激素难治性前列腺癌组织中雄激素受体蛋白表达的研究

背景与目的:近来有研究报道,在激素难治性前列腺癌(hormonerefractoryprostatecarcinoma,HRPC)中发现有雄激素受体(androgenreceptor,AR)基因扩增,并提出AR基因扩增可能是导致激素治疗失败的一个新的分子机制。本研究拟对前列腺癌在激素治疗前和治疗失败后的AR蛋白表达作定量测定,进一步探讨AR表达与HRPC发生的关系。方法:采用放射配体结合分析方法测定28例晚期前列腺癌患者在激素治疗前以及治疗失败后原发癌组织中的AR蛋白含量。结果:28例前列腺癌在治疗前、后癌组织中的AR蛋白平均水平分别为(390.0±204.1)和(690.4±444.0)fmol/mgProtein,两者间差异有显著性(P<0.001)。其中10例在治疗后12个月内复发,其AR蛋白平均水平在治疗前、后分别为(398.2±199.5)和(448.2±274.1)fmol/mgProtein两者间差异无显著性(P>0.20),其余18例的AR蛋白平均水平在治疗前、后分别为(386.4±212.3)和(824.9±468.6)fmol/mgProtein,两者间差异有显著性(P<0.001)。结论:AR蛋白水平升高可能是前列腺癌对激素治疗不敏感的原因之一。     

PlncRNA-1在雄激素非依赖去势抵抗型前列腺癌细胞中的作用

目的:探讨长链非编码RNA PlncRNA-1在雄激素非依赖的前列腺癌细胞中的作用。方法:选取雄激素依赖的前列腺癌细胞系LNCaP及雄激素非依赖的前列腺癌细胞系C4-2,应用实时定量PCR技术检测两种细胞系中PlncRNA-1的表达差异。RNA干涉技术沉默PRNCR1的表达,检测AR表达变化。流式细胞术检测细胞周期及凋亡的变化。MTT实验检测对细胞增殖的影响。细胞侵袭实验检测细胞侵袭能力的变化。结果:PlncRNA-1在雄激素非依赖的细胞系C4-2中高表达。沉默其表达可以明显降低前列腺癌细胞中AR的表达,抑制前列腺癌细胞的细胞周期、增殖及细胞的侵袭能力,并促进细胞的凋亡。结论:PlncRNA-1在前列腺癌细胞中通过调节AR,影响细胞的增殖、凋亡及细胞的侵袭能力,PlncRNA-1可能在前列腺癌CRPC进展中发挥着重要作用。     

三氧化二砷重新激活PC-3前列腺癌细胞雄激素受体基因的表达

目的探讨三氧化二砷（As2O3）重新激活PC-3前列腺癌细胞雄激素受体（AR）基因的表达。方法用免疫组织化学的方法，对用浓度为2mg／L的As2O3处理前后的PC-3前列腺癌细胞和阳性对照的LNCaP前列腺癌细胞（AR）基因的表达情况进行检测分析。结果As2O3处理过的PC-3前列腺癌细胞AR的阳性表达率明显增高（P〈0．05）。结论As2O3可以重新激活PC-3前列腺癌细胞（AR）基因的表达。     

