Expression of pro-angiogenic growth factors VEGF,EGF and bFGF and their topographical relation to neovascularisation in prostate cancer

The aim of the study was to quantify the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in prostate cancer and adjacent non-tumorous tissue in a standardized experimental set-up and to evaluate the paracrine effects of three endothelial stimuli on neovascularisation. Immunohistochemical staining of prostate cancer (PCa) specimens for VEGF, bFGF, EGF and the endothelial marker CD31 was performed (n=56). Sections were analyzed for growth factor-positive cancer/epithelial cells as well as staining intensity in (I) malignant and (II) non-tumorous tissue. Within PCa the topographic relationship (TR) of maximum microvessel density (MWD) and maximum expression of each growth factor was assessed. The number of VEGF- and EGF-positive cells in PCa was significantly enhanced compared with non-tumorous tissue (p<0.0001), whereas there was no difference in staining intensity. In contrast, the staining intensity of bFGF sections revealed a stronger expression in non-tumorous tissue compared with PCa (p<0.0001). In benign glands, VEGF, EGF and bFGF expression is chiefly restricted to basal cells. VEGF and EGF displayed a close TR in 65 and 57% of cases, respectively, whereas bFGF revealed a close TR in only 43% of PCa specimens. The results outline the relationship of the investigated growth factors and angiogenesis in PCa, which is strongest for VEGF and EGF. The relevance of VEGF and EGF is underlined by the increased number of positive cancer cells. Although previously reported to be a pro-angiogenic growth hormone, bFGF appears to play an assimilably minor role in the angiogenesis of PCa.This project was supported by a grant from the Reinhard Nagel Trust, Germany     

Endopeptidase 24.11 activity in the human prostate cancer cell lines LNCaP and PPC-1

Human endopeptidase 24.11 (EP) occurs in greatest abundance on terminally differentiated prostate cells; thus, loss of EP could mark dedifferentiation of prostate epithelium. To identify laboratory models that would permit continuous work on the biochemistry and hormonal regulation of EP, we examined the well-differentiated LNCaP and poorly differentiated PPC-1 human prostate cancer cell lines. Ultrastructural analysis revealed that LNCaP secretes electron-dense material that resembles the particulate matter of seminal plasma, which is associated with endopeptidase activity. LNCaP medium contained EP activity while PPC-1 medium did not. Whether the apparent deletion of EP from the PPC-1 cell line is characteristic of poorly differentiated prostate adenocarcinoma is not yet clear. However, it may be relevant to the carcinogenic process that EP can limit growth of lung small carcinomas by inactivating cell growth-promoting bombesin-like peptides. Because bombesin has been identified in aggressive human prostate cancers, loss of EP in PPC-1 could represent a necessary step in transformation to aggressive phenotype. The combination of LNCaP and PPC-1, which offers well-differentiated and poorly differentiated cancer phenotypes, appears well suited to studying the relevance of EP in prostate cancer biology.     

ERCC 1在前列腺癌PC-3细胞和组织样本中的表达及其临床意义

目的 探讨切除修复交叉互补基因1(ERCC 1)在前列腺癌（PCa）PC-3细胞和前列腺样本中的表达情况及与预后的关系。方法 Western blot检测小干扰核糖核酸（siRNA）ERCC 1在转染PC-3细胞后ERCC 1蛋白的表达水平,MTT法检测细胞增殖活力,Transwell试验检测细胞迁移和侵袭能力。免疫组化（IHC）检测80例PCa组织及30例前列腺增生（BPH）组织中ERCC 1蛋白的表达水平,分析ERCC 1与PCa临床病理特征及其预后关系。结果 siRNA ERCC 1质粒转染PC-3细胞后Western blot检测证实ERCC 1表达水平明显减低。Transwell试验结果显示siRNA干扰表达ERCC 1后PC-3细胞迁移和侵袭能力下降,差异有统计学意义（P<0.05）。IHC结果提示ERCC 1在PCa样本中阳性表达率为71.3%(57/80),高表达率为23.8%(19/80,IRS≥6);在BPH样本中阳性表达率为10%(3/30),均为低表达（IRS<6）。ERCC 1表达与PCa患者术前PSA值,Gleason评分,病理分期（pT）,淋巴...     

