Analysis of vitamin D-regulated gene expression in LNCaP human prostate cancer cells using cDNA microarrays

BACKGROUND: 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] exerts growth inhibitory, pro-differentiating, and pro-apoptotic effects on prostate cells. To better understand the molecular mechanisms underlying these actions, we employed cDNA microarrays to study 1,25(OH)2D3-regulated gene expression in the LNCaP human prostate cancer cells. METHODS: mRNA isolated from LNCaP cells treated with vehicle or 50 nM 1,25(OH)2D3 for various lengths of time were hybridized to microarrays carrying approximately 23,000 genes. Some of the putative target genes revealed by the microarray analysis were verified by real-time PCR assays. RESULTS: 1,25(OH)2D3 most substantially increased the expression of the insulin-like growth factor binding protein-3 (IGFBP-3) gene. Our analysis also revealed several novel 1,25(OH)2D3-responsive genes. Interestingly, some of the key genes regulated by 1,25(OH)2D3 are also androgen-responsive genes. 1,25(OH)2D3 also down-regulated genes that mediate androgen catabolism. CONCLUSIONS: The putative 1,25(OH)2D3 target genes appear to be involved in a variety of cellular functions including growth regulation, differentiation, membrane transport, cell-cell and cell-matrix interactions, DNA repair, and inhibition of metastasis. The up-regulation of IGFBP-3 gene has been shown to be crucial in 1,25(OH)2D3-mediated inhibition of LNCaP cell growth. 1,25(OH)2D3 regulation of androgen-responsive genes as well as genes involved in androgen catabolism suggests that there are interactions between 1,25(OH)2D3 and androgen signaling pathways in LNCaP cells. Further studies on the role of these genes and others in mediating the anti-cancer effects of 1,25(OH)2D3 may lead to better approaches to the prevention and treatment of prostate cancer.     

Interleukin-4 stimulates androgen-independent growth in LNCaP human prostate cancer cells

BACKGROUND: Clinical data showed that the levels of interleukin-4 (IL-4) are significantly elevated in serum of patients with ablation resistant prostate cancer. Previous studies demonstrated that IL-4 enhances androgen receptor (AR) activation mediated by NF-kappaB in the absence or in the very low levels of androgen in prostate cancer cells. In this study, the role of IL-4 in promoting the growth of androgen-independent prostate cancer cells was examined. METHODS: LNCaP cells were transfected with a full-length IL-4 cDNA and stable clones expressing IL-4 were selected. The growth of LNCaP cells expressing IL-4 was analyzed in vitro and in vivo both in the presence and absence of androgen. RESULTS: Overexpression of IL-4 enhances the growth of androgen-sensitive LNCaP cells in culture media containing charcoal-stripped FBS condition (CS-FBC), and increases the sensitivity of LNCaP cells in response to androgen stimulation. The DHT-mediated cell growth could not be blocked by bicalutamide in IL-4 overexpressing LNCaP cells, but can be neutralized by bicalutamide in parental LNCaP and neo control cells. Furthermore, overexpression of IL-4 stimulates tumor growth of androgen-sensitive LNCaP cells both in intact and castrated male mice. CONCLUSIONS: Overexpression of IL-4 increases the sensitivity of androgen-sensitive LNCaP prostate cancer cells in response to androgen stimulation and enhances the growth of LNCaP cells both in the presence and absence of androgen in vitro and in vivo. These studies suggest that IL-4 plays an important role in promoting androgen-independent prostate cancer growth.     

Emodin induces apoptosis in human prostate cancer cell LNCaP

AIM: To elucidate effects and mechanisms of emodin in prostate cancer cells. METHODS: Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor (AR), prostate-specific antigen (PSA), p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. RESULTS: In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax /Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. CONCLUSION: In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.     

Efficient DNA-mediated gene transfer into prostate cancer cell line LNCaP

BACKGROUND: Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in men. In the beginning stages of the disease, prostate cancer is dependent on androgens for growth. The only common and readily available cell line to study this phase of prostate cancer in vitro is LNCaP, which was originally derived from a lymph node metastatis of a human prostatic adenocarcinoma. However, DNA-mediated gene transfer, a common and key procedure in cellular and molecular studies, is very inefficient for LNCaP cells, and this limits the utility of these cells in investigations of the molecular mechanisms of prostate carcinogenesis. METHODS: In search of a simple, reproducible, and cost-effective method for introducing DNA into LNCaP cells, we adopted and optimized two methods of transient transfection into LNCaP cells: a modified calcium phosphate (CaPO(4)) coprecipitation procedure and polyethylenimine (pEI)-mediated transfection. RESULTS: When compared with the liposome-mediated transfection that was previously used for LNCaP cells, we find that the most efficient of these techniques is the modified CaPO(4) coprecipitation procedure. For experiments in which calcium exposure of the cells is not desirable, the pEI procedure provides a less efficient, but reproducible and cost-effective alternative. CONCLUSIONS: These two new DNA-mediated gene transfer methods should facilitate gene expression studies in LNCaP cells and thereby aid in the study of the androgen-dependent phase of prostate cancer.     