雄激素非依赖性前列腺原位癌动物模型建立及Pim-1表达研究

目的建立雄激素非依赖性前列腺原位癌动物模型,研究Pim-1基因及蛋白在该动物模型的表达。方法采用原位种植包埋法和外科手术去势技术,分别建立雄激素依赖、去势3d和雄激素非依赖性前列腺原位癌动物模型。分别采用基因芯片技术、酶联免疫法和免疫组织化学等实验方法,研究3组肿瘤组织中pim-1基因和蛋白表达的变化。结果 Affymetrix表达谱芯片技术检测结果显示,雄激素非依赖组Pim-1表达高于雄激素依赖组,差异有统计学意义,差异倍数为2.307 71;去势3d组与雄激素依赖组之间差异无统计学意义,差异倍数为1.108 67。ELISA检测结果显示,去势3d组血清睾酮浓度为（2.27±0.035）ng/mL,与雄激素依赖组的（9.02±0.99）ng/mL比较,差异有统计学意义,t=19.28,P〈0.01;雄激素非依赖组为（0.29±0.068）ng/mL,与雄激素依赖组比较,差异有统计学意义,t=24.87,P〈0.01;空白对照组为（9.23±0.78）ng/mL,与雄激素依赖组比较,差异无统计学意义,t=0.45,P=0.998。去势3d组PSA浓度为（0.17±0.032）ng/mL,与雄激素依赖组的（0.48±0.025）ng/mL比较,差异有统计学意义,t=21.82,P〈0.05;雄激素非依赖组为（0.87±0.023）ng/mL,与雄激素依赖组比较,差异有统计学意义,t=31.53,P〈0.05;空白对照组为0ng/mL,与雄激素依赖组比较,差异有统计学意义,t=41.80,P〈0.01。免疫组织化学结果显示,雄激素非依赖组Pim-1蛋白表达量为0.024±0.001 9,明显高于雄激素依赖组的0.017±0.002 1,差异有统计学意义,t=8.27,P〈0.05;去势3d组为0.018±0.001 3,与雄激素依赖组比较,差异无统计学意义,t=1.17,P=0.252。结论成功建立雄激素非依赖性前列腺原位癌动物模型,Pim-1与雄激素非依赖性前列腺癌有高度相关性。     

LncRNA PCGEM1和雄激素受体在前列腺癌中的共定位表达及临床意义

背景与目的：长链非编码RNA(long non-coding RNA,lncRNA)可参与肿瘤调控,有研究提示lncRNA PCGEM1可能影响雄激素受体(androgen receptor,AR)通路。本研究拟检测前列腺癌lncRNA PCGEM1和AR的表达情况,探讨其在前列腺癌中的表达及意义。方法：构建RNA核酸探针,应用荧光原位杂交(lfuores-cencein situ hybridization,FISH)技术检测前列腺癌lncRNA PCGEM1的表达情况,并使用荧光免疫组织化学技术检测前列腺癌AR的表达;采用RNA-pull down技术检测PCGEM1和AR的共同作用。结果：依赖AR的LNCaP细胞的PCGEM1表达水平显著高于非AR依赖性的PC3和DU145细胞,差异有统计学意义(P<0.01)。前列腺癌组织中PCGEM1的表达与AR表达程度显著相关。与良性前列腺肿瘤相比,转移性肿瘤中的PCGEM1细胞核内定位显著不同,雄激素去势显著影响PCGEM1的表达及细胞内定位。而共定位检测发现,PCGEM1与AR之间存在一定程度的共定位现象。结论：本研究通过FISH研究发现,PCGEM1表达在雄激素去势条件下显著上调,而在转移性前列腺癌中,PCGEM1和AR的共定位表达表明,lncRNA PCGEM1和AR相互作用可能共同参与调控前列腺癌的恶性进展及转移。     

前列腺癌患者血清睾酮水平、前列腺雄激素受体表达与精索静脉曲张相关研究

目的：研究血清睾酮水平、前列腺雄激素受体（AR）表达与精索静脉曲张的相关性,为临床前列腺疾病诊治提供一定理论依据。方法选取未经药物治疗的64例前列腺癌患者,其中实验组为32例前列腺癌合并精索静脉曲张患者,对照组为32例前列腺癌不伴精索静脉曲张患者,记录两组患者一般情况,并行血清睾酮水平、前列腺雄激素受体检测及病理组织学检查。结果对照组血清睾酮水平为（5.89±1.32）ng/ml,实验组血清睾酮值为（6.07±1.16）ng/ml,差异无统计学意义（P﹥0.05）。实验组前列腺雄激素受体的阳性表达率为62.5%,低于对照组84.4%,差异有统计学意义（P﹤0.05）。AR的表达均与伴精索静脉曲张前列腺癌分期呈负相关（r=-0.318, P﹤0.05）。结论前列腺雄激素受体表达在前列腺癌精索静脉曲张的发病及病情进展中发挥了重要的作用。     