微小RNA-494通过抑制Survivin诱导前列腺癌PC-3细胞凋亡

目的 观察微小RNA( miRNA) -494对人前列癌PC-3细胞中Survivin基因的表达抑制及对PC-3细胞增殖与凋亡的影响.方法 TargetScan 5.2预测miRNA-494与Survivin mRNA配对评分为mirSVR score:-0.4916、PhastCons score:0.4876;定量聚合酶链反应(qPCR)分析PC-3相对于正常前列腺上皮细胞株RWPE-1中miRNA-494的上下调情况;构建过表达miRNA-494的腺病毒载体,应用逆转录-聚合酶链反应(RT-PCR)、Western blot、噻唑蓝(MTT)法、流式细胞仪分析分别检测感染前后Survivin基因及蛋白的表达差异、细胞增殖及生长周期和凋亡变化.结果 miRNA-494在PC-3中的表达为RWPE-1中的(0.547±0.032)倍;Ad-pre-miRNA-494构建成功;实验组细胞增殖抑制呈现时间依赖性;和对照组比较,72 h后实验组Survivin蛋白表达为(21.69±4.62)％,与空载体组比较,差异有统计学意义(P＜0.01),流式细胞仪检测结果显示实验组PC-3细胞G2/M期阻滞率及细胞凋亡率显著增加.结论 miRNA-494通过抑制前列腺癌PC-3细胞中Survivin基因的表达而诱导前列腺癌PC-3凋亡.     

B细胞特异性莫洛尼氏白血病毒插入位点1沉默对前列腺癌PC-3细胞增殖迁移的影响及对同源性磷酸酶和张力蛋白基因/磷酸化蛋白激酶B通路的作用

目的:探讨B细胞特异性莫洛尼氏白血病毒插入位点1(Bmi-1)沉默对前列腺癌PC-3细胞增殖迁移的影响及对同源性磷酸酶和张力蛋白基因(PTEN)/磷酸化蛋白激酶B(pAkt)通路的作用。方法:将Bmi-1小干扰RNA(siRNA)序列及阴性对照序列转入PC-3细胞,设为Si-Bmi-1组和Si-NC组,稳定转染Bmi-...     

微小RNA-23a-p21活化激酶6通路对前列腺癌细胞迁移与侵袭能力的影响

目的 探讨调控p21活化激酶6(PAK6)的微小RNA(miRNA)与前列腺癌侵袭及迁移的关系.方法 生物信息学预测靶向PAK6的miRNA,转染miRNA模拟物及抑制剂,Western blot 法及实时定量逆转录-聚合酶链反应( Real-time PCR)检测PC-3细胞PAK6的表达,荧光素报告酶实验检测预测miRNA对PAK6的调控,并行体外迁移及侵袭实验,检测转染miRNA对PC-3细胞迁移及侵袭能力的影响.结果 生物信息学预测miRNA-23a可能是调控PAK6的靶miRNA. Western blot检测转染miRNA-23a组PAK6表达量下降79％,而转染miRNA-23a抑制剂组PAK6表达量上升40％,差异均有统计学意义(P＜0.05).荧光素报告酶实验结果显示,转染miRNA-23a后野生型PAK6组荧光素酶活性下降32％,而突变组差异无统计学意义(P＞0.05).Real-time PCR检测3组PAK6 mRNA的表达,差异无统计学意义(P＞0.05).体外迁移及侵袭实验示,转染miRNA-23a组迁移细胞数减少57％,侵袭的细胞数减少91％.结论 微小RNA-23a通过下调靶蛋白PAK6的表达从而引起前列腺癌迁移及侵袭能力下降.     

