肌酸激酶B在前列腺细胞中的表达及临床意义

目的：探讨肌酸激酶B（脑型肌酸磷酸激酶,CKB）在前列腺良性及恶性细胞中的表达差异及临床意义。方法：采用实时定量PCR方法检测CKB在BPH1与LNCaP细胞中的表达差异,采用电泳仪法进行血标本CKB检验,最后采用半定量PCR方法研究CKB与雄激素的相关性。结果：CKB在LNCaP的表达量是BPH1细胞中的12．3倍,血检验发现,CKB在未经内分泌治疗前列腺癌组阳性率（5／10）显著高于前列腺增生组（0／10）,差异有统计学意义（P〈0．05）,内分泌治疗前列腺癌组CKB阳性率（4／37）与前列腺增生组阳性率（0／10）相比差异无统计学意义（P〉0．05）。CKB在比卡鲁胺（bicalutamide,Casodex）阻断组LNCaP细胞中的表达明显低于无比卡鲁胺阻断LNCaP细胞。结论：CKB在恶性细胞中高表达,对未经内分泌治疗前列腺癌患者有一定诊断价值,并且其表达受雄激素的调节,可能在前列腺癌的发生发展中起重要作用。     

Fas、FasL在前列腺癌表达的研究

为探讨Fas、FasL在前列腺癌发生、发展中的作用 ,应用免疫组化SP方法对 49例前列腺癌 (Pca)组织、2 2例前列腺增生症 (BPH)组织和 14例正常前列腺 (NP)组织中Fas、FasL的表达进行检测。结果表明 :①Fas、FasL表达于所有被检测的前列腺良、恶性病变及正常组织中 ;②Fas在BPH中表达强度最强 ,在Pca中表达强度最弱 (P <0 .0 5 ) ;③FasL在三组间表达强度的差异无显著性意义 (P <0 .0 5 ) ;④ 17例雄激素依赖性前列腺癌(AD Pca)组织中Fas表达强度比 9例雄激素非依赖性前列腺癌 (AI Pca)显著。由此推论Fas系统与前列腺癌发生、发展有关。     

125I标记雄激素受体TFO抗前列腺癌细胞增殖的研究

目的探讨反基因放射治疗对雄激素受体（AR）表达和前列腺癌细胞增殖的影响。方法用Iodogen法对AR三螺旋形成寡核苷酸（TFO）进行直接^125I标记，经脂质体介导转染LNCaP前列腺癌细胞株，于转染后24和48h分别采用四甲基偶氮唑蓝（MTT）方法测定癌细胞增殖活性，RT-PCR方法检测ARmRNA表达，免疫组织化学方法检测AR蛋白表达。结果^125I-TFO的标记率为63．7％，放化纯为95．6％，比活度为80．1kBq／μg。相同TFO浓度下，^125I-TFO组LNCaP细胞的AR表达水平显著低于TFO组（P〈0．01），^125I-TFO对LNCaP细胞增殖的抑制率显著高于TFO（P〈0．01）。结论反基因放射治疗对AR表达和前列腺癌细胞增殖的抑制作用明显强于单纯的反基因治疗。     

雄激素受体三螺旋形成寡核苷酸抗前列腺癌细胞增殖的研究

目的　探讨反基因策略对雄激素受体(AR)表达和前列腺癌细胞增殖的影响。方法　针对AR基因2 4 4 7～2 4 6 1核苷酸序列设计并合成三螺旋形成寡核苷酸(TFO) ,经脂质体介导转染LN CaP前列腺癌细胞株,于转染后2 4、4 8、72h分别采用3H 胸腺嘧啶核苷(TdR)参入实验测定癌细胞增殖活性,用逆转录聚合酶链反应(RT PCR)方法检测ARmRNA表达,用放射配基结合分析法(RBA)单点法检测AR蛋白质表达,并与反义寡核苷酸(ASON)相比较。结果　2 4、4 8、72hTFO组LNCaP细胞的AR表达水平明显低于ASON组(P <0 0 5 ) ,TFO对LNCaP细胞增殖的抑制率显著高于ASON(P <0 0 5 )。结论　该TFO能有效抑制AR表达和前列腺癌细胞增殖,可用于反基因放射治疗的研究     