雄激素受体基因微卫星多态性与前列腺癌的生物学行为

目的探讨雄激素受体(AR)基因CAG微卫星多态性与前列腺癌(PC)生物学行为的关系.方法采用聚合酶链反应-单链构象多态性分析法(PCR-SSCP),对37例中国人、57例美国白人以及19例美国黑人PC患者共113例PC标本,行AR基因CAG微卫星数量测定,并进行不同病理期别、细胞分化级别以及种族间的比较.结果PC病变C期和D期的AR基因CAG数量均少于B期,其均数分别为中国人22.14±2 64与25.04±1.88(P＜0.01)、美国白人21.97±3.31与23.74±2.49(P＜0.05)、美国黑人19.46±3.14与21.75±1.10(P＜0.05),差异显著.短的AR基因CAG(数量＜22)的分布分别是中国人B期病变占13.0%(3/23)、C～D期28.6%(4/14),美国白人B期14.8%(4/27)、C～D期36.7%(11/30),美国黑人B期25.0%(1/4)、C～D期80.0%(12/15);其长的AR基因CAG(数量≥22)的分布比例与之相反,差异显著(P＜0.05).AR基因CAG数量随着肿瘤细胞分化程度的降低而减少,在高、中、低分化PC中其均数分别为中国人25.60±1.67、24.29±3.45、23.64±2.56;美国白人23.81±2.81、23.11±1.79、21.44±4.03;美国黑人21.00±0.00、21.33±0.47、19.57±2.56,其高分化与低分化PC之间在中国人和美国白人患者中均有显著性差异(P＜0.05).短的AR基因CAG在高、中、低分化PC的分布分别是中国患者0%(0/5)、28.6%(2/7)、60.0%(15/25);美国白人14.3%(3/21)、22.2%(4/18)、50.0%(9/18);美国黑人0%(0/2)、33.3%(1/3)、71.4%(10/14),而长的AR基因CAG分布结果则相反,其差别在低分化与高分化组间存在显著性差异(P＜0.05).美国黑人、白人前列腺癌患者肿瘤病变及分化程度比较显示美国黑人患者中病变外侵或转移(C～D期)达78.9%(15/19),多于白人患者的52.63%(30/57);其低分化肿瘤占73.7%(14/19),多于白人患者的31.6%(18/57);两者AR基因CAG均数相比,美国黑人患者(19.89±2.95)明显少于白人(22.95±2.97).结论AR基因CAG微卫星的多寡可能是构成前列腺癌不同生物学特性的因素之一.含有短雄激素受体基因CAG微卫星的前列腺癌可具有较高的侵袭特性.     

5-氮杂-2’-脱氧胞苷对人前列腺癌裸鼠移植瘤生长及雄激素受体表达影响的研究

目的探讨5-氮杂-2’-脱氧胞苷(5-Aza-2’deoxycytidine,5-Aza-CdR)对LNCaP荷瘤裸鼠的肿瘤生长及雄激素受体(androgen receptor,AR)表达的影响。方法裸鼠皮下接种LNCaP细胞,建立人前列腺癌裸鼠移植瘤模型,待肿瘤长至300 mm3大小时,荷瘤裸鼠腹腔内注射5-Aza-CdR,0.25 mg/kg,隔天1次。对照组给予生理盐水注射。每3天测量肿瘤体积1次,共观察24天。实验结束时处死裸鼠,取出肿块称重后,肿瘤组织抽提RNA和蛋白质,分别应用实时定量PCR和western blot方法,检测AR在mRNA和蛋白质水平上的表达。结果 LNCaP荷瘤裸鼠经5-Aza-CdR治疗后,与对照组相比,肿瘤生长缓慢。第24天观察结束时,5-Aza-CdR处理组肿瘤体积[(982±83)mm3]显著低于对照组[(1 867±195)mm3](P<0.01)。移植瘤重量与对照组相比明显下降[(796±178)mg vs(1 267±252)mg],抑瘤率为37.2%,差异有统计学意义(P<0.01)。5-Aza-CdR组游离PSA(FPSA)浓度相对对照组显著下降,分别为(40.25±13.3)ng/mL和(79.7±13.2)ng/mL,差异有统计学意义(P<0.01)。荷瘤裸鼠经5-Aza-CdR处理后,AR在mRNA水平上的表达下降为对照组的0.5倍,在蛋白质水平上的表达也显著降低(P<0.01)。结论 5-Aza-CdR能够有效抑制LN-CaP荷瘤裸鼠移植瘤的生长,并显著降低AR基因表达,为前列腺癌的去甲基化治疗提供了理论依据。     