RNAi抑制PSA表达对前列腺癌22RV1细胞增殖和侵袭力的影响

目的 研究PSA表达对去势抵抗性前列腺癌22RV1细胞增殖和侵袭的影响.方法 构建含有针对PSA基因特异性短发夹RNA(shRNA,分别为shPSA1、shPSA2、shPSA3、shPSA4)和阴性对照序列(NC)的慢病毒载体并由慢病毒颗粒包装后,分别感染去势抵抗性前列腺癌22RV1细胞并筛选稳定表达PSA特异性shRNA的shPSA1-、shPSA2-、shPSA3-、shPSA4-和表达NC序列的NC-22RV1细胞.分别应用实时荧光定量聚合酶链反应和Western blot检测shRNA对22RV1细胞中PSA mRNA及蛋白表达的影响;采用CCK-8法、Transwell侵袭试验和流式细胞术检测PSA基因沉默后的22RV1细胞增殖、侵袭和凋亡情况.结果 与NC-22RV1细胞相比,shPSA3-22RV1细胞的基因和蛋白表达水平的沉默效果最好;shPSA2组在24、48、72 h的吸光度值分别为(0.1564±0.0225)、(0.2438±0.0358)、(0.3641±0.0205),shPSA3组分别为(0.1069±0.0120)、(0.1588±0.0192)、(0.2534±0.0289),与NC组比较差异均有统计学意义(P<0.01);shPSA1、shPSA2、shPSA3组侵袭力分别为NC组的77.35%(P=0.002)、54.35%(P<0.001)、42.21%(P<0.001);shPSA2、shPSA3组凋亡率分别为(27.97±4.28)%(P=0.001)、(43.03±3.19)%(P<0.001),高于NC组[(16.77±0.59)%].结论 PSA具有促进去势抵抗性前列腺癌22RV1细胞的增殖和侵袭效应,抑制PSA表达后前列腺癌22RV1细胞凋亡增加.     

微小RNA-101对前列腺癌干细胞EZH2表达及增殖迁移的影响

目的 调控前列腺癌干细胞(PCSCs)中微小RNA-101(miR-101)表达,观察细胞中EZH2表达及其增殖迁移能力的变化.方法 通过流式分选从LNcap细胞株中获取PCSCs;采用实时荧光定量聚合酶链反应(qRT-PCR)、Western blot、双荧光素酶、细胞凋亡、细胞周期、细胞迁移实验观察miR-101对前列腺癌干细胞EZH2表达及增殖迁移能力影响.结果 在PCSCs中转染miR-101后,实验组较未处理组EZH2表达量下降46％ (P ＜0.05);双荧光素酶实验证实PCSCs中miR-101直接调控EZH2;凋亡实验中,实验组细胞凋亡率为(3.79±0.24)％,较对照组凋亡率明显增加(P＜0.05);周期实验中,实验组细胞G1期比例为(82.95±0.78)％,较对照组比例增加(P＜0.05),细胞被阻滞在G1/S期;迁移试验中,细胞迁移率为0.38±0.01,明显低于对照组(P＜0.05).结论 过表达miR-101能下调前列腺癌干细胞中EZH2的表达并能降低细胞的增殖迁移能力.     

