Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).     

Evaluation of NUF2 and GMNN Expression in Prostate Cancer: Potential Biomarkers for Prostate Cancer Screening

Background:Prostate cancer (PC) is one of the most abundant cancers among men, and In Iran, has been responsible for 6% of all deaths from cancer in men. NUF2 and GMNN genes are considered as loci of susceptibility to tumorigenesis in humans. Alterations in expression of these genes have been reported in various malignancies. The aim of our study was to test whether different NUF2 and GMNN expression levels are associated with PC incidence and hence, might be considered as new molecular tools for PC screening.Methods:Biopsy samples from 40 PC patients and 41 healthy Iranian men were used to determine the relative gene expression. After RNA extraction and cDNA synthesis, samples were analyzed using TaqMan Quantitative Real time PCR. Patients’ background information, included smoking habits and family histories of PC, were recorded. Stages and grades of their PC were classified by the TNM tumor, node, metastasis (TMN) staging system based on standard guidelines.Results:NUF2 expression did not significantly differ between the groups, while GMNN expression was significantly greater in the PC specimens than in the controls.Conclusion:Regarding the significant role of GMNN in various tumor phenotypes, and its importance in PC progression, the alteration in GMNN expression in PC samples vs. controls indicate that the genetic profiling of this cancer might be considered to personalize therapy for each patient in the future.Key Words: Family history, Geminin (GMNN), Tumor staging, NUF2, Prostate cancer     

BK Virus as a Cofactor in the Etiology of Prostate Cancer in Its Early Stages

Prostate cancer has been projected to cause almost 10% of all male cancer deaths in the United States in 2007. The incidence of mutations in the tumor suppressor genes Rb1 and p53, especially in the early stages of the disease, is low compared to those for other cancers. This has led to the hypothesis that a human virus such as BK virus (BKV), which establishes a persistent subclinical infection in the urinary tract and encodes oncoproteins that interfere with these tumor suppressor pathways, is involved. Previously, we detected BKV DNA in the epithelial cells of benign and proliferative inflammatory atrophy ducts of cancerous prostate specimens. In the present report, we demonstrate that BKV is present at a much lower frequency in noncancerous prostates. Additionally, in normal prostates, T-antigen (TAg) expression is observed only in specimens harboring proliferative inflammatory atrophy and prostatic intraepithelial neoplasia. We further demonstrate that the p53 gene from atrophic cells expressing TAg is wild type, whereas tumor cells expressing detectable nuclear p53 contain a mix of wild-type and mutant p53 genes, suggesting that TAg may inactivate p53 in the atrophic cells. Our results point toward a role for BKV in early prostate cancer progression.     

Antioxidant Enzymes and Lipid Peroxidation in Different Stages of Breast Cancer

Oxidative stress resulting from an imbalance between pro-oxidants and anti-oxidants seems to play an important role in human breast carcinogenesis. There are conflicting reports regarding the tissue levels of malondialdehyde (MDA), ascorbic acid and superoxide dismutase (SOD) in breast cancer patients whereas few blood values have been reported. The present study was carried out to observe the changes in serum MDA, serum SOD and plasma ascorbic acid with the stage-wise progression of the disease. Serum MDA and serum SOD levels were found to be increased gradually from Stage I to Stage IV as compared to control group (?p<0.001). The maximum rise was in Stage IV patients. In contrast, mean plasma ascorbic acid levels were low in all stages compared to control group (?p<0.001). The decrease was more pronounced in Stage III and Stage IV. The study would be of immense help for establishing blood based biochemical marker in breast cancer patients.     

Reduced Kv1.3 Potassium Channel Expression in Human Prostate Cancer

The presence of Kv1.3 voltage-gated potassium channels in rat and human prostate epithelial cells has been previously reported. We examined, by immunohistochemistry, Kv1.3 levels in 10 normal human prostate, 18 benign prostatic hyperplasia (BPH) and 147 primary human prostate cancer (Pca) specimens. We found high epithelial expression of Kv1.3 in all normal prostate, 16 BPH and 77 (52%) Pca specimens. Compared to normal, Kv1.3 levels were reduced in 1 (6%) BPH specimen and in 70 (48%) Pca specimens. We found a significant inverse correlation between Kv1.3 levels and tumor grade (r = −0.25, P = 0.003) as well as tumor stage (r = −0.27, P = 0.001). Study of an additional 30 primary Pca specimens showed that 15 (50%) had reduced Kv1.3 immunostaining compared to matched normal prostate tissue. Our data suggest that in Pca reduced Kv1.3 expression occurs frequently and may be associated with a poor outcome.     