雄激素阻断治疗后前列腺癌组织中肿瘤干细胞比例变化

目的 研究雄激素阻断治疗（androgen deprivation therapy ,ADT）对前列腺癌组织中肿瘤干细胞比例的影响,探讨激素非依赖性前列腺癌形成的机制。方法 实验分为ADT治疗组和无ADT治疗组,应用免疫荧光技术比较两组前列腺癌患者术前术后标本中肿瘤干细胞的比例变化。结果 无ADT组术前穿刺标本和前列腺根治切除术后标本肿瘤干细胞比例差异无统计学意义[（3.56±1.33）%vs. （3.78±1.39）%, n=9, t=-0.686, P=0.512] ,在ADT组,接受雄激素阻断治疗3月后,与穿刺标本比较,根治切除标本中前列腺癌肿瘤干细胞比例明显升高[（3.44±1.81）% vs. （9.22±1.71）% ,n=9,t=-6.353, P=0.000]。在癌旁组织,也可见ADT后干细胞标志的增加。结论 雄激素可能参与前列腺和前列腺癌组织中干细胞的分化;雄激素非依赖性前列腺癌的形成可能与前列腺癌肿瘤干细胞有关。     

胃癌雄激素受体的研究

自1977年Kiang等[1]在胃癌组织中首次发现雌激素受体(ER)后,人们开始逐渐发现性激素受体不仅在性激素依赖器官中存在[2],后来又发现胃癌组织中存在孕激素受体(PGR)以及雄激素受体(AR).近年来有关非性激素靶器官肿瘤胃癌中ER、PGR和AR的研究较多.本文对胃癌中AR的表达作用机制、检测方法以及其与胃癌的临床生物学行为的相关性进行综述.     

Role of SRC-1 in the promotion of prostate cancer cell growth and tumor progression

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor-negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.     

Programmed cell death during regression of PC-82 human prostate cancer following androgen ablation

To study the mechanism of regression of human prostatic cancer following androgen ablation, the androgen-responsive PC-82 human prostatic adenocarcinoma xenograft was used as a model system. Castration of male nude mice bearing PC-82 xenografts results in a 50% tumor regression by 2 wk following androgen ablation. This regression is due to a sequence of biochemical and morphological events that results in both the cessation of cell proliferation and activation of programmed death or apoptosis of the androgen-dependent prostatic cancer cells. Associated with this response are an enhanced expression of the transforming growth factor beta 1 gene, a potent inhibitor of cell proliferation, and testosterone-repressed prostatic message 2 (designated TRPM-2), a programmed cell death-associated gene. Fragmentation of tumor DNA into nucleosomal oligomers and histological appearance of apoptotic bodies are characteristic early events that preceded the dramatic reduction in tumor volume following androgen ablation. These results suggest that androgen-dependent human prostatic cancer cells, like normal prostatic cells, retain the ability to inhibit proliferation and to activate programmed cell death in response to androgen ablation. Clarification of the biochemical pathway involved in the activation of this programmed cell death should identify new targets of therapy for even androgen-independent human prostatic cancer.     