瘦素对人乳腺癌细胞系MCF-7增殖的影响及其作用机制探讨

目的 观察瘦素对人乳腺癌细胞系MCF-7增殖的影响，探讨其作用机制。方法 免疫荧光检测人乳腺癌细胞系MCF-7瘦素受体（OB．Rb）的表达。于MCF-7细胞中分别加入不同浓度的瘦素（0、10、50、100、150μg／L），作用不同的时间（6、12、24h），噻唑蓝（MTr）法检测各组细胞的增殖情况，同法分别检测STAT3抑制剂AG490和ERK1／2抑制剂PD98059对瘦素刺激MCF-7细胞增殖的影响。采用免疫印迹法（Westernblot）检测瘦素（100μg／L）作用于MCF-7细胞不同时间（0、20、40、60min）STAT3和ERK1／2的磷酸化水平。结果 免疫荧光检测证实MCF-7细胞存在瘦素受体（0B—Rb）表达。瘦素能明显促进MCF-7细胞的增殖，并呈时间和剂量依赖性，于100μg／L瘦素作用24h时增殖效应最大，100μg／L组与150μg/L组差异无统计学意义（P〉0．05）；不同浓度的AG490与PD98059均可明显抑制瘦素对MCF-7细胞的增殖，随着AG490与PD98059浓度增高，抑制作用逐渐增强；MCF-7细胞经100ng／ml瘦素处理后，随时间延长STAT3和ERK1／2磷酸化程度逐渐增高，且持续至少达60min。结论 瘦素可能通过激活STAT3和ERK信号通路促进入乳腺癌细胞系MCF-7的增殖。     

性激素受体及相关生长因子在前列腺基质细胞中的表达

目的：探讨前列腺基质增生的发病机制与性激素以及相关生长因子的关系。方法：应用RT－PCR的方法研究了在人前列腺不同细胞类型中Smoothelin的表达，研究了雄、雌激素受体及相关生长因子在前列腺基质细胞中的表达，以及它们在前列腺基质细胞分化中表达的变化。结果：Smoothelin是前列腺平滑肌细胞特异性的标记蛋白；雄激素受体（AR）、碱性成纤维细胞生长因子（bFGF)、角化细胞生长因子（KGF）主要在前列腺成纤维细胞中表达，而雌激素受体（ER）、转移生长因子β1（TGFβ1）主要在平滑肌细胞中表达。结论：前列腺基质增生与雌激素受体和转移生长因子β1的过度表达密切相关。     

Vitamin D3 analogue inhibits keratinocyte growth factor signaling and induces apoptosis in human prostate cancer cells.

BACKGROUND: Prostate cancer is a worldwide significant health care problem, due to its high incidence and mortality. In particular, androgen-independent tumors have the worst prognosis, because they are refractory to almost all kinds of available therapy. Hence, there is the need of new treatment opportunities targeting androgen-independent, growth factor-mediated, tumor signaling. One of these new promising opportunities is vitamin D3 and its related analogues. METHODS: We investigated the effect of a vitamin D3 analogue, analogue (V), on proliferation of several human prostate cancer cells in basal condition and after treatment with KGF, one of the intraprostatic growth factors that might participate in the progression of prostate cancer. In addition, in the androgen-independent cell line DU 145, we also studied the effect of analogue (V), KGF, and their mutual interaction on protein tyrosine phosphorylation, bcl-2 expression and apoptosis. RESULTS: Overall, we found that analogue (V) dose-dependently decreased basal and KGF-induced prostate cancer cell growth, although to a different extent. Maximal effect was obtained in DU 145 cells. In these cells, KGF stimulated tyrosine phosphorylation of a protein corresponding to its receptor, induced bcl-2 expression, and prolonged cell survival. Analogue (V) not only counteracted all these KGF-mediated events, but also decreased basal bcl-2 expression, therefore, allowing DU 145 cells to undergo an apoptotic program. CONCLUSIONS: Our results indicated that in prostate cancer cells analogue (V) decreased basal and KGF-induced cell proliferation. This effect, at least in DU 145 cells, is in part mediated by negative interactions with cell survival and KGF signaling.     