Suicide gene therapy for prostate cancer using a replication-deficient adenovirus containing the herpesvirus thymidine kinase gene

Current therapies for localized prostate cancer include radical prostatectomy, local radiation therapy, and cryoablation and are associated with a high rate of cure and acceptable morbidity. However, for men who have failed primary curative attempts or have metastatic disease, no effective therapy associated with acceptable morbidity exists. “Suicide” gene therapy delivered alone or in combination with other forms of treatment could potentially provide simultaneous efficacy against localized and systemic disease via the generation of cytotoxic activity and/or systemic immunity to the cancer. In this article we discuss our preclinical and clinical experience with a herpes-simplex-virus thymidine kinase/ganciclovir gene-therapy protocol for prostate cancer.     

环巴胺对人前列腺癌LNCaP细胞增殖和凋亡作用及对PCA3基因表达的影响

目的:探讨环巴胺对人前列腺癌LNCaP细胞增殖和凋亡的作用及对PCA3基因表达的影响。方法:不同浓度环巴胺(1、5、10、15μmol/L)干预LNCaP细胞,以只加入RPMI 1640培养液为空白对照组,分别在不同作用时间(24、48、72h),用MTT法检测其对细胞增殖的抑制、流式细胞术观察细胞凋亡率变化、Hoechst染色观察凋亡细胞形态变化、实时荧光定量逆转录聚合酶链反应(FQ-RT-PCR)观察对PCA3基因表达的影响。结果:5、10、15μmol/L环巴胺对LNCaP细胞增殖均有显著抑制作用,与空白对照组相比差异有显著性(P0.01),10μmol/L组于48h达到半数抑制量(IC50);10、15μmol/L组24、48、72hLNCaP细胞的凋亡率分别为37.21%、57.38%、57.98%和21.16%、71.31%、72.90%,与空白对照组相比差异均有显著性(P0.01)。随着环巴胺浓度增大和作用时间的延长,LNCaP细胞凋亡显著增加。PCA3基因的表达随着环巴胺浓度的上升呈现明显的递减趋势,较空白对照组显著降低(P0.01)。浓度为10μmol/L时,不同作用时间PCA3基因的表达均极低。结论:环巴胺浓度为10、15μmol/L作用48、72h时能够明显抑制LNCaP细胞的增殖,诱导细胞凋亡,并显著下调LNCaP细胞PCA3基因的表达。     

Induction of apoptosis by mifepristone and tamoxifen in human LNCaP prostate cancer cells in culture

BACKGROUND: Published data indicate that antiprogestins and antiestrogens could inhibit prostate cancer cell growth in vitro and in vivo. The main objective of the present studies was to explore the role of bcl(2) and TGFbeta(1) for induction of apoptosis in LNCaP prostate cancer cells growing in culture as a treatment response to the antiprogestin, mifepristone, and the antiestrogen, 4-hydroxytamoxifen. METHODS: In vitro cell viability (cytotoxicity), DNA fragmentation, and changes in the expression of bcl(2) and TGFbeta(1) proteins were assessed using the sulforhodamine B protein dye-binding assay, specific ELISA, and competitive inhibition assays. RESULTS: Both steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of viable cells was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl(2), and induction of TGFbeta(1) protein. Abrogation of the mifepristone- and 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta(1)-neutralizing antibody and by the addition of mannose-6-phosphate confirmed the correlation between induction of active TGFbeta(1) and subsequent prostate cancer cell death. The effect of mifepristone was not significantly reduced or prevented by occupying the progesterone or glucocorticoid receptors by their corresponding high-affinity native ligands. On the contrary, the effect of a combination of mifepristone with progesterone or hydrocortisone on the increase in DNA fragmentation, bcl(2) downregulation, and induction of TGFbeta(1) protein was additive and significantly different (P < 0.05) from the effect of mifepristone monotherapy. CONCLUSIONS: Our data suggest that mifepristone and tamoxifen are effective inducers of apoptosis and may represent nonandrogen-ablation, novel therapeutic approaches to overcome a potential intrinsic apoptosis resistance of androgen-independent prostate cancer cells.     

