠杆菌BL21(DE3)后IPTG诱导表达,获得的包含体经Ni2+柱纯化,透析复性后通过pull-down及ELISA检测纯化蛋白与Bo NT/AHc的相互作用。结果与结论:SDS-PAGE分析结果显示得到相对分子质量约27×103的蛋白特异表达条带,表达量为1.39 mg/L,包含体占73.4%;纯化和复性得到的目的蛋白纯度约96%,复性率约3%;pull-down及ELI-SA实验结果证明FGFR3D23可以与Bo NT/AHc特异性地相互作用。     

去甲基化酶JARID1D在前列腺癌侵袭和转移中的作用与机制

目的 探索去甲基化酶JARID1D在前列腺癌侵袭和转移中的作用与机制,以期为前列腺癌的转移防治提供新的预测指标及治疗靶点。方法 选择前列腺癌细胞系22RV1和DU145转染慢病毒,以敲低JARID1D的表达,获得细胞系sh-JARID1D,并通过qRT-PCR及Western Blot检测转染后22RV1和DU145细胞中JARID1D的表达情况。通过Transwell实验检测JARID1D低表达后细胞侵袭能力的变化。通过裸鼠尾静脉注射sh-JARID1D细胞系建立体内实验转移模型,观察敲低JARID1D表达后体内肿瘤细胞的转移能力的变化。通过qRT-PCR及Western Blot检测JARID1D对转移相关基因基质金属蛋白酶2(MMP2)表达的影响。结果 Transwell实验显示JARID1D低表达的前列腺癌细胞侵袭能力增强(22RV1细胞系P<0.01,DU145细胞系P<0.001);体内实验显示JARID1D低表达可显著增强肿瘤细胞的体内转移潜能(22RV1转移模型P<0.01,DU145转移模型P<0.01)。进一步实验表明降低JARID1D表达,...     

西达本胺对人前列腺癌DU145及PC3细胞凋亡的影响

目的:研究西达本胺对前列腺癌DU145、PC3细胞生长抑制及凋亡调控的作用。方法:设置西达本胺浓度分别为4、8、16、32和64μmol/L的5个处理组和对照组(未加西达本胺),分别处理DU145、PC3细胞不同时间,采用MTT法检测细胞的增殖抑制情况,用倒置显微镜观察细胞形态,用流式细胞仪进行细胞凋亡分析,用免疫印迹法检测细胞内Bax、Bcl-2、caspase-3、caspase-9的表达水平。结果:与对照组比较,西达本胺处理组可使细胞明显变圆、体积缩小、脱壁细胞增多;MTT法检测显示,随着西达本胺浓度的增加和作用时间的延长,对DU145、PC3细胞的增殖抑制作用增强,并呈时间、剂量正相关(P<0.01);流式细胞术显示,对照组和16、64μmol/L西达本胺组细胞凋亡率分别为42.24%、50.23%;随着西达本胺浓度的增加,Bcl-2的表达呈下调趋势,Bax、caspase-3、caspase-9的表达呈上调趋势,呈剂量正相关。结论:西达本胺对前列腺癌DU145、PC3细胞生长具有明显的抑制作用,作用机制可能与Bax、Bcl-2、caspase-3及caspase-9凋亡信号通路有关。     

细胞质内微环境直接影响FGFR1酪氨酸激酶介导的SNT1细胞特异性磷酸化

成纤维细胞生长因子受体(FGFR)介导的SNT1(亦称为FRS2)底物磷酸化具有宿主细胞以及受体特异性。为探明这种宿主细胞特异性的决定因素,我们构建了1个FGFR2Ⅲb/R1嵌合受体。该嵌合受体具有1个FGFR2Ⅲb的胞外片段及1个FGFR1蛋白质酪氨酸激酶片断。当表达在3T3细胞(内源性受体为FGFR1并能强烈响应FGFR1的信号)以及DTE-R1/100细胞时,该嵌合受体能即刻诱导SNT1磷酸化。DTE-R1/100细胞为经长期培养的带有外源性FGFR1的非恶性前列腺肿瘤上皮细胞(DTE)并已获得未转化DTE细胞所不具备的FGFR1信号响应性。与此相反,当表达在非转化DTE细胞或未经长期培养的FGFR1转化细胞(DTE-R1)时,FGFR2Ⅲb/R1嵌合受体则无法诱导SNT1磷酸化。我们曾报导DTE细胞对FGFR1介导的SNT1磷酸化活力及其刺激细胞生长信号的响应性是一种获得性的性质,这种性质的获得与细胞恶化是紧密联系在一起的。在此我们进一步证明FGFR介导的SNT1磷酸化具有宿主细胞特异性。这些结果表明细胞内围绕着激酶的微环境而不是细胞外环境决定了SNT1是否可为FGFR1所磷酸化。而且,长期受外源性FGFR1刺激诱发DTE细胞内微环境的变化,从而使表达在DTE细胞里的FGFR1激酶可强烈地磷酸化SNT1。     