骨髓间充质干细胞向前列腺癌趋向转移的研究

目的探讨人骨髓间充质干细胞（hMSCs）在体内向前列腺癌微环境趋向转移的特性。方法应用人前列腺癌细胞株PC-3异种皮下种植建立前列腺癌严重联合免疫缺陷小鼠（SCID）模型，通过密度梯度离心和贴壁法分离、培养hMSCs，将体外4,6-联脒-2-苯基吲哚（DAPI）标记的4～6代hMSCs经尾静脉和肿瘤周围注射入荷瘤SCID鼠体内。分别在经尾静脉注射后17d和肿瘤周围注射后7、10、14d处死小鼠，收集肿瘤和肝、肺、脾、肾等脏器作冰冻切片和石蜡切片。在荧光显微镜下观察肿瘤及各脏器中hMSCs的分布情况。结果前列腺癌SCID鼠模型成瘤率83．3％，经尾静脉注射DAPI标记的hMSCs后17d和肿瘤周围注射DAPI标记的hMSCs后7、10、14d，荧光显微镜下显示，前列腺癌组织中可见DAPI标记的hMSCs的核呈蓝色荧光。而肝、肺、脾、肾等脏器中均未见hMSCs的存在。结论hMSCs在体内具有向前列腺癌微环境趋向转移的特性。     

RPS7 promotes cell migration through targeting epithelial-mesenchymal transition in prostate cancer

Objectives

Small ribosomal protein subunit 7 (RPS7) is an important structural components of the ribosome involved in protein synthesis, previous studies demonstrated that RPS7 was associated with several malignancies, but the role of RPS7 in prostate cancer (PCa) remains unclear. To decipher such a puzzle, in the current study, we deciphered the role and mechanism of RPS7 during the progression of PCa.

微小RNA-138-5p在前列腺癌细胞中的表达及对其侵袭能力的影响

目的:探讨微小RNA(miR)-138-5p在前列腺癌(PCa)中的表达及对前列腺癌侵袭、迁移、凋亡等能力的影响。方法:miR-138-5p模拟物转染正常前列腺上皮细胞(RWPE)-1、DU145细胞,实时定量反转录聚合酶链反应(RT-qPCR)用于检测转染前后细胞miR-138-5p的表达。细胞计数试剂盒(CCK-8...     

Stimulation of human prostate cancer cell lines by factors present in human osteoblast-like cells but not in bone marrow

Secondary deposits of prostate tumours are frequently found in the skeleton where they produce osteoblastic lesions. In this study both osteoblast-like cells and bone marrow from the proximal femur have been cultured to determine whether or not they can release factors which could support the growth of secondary prostate tumours. Media conditioned by both osteoblast-like cells (OBCM) and bone marrow were examined for their potential to stimulate prostate carcinoma cell lines. Whilst the results obtained demonstrated that OBCM could enhance the growth of both the hormone sensitive (LNCaP) and hormone unresponsive (PC-3 and DU-145) prostate carcinoma cell lines, no proliferative effect could be shown on cell lines derived from cancers of the breast, bladder, and liver. Significantly, media conditioned by either bone marrow or human skin fibroblasts also had no effect on the growth of prostate carcinoma cell lines. This study supports the possibility that the proliferation of prostate cancer cells at secondary skeletal sites, in vivo, may be due to osteoblast derived factors. © 1995 Wiley-Liss, Inc.     

双氢睾酮对人前列腺癌细胞株PC-3细胞生长影响的体外研究

目的:探讨双氢睾酮(Dihydrotestosterone,DHT)对前列腺癌PC-3细胞株生长的影响。方法:采用MTT方法,利用不同浓度的DHT刺激非激素依赖性前列腺癌PC-3细胞株,在培养1、2、3、4天分别测定细胞活性。结果:在加用DHT后的第1天,10-8mol/L组细胞生长速度明显加快,MTTOD值与对照组比较差异有统计学意义(P<0.05)。在第4天,分别加用10-9mol/L、10-8mol/L、10-7mol/L、10-6mol/LDHT,MTTOD值与对照组比较差异均有统计学意义(P<0.05),10-5mol/LDHT组与对照组OD值比较差异无统计学意义(P>0.05)。结论:非激素依赖性前列腺癌PC-3细胞生长受雄激素的影响,低浓度DHT可促进PC-3细胞的生长。     

