γ-干扰素对前列腺癌细胞粘附和侵袭能力影响的初步研究

目的：探讨γ-干扰素对前列腺癌细胞粘附和侵袭行为的影响。方法：用纤维粘连蛋白和层粘连蛋白处理细胞培养板，检测γ-干扰素对前列腺癌细胞系LNCaP和PC-3粘附作用的影响；用Transwell小室，以Matrigel和纤维粘连蛋白构建基底膜，检测γ-干扰素对前列腺癌细胞侵袭人工基底膜能力的影响；应用Western-blot法检测γ-干扰素对前列腺癌细胞膜粘连蛋白-2（annexin-2）表达的影响。结果：γ-干扰素未处理的两种人前列腺癌细胞系细胞LNCaP、PC-3粘附率分别为46％和40％，γ-干扰素处理的两种细胞系细胞LNCaP、PC-3粘附率分别为21％和23％，同种细胞系γ-干扰素处理组与未处理组间差异有显著性（P均〈0．05）。在相同细胞系γ-干扰素处理组较未处理组前列腺癌细胞24h侵袭能力明显降低（P均〈0．05）。Western印迹结果表明γ-干扰素可显著抑制annexin-2蛋白的表达（P〈0．05）。结论：γ-干扰素可能通过下调annexin-2的表达来抑制前列腺癌细胞的粘附和侵袭。     

环氧化酶2在不同前列腺癌细胞系中的表达

目的:研究环氧化酶2(COX-2)在不同前列腺癌细胞系中的表达,探讨COX-2在前列腺癌侵袭进展及转移潜能获得机制中的可能作用。方法:应用Western印迹及RT-PCR鉴定LNCaP及其亚细胞系C4-2和AR-CaP亚细胞系IF11、IA8,以及PC-3细胞中COX-2的表达情况,并初步分析其在不同特性前列腺癌细胞系转移侵袭过程中的作用。结果:Western印迹结果显示:COX-2蛋白在PC-3细胞中表达相对较高,在IF11、IA8、LNCaP和C4-2细胞中表达缺失,差异具有统计学意义(P<0.05)。COX-2mRNA表达结果同蛋白一致。结论:不同来源、不同转移潜能的前列腺癌细胞株中COX-2表达存在差异。高表达COX-2可能在PC-3细胞高侵袭转移潜能获得方面起着一定作用,而与其他细胞系转移作用无关。     

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。     

基质细胞衍生因子-1α对人前列腺癌PC-3细胞系失巢凋亡的影响

目的：探讨基质细胞衍生因子-lα（stromal cell-derived factor-lα,SDF-lα）对人前列腺癌PC-3细胞失巢凋亡的影响。方法：悬浮培养诱导细胞失巢凋亡,使用0μg／L、50μg／L、100μg／L的SDF-1α作用于悬浮培养的PC-3细胞;天后,采用CCK8法检测PC-3细胞增殖活性变化;Western-blot方法检测促凋亡蛋白Bmf及抗凋亡蛋白Bcl-xl表达变化。结果：PC-3细胞悬浮培养3天后,细胞存活率为（25．19±0．43）％,提示大部分悬浮培养的细胞发生失巢凋亡;50μg／L、100μg／L的SDF-α处理后,与对照组相比,细胞存活率提高,分别为（37．60±6．24）％（P〈0．05）和（33．66±5．51）％（P〉0．05）;悬浮培养的PC-3细胞经SDF-lα处理后,其促凋亡蛋白Bmf表达减低;抗凋亡蛋白Bcl-xl表达增高。结论：SDF-lα可抑制人前列腺癌PC-3细胞发生失巢凋亡,其机制可能与下调Bmf和上调Bcl-xl蛋白表达相关。     

葡萄籽提取物对前列腺癌LNCaP与PC-3细胞VEGF和三种性激素的影响

目的：研究葡萄籽提取物（GSE）对雄激素依赖性前列腺癌LNCaP细胞和雄激素非依赖性前列腺癌PC-3细胞血管内皮生长因子（VEGF）和三种性激素睾酮（T）、孕酮（P）、雌二醇（E2）的影响.方法：实验分为四组：A1组：LNPCa细胞应用正常细胞培养液培养;A2组：LNCaP细胞应用含30μg/ml GSE的培养液培养;B1组：PC-3细胞应用正常细胞培养液培养;B2组：PC-3细胞应用含300 μg/ml GSE的培养液培养.常规培养48 h后收集四组细胞的上清液,用定量酶联榆测法和化学发光酶免疫检测法检测VEGF和三种性激素T、P、E2的浓度变化.结果：与A1组相比,A2组VEGF含量降低（P〈0.05）,T含量升高（P＜0.05）,E2含量无明显变化（P〉0.05）,P未检测到;与B1组相比,B2组VEGF含量显著降低（P〈0.01）,E2含量升高（P〈0.05）,T和P含量无明显变化（P〉0.05）.结论：GSE在体外能降低前列腺癌LNCaP与PC-3细胞VEGF的含量,提示其可能具有抑制前列腺癌侵袭转移的作用.     