WFS1在大鼠胰腺不同发育阶段的表达

目的:研究WFS1在胰腺发育不同阶段的表达和细胞定位.方法:运用Western Blot技术检测WFS1在大鼠胰腺发育不同阶段的蛋白表达水平;运用免疫荧光检测不同时期WFS1在胰腺的定位.结果:Western Blot结果显示WFSl的蛋白表达量胚胎后期高于新生期,成年期表达量上升;免疫荧光结果显示在不同发育时期WFS1与胰岛β细胞共表达.结论:WFS1在胚胎发育中后期的高表达可能与胰岛形成及功能完善有关,并且可能参与了胰岛重塑.     

Distinct Phenotypes of Human Prostate Cancer Cells Associate with Different Adaptation to Hypoxia and Pro-Inflammatory Gene Expression

Hypoxia and inflammation are strictly interconnected both concurring to prostate cancer progression. Numerous reports highlight the role of tumor cells in the synthesis of pro-inflammatory molecules and show that hypoxia can modulate a number of these genes contributing substantially to the increase of cancer aggressiveness. However, little is known about the importance of the tumor phenotype in this process. The present study explores how different features, including differentiation and aggressiveness, of prostate tumor cell lines impact on the hypoxic remodeling of pro-inflammatory gene expression and malignancy. We performed our studies on three cell lines with increasing metastatic potential: the well differentiated androgen-dependent LNCaP and the less differentiated and androgen-independent DU145 and PC3. We analyzed the effect that hypoxic treatment has on modulating pro-inflammatory gene expression and evaluated the role HIF isoforms and NF-kB play in sustaining this process. DU145 and PC3 cells evidenced a higher normoxic expression and a more complete hypoxic induction of pro-inflammatory molecules compared to the well differentiated LNCaP cell line. The role of HIF1α and NF-kB, the master regulators of hypoxia and inflammation respectively, in sustaining the hypoxic pro-inflammatory phenotype was different according to cell type. NF-kB was observed to play a main role in DU145 and PC3 cells in which treatment with the NF-kB inhibitor parthenolide was able to counteract both the hypoxic pro-inflammatory shift and HIF1α activation but not in LNCaP cells. Our data highlight that tumor prostate cell phenotype contributes at a different degree and with different mechanisms to the hypoxic pro-inflammatory gene expression related to tumor progression.     

帕比司他逆转前列腺癌细胞hepaCAM基因表达机制研究

目的:研究帕比司他(Panobinostat)逆转抑癌基因肝细胞粘附分子(hepatocyte cell adhesion molecule,hepaCAM)表达,协同hepaCAM抑制前列腺癌(prostate cancer,PCa)细胞生长。方法:不同浓度帕比司他作用于体外培养的PC3细胞,首先采用MTT法检测帕比司他对细胞增殖的影响,RT-PCR和Western blot法检测hepaCAM、组蛋白去乙酰化酶(histone deacetylases, HDACs)以及乙酰化组蛋白H3 赖氨酸9(Ac-H3K9)的表达变化。接着用不同因素处理细胞,MTT法检测细胞增殖活性,流式细胞术检测细胞周期改变,RT-PCR和Western blot法检测细胞周期调节因子cyclinD1 和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的基因表达。结果:帕比司他抑制PC3细胞生长与作用浓度增加和作用时间呈正相关, hepaCAM mRNA、蛋白以及细胞核中Ac-H3K9表达随帕比司他浓度升高而增高,HDAC1、 HDAC3、 HDAC4 mRNA和蛋白的表达随浓度升高而降低;单独过表达hepaCAM腺病毒和单独使用帕比司他组PC3细胞生长明显受到抑制且随作用时间延长抑制率增加,两者联用更为明显,差异有统计学意义(P < 0.05);与单独hepaCAM 腺病毒组和帕比司他组相比,两者联用S期细胞比例明显增高,差异有统计学意义(P < 0.05),且可进一步下调cyclinD1、 PCNA mRNA和蛋白的表达,差异有统计学意义(P < 0.05)。结论:帕比司他可通过抑制HDACs活性,加强PCa PC3细胞组蛋白H3 N-端的赖氨酸残基乙酰化,逆转hepaCAM表达;hepaCAM腺病毒和帕比司他联用可通过阻滞细胞周期于S期协同抑制PC3 细胞生长,作用机制可能与下调cyclinD1 和PCNA 的表达有关。这对揭示抑癌基因hepaCAM在肿瘤缺失的原因及为将帕比司他应用于临床治疗肿瘤提供了科学支持。     