Aberrant expression of E-cadherin and beta-catenin in human prostate cancer

Cadherin-catenin complexes play a key role in embryonic development, and are associated with carcinogenesis and metastasis. We studied the expression of the major members of the family, including E-cadherin and β-catenin in prostate cancer (PC), and correlated with Gleason grade and pathologic stage. Immunohistochemistry was performed on serial sections of paraffinized radical prostatectomy specimens to evaluate E-cadherin (n = 16) and β-catenin (n = 17) expression using heat induced epitope retrieval. Benign appearing prostate epithelium was used as an internal control in each specimen. Two pathologists independently reviewed and scored the intensity and extent of immunostaining using a semiquantitative scale. The Mantel-Haenszel method, stratified by reviewer, was used to test for an association among Gleason score, pathologic stage, and the expression of E-cadherin or β-catenin in PC. Gleason grade ≥7 cancers showed significantly lower expression of E-cadherin and β-catenin compared to Gleason grade <7 PC, P = 0.015 and 0.025, respectively. In addition, β-catenin was down regulated in 4 of 5 (80%) specimens with identifiable high-grade prostatic intraepithelial neoplasia and had demonstrable nuclear staining in higher grade PC (P = 0.0001). However, E-cadherin and β-catenin membranous or nuclear expressions were not significantly associated with final pathologic stage of the specimens (P values >0.05). Overall, the expression of E-cadherin and β-catenin is significantly down regulated in PC compared to surrounding benign appearing prostate, which correlates with increasing Gleason grade. Furthermore, nuclear localization of β-catenin in high grade PC may be a useful biomarker for aggressive PC.     

EN2蛋白在前列腺癌组织的表达及其意义

目的 探讨同源异型盒蛋白EN2在前列腺癌的表达差异及其意义.方法 选取48例前列腺癌、22例高级别前列腺内瘤病(HGPIN)及48例前列腺增生症(BPH),采用免疫组织化学法检测上述组织中的EN2蛋白表达差异.选取其中14例前列腺癌及15例BPH,采用Western blot法检测EN2蛋白表达差异.结果 免疫组织化学法显示EN2蛋白在前列腺癌中表达增高,主要见于前列腺腺上皮细胞质内及部分细胞核.前列腺癌、HGPIN和BPH组织中EN2蛋白表达阳性率分别为87.50％、31.81％和27.08％,采用Kruskal-Wallis H检验,差异有统计学意义(P＜0.05).EN2在高、中、低分化前列腺癌患者中阳性率分别为75.00％、88.24％和88.90％.Western blot法检测显示EN2蛋白在前列腺癌组织的表达比BPH组织增高3倍.结论 同源异型盒蛋白EN2在前列腺癌表达增高,其中以中、低分化腺癌中明显,并能为前列腺上皮细胞分泌.     

Pentosan polysulfate (Elmiron): in vitro effects on prostate cancer cells regarding cell growth and vascular endothelial growth factor production

BACKGROUND: Elmiron (ALZA Corp, Mountain View, CA) is the only Food and Drug Administration-approved oral therapy for interstitial cystitis. We hypothesized that Elmiron would affect the growth of prostate cancer in vitro. METHODS: Prostate cancer cell lines (LnCaP, PC3, and DU145) were treated with Elmiron. Cell viability was measured by MTT (3-4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), whereas vascular endothelial growth factor (VEGF) was measured by a commercial enzyme-linked immunosorbent assay. RESULTS: Inhibition of cell growth was observed in all cell lines tested. LnCaP exhibited a mean inhibition of 12% +/- 7% at 24 hours (P = .025) and 20% +/- 15% at 72 hours (P < .001). PC3 exhibited a mean inhibition of 26% +/- 13% at 24 hours (P < .001) and 44% +/- 5% at 72 hours (P < .001). DU145 exhibited a mean inhibition of 9% +/- 6% at 24 hours (P < .015) and 30% +/- 5% at 72 hours (P < .001). PC3 cells exhibited a significant reduction in VEGF levels (P < .001). CONCLUSIONS: The reductions in cell growth and VEGF indicate that Elmiron may act as an antiangiogenic agent and may have application in the treatment of prostate cancer.