c-erbB-2基因在维甲酸诱导前列腺癌细胞DU-145凋亡中的作用

目的：克隆全反式维甲酸诱导前列腺癌细胞系DU—145细胞产生凋亡的相关基因。探讨前列腺癌细胞产生凋亡的分子机理。方法：应用全反式维甲酸诱导前列腺癌DU—145细胞凋亡，采用基于PCR的改良消减杂交技术克隆与凋亡相关的基因。结果：在前列腺癌DU—145细胞凋亡过程中，克隆出包括c—erbB—2基因在内的多个基因序列。结论：全反式维甲酸诱导的前列腺癌细胞DU—145凋亡有多基因参与其中，其中c—erbB—2等基因与前列腺癌细胞凋亡关系密切。     

人前列腺癌cDNA文库的构建和筛选

目的:PC1基因是与前列腺癌相关的新基因,在雄激素非依赖和转移的前列腺癌晚期细胞系C42中高表达。为了进一步研究PC1基因的功能及作用机制,构建C42细胞的cDNA文库,寻找与前列腺癌相关基因PC1相互作用的蛋白。方法:从前列腺癌细胞系C42中提取总RNA,进而分离poly(A)+RNA,用poly(A)+RNA进行反转录并以SMARTⅢTM和CDSⅢoligo(dT)为引物进行PCR扩增,得到两端具有同源臂的PCR片段,以此同源臂为基础在酵母中实现同源重组。通过文库片段、线性化的pGADT7Rec和诱饵质粒pGBKT7PC1C共转化酵母AH109菌株,在文库构建的同时进行与PC1相互作用蛋白的筛选;或先将文库片段,线性化的pGADT7Rec转化AH109,再利用AH109和Y187两种酵母菌株的接合生殖进行筛选。最后用Far Western印迹方法进一步从体外论证了PC1蛋白可与自身相互作用形成二聚体。结果:构建了具有基因多样性和库容量足够大的人前列腺癌cDNA文库,双链cDNA片段的长度大小范围为250～5000bp。共转化的效率为4.3×105,重组效率为1.9×106,筛选的克隆数为4.3×105。接合法筛选时的接合效率为32%,筛选的克隆数为1.0×106。筛选到4个与PC1蛋白相互作用的阳性克隆。从体外证明了PC1蛋白可形成二聚体。结论:此文库的多样性和库容量均符合筛选需求。可用于前列腺癌相关基因     

抑制细丝蛋白A促进前列腺癌C4-2细胞迁移

目的 探讨细丝蛋白A(FLNA)对前列腺癌细胞迁移的影响。方法 设计并构建靶向FLNA的shRNA敲低质粒pSIH-H1-shFLNA。包装慢病毒后感染前列腺癌C4-2细胞,获得敲低FLNA的稳定细胞系C4-2-shFLNA。Western印迹检测前列腺癌C4-2细胞中FLNA蛋白表达和PC-1蛋白表达;实时荧光定量PCR(qPCR)检测FLNA以及PC-1 mRNA表达水平;Western印迹检测敲低FLNA对磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B(AKT)信号通路的影响;划痕实验和Transwell实验检测C4-2细胞体外迁移能力。结果 前列腺癌C4-2细胞敲低FLNA后FLNA mRNA和蛋白水平明显下调,PC-1 mRNA水平和蛋白水平表达增加,同时AKT磷酸化水平显著升高。划痕及Transwell实验证实在前列腺癌C4-2细胞中敲低FLNA后细胞迁移能力增强。结论 前列腺癌细胞中敲低FLNA能够激活PI3K/AKT信号通路,促进前列腺癌细胞迁移。     

喉癌淋巴结转移相关分子标志物的研究进展

喉癌是耳鼻咽喉-头颈外科的常见恶性肿瘤，在头颈部恶性肿瘤中发病率较高，占耳鼻咽喉恶性肿瘤的11.6％-22％，占全身肿瘤的2％，复发和转移是导致喉癌死亡率居高不下的主要原因，淋巴结转移是喉癌转移的早期事件和独立的预后因素。喉癌淋巴结转移过程可能涉及到许多分子作用机制和信号传导途径，包括肿瘤淋巴管生成相关基因、细胞粘附分子、细胞外基因相关蛋白、细胞因子、肿瘤转移相关基因等。本文综述喉癌淋巴结转移的相芙分子标志物的研究进展。     