Programmed death of nonproliferating androgen-independent prostatic cancer cells

Androgen ablation induces an energy-dependent process of programmed death in nonproliferating androgen-dependent prostatic cancer cells which involves fragmentation of genomic DNA into nucleosomal oligomers catalyzed by nuclear Ca2+, Mg(2+)-dependent endonuclease enzymes activated following a sustained elevation in intracellular free Ca2+ (Cai). In contrast, androgen-independent prostatic cancer cells are not induced to undergo such programmed cell death by androgen ablation. One explanation for the inability of androgen ablation to induce programmed death of androgen-independent prostatic cancer cells is that such ablation does not result in a sustained elevation in Cai in these cells. This raises the issue of whether androgen-independent prostatic cancer cells can be induced to undergo programmed death if an elevation in the Cai is sufficiently sustained by nonhormonal means. To test this possibility, androgen-independent, highly metastatic Dunning R-3327 AT-3 rat prostatic cancer cells were chronically exposed in vitro to the calcium ionophore ionomycin to sustain an elevation in their Cai. These studies demonstrated that an elevation of Cai as small as only 3-6-fold above baseline can induce the death of these cells if sustained for greater than 12 h. Temporal analysis demonstrated that the death of these cells does not require cell proliferation and involves Ca(2+)-induced fragmentation of genomic DNA into nucleosome-sized pieces as the commitment step in this process. These results demonstrate that even nonproliferating androgen-independent prostatic cancer cells can be induced to undergo programmed cell death if a modest elevation in the Cai is sustained for a sufficient time. These observations identify Cai as a potential target for therapy for androgen-independent prostatic cancer cells.     

Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor

This study discovered that Id-1 expression in androgen-dependent prostate cancer decreased immediately after androgen deprivation but increased after longer androgen deprivation both in vivo and in vitro. Id-1 expression in androgen-independent LNCaP cells was about 6 fold as that in their parental cells. As was the case with LNCaP cells, when androgen receptor (AR) was introduced into AR-negative PC-3 cells, dihydrotestosterone inhibited while flutamide increased Id-1 expression. Thus, Id-1 expression in androgen-dependent prostate cancer was negatively regulated by androgen in a receptor-dependent way. The re-increased Id-1 might partially contribute to the emergence of androgen-independent prostate cancer after longer androgen deprivation therapy.     

Androgen receptor-dependent PSA expression in androgen-independent prostate cancer cells does not involve androgen receptor occupancy of the PSA locus

It is widely suspected that androgen-independent prostate cancer growth depends on androgen receptor signaling via ill-defined mechanisms. Prostate-specific antigen (PSA) expression is often used to measure androgen receptor activity in cells and prostate cancer progression in patients. In the present study, we have compared androgen receptor activity using PSA and human male germ cell-associated kinase (hMAK), as read-outs in androgen-dependent LNCaP and androgen-independent C4-2B cells. As expected, very little PSA and hMAK expression were detected in LNCaP cells in the absence of androgens, whereas substantial expression of PSA was observed only in C4-2B cells under the same conditions. The addition of dihydrotestosterone to the culture medium increased the expression of both genes in both cell types. Comprehensive chromatin immunoprecipitation analysis of the entire PSA locus and an androgen-response element in hMAK unexpectedly revealed that androgen receptor was not occupying any site in the absence of dihydrotestosterone in either cell type. In line with the expression data, and in the absence of dihydrotestosterone, histone acetylation and RNA polymerase II occupancy was substantial at the PSA locus in C4-2B but not in LNCaP cells. In the presence of dihydrotestosterone, androgen receptor was found to occupy mainly the enhancer region of PSA in both cell types, accompanied with increases in histone acetylation and RNA polymerase II occupancy. Although the androgen receptor was not directly involved in the androgen-independent expression of PSA in C4-2B cells, small interfering RNA knock-down of androgen receptor significantly reduced PSA expression in both the presence and absence of dihydrotestosterone. In contrast, hMAK expression was decreased only in the presence of dihydrotestosterone after androgen receptor knock-down. We conclude that androgen-independent expression of PSA in C4-2B cells does not rely on the direct occupancy of the androgen receptor at the PSA locus, but is nevertheless affected indirectly via unknown androgen receptor-dependent mechanism(s) that influence the expression from some but not all androgen receptor target genes.     