应用组织芯片技术研究Survivin基因蛋白在前列腺肿瘤组织中的表达及其意义

目的:应用组织芯片技术分析Survivin基因蛋白在人类前列腺癌组织、前列腺正常组织及前列腺良性痛变组织中的表达情况.方法:采用兔抗人survivin单克隆抗体的免疫组织化学ABC法,研究Survivin在不同前列腺组织的表达,并分析Survivin在不同前列腺组织中的表达差异.结果:免疫组化结果显示,前列腺癌组织与前列腺良性病变组织及正常前列腺组织中Survivin的表达相比呈显著性差异(P<0.05).结论:Survivin在前列腺癌组织中呈高表达,提示其可能对前列腺癌的发生或发展有重要作用.     

Thioredoxin Reductase 1 Expression and Castration-recurrent Growth of Prostate Cancer

INTRODUCTION: Many genes are differentially expressed between androgen-dependent and androgen-independent prostate cancer (CaP). Differential expression analysis and subtractive hybridization previously identified nine genes expressed in intact mice bearing CWR22 tumors and castrated mice bearing recurrent CWR22 tumors but not in regressed tumors. The objectives of this study were to develop an immunostaining method to dual-label foci of proliferating tumor cells [the origin of castration-recurrent CaP (CR-CaP)], to determine which of the nine candidate proteins were differentially expressed in proliferating versus nonproliferating cells at the onset of growth after castration, and to test preclinical findings using clinical specimens of androgen-stimulated benign prostate (AS-BP) and CaP (AS-CaP) and CR-CaP. METHODS: Paraffin-embedded, bromodeoxyuridine-injected CWR22 tumors were hydrated, antigen-retrieved using high heat and high pressure, labeled for each of the nine antigens of interest, visualized using peroxidase, and counterstained with hematoxylin. Mean optical density was calculated for proliferating and nonproliferating areas using automated (nuclear staining) or manual (cytoplasmic staining) image analysis. Prostate tissue microarray sections were immunostained and visually scored. RESULTS: Immunohistochemistry revealed higher nuclear expression of thioredoxin reductase 1 (TrxR1) in proliferating cells than nonproliferating cells (P < .005). There were no statistical differences between cell types in the expression of other proteins. TrxR1 expression was higher (P < .01) in CR-CaP compared with AS-BP or AS-CaP. CONCLUSIONS: Increased TrxR1 expression in CR-CaP was consistent with increased TrxR1 and BrdU expression at the onset of growth in the CWR22 model. Thioredoxin reductase 1 should be targeted in an attempt to delay or prevent CaP recurrence after castration.     

水稻U-Box蛋白质在不同发育时期的表达分析

真核生物基因组中广泛存在U-Box基因,其编码蛋白大部分是泛素系统中决定底物特异性识别的E3蛋白,其构象与RING-finger极其相似．U-Box蛋白质能促进底物蛋白泛素化降解,对细胞内异常蛋白的降解及质量控制方面发挥着重要的作用．水稻基因组中有77个U-Box蛋白质,系统了解它们的表达可为功能研究提供数据．制备针对水稻U-Box蛋白质的抗体,了解水稻中U-Box蛋白质在不同发育时期的表达信息,为功能研究积累数据．选取了4个水稻U-Box蛋白质,其共同结构特点为U-Box结构在N端,C端有ARM结构．用计算机软件预测抗原决定簇,细菌体系体外表达、纯化U-Box蛋白质的片段,免疫动物制备多克隆抗体,用Western blotting检测U-Box蛋白质在水稻品种93-11苗期地上部和地下部、分蘖期根和茎、孕穗期剑叶和幼穗、开花期剑叶和穗子、成熟期剑叶和种子中的表达,并与EST数据库中公布的U-Box蛋白质EST数据进行了比较分析．体外克隆表达后,获得了纯化的蛋白质,制备的抗体特异性强,蛋白质印迹(Western blotting)检测可见一条明显的主带,其中Os06g01304和Os12g38210两个蛋白质的表观分子质量与预测分子质量相符,Os01g66130和Os08g01900两个蛋白质的表观分子质量低于预测分子质量．4个U-Box蛋白质在水稻生长发育的不同时期或部位基本上是组成型表达,且表达量接近．对NCBI上公布的来自274个文库100万条以上的EST进行分析,可以看出4个U-Box蛋白质EST的数量分布大致均匀,与Western blotting结果揭示的组成型表达平行,与ATPase、HSP81-3、EGF-1 alpha和RuBisCo等对照基因相比,U-Box基因的EST数目相对很少,说明它们属于低丰度转录的基因．选取了4个水稻U-Box蛋白质,通过抗原决定簇预测,表达片段蛋白,制备了特异性抗体,证明了这一技术路线的可行性．利用抗体对水稻不同发育时期材料进行蛋白质表达谱研究,发现这些U-Box蛋白质呈组成型表达,与EST数据揭示的结果具有平行性．所制备的抗体也为相关功能研究,如免疫共沉淀、ChIP-on-chip、Pull-down以及在抗病、抗逆反应中U-Box蛋白质的表达等,积累了 资源．     

