慢病毒介导的稳定表达LBH的前列腺癌细胞株的建立

目的:构建稳定表达LBH基因的人前列腺癌细胞株PC-3M-LBH,探讨LBH基因对PC-3M细胞增殖能力的影响。方法:构建表达LBH基因的重组慢病毒载体并制备出相应的慢病毒,感染低表达LBH基因的人前列腺癌PC-3M细胞后,经嘌呤霉素筛选获得细胞克隆;实时荧光定量PCR和蛋白印迹法(Western-Blot)分别检测细胞株中LBH的mRNA、蛋白表达水平;采用CCK-8法检测表达LBH基因后细胞增殖能力的改变。结果:成功构建了重组慢病毒表达质粒p Lenti-LBH并包装出了慢病毒,感染前列腺癌细胞后经嘌呤霉素筛选得到PC-3M-LBH细胞株;PC-3M-LBH细胞株中LBH基因的mRNA和蛋白表达显著上调;相对母细胞和NC对照组,PC-3M-LBH细胞在接种后第4天即出现明显的生长抑制,到第6天其生长抑制率达到19.7%。结论:构建的细胞株能稳定表达LBH基因,该基因的表达能显著抑制前列腺癌PC-3M细胞的体外增殖。     

敲低前列腺癌相关基因PC-1的表达抑制前列腺癌细胞C4-2雄激素非依赖性生长

目的:研究敲低癌基因D52家族成员PC-1的表达对前列腺癌细胞雄激素非依赖性生长的影响。方法:利用RNA干扰技术构建PC-1稳定低表达的C4-2细胞株;利用四唑盐(MTT)比色实验检测敲低PC-1基因表达对C4-2细胞雄激素非依赖生长的影响。结果:敲低PC-1表达抑制前列腺癌C4-2细胞的生长,并降低了C4-2细胞雄激素非依赖性生长的能力。结论:PC-1基因参与了前列腺癌向雄激素非依赖阶段发展和维持的过程,为进一步研究PC-1在促进前列腺癌细胞发生发展过程中的作用奠定了基础。     

选择性磷酸二酯酶4 抑制剂ZL-n-91 对人前列腺癌PC-3 细胞和裸鼠移植瘤的影响

目的:研究选择性磷酸二酯酶4抑制剂ZL-n-91对人前列腺癌PC-3细胞人前列腺癌PC-3细胞和裸鼠移植瘤的影响。方法:将不同浓度的ZL-n-91(10,50,100,200μM)分别处理体外培养的前列腺癌PC-3细胞24 h和48 h,用CCK-8法测定ZL-n-91对前列腺癌PC-3细胞增殖的影响;将人前列腺癌PC-3细胞皮下种植裸鼠,待瘤体积达到100~200 mm3,口服ZL-n-91,定期测定动物体重、肿瘤体积、肿瘤重量、计算抑瘤率;采用免疫组织化学检测肿瘤组织Ki67的表达。结果:ZL-n-91能抑制PC-3细胞的增殖,药物浓度为100,200μM时,作用24 h后其抑制率分别达23.7%、58.1%,作用48 h后其抑制率分别达36.5%、70%。与对照组比较差异有统计学意义(P0.001),并且其抑制增殖作用呈浓度和时间依赖性。荷瘤裸鼠经ZL-n-91治疗12天开始,给药组小鼠肿瘤体积明显小于对照组(P0.05),给药32天时,给药组肿瘤体积和重量约为对照组的1/2倍,其抑瘤率高达45.96%;免疫组化检测肿瘤组织中Ki67的表达,定量分析对照组和给药组阳性率分别为(40.7±0.18)%和(18.11±0.06)%,结果显示给药组小鼠肿瘤的增殖明显弱于对照组(P0.001)。结论:ZL-n-91对人前列腺癌PC-3细胞及移植瘤的生长有明显的抑制作用。     