TGF-β/Smads通路关键蛋白在5种前列腺癌细胞株中表达的鉴定及意义

目的:对多种不同人前列腺癌细胞株TGF-β/Smads信号通路的"开放"或"关闭"状态进行鉴定,初步探讨此通路在前列腺癌侵袭、转移中的作用及可能的机制。方法:用Western印迹法检测LNCaP、PC-3、DU145及AR-CaP亚细胞系IF11、IA8细胞中TGF-β/Smads通路的关键蛋白TGF-βⅡ型受体(TβRⅡ)、Smad2/3、磷酸化Smad2(p-Smad2)、Smad4的差异表达。结果:TβRⅡ在PC-3、DU145、IF11、IA8中表达较高,在LNCaP中表达极低;Smad2/3在所有细胞中表达均较高,但活性成分p-Smad2仅在PC-3、DU145中表达;Smad4在LNCaP、PC-3、DU145中表达较高,IF11、IA8中表达缺失。结论:不同转移潜能的前列腺癌细胞株TGF-β/Smads通路的"开闭"状态存在差异,仅PC-3、DU145细胞处于开放状态;前列腺癌细胞可能通过不同的方式改变TGF-β/Smads通路状态参与晚期肿瘤的侵袭、转移过程。     

miR-152对前列腺癌细胞株迁移和侵袭能力的影响

目的研究miR-152在前列腺癌、前列腺正常组织中的表达情况及其在前列腺癌细胞系中的作用。方法采用TaqMan荧光定量RT—PCR方法检测8例前列腺癌和8例前列腺正常组织的样本中miR-152的表达水平。运用Transwell细胞迁移实验及侵袭实验评估miR一152对前列腺细胞系PC-3和DU145细胞功能的影响。结果与正常前列腺组织相比,miR-152在前列腺癌组织中的表达水平显著下调（P〈0．05）。体外实验中上调miR152的表达可以显著降低前列腺癌细胞的迁移和侵袭能力（P〈O．05）。结论miR-152在前列腺癌中可作为一种肿瘤抑制因子,影响前列腺癌细胞的迁移和侵袭能力。     

多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”特性鉴定及其意义

目的:对多种转移潜能不同的人前列腺癌细胞“上皮细胞间质转化态”(EMT)特性进行鉴定,并从粘附因素和细胞骨架蛋白角度分析其骨转移潜能获得的分子机制。方法:用W estern印迹法鉴定LNCaP及其亚细胞系C4、C4-2和ArCaP亚细胞系IF11、IA8,以及PC-3、Du145等细胞中上皮型钙粘素(E-cadherin)、神经型钙粘素(N-cadherin)和波形纤维蛋白(V im entin)的表达差异情况,并分析其在前列腺癌转移过程中的作用。结果:E-cad-herin在PC-3、LNCaP、C4、C4-2中表达较高,但在Du145、IF11、IA8中表达极低;而V im entin的表达情况恰恰与E-cadherin相反;N-cadherin在IF11、IA8细胞中呈现显著的高表达状态。结论:转移潜能不同的人前列腺癌细胞株之间存在EMT表型的表达差异,其中PC-3、LNCaP、C4、C4-2是未发生EMT改变的细胞,Du145、IF11、IA8却是EMT化的细胞。EMT表型差异蛋白在解释前列腺癌转移机制方面占据着重要地位。     

RNAi对前列腺癌细胞水通道蛋白-1的影响

目的探讨RNA干扰技术沉默AQP1基因对前列腺癌PC-3细胞AQP1蛋白表达影响。方法设计合成针对前列腺癌PC-3细胞AQP1基因的小干扰RNA并构建高效表达载体,脂质体法转染PC-3细胞,Western Blot检测AQP1蛋白表达的变化。结果 AQP1在前列腺癌PC-3细胞表达,针对AQP1基因siRNA高效表达载体能够抑制AQP1蛋白在PC-3细胞的表达,AQP1蛋白相对表达量为：转染组0.54±0.04,正常对照组0.82±0.08,差异有显著性（P〈0.01）。结论通过抑制AQP1基因的表达,有效抑制了前列腺癌PC-3细胞AQP1蛋白表达。     