Suicide gene therapy toxicity after multiple and repeat injections in patients with localized prostate cancer

PURPOSE: We assess risks, toxicity and side effects of multiple and repeat in situ suicide gene therapy in patients with localized prostate cancer. MATERIALS AND METHODS: The study population comprised patients with localized prostate cancer receiving multiple and/or repeat intraprostatic injections of a replication deficient adenovirus containing the herpes simplex virus thymidine kinase (HSV-tk) gene. Intravenous ganciclovir or oral valaciclovir was given for 14 days after injection. Patients were recruited from 4 different clinical protocols in studies of toxicity and efficacy of suicide gene therapy, and closely monitored for toxicity and side effects during and after treatment. Toxicity was graded according to the Cancer Therapy Evaluation Program common toxicity criteria published by the National Cancer Institute. RESULTS: A total of 52 patients were treated under these clinical protocols with a total of 76 gene therapy cycles. Toxic events were recorded in 16 of 29 patients (55.2%) who were given multiple viral injections into the prostate, 7 of 20 (35%) who received 2 cycles of "suicide" gene therapy and 3 of 4 (75%) who received a third course of gene therapy. All toxic events after multiple or repeat injections were mild (grades 1 to 2) and resolved completely once the therapy course was terminated. No additive toxicity was noted in patients receiving repeat gene therapy cycles. Mean followup was 12.8 months (range 3 to 34). Preliminary results for 28 patients in 2 clinical protocols indicated a mean decrease of 44% in PSA in 43%. CONCLUSIONS: Direct injection into the prostate of a replication defective adenovirus containing the HSV-tk gene followed by intravenous ganciclovir is safe even in repeat cycles.     

High-malignancy orthotopic nude mouse model of human prostate cancer LNCaP.

BACKGROUND: An animal model of human prostate cancer LNCaP demonstrating high rates of spontaneous metastasis from the orthotopic site after tumor implantation would be very valuable for mechanistic and drug discovery studies. We previously developed microsurgical techniques to implant histologically intact tumor tissues orthotopically in nude mice in order to develop high metastatic mouse models of human cancer. METHODS: Intact tissue of the androgen-dependent human prostate cancer cell line, LNCaP, was implanted on the ventral lateral lobes of the prostate gland by surgical orthotopic implantation (SOI) in a series of 20 nude mice. Mice were autopsied, and histopathological examination of primary tumors and relevant organs was done to identify and quantitate micrometastasis. RESULTS: Eighteen of 20 animals transplanted with LNCaP by SOI had tumor growth. Mean primary tumor weight in the prostate was 9.24 g at time of necropsy. Sixty-one percent of the transplanted animals had lymph node metastasis. Forty-four percent had lung metastasis. Mean survival time was 72 days, indicating a high degree of malignancy of the tumor. CONCLUSIONS: The extensive and widespread lung metastasis as well as lymph node metastasis following orthotopic implantation of LNCaP in nude mice and the short survival time provide a high-malignancy nude model of the LNCaP human prostate tumor.     

p16基因对前列腺癌细胞的体外作用研究

目的：研究可控性表达的p16基因对前列腺癌细胞的生长抑制作用。方法：通过可诱导的真核表达载体pMDNA-3-p16将外源野生型p16基因转染至该基因表达功能丧失的人前列腺癌细胞PC3中,使用ZnSO4作为诱导物,观察p16基因的可控性表达及其对前列腺癌细胞生物学特性的影响。 结果：转染了野生型p16基因的人膀胱癌细胞PC3-pMDNA3-p16经ZnSO4诱导后,开放了p16基因的转录和翻译,p16蛋白得到高水平表达,细胞生长速度及集落形成率均有了部分抑制,细胞周期分析可见G1期增加,S期下降。结论：P16基因与前列腺癌的发生、发展有关,为用p16基因进行前列腺癌的基因治疗提供了实验依据。     

Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model

BACKGROUND: The acquisition of an androgen-independent phenotype is the most serious issue of prostate cancer treatment. Although several experimental cell models have been reported for studying androgen independence, they have limited applications related to hormone-refractory prostate cancer. To investigate the molecular mechanism of androgen-independent growth of prostate cancer, we established a useful LNCaP cell model that resembles the clinical scenario of hormone-refractory prostate cancer. METHODS: Androgen-sensitive LNCaP parental cells were continuously maintained in a regular cell-culture medium, that is, phenol red-positive RPMI 1640 medium supplemented with 5% fetal bovine serum and 1% glutamine. Upon passage, the androgen responsiveness of those cells decreased, to a level lower than that of parental cells. We examined the growth properties and androgen responsiveness of these different LNCaP cells in vitro and in vivo. Cytogenetic characteristics and expression of androgen receptors (ARs) and prostate-specific antigen (PSA) were determined. RESULTS: Upon continuous passage, the biological behavior of parental C-33 cells (passage number less than 33) was altered. C-81 cells (passage number higher than 81) clearly exhibited more aggressive growth and lower androgen responsiveness than C-33 and C-51 cells (passage number between 35 and 80) in vitro and in vivo. Nevertheless, all these cells expressed a similar level of functional AR protein as well as a similar genetic profile. Moreover, in a steroid-reduced culture condition, C-81 cells secreted a higher level of PSA than C-33 cells. CONCLUSIONS: Our LNCaP cell model closely recapitulates the progression of human prostate cancer from the androgen-responsive to the hormone-refractory state under the androgen nondeprived condition. This cell model may provide the opportunity to understand the molecular mechanisms associated with the acquisition of androgen independence during human prostate cancer progression.     

9-顺维甲酸对前列腺癌细胞系LNCaP中同源盒基因NKX3.1表达的影响(英文)

目的:研究9-顺维甲酸对前列腺癌细胞系 LNCaP 中同源盒基因 NKX3.1表达的调节作用。方法:采用流式细胞术、反转录 PCR 和 Western Blot 技术检测9-顺维甲酸对 LNCaP 细胞周期及 NKX3.1表达的影响;构建和转染 NKX3.1启动子-报告基因质粒及其缺失突变体,通过报告基因活性测定,鉴定 NKX3.1启动子中受9顺-维甲酸调控的区域。结果:通过转染及报告基因检测,发现9-顺维甲酸在 LNCaP 细胞中可明显提高 NKX3.1启动子的活性:RT-PCR 和 Western Blot 结果显示,9-顺维甲酸可提高 NKX3.1mRNA 和蛋白的表达,并呈剂量依赖性;通过启动子缺失突变分析,发现 NKX3.1基因上游-936至-921区明显受9-顺维甲酸诱导调节;流式细胞术分析细胞周期的结果显示,9-顺维甲酸可阻止 LNCaP 细胞于 G_1期,减少 G_2/M 期细胞。结论:9-顺维甲酸作为诱导分化剂可阻止 LNCaP 细胞于 G_1期,减少细胞有丝分裂,并明显上调前列腺特异的抑癌基因 NKX3.1的表达。     

C16 ceramide accumulates following androgen ablation in LNCaP prostate cancer cells

BACKGROUND: Adenocarcinoma of the prostate is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related deaths among men in the United States. The most successful therapies to date for this tumor have involved some form of androgen ablation. However, these therapies become ineffective as the tumor evolves to an androgen-insensitive state. Ceramide is a lipid second messenger that has been shown to mediate growth arrest or cell death when added exogenously to prostate cancer cells. As a first step toward understanding the events that lead to the transition of prostate cancer cells to an androgen-independent state, we considered investigating the effect of androgen ablation on endogenous ceramide levels in androgen-sensitive and androgen-insensitive prostate cancer cells. METHODS: To investigate the mechanisms of growth arrest/apoptosis in androgen-sensitive (LNCaP) and insensitive (DU-145, PC-3) cells, we used various methods including nonyl acridine orange (NAO) staining, propidium iodide (PI) staining/cell-cycle analysis, lipid analysis, and Western blotting assays. RESULTS: In this study, we demonstrate that androgen ablation drives G(0)/G(1)-phase cell-cycle arrest followed by progressive apoptosis in vitro, in LNCaP cells. Lipid analysis indicated an increase in C16 ceramide, which was generated via the de novo pathway as revealed by blockade of ceramide synthase by fumonisin B1. The addition of 5alpha-dihydrotestosterone (DHT) or fumonisin B1 rescued LNCaP cells from apoptosis induced by androgen ablation, and decreased levels of intracellular C16 ceramide. Neither apoptosis nor an increase in C16 ceramide was observed in androgen-independent cell lines following androgen ablation.