不同发育阶段雄性小鼠睾丸组织mEppin基因mRNA的表达差异研究

目的实时荧光定量PCR检测雄性小鼠不同发育阶段睾丸组织中m Eppin基因的mRNA表达差异,为探究m Eppin基因表达与雄性生殖相关性提供生物学数据。方法分别选取胚胎期、50日龄、90日龄雄性ICR小鼠各6只,共18只,取各组小鼠睾丸组织提取总RNA,实时荧光定量PCR方法检测m Eppin基因mRNA表达水平。结果不同发育阶段小鼠睾丸m Eppin基因表达水平差异明显。胚胎组小鼠中没有检测到m Eppin基因mRNA表达,50日龄小鼠和90日龄小鼠中均检测到m Eppin基因mRNA有较高表达水平。其中,90日龄小鼠睾丸中m Eppin基因mRNA的相对表达水平是50日龄小鼠的2.65倍。结论本研究结果表明m Eppin基因mRNA的表达在小鼠睾丸组织中具阶段特异性。     

RNA结合蛋白QKI-5在前列腺癌中的表达分析

目的:检测前列腺癌及癌旁正常前列腺组织中RNA结合蛋白QKI-5的表达情况并分析其临床意义。方法:收集前列腺癌及与之匹配的癌旁组织124例,通过免疫组化染色、Western blot、实时PCR方法检测其QKI-5的表达水平,并分析QKI-5的表达与前列腺癌临床病理特征的关系。结果:前列腺癌组织中QKI-5蛋白及m RNA表达水平均明显低于癌旁正常前列腺组织,并且随着Gleason评分的增高而降低(P0.05)。前列腺癌组织中QKI-5的表达与其Gleason评分(r=-0.939,P0.05)、TNM分期(r=-0.913,P0.05)、血清PSA值(r=-0.743,P0.05)均密切相关。结论:QKI-5在前列腺癌的发生发展过程中可能起抑癌基因的作用,并可能作为前列腺癌的诊断、病情分析和预后评估的参考指标。     

骨形态发生蛋白7 在前列腺癌中的表达

目的:探讨骨形态发生蛋白( BMP-7)在前列腺癌组织中的表达及其与临床分期之间的关系.方法:应用免疫印迹法检测30例前列腺癌患者及30例前列腺良性增生患者前列腺组织中BMP-7的表达情况.结果:前列腺癌组织中BMP-7的表达显著高于前列腺良性增生组织,且BMP-7的表达随前列腺癌的临床分期、Gleason分级增高而增加.结论:BMP-7在前列腺癌中的表达明显增高,其表达量与临床分期相关,前列腺癌组织中BMP-7的表达增高提示预后不佳.     

PTEN蛋白表达与前列腺癌病理分期的关系

目的：探讨PTEN蛋白在前列腺癌组织中的表达及其临床意义。方法：应用免疫组织化学S-P方法,检测前列腺癌及良性前列腺增生组织中PTEN蛋白的表达。结果：PTEN蛋白表达阳性随肿瘤细胞病理分级、临床分期的增高,PTEN蛋白阳性表达率降低。结论：PTEN蛋白异常表达在前列腺癌的进展中有重要作用,检测PTEN蛋白的表达有助于判断病情及预后。     

PTEN蛋白表达与前列腺癌病理分期的关系

目的:探讨PTEN蛋白在前列腺癌组织中的表达及其临床意义。方法:应用免疫组织化学S-P方法,检测前列腺癌及良性前列腺增生组织中PTEN蛋白的表达。结果:PTEN蛋白表达阳性随肿瘤细胞病理分级、临床分期的增高,PTEN蛋白阳性表达率降低。结论:PTEN蛋白异常表达在前列腺癌的进展中有重要作用,检测PTEN蛋白的表达有助于判断病情及预后。     

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。