维生素E对前列腺癌微小染色体维持蛋白4的抑制作用及信号通路

目的探讨维生素E调节前列腺癌细胞DNA复制的新分子机制及信传导通路。方法利用基因转染、免疫印迹、实时定量聚合酶链反应（PCR）、免疫沉淀等方法研究不同浓度、不同时间琥珀酸维生素E（VES）对前列腺癌细胞内的微小染色体维持蛋白4（MCM4）的mRNA及蛋白表达水平的影响。结果YES可抑制前列腺癌MCM4mRNA的表达，并存在时效与量效关系，20μmol／LVES处理LNCaP细胞36h，MCM4的mRNA表达水平较对照组下降24．5％（P〈0．05），作用96h下降达60．3％；随着VES浓度的增加，MCM4mRNA的表达水平受抑制的程度逐步加深。Westernblot表明VES可抑制MCM4蛋白的表达。过表达E2F1可拮抗VES对MCM4的抑制作用。VES抑制Rb磷酸化，增加Rb与E2F1的结合程度。BrdU摄入试验表明20μmol／L与40μmol／L VES处理LNCaP细胞24h后，癌细胞内DNA合成较对照组下降63．4％与71．6％（P〈0．05）；48h后分别达74．8％与81．5％（P〈0．05）。结论我们识别了维生素E抑制前列腺癌细胞DNA复制的一种新机制是下调MCM4的表达，E2F1-Rb蛋白参与这一信号传导通路。     

Down-regulation of prostasin serine protease: a potential invasion suppressor in prostate cancer.

BACKGROUND: Prostasin is a serine protease predominantly expressed in normal prostate epithelial cells. The biological function of prostasin has not been determined. METHODS: Western blot and RT-PCR analyses were used to examine the expression of prostasin in prostate cancer cell lines. Immunohistochemistry was used to evaluate prostasin protein expression in human prostate cancer. An in vitro Matrigel invasion assay was used to test the invasiveness of prostate cancer cell lines forced to express recombinant prostasin. RESULTS: Both prostasin protein and mRNA were found to be expressed in normal human prostate epithelial cells and a non-invasive human prostate cancer cell line, the LNCaP, but neither was found in invasive human prostate cancer cell lines DU-145 and PC-3. Prostasin mRNA expression was absent in invasive prostate cancer cell lines of a transgenic mouse model. Immunohistochemistry analysis showed that prostasin protein expression is down-regulated in high-grade prostate cancer. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68 and 42%, respectively. CONCLUSIONS: Our data indicate that prostasin may be implicated in normal prostate biology and is able to suppress prostate cancer invasion in vitro.     

凋亡抑制基因XIAP在前列腺癌细胞中的表达及与临床病理特征的关系

目的 :研究XIAP基因在前列腺癌细胞系和前列腺癌组织的表达情况 ,及其与前列腺癌临床病理特征的关系。　方法 :应用RT PCR检测前列腺癌组织、正常前列腺组织和前列腺癌细胞株PC 3,DU 14 5 ,LNCaP细胞XIAP基因的表达 ,并通过免疫组化SP法检测 5 6例前列腺癌组织标本XIAP蛋白的表达情况。　结果 :XIAP基因在前列腺癌组织和前列腺癌细胞株PC 3,DU 14 5 ,LNCaP细胞高表达 ,正常前列腺组织无表达。在前列腺癌组织和癌旁组织中 ,XIAP蛋白阳性检出率分别为 5 3.6 % (30 / 5 6 )和 2 1.5 % (12 / 5 6 ) (P <0 .0 1) ;不同分期、分级组XIAP阳性检出率相比差异无显著性 (P >0 .0 5 )。　结论 :凋亡抑制基因XIAP与前列腺癌相关 ,在前列腺癌发生过程中具有重要的作用 ,有可能成为前列腺癌治疗的靶标。     

Suppression of the growth and invasiveness of human prostate cancer cells in vitro by neuropeptide antagonist substance P analogues