人前列腺癌细胞株PC-3中类肿瘤干细胞的培养分离和初步鉴定

目的 从人前列腺癌细胞系PC-3中分离前列腺癌类干细胞并进行初步鉴定,为前列腺癌干细胞的进一步研究奠定基础.方法分别用含血清及无血清培养基培养前列腺癌PC-3细胞,流式细胞仪检测两种不同培养条件下前列腺癌类干细胞的比例,细胞免疫荧光鉴定前列腺癌类干细胞,MTT法测定并比较前列腺癌类干细胞与前列腺癌PC-3细胞的增殖能力及倍增时间,观察前列腺癌类干细胞诱导分化过程及其分化后细胞的双重免疫荧光表达.结果 少量PC-3细胞能在添加了人表皮生长因子(EGF)、人碱性成纤维生长因子(bFGF)和人白血病抑制因子(LIF)的无血清培养基中存活并形成悬浮细胞球团,无血清培养基中前列腺癌类干细胞比例(1.43%)显著高于含血清培养基(0.41%,P<0.05).前列腺癌类干细胞的增殖能力(培养7d的细胞数为9.6×10~5个)强于前列腺癌细胞(培养7d的细胞数为3×10~5个,P<0.000 5),倍增时间(21.90h)短于前列腺癌细胞(28.38h,P<0.000 5),免疫荧光显示前列腺癌类干细胞表达CD133;前列腺癌类干细胞球可在含血清培养基中贴壁分化,且双重荧光检测显示其同时低表达CD44和CD133.结论 体外培养的前列腺癌细胞系PC-3中含有少量能表达前列腺癌干细胞表面标志物的前列腺癌类干细胞,并可通过无血清培养基富集这类类肿瘤干细胞.     

急性非淋巴细胞性白血病细胞共刺激分子的表达

目的：检测急性非淋巴细胞白血病患者白血病细胞表面MHC—Ⅱ类分子和共刺激分子的表达情况。方法：采集骨髓中白血病细胞大于70％的27例初诊或复发息者骨髓细胞。荧光抗体标记。应用流式细胞仪进行HLA—DR、CD80(B7-1)和CI)和(B7—2)免疫标记检测。结果：27例病人除M3型表达明显低于其他各型外。HLA—DR抗原的表达均较高。其中M2型最高为90％；CD80阳性率很低，最高为M1型只有5％。其余均在l％～3％之间。CD86的表达高于CD80，最高为M5型为48％，最低为M6型为11％。结论：白血病细胞表面共刺激分子表达以CD80缺乏为主。     

Examining the relationship between Cu-ATSM hypoxia selectivity and fatty acid synthase expression in human prostate cancer cell lines

INTRODUCTION: Positron emission tomography (PET) imaging with copper (II)-diacetyl-bis(N4-Methylthiosemicarbazone)(Cu-ATSM) for delineating hypoxia has provided valuable clinical information, but investigations in animal models of prostate cancer have shown some inconsistencies. As a defense mechanism in prostate cancer cells, the fatty acid synthesis pathway harnesses its oxidizing power for improving the redox balance despite conditions of extreme hypoxia, potentially altering Cu-ATSM hypoxia selectivity. METHODS: Human prostate tumor-cultured cell lines (PC-3, 22Rv1, LNCaP and LAPC-4), were treated with a fatty acid synthase (FAS) inhibitor (C75, 100 microM) under anoxia. The 64Cu-ATSM uptake in these treated cells and nontreated anoxic cells was then examined. Fatty acid synthase expression level in each cell line was subsequently quantified by ELISA. An additional study was performed in PC-3 cells to examine the relationship between the restoration of 64Cu-ATSM hypoxia selectivity and the concentration of C75 (100, 20, 4 or 0.8 microM) administered to the cells. RESULTS: Inhibition of fatty acid synthesis with C75 resulted in a significant increase in 64Cu-ATSM retention in prostate tumor cells in vitro under anoxia over 60 min. Inhibition studies demonstrated higher uptake values of 20.9+/-3.27%, 103.0+/-32.6%, 144.2+/-32.3% and 200.1+/-79.3% at 15 min over control values for LAPC-4, PC-3, LNCaP and 22Rv1 cells, respectively. A correlation was seen (R2=.911) with FAS expression plotted against percentage change in 64Cu-ATSM uptake with C75 treatment. CONCLUSIONS: Although Cu-ATSM has clinical relevance in the PET imaging of hypoxia in many tumor types, its translation to the imaging of prostate cancer may be limited by the overexpression of FAS associated with prostatic malignancies.     