细胞质内微环境直接影响FGFR1酪氨酸激酶介导的SNT1细胞特异性磷酸化

成纤维细胞生长因子受体（FGFR）介导的SNT1（亦称为FRS2）底物磷酸化具有宿主细胞以及受体特异性。为探明这种宿主细胞特异性的决定因素,我们构建了1个FGFR2IIIb/R1嵌合受体。该嵌合受体具有1个FGFR2IIIb的胞外片段及1个FGFR1蛋白质酪氨酸激酶片段。当表达在3T3细胞（内源性受体为FGFR1并能强烈响应FGFR1）的信号）以及DTE－R1／100细胞时,该嵌合受体能即刻诱导SNT1磷酸化。DTE－R1／100细胞为经长期培养的带有外源性FGFR1的非恶性前列腺肿瘤上皮细胞（DTE）并已获得未转化DTE细胞所不具备的FGFR1信号响应性。与此相反,当表达在非转化DTE细胞或未经长期培养的FGFR1(DTE-R1)锂,FGFR2IIIb/R1嵌合受体则无法诱导SNT1磷酸化。我们曾报导DTE细胞对FGFR1介导的SNT1磷酸化活力及其刺激细胞生长信号的响应性是一种获得性的性质,这种性质的获得与细胞恶化是紧密联系在一起的。在此我们进一步证明FGFR介导的SNT1磷酸化具有宿主细胞特异性。这些结果表明细胞内围绕着激酶的微环境而不是细胞外环境决定了SNT1是否可为FGFR1所磷酸化。而且,长期受外源性FGFR1刺激诱发DTE细胞内微环境的变化,从而使表达在DTE细胞里的FGFR1激酶可强烈地磷酸化SNT1。     

哺乳动物PUF家族蛋白PUMILIO1的伴侣蛋白研究

目的:探索NIH3T3和DU145细胞中PUM1蛋白的伴侣蛋白。方法:收集NIH3T3和DU145细胞进行免疫沉淀实验,实验组和对照组分别使用抗PUM1抗体和抗Goat-Ig G抗体。通过银染实验观察实验组与对照组是否有差异性条带,通过质谱鉴定和Mascot软件对差异性条带所包含的蛋白进行分析,鉴定PUM1蛋白在细胞中发挥作用时的可能互动蛋白。结果:在NIH3T3和DU145细胞银染实验中,实验组凝胶泳道均在约37 KD处出现差异性条带。对NIH3T3细胞中差异性条带进行质谱分析,通过Mascot软件肽质量指纹谱搜索,将有真实差异的蛋白评分最低阈值设为65,P0.05,结果显示肽段评分最高的蛋白为NDFIP2蛋白。与NCBInr数据库比对,检测到针对NDFIP2的肽段的覆盖率为40%。结论:NDFIP2与哺乳动物PUM1蛋白在细胞内关联,可能是PUF家族蛋白的伴侣蛋白。     

Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell

Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE‐based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys‐regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF‐κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism‐based chemopreventive strategy for prostate cancer.     

Elevated expression of ERK 2 in human tumor cells chronically treated with PD98059

We examined the effect of chronic exposure of tumor cells to a mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK) kinase inhibitor, PD98059, on cell proliferation was investigated. Human renal carcinoma cells (ACHN) and prostatic carcinoma cells (DU145) were cultured in the presence of PD98059 for more than 4 weeks (denoted ACHN (PD) cells and DU145 (PD) cells, respectively) and proliferation and signal transduction pathways were examined. PD98059 significantly inhibited the proliferation of parental cells. However, PD98059 failed to inhibit proliferation of ACHN (PD) and DU145 (PD) cells significantly. Expression of ERK 1 and 2 was elevated in these cells. These phenotypes were reversible. Downregulation of ERK 2, but not ERK 1, by small interfering RNA significantly inhibited the proliferation of ACHN (PD) and DU145 (PD) cells. Taken together, chronic exposure of tumor cells to PD98059 induced elevated expression of ERK 2, which was associated with decreased sensitivity of cellular proliferation to PD98059.     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

miR-155 通过p53/p21 调控前列腺癌细胞周期

目的:探讨miR-155对前列腺癌细胞周期的影响及其分子机制。方法:通过转染anti-miR-155抑制前列腺癌DU145和PC-3细胞中miR-155水平后,采用流式细胞术观察细胞周期的变化,western blot和RT-PCR观察p53和p21蛋白及CDK2和cyclin蛋白和m RNA表达的变化。结果:与对照组相比,DU145和PC-3细胞转染anti-miR-155后,G2/M期细胞阻滞,S期细胞数比例显著增加(P0.05),p53和p21蛋白和m RNA表达水平显著增加(P0.01),CDK2和cyclin E蛋白和m RNA表达均显著降低(P0.01)。结论:miR-155可影响人前列腺癌细胞的周期,可能与其调节p53、p21及其下游的CDK2和cyclin E的表达相关。     