华蟾素注射液对前列腺癌PC3细胞株增殖及VEGF及其受体KDR表达的影响

目的:探讨华蟾素注射液对前列腺癌PC3细胞的生长抑制作用及其对VEGF及KDR表达水平的影响。方法:体外培养前列腺癌PC3细胞株,采用MTT(四甲基偶氮唑蓝)法检测不同浓度华蟾素注射液对前列腺癌PC3细胞增殖的影响;采用FCM法分析该药对PC3细胞周期分布的影响;蛋白印迹法检测药物处理PC3细胞后,细胞中VEGF及KDR蛋白表达的变化。结果:不同浓度浓度的华蟾素注射液对前列腺癌PC3细胞具有抑制作用(P<0.05),并呈一定的时间剂量依赖性;流式细胞仪检测显示,华蟾素可阻滞细胞进程于S期(P<0.05);蛋白印迹法检测结果显示,华蟾素注射液能使VEGF及KDR蛋白表达下降,并呈浓度依赖性。结论:华蟾素注射液能抑制PC3细胞的增殖作用,其机制可能与下调VEGF及KDR表达有关。     

P53参与抑制PC-1基因转录的结构域分析

目的:分析P53分子参与抑制PC-1基因转录的结构域。方法:构建P53分子突变体;将前列腺癌LNCaP细胞瞬时转染野生型或突变型p53及含PC-1启动子的荧光素酶表达载体p4939,分析PC-1启动子的转录活性。结果:P53分子N端转录激活域及富含脯氨酸功能域突变体没有减弱对PC-1启动子的转录抑制作用,而特异性DNA结合域上175、273位突变体和C端339～346位氨基酸的缺失突变体都减弱了对PC-1启动子转录的抑制作用;另外P53分子第175和273位突变体在前列腺癌细胞中对野生型P53的转录抑制功能表现出显性负效应。结论:P53分子特异性DNA结合域和C端结构域参与对PC-1基因启动子的转录抑制,而P53突变体的显性负效应可能是PC-1在前列腺癌进展中表达失调的因素之一。     

前列腺癌细胞系中环状RNA circRNA-1565的表达及鉴定

目的:探讨前列腺癌细胞系中的环状RNA circRNA-1565的表达及鉴定。方法:培养前列腺正常上皮细胞(RWPE-1)、4种前列腺癌细胞(22RV1、LNCap、PC-3、DU145),提取总RNA,采用RT-PCR检测不同细胞系中circRNA-1565的表达,并对其进行成环性验证及细胞内亚定位实验,进行差异比较。结果:circRNA-1565在正常前列腺细胞系(RWPE-1)中表达量极低,在转移性前列腺癌细胞系中高表达,在非转移性前列腺癌细胞系中低表达。且circRNA-1565是一条主要定位于细胞质内的具有有效环状结构的RNA分子。结论:circRNA-1565在不同前列腺癌细胞系中存在差异表达,可能会通过mi RNA sponge途径发挥生物学作用。     

外源高表达PC-1蛋白N端43个氨基酸可加速人前列腺癌细胞系C4-2的生长

研究PC-1蛋白N端43个氨基酸表达对人前列腺癌细胞C4—2生长的影响。用DNA重组技术将含PC—1蛋白N端43个氨基酸的DNA序列正向克隆到真核表达载体pIRES2-EGFP中,采用脂质体法将重组质粒稳定转染进C4—2细胞中,RT—PCR分析外源序列的转录情况,固相ELISA法测定PC—1蛋白N端43个氨基酸的表达,MTT实验分析细胞的生长速度。结果获得了稳定转染PC—J基因N端43个氨基酸的前列腺癌细胞株,在该细胞株中外源PC—1蛋白N端43个氨基酸得到高表达,细胞生长速度较对照细胞加快了38％。结果表明外源PC—I基因N端43个氨基酸高表达可提高人前列腺癌细胞C4—2的生长速度,推论PC—J基因高表达可能在人前列腺癌的发展中起一定的促进作用。     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

FABP5基因重组突变体蛋白抗前列腺癌细胞活性分析

为构建脂肪酸结合蛋白5 (FABP5)突变体的原核表达体系,评价突变体蛋白质体外抗前列腺癌细胞的活性.利用定点突变技术,突变FABP5蛋白脂肪酸结合的3个关键位点,并构建原核表达体系,对重组蛋白质进行原核表达、分离纯化.通过细胞毒性、细胞划痕和细胞侵袭试验,评价重组FABP5突变体蛋白质对前列腺癌细胞22RV1和PC3增殖、迁移和侵袭的影响.结果 显示定点突变后的DNA与表达载体pQE32连接并转入大肠杆菌(Escherichia coli) BL21(DE3),经序列测定正确,构建了重组表达工程菌,并诱导表达的重组蛋白经亲和层析纯化获得纯度较高的重组蛋白;三突变体对细胞的增殖、迁移和侵袭抑制作用比3个单突变体和3个双突变体抑制效果明显;单突变体和双突变体组内对细胞的增殖、迁移和侵袭抑制作用差异较小;而野生型重组蛋白质对两株细胞的增殖、迁移和侵袭具有促进作用.本研究从所有突变体中筛选出FABP5的三突变体重组蛋白质对前列腺癌细胞抑制作用较好,为后续开发去势抵抗性前列腺癌(CRPC)蛋白质药物提供参考.     