前列腺癌miRNA生物标志物及其与辐射敏感性的研究进展

前列腺癌是男性泌尿系统最常见的恶性肿瘤之一。miRNA是近年来发现的一类长度为19~23 bp核苷酸的非编码小分子RNA,参与调节许多重要的细胞生物学行为,甚至在肿瘤的发生中发挥关键作用。前列腺癌基于血液miRNA生物标志物的研究不断涌现,但未发现特异性细胞外miRNA标志物。研究前列腺癌miRNA表达规律、作用机制,对深入探讨前列腺癌的发病机制、探索新的诊断和治疗途径意义重大。笔者主要综述针对健康人群、前列腺癌患者、转移性前列腺癌患者中不同miRNA丰度的不同,及前列腺癌miRNA对辐射敏感性的调节作用,试图探索miRNA作为前列腺癌生物标志物的线索。     

Inhibition of cell growth induced by photosensitizer PP(Arg)2-mediated photodynamic therapy in human breast and prostate cell lines. Part I

Photodynamic therapy can become an effective alternative method to surgery. The experiments reveal that using low photosensitizer doses and relatively low energy doses allow us to obtain effective results after PDT (to limit formation of colonies by investigated cancer cells). The prostate and breast cancer cell lines were investigated: MCF-7, a human breast cancer responsive to androgen therapy; MDA-MB231, a more aggressive human breast cancer non-responsive to androgen therapy; LNCaP, a lymphonodal metastasis of prostate carcinoma responsive to androgen therapy; DU-145, a human prostate cancer non-responsive to androgen therapy. Clonogenic assay shows that certain PP(Arg)(2) and light energy low doses stimulate the researched colony-forming cancer cells growth. Some low energy doses used during PP(Arg)(2)-mediated PDT also cause the increase in the colony-forming tumor cells. Among investigated cancer lines, MCF-7 exhibited the biggest sensibility towards PP(Arg)(2) and LNCaP the smallest one. PP(Arg)(2) based PDT is an effective method in colony growth limitation of breast cancer cell lines: MCF-7, MDA-MB231 and prostate cancer cell lines: LNCaP, DU-145.     

Heat shock protein 90 (Hsp90) chaperone complex inhibitor,Radicicol, potentiated radiation-induced cell killing in a hormone-sensitive prostate cancer cell line through degradation of the androgen receptor

Until now, there has not been enough information on how androgens or androgen deprivation may influence the response of cancer cells to radiation. In this study, the effect of dihydrotestosterone (DHT) on cellular proliferative activity and radiosensitivity was examined in a hormone-sensitive human prostate cancer cell line, LNCaP. In addition, the study also examined how a heat shock protein 90 (Hsp90) chaperone complex inhibitor modified the effect of DHT on the radiosensitivity of the cells, because binding of the androgen receptor (AR) to Hsp90 is required to maintain the stability and functioning of AR. The hormone-sensitive human prostate cancer cell line, LNCaP, was used. Radicicol was used as one of the known Hsp90 chaperone complex inhibitors, and the cells were incubated in the presence of this compound at a concentration of 500 nM. Cellular radiosensitivity was determined by the clonogenic assay; the changes in the protein expression were examined by Western blotting or immunofluorescence. DHT at a concentration of 1 nM caused enhancement of the proliferative activity and reduction of the radiosensitivity of the cells. Radicicol at a concentration of 500 nM abolished the DHT-induced decrease in cellular radiosensitivity and potentiated the radiation-induced cell killing synergistically. Consistent with the changes in the cellular radiosensitivity, radicicol degraded AR, Raf-1 and HER2/neu via reduced binding of AR to Hsp90, although selective degradation of HER2/neu caused by Herceptin, a monoclonal antibody against HER2, did not affect the cellular radiosensitivity. The results suggest that the Hsp90 chaperone complex may be a potential molecular target for potentiation of radiation-induced cell killing in a hormone-sensitive prostate cancer cell line.     