KHC-4 inhibits β-catenin expression in prostate cancer cells

ABSTRACT

KHC-4 is a 2-phenyl-4-quinolone analogue that exhibits anticancer activity. Aberrant activation of β-catenin signaling contributes to prostate cancer development and progression. Therefore, targeting β-catenin expression could be a useful approach to treating prostate cancer. We found that KHC-4 can inhibit β-catenin expression and its signaling pathway in DU145 prostate cancer cells. Treatment with KHC-4 decreased total β-catenin expression and concomitantly decreased β-catenin levels in both the cytoplasm and nucleus of cells. KHC-4 treatment also inhibited β-catenin expression and that of its target proteins, PI3K, AKT, GSK3β and TBX3. We monitored the stability of β-catenin with the proteasomal inhibitor, MG132, in DU145 cells and found that MG132 reversed KHC-4-induced proteasomal β-catenin degradation. We verified CDK1/β-catenin expression in KHC-4 treated DU145 cells. We found that roscovitine treatment reversed cell proliferation by arresting the cell cycle at the G2/M phase and β-catenin expression caused by KHC-4 treatment. We suggest that KHC-4 inhibits β-catenin signaling in DU145 prostate cancer cells.     

The effect of tyrosine kinase inhibitors, tyrphostins: AG1024 and SU1498, on autocrine growth of prostate cancer cells (DU145)

It is well established that autocrine growth of human prostate cancer cell line DU145 is dependent on TGF (EGF)/EGFR loop. However, the participation of several other growth factors in proliferation of DU145 cells has been also proposed. We employed two selective tyrosine kinase inhibitors (tyrphostins): AG1024 (an IGFIR inhibitor) and SU1498 (a VEGFR2 inhibitor) for growth regulation of DU145 cells, cultured in chemically defined DMEM/F12 medium. Both the tested compounds inhibited autocrine growth of DU145 cells at similar concentration values (IC50 approximately 2.5 microM). The tyrphostins arrested cell growth of DU145 in G1 phase, similarly as inhibitors of EGFR. However, in contrast to selective inhibitors of EGFR, neither AG1024, nor SU1498 (at concentration < or =10 microM) decreased the viability of the investigated cells. These results strongly suggest that autocrine growth of DU145 cells is stimulated by, at least, three autocrine loops: TGFalpha(EGF)/EGFR, IGFII/IGFIr and VEGF/VEGFR2(VEGFR1). These data support the hypothesis of multi-loops growth regulation of metastatic prostate cancer cell lines.     

Index

Anticancer role of oxindole compounds is well documented. Here, we synthesized new derivatives of 3-hydroxy-2-oxindole functionalized at position 3 (1a–f) which are expected to have antiproliferative activity in cancer cells. Human prostate cancer cell line (DU145) was treated with the synthesized derivatives at 40-μM concentration for 24, 48, and 72 h. Compounds 1-ethyl-3-hydroxy-1,1′,3,3′-tetrahydro-2H,2′H-3,3′-biindole-2,2′-dione (1d), 5-bromo-1-ethyl-3-hydroxy-1,1′,3,3′-2H,2′H-3,3′-biindole-2,2′-dione (1e), and 5-chloro-1-ethyl-3-hydroxy-1,1′,3,3′-tetrahydro-2H,2′H-3,3′-biindole-2,2′-dione (1f) were found to significantly reduce DU145 cell viability at 48 and 72 h whereas no significant changes were observed up to 24 h. The compounds 1e and 1f showed the most cytotoxicity effect and had a similar antiproliferative activity on DU145 cell line. They have halogen and ethyl substitutions at positions 5 and 1, respectively. The IC₅₀ of compound 1e for DU145 and A375 cells at 48 h was determined. The apoptotic effects and cell cycle progression of compound 1e at 1/2 × IC₅₀ (55 μM) concentration in DU145 cells were investigated by nuclei staining, comet assay, flow cytometry, and scanning electron microscopy (SEM). The results obtained showed that this compound increased the percentage of tail DNA, increased the occurrence of the sub-G1 phase, and induced G2M arrest and apoptosis in DU145 cells after exposure for 48 h to a 55-μM concentration. The SEM images revealed cell contraction at 24 h, cell condensation, plasma membrane blebbing, and formation of apoptotic bodies at 48 and 72 h. These observations suggest that the antiproliferative activity of compound 1e may be to induce apoptosis in DU145 cells.