PC-1在前列腺癌细胞中促进c-myc基因的表达

前列腺癌相关基因PC-1(Prostate and colon gene1)是属于癌基因D52家族成员,具有促进前列腺癌细胞雄激素非依赖性生长的功能。为了研究PC-1发挥这种生物功能的分子机制,文章在PC-1高表达的LNCaP-pc-1及对照LNCaP-zero细胞中,利用RT-PCR和Western blotting等方法检测c-myc基因表达;提取两细胞胞质和胞核蛋白,利用Western blotting分析c-myc上游调节蛋白β-catenin变化;利用c-Myc蛋白抑制剂10058-F4作用前列腺癌细胞C4-2,Western blotting检测PC-1蛋白表达变化。发现PC-1促进c-myc基因表达,并促进β-catenin入核;c-Myc蛋白抑制剂10058-F4可抑制PC-1的表达。结果表明:PC-1在前列腺癌中促进c-myc基因的表达,并且这种促进作用可能是通过Wnt/β-catenin信号通路实现的。同时,PC-1与c-Myc蛋白间可相互促进,进一步促进前列腺癌细胞雄激素非依赖性生长。     

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post-messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock-induced gene expression, leading to abundant HSP induction in vitro or in vivo.     

miR-182在前列腺癌组织中的表达及其对前列腺癌细胞增殖和凋亡的影响

目的:观察前列腺癌组织及不同前列腺癌细胞系中miR-182的表达,并探讨下调其表达对前列腺癌细胞增殖和凋亡的影响及机制。方法:采用实时荧光定量PCR(q RT-PCR)检测30例前列腺癌组织和30例相应的癌旁组织以及前列腺正常上皮RWPE-1细胞、前列腺癌PC-3、LNCa P和DU145细胞中miR-182的表达,进一步采用Lipfectamine 2000脂质体转染miRNA-182 inhibitor和阴性对照miRNA于PC-3细胞后,通过噻唑蓝(MTT)比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,免疫印迹(Western blot)法检测转录因子FOXO1、血管内皮生长因子(VEGF)和抑癌基因p53蛋白的表达。结果:miR-182在前列腺癌组织中的表达明显高于癌旁组织(P0.05);miR-182在前列腺癌细胞系PC-3、LNCa P和DU145中的表达均高于前列腺正常上皮细胞RWPE-1(P0.05),其中PC-3细胞中miR-182表达水平最高。转染miRNA-182 inhibitor至PC-3细胞成功下调miR-182表达后,细胞的增殖能力明显受到抑制,细胞凋亡能力明显增强,FOXO1表达水平显著升高,VEGF和p53的表达明显降低,差异均具有统计学意义(P0.05)。结论:miR-182在前列腺癌组织及细胞中呈高表达,下调miR-182的表达可能通过增加FOXO1的表达并减少VEGF和p53的表达,抑制前列腺癌细胞增殖并诱导细胞凋亡。     

Quercetin inhibits invasion, migration and signalling molecules involved in cell survival and proliferation of prostate cancer cell line (PC-3)

Urokinase-type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis including prostate cancer. uPA activation is mediated by transactivation of uPAR and epidermal growth factor receptor (EGF-R) in prostate cancer progression. Prostate cancer (PC-3) cells have highly invasive capacity and they express uPA and uPAR gene. PC-3 cells are treated with quercetin, which inhibits invasion and migration of PC-3 cells. Quercetin downregulates uPA, uPAR and EGF, EGF-R mRNA expressions. Quercetin inhibits cell survival factor β-catenin, NF-κB and also proliferative signalling molecules such as p-EGF-R, N-Ras, Raf-1, c.Fos c.Jun and p-c.Jun protein expressions. But quercetin increased p38 mitogen-activated protein kinase protein expression. Our results suggest that quercetin inhibit migration and invasion of prostate cancer cells. It shows the value for treatment of invasive and metastasis type of prostate cancer.     