Heat shock protein 90 (Hsp90) chaperone complex inhibitor, radicicol, potentiated radiation-induced cell killing in a hormone-sensitive prostate cancer cell line through degradation of the androgen receptor

Until now, there has not been enough information on how androgens or androgen deprivation may influence the response of cancer cells to radiation. In this study, the effect of dihydrotestosterone (DHT) on cellular proliferative activity and radiosensitivity was examined in a hormone-sensitive human prostate cancer cell line, LNCaP. In addition, the study also examined how a heat shock protein 90 (Hsp90) chaperone complex inhibitor modified the effect of DHT on the radiosensitivity of the cells, because binding of the androgen receptor (AR) to Hsp90 is required to maintain the stability and functioning of AR. The hormone-sensitive human prostate cancer cell line, LNCaP, was used. Radicicol was used as one of the known Hsp90 chaperone complex inhibitors, and the cells were incubated in the presence of this compound at a concentration of 500 nM. Cellular radiosensitivity was determined by the clonogenic assay; the changes in the protein expression were examined by Western blotting or immunofluorescence. DHT at a concentration of 1 nM caused enhancement of the proliferative activity and reduction of the radiosensitivity of the cells. Radicicol at a concentration of 500 nM abolished the DHT-induced decrease in cellular radiosensitivity and potentiated the radiation-induced cell killing synergistically. Consistent with the changes in the cellular radiosensitivity, radicicol degraded AR, Raf-1 and HER2/neu via reduced binding of AR to Hsp90, although selective degradation of HER2/neu caused by Herceptin, a monoclonal antibody against HER2, did not affect the cellular radiosensitivity. The results suggest that the Hsp9O chaperone complex may be a potential molecular target for potentiation of radiation-induced cell killing in a hormone-sensitive prostate cancer cell line.     

PET/CT Imaging and Radioimmunotherapy of Prostate Cancer

Prostate cancer is a common cancer in men and continues to be a major health problem. Imaging plays an important role in the clinical management of patients with prostate cancer. An important goal for prostate cancer imaging is more accurate disease characterization through the synthesis of anatomic, functional, and molecular imaging information. Positron emission tomography (PET)/computed tomography (CT) in oncology is emerging as an important imaging tool. The most common radiotracer for PET/CT in oncology, (18)F-fluorodeoxyglucose (FDG), is not very useful in the imaging of prostate cancer. However, in recent years other PET tracers have improved the accuracy of PET/CT imaging of prostate cancer. Among these, choline labeled with (18)F or (11)C, (11)C-acetate, and (18)F-fluoride has demonstrated promising results, and other new radiopharmaceuticals are under development and evaluation in preclinical and clinical studies. Large prospective clinical PET/CT trials are needed to establish the role of PET/CT in prostate cancer patients. Because there are only limited available therapeutic options for patients with advanced metastatic prostate cancer, there is an urgent need for the development of more effective treatment modalities that could improve outcome. Prostate cancer represents an attractive target for radioimmunotherapy (RIT) for several reasons, including pattern of metastatic spread (lymph nodes and bone marrow, sites with good access to circulating antibodies) and small volume disease (ideal for antigen access and antibody delivery). Furthermore, prostate cancer is also radiation sensitive. Prostate-specific membrane antigen is expressed by virtually all prostate cancers, and represents an attractive target for RIT. Antiprostate-specific membrane antigen RIT demonstrates antitumor activity and is well tolerated. Clinical trials are underway to further improve upon treatment efficacy and patient selection. This review focuses on the recent advances of clinical PET/CT imaging and RIT of prostate cancer.     

The value and limitations of contrast-enhanced transrectal ultrasonography for the detection of prostate cancer

Objectives

To evaluate the role of contrast-enhanced transrectal ultrasonography (CE-TRUS) for detecting prostate carcinoma.