Neuropeptides may be essential to the growth and progress of prostate cancer, particularly during androgen-independent cell development. To determine whether neuropeptide antagonists substance P analogues can be used as therapeutic agents in the treatment of prostate cancer, their effects on the growth and invasiveness of established human prostate cancer cell lines were examined. The effects of [d-Arg(1), d-phe(5), d-Trp(7,9), Leu(11)]substance P and [Arg(6), d-Trp(7,9), MePhe(8)]substance P(6-11) on two androgen-independent cell lines (PC-3 and DU-145) and an androgen-dependent cell line (LNCaP) were studied. The cytotoxicity of substance P analogues was assessed based on their effects on DNA synthesis and cell proliferation by [(3)H]thymidine incorporation and cell growth assays, respectively. Inhibition of the invasiveness of prostate cancer cells was estimated based on the extent of cell penetration of reconstituted basement membrane. Substance P analogues inhibited DNA synthesis and cell proliferation of prostate cancer cells dose dependently but complete recovery was achieved by the addition of bombesin or substance P. [d-Arg(1), d-phe, d-Trp(7,9), Leu(11)]substance P inhibited the invasiveness of PC-3 cells. Neuropeptide antagonists substance P analogues have been found useful as therapeutic agents for prostate cancer. Their action occurs primarily through the inhibition of Ca(2+) mobilizing neuropeptides such as bombesin and substance P.     

Prostasin基因在前列腺细胞株中表达情况研究

目的 探讨Prostasin在前列腺癌中的功能。方法 运用RT-PCR方法检测人前列腺增生细胞株BPH-1，无转移能力前列腺癌细胞株LNCaP，及有转移能力的前列腺癌细胞株PC-3、DU-145中Prostasin基闪表达情况。结果 Prostasin基斟在BPH-l和LNCaP中正常表达，在PC-3、DU-145中低表达。BPH-l中Prostasin的表达与DU-145和PC-3比较差异有显著性，同样LNCaP中Prostasin的表达与DU-145和PC-3比较差异亦有显著性（P〈0．01）。BPH-1与LNCaP之间表达差异无显著性，DU-145和PC-3之间表达差异亦无显著性，（P〉0.05）。结论 ProStasin可能是前列腺癌的转移抑制剂。     

Expression of gonadotropin-releasing hormone (GnRH) and GnRH receptor mRNA in prostate cancer cells and effect of GnRH on the proliferation of prostate cancer cells

The purpose of this study was to determine the production of gonadotropin-releasing hormone (GnRH), the co-occurrence of GnRH receptors in prostate cancer cells, and the effect of GnRH on prostate cancer cell proliferation. Four human prostate cancer cell lines were studied. LNCaP is an androgen sensitive prostate cancer cell line, DU-145 and PC-3 are androgen resistant, and TSU-Pr1 is uncharacterized. The expression of GnRH and GnRH receptor mRNAs were assessed by in situ hybridization and the effect of exogenous GnRH on proliferation of prostate cancer cells was measured by thymidine incorporation assay. GnRH mRNA expression, determined by in situ hybridization, was found in 83.48% of the LNCaP, 89.7% of the TSU-Pr1, 86.2% of the PC-3 and 95.3% of the DU-145. Signals of GnRH receptor mRNA were detected in more than 95% of the cells of all four cell lines. The proliferation of the prostate cancer cells grown in media supplemented with peptide hormone lacking charcoal-stripped serum was significantly (P < 0.05) suppressed. No significant effect of GnRH on the proliferation of all four prostate cancer cells was observed. In summary, prostate cancer cells produced GnRH and its receptors, and exogenous GnRH treatment did not affect the prostate cancer cell proliferation. The existence of GnRH and GnRH receptor mRNA in the same cell suggests that the role of GnRH produced by prostate cancer cells would be autocrine. Received: 21 August 1997 / Accepted 15 April 1998     

Characterization of human prostate and breast cancer cell lines for experimental T cell-based immunotherapy