The use of LC-MS to identify differentially expressed proteins in docetaxel-resistant prostate cancer cell lines

Docetaxel is a taxane-derived chemotherapy drug that has been approved for treatment of prostate cancer. While docetaxel is frequently used as a treatment for hormone-refractory prostate cancer, a subset of patients either do not respond to this treatment or those that do respond eventually become resistant to the drug over time. Resistance to docetaxel is complex and multi-factoral and further understanding of the cellular biochemistry underlying resistance is vital to improve treatment efficacy. To identify proteins altered in the resistant phenotype, three parental cell lines DU145, 22RV1 and PC-3, as well as their docetaxel resistant sub-lines, were subjected to quantitative label-free LC-MS proteomic profiling. A total of 189 significant (p < 0.05) protein abundance changes were identified in the DU145 resistant sub-lines, 254 in the 22RV1 sub-lines, and 51 and 72 in the 8 and 12 nM resistant PC-3 sub-lines, respectively. From these, 29 proteins demonstrated a significant (p < 0.05) fold change across two or more resistant variants. These included proteins indicative of an epithelial-to-mesenchemyl transition as well as altered heat shock response elements.     

Cell death induced by MPPa-PDT in prostate carcinoma in vitro and in vivo

Lack of effective photosensitizers has become a major limit for extensive application of photodynamic therapy. In this study, the photocytotoxicity and mode of death induced by a newly developed photosensitizer MPPa, a derivative of chlorophyll a, were investigated in PC-3M cell line, a highly metastatic variant of poorly differentiated androgen-independent proctanec adenocarcinoma PC-3. MTT reduction assay was used to measure cytotoxicity in both PC-3M and HUVEC, after which a flow cytometer was used to measure apoptotic rate and cell cycle, and then Caspase-3, -8, -9 were investigated. Finally, an animal model was set up to embody the curative effect and for histopathological examinations. The photocytotoxicity of MPPa showed both light- and drug-dose dependent characteristics and no significant dark cytotoxicity was observed in PC-3M cells. In HUVEC, MPPa exhibited an obviously low cytotoxicity. By other in vitro studies, we found MPPa-PDT induced apoptotic mainly via the mitochondrial/Caspase-9/Caspase-3 pathway and could restrain the cell cycle progression from the more sensitive G0/G1-phases. In vivo, the tumour growth was significantly inhibited after PDT, and many apoptotic cells could be seen by histopathological examinations. These results indicate the death way of cells induced by MPPa is mainly via mild apoptotic and the cure effect is obvious, suggesting that MPPa is a potential photosensitizer of photodynamic therapy for prostate cancer.     

Alpha-tomatine induces apoptosis and inhibits nuclear factor-kappa B activation on human prostatic adenocarcinoma PC-3 cells

Background

Alpha-tomatine (α-tomatine) is the major saponin in tomato (Lycopersicon esculentum). This study investigates the chemopreventive potential of α-tomatine on androgen-independent human prostatic adenocarcinoma PC-3 cells.

Methodology/Principal Findings

Treatment of highly aggressive human prostate cancer PC-3 cells with α-tomatine resulted in a concentration-dependent inhibition of cell growth with a half-maximal efficient concentration (EC₅₀) value of 1.67±0.3 µM. It is also less cytotoxic to normal human liver WRL-68 cells and normal human prostate RWPE-1 cells. Assessment of real-time growth kinetics by cell impedance-based Real-Time Cell Analyzer (RTCA) showed that α-tomatine exhibited its cytotoxic effects against PC-3 cells as early as an hour after treatment. The inhibitory effect of α-tomatine on PC-3 cancer cell growth was mainly due to induction of apoptosis as evidenced by positive Annexin V staining and decreased in mitochondrial membrane potential but increased in nuclear condensation, polarization of F-actin, cell membrane permeability and cytochrome c expressions. Results also showed that α-tomatine induced activation of caspase-3, -8 and -9, suggesting that both intrinsic and extrinsic apoptosis pathways are involved. Furthermore, nuclear factor-kappa B (NF-κB) nuclear translocation was inhibited, which in turn resulted in significant decreased in NF-κB/p50 and NF-κB/p65 in the nuclear fraction of the treated cells compared to the control untreated cells. These results provide further insights into the molecular mechanism of the anti-proliferative actions of α-tomatine.

Conclusion/Significance

α-tomatine induces apoptosis and inhibits NF-κB activation on prostate cancer cells. These results suggest that α-tomatine may be beneficial for protection against prostate cancer development and progression.