Methods

Sixty-five patients with elevated serum prostate-specific antigen (PSA) and/or abnormal digital rectal examination (DRE) were assessed using transrectal ultrasound (TRUS) and CE-TRUS. In all the patients, CE-TRUS was performed with intravenous injection of contrast agent (SonoVue, 2.4 ml) before biopsy. The cancer detection rates of the two techniques were compared. False-positive and false-negative findings related to CE-TRUS were analyzed in comparison to the pathological results of biopsy or radical prostatectomy. The targeted biopsy to abnormal CE-TRUS areas was also compared to systematic biopsy.

Results

Prostate cancer was detected in 29 of the 65 patients. CE-TRUS showed rapid focal enhancement or asymmetric vessels of peripheral zones in 28 patients; 23 of them had prostate cancer. CE-TRUS had 79.3% sensitivity, compared to 65.5% of TRUS (P < 0.05). There were five false-positive and six false-negative findings from CE-TRUS. Benign prostate hyperplasia, and acute and chronic prostatitis were important causes related to the false-positive results of CE-TRUS. Prostate cancer originating from the transition zone or peripheral zone with lower PSA levels, small-size foci, and moderately or well-differentiated tumor was missed by CE-TRUS. The cancer detection rate of targeted biopsy (75%, 33/44 cores) was significantly higher than one of systematic biopsy (48.2%, 162/336) in those 28 cases (P < 0.05). In addition, no significant correlation was found between the cancer detection rate with CE-TRUS and serum PSA levels.

Conclusion

CE-TRUS may improve the detection rate of prostate cancer through targeted biopsy of contrast-enhanced abnormalities. Our findings indicate that systematic biopsies should not be eliminated on the basis of false-positive and false-negative findings related to CE-TRUS.     

Prostate cancer screening: the clinical value of diffusion-weighted imaging and dynamic MR imaging in combination with T2-weighted imaging

PURPOSE: To evaluate the clinical value of diffusion-weighted imaging (DWI) and dynamic MRI in combination with T2-weighted imaging (T2W) for the detection of prostate cancer. MATERIALS AND METHODS: A total of 83 patients with elevated serum prostate specific antigen (PSA) levels (>4.0 ng/mL) were evaluated by T2W, DWI, and dynamic MRI at 1.5 T prior to needle biopsy. The data from the results of the T2W alone (protocol A), combination of T2W and DWI (protocol B), and the combination of T2W+DWI and dynamic MRI (protocol C) were entered into a receiver operating characteristic (ROC) curve analysis, under results of systemic biopsy as the standard of reference. RESULTS: Prostate cancer was pathologically detected in 44 of the 83 patients. The sensitivity, specificity, accuracy, and the area under the ROC curve (Az) for the detection of prostate cancer were as follows: 73%, 54%, 64%, and 0.711, respectively, in protocol A; 84%, 85%, 84%, and 0.905, respectively, in protocol B; and 95%, 74%, 86%, and 0.966, respectively, in protocol C. The sensitivity, specificity, and accuracy were significantly different between the three protocols (P < 0.01). CONCLUSION: In patients with elevated serum PSA levels, the combination of T2W, DWI, and dynamic MRI may be a valuable tool for detecting prostate cancer and avoiding an unnecessary biopsy without missing prostate cancer.     

Prostate cancer: 1.5 T endo-coil dynamic contrast-enhanced MRI and MR spectroscopy—correlation with prostate biopsy and prostatectomy histopathological data

Purpose

To investigate diagnostic accuracy of detection of prostate cancer by magnetic resonance: to evaluate the performance of T2WI, DCEMRI and CSI and to correlate the results with biopsy and radical prostatectomy histopathological data.

Materials and methods

43 patients, scheduled for radical prostatectomy, underwent prostate MR examination. Prostate cancer was identified by transrectal ultrasonographically (TRUS) guided sextant biopsy. MR examination was performed at 1.5T with an endorectal MR coil. Cancer localisation was performed on sextant-basis - for comparison between TRUS biopsy, MR techniques and histopathological findings on prostatectomy specimens.

Results

Prostate cancer was identified in all 43 patients by combination of the three MR techniques. The detection of prostate cancer on sextant-basis showed sensitivity and specificity: 50% and 91% for TRUS, 72% and 55% for T2WI, 49% and 69% for DCEMRI, and 46% and 78% for CSI.

Conclusion

T2WI, DCEMRI and CSI in combination can identify prostate cancer. Further development of MR technologies for these MR methods is necessary to improve the detection of the prostate cancer.