In vivo effects of the human type I insulin-like growth factor receptor antibody A12 on androgen-dependent and androgen-independent xenograft human prostate tumors.

PURPOSE: The type I insulin-like growth factor receptor (IGF-IR) and its ligands have been shown to play a critical role in prostate carcinoma development, growth, and metastasis. Targeting the IGF-IR may be a potential treatment for prostate cancer. A fully human monoclonal antibody, A12, specific to IGF-IR, has shown potent antitumor effects in breast, colon, and pancreatic cancers in vitro and in vivo. In this study, we tested the in vivo effects of A12 on androgen-dependent and androgen-independent prostate tumor growth. EXPERIMENTAL DESIGN: Androgen-dependent LuCaP 35 and androgen-independent LuCaP 35V prostate tumors were implanted s.c. into intact and castrated severe combined immunodeficient mice, respectively. When tumor volume reached about 150 to 200 mm(3), A12 was injected at 40 mg/kg body weight thrice a week for up to 5 weeks. RESULTS: We find that A12 significantly inhibits growth of androgen-dependent LuCaP 35 and androgen-independent LuCaP 35V prostate xenografts, however, by different mechanisms. In LuCaP 35 xenografts, A12 treatment induces tumor cell apoptosis or G(1) cycle arrest. In LuCaP 35V xenografts, A12 treatment induces tumor cell G(2)-M cycle arrest. Moreover, we find that blocking the function of IGF-IR down-regulates androgen-regulated gene expression in androgen-independent LuCaP 35V tumor cells. CONCLUSIONS: Our findings suggest that A12 is a therapeutic candidate for both androgen-dependent and androgen-independent prostate cancer. Our findings also suggest an IGF-IR-dependent activity of the androgen receptor in androgen-independent prostate cancer cells.     

Inhibition of interleukin-6 with CNTO328, an anti-interleukin-6 monoclonal antibody, inhibits conversion of androgen-dependent prostate cancer to an androgen-independent phenotype in orchiectomized mice

Initially, prostate cancer is androgen dependent. However, most cases progress to an androgen-independent state through unknown mechanisms. Interleukin-6 (IL-6) has been associated with prostate cancer progression including activation of the androgen receptor (AR). To determine if IL-6 plays a role in the conversion of prostate cancer from androgen dependent to androgen independent, we established androgen-dependent LuCaP 35 human prostate cancer xenografts in nude mice, castrated the mice, and blocked IL-6 activity using a neutralizing antibody (CNT0328) for a period of 18 weeks. IL-6 inhibition increased survival of mice and inhibited tumor growth, as reflected by decreased tumor volume and prostate-specific antigen levels, compared with that in mice receiving isotype control antibody. To test the effect of IL-6 inhibition on the conversion from androgen dependent to androgen independent, tumor cells from the treated mice were assessed for their androgen dependence both in vitro and by implanting them into sham-operated or orchiectomized mice. Tumor cells derived from the isotype-treated animals converted to androgen-independent state, whereas tumor cells from the anti-IL-6 antibody-treated mice were still androgen dependent in vitro and in vivo. Although there was no difference in AR levels between the androgen-independent and androgen-dependent tumors, IL-6 inhibition promoted both apoptosis and inhibited cell proliferation in tumors and blocked the orchiectomy-induced expression of histone acetylases, p300 and CBP, which are AR cofactors. These data show that IL-6 contributes to the development of androgen independence in prostate cancer and suggest that it mediates this effect, in part, through modulation of p300 and CBP.