BACKGROUND: In order to develop experimental immunotherapy for prostate and breast cancer it is of outmost importance to have representative target cell lines that through human leukocyte antigen (HLA) class I molecules present relevant levels of peptides from tumor-associated antigens for cytotoxic T lymphocyte (CTL) recognition. METHODS: We sequenced the HLA-A and HLA-B loci of eight commonly used prostate and breast cancer cell lines and analyzed the surface expression of HLA-ABC, HLA-DR, CD40, CD80, CD86, and CD54 by flow cytometry. We also analyzed the cell lines for mRNA expression from 25 genes reported to be specifically or preferentially expressed by prostate cells. RESULTS: Among the analyzed cell lines we found that LNCaP, PC-346C and MCF-7 are HLA-A*0201 positive. However, the HLA-A2 expression level is low and only MCF-7 upregulates HLA-A2 in response to IFN-gamma stimulation. MCF-7 also expresses high levels of CD54, which further improve its value as a CTL target cell line. On the other hand, LNCaP and PC-346C express 25 and 23 out of 25 prostate-related genes, respectively, while MCF-7 expresses 16 out of 25 genes. CONCLUSIONS: None of the analyzed prostate cancer cell lines are optimal CTL target cells. However, MCF-7 could in many cases be used as a complement to HLA-A*0201 positive prostate cancer cells. The LNCaP and PC-346C cell lines are rich sources of prostate-related antigens that may be valuable for cancer vaccine development.     

Fibronectin influences cellular proliferation and apoptosis similarly in LNCaP and PC-3 prostate cancer cell lines

The mechanisms responsible for the emergence of clinically advanced prostate cancer (PC) are incompletely understood. Recent studies suggest that altered tumoral apoptosis with disordered cell proliferation sustains advanced disease and may account for the phenomena of anti-androgen therapeutic resistance. Previous inquiry has focused primarily on faulty intracellular mechanisms with limited scrutiny of the extracellular matrix including fibronectin and collagen type 4. We evaluated cell proliferation with Ki-67 immunoassay/image analysis and apoptosis by TUNEL staining and Bcl-2 immunoassay/image analysis in LNCaP and PC-3 human PC cell lines at baseline and following propagation on fibronectin and collagen type 4-coated coverslip substrate. Cell cultures showed differing proliferative and apoptosis characteristics at baseline, with the LNCaP cell line showing relatively higher proliferation and apoptosis rates than the PC-3 cell line. Cell proliferation and apoptosis were statistically significantly decreased in both cell lines following propagation on fibronectin. Bcl-2 expression was significantly increased among both cell lines following propagation on fibronectin. In contrast, cell proliferation, apoptosis, and Bcl-2 expression showed insignificant changes in both cell lines following uncoated coverslip and collagen type 4 matrix propagation. Our findings showed that fibronectin influences cell proliferation, apoptosis, and Bcl-2 expression similarly among LNCaP and PC-3 PC cell lines. It is likely that the altered rates are independent of the androgen status of the cell line and are mediated through a nonhormonal mechanism.     

Synthetic retinoid CD437 induces S-phase arrest and apoptosis in human prostate cancer cells LNCaP and PC-3

BACKGROUND: Exposure of prostate carcinoma cell lines to retinoids, which function through the classical retinoic acid nuclear receptor, (RARs) or retinoid X receptors (RXRs), results in minimal cytostatic inhibition of cell proliferation. METHODS: Growth inhibition and various regulatory responses were investigated in two human prostate carcinoma cell lines (LNCaP and PC-3) treated with or without a synthetic retinoid, CD 437. RESULTS: Incubation of prostate carcinoma cell lines with a novel retinoid CD437 resulted in the marked inhibition of proliferation. LNCaP and PC-3 possessed IC50 values for CD437 of 375 nM and 550 nM, respectively. Incubation with 1 microM CD437 for 24 hr resulted in 100% and 60% inhibition of growth in LNCaP and PC-3 cells, respectively. Simultaneously, cell flow cytometric analyses revealed a dramatic increase of the cell population in S phase, in both LNCaP (from 38.6% up to 86.7%) and PC-3 (27.9% to 55.7%), and a decreased proportion of cells in G2 phase, in LNCaP (from 23.7% down to 1.2%) and PC-3 (14.9% to 2.2%), indicating a significant S-phase arrest. The cell growth inhibition and S-phase arrest in these cells were followed by apoptosis, as revealed by the acquisition of the characteristic cell morphology including the appearance of apoptotic bodies, and further confirmed by cellular DNA fragmentation. CD437-induced-S phase arrest was associated with upregulated mRNA levels of p21waf1/cip1/sdi1 in both LNCaP (p53+/+) and PC-3 (53-/-) cells. CONCLUSIONS: CD437 represents a unique retinoid that induces S-phase arrest and apoptosis in both androgen-dependent (LNCaP) and -independent (PC-3) human prostate cancer cells, suggesting a potential role of CD437 in the treatment of human prostate cancer.