Stable expression of full length human androgen receptor in PC-3 prostate cancer cells enhances sensitivity to retinoic acid but not to 1alpha,25-dihydroxyvitamin D3

BACKGROUND: PC-3 prostate cancer cell growth is inhibited by 1alpha,25-dihydroxyvitamin D(3) (1,25 D) and retinoids, but not to the same extent as the androgen receptor (AR) positive LNCaP prostate cancer cells. Previous reports suggest a role for AR in growth inhibition of LNCaP cells by 1,25 D and retinoids. PC-3 cells do not express AR. We therefore asked whether re-expression of AR would enhance the response of PC-3 cells to 1,25 D and retinoids. METHODS: PC-3 cells were stably transfected with wild type human AR cDNA. Pooled cells expressing AR protein at levels comparable to LNCaP cells were used to analyze response to 1,25 D, retinoids, androgens, and anti-androgens. RESULTS: AR re-expression in PC-3 cells restored response to androgens and anti-androgens, but growth inhibition by 1,25 D was not significantly altered. However, cells were sensitized to low levels of retinoids, and, in contrast to the parental PC-3 cells, sub-optimal levels of 1,25 D and retinoids caused additive growth inhibition. CONCLUSIONS: Restoring AR expression and activity in PC-3 cells results in enhanced sensitivity to low levels of retinoids while the response to 1,25 D remains unaltered. Thus, the involvement of AR in prostate cancer growth inhibition by 1,25 D appears to be cell line specific.     

Differential localization of MT1-MMP in human prostate cancer tissue: role of IGF-1R in MT1-MMP expression

BACKGROUND: MT1-MMP is a metalloproteinase involved in prostate cancer metastasis. The IGF-1R is a tyrosine kinase receptor involved with tumor progression and metastasis. The purpose of this investigation was to examine MT1-MMP and IGF-1R expression and localization in prostate cancer tissues and explore the role of IGF-1R in regulating MT1-MMP in prostate cancer cell lines. METHODS: Immunohistochemistry was utilized to study MT1-MMP and IGF-1R expression in human prostate tissues. IGF-1R regulation of MT1-MMP expression was determined by gene promoter analysis, quantitative RT-PCR and Western blot analysis following pharmacological inhibition of the receptor in PC-3N cells and treatment of LNCaP cells with androgen and IGF-1. RESULTS: MT1-MMP expression was high in the apical regions of the luminal cells in PIN and prostate cancer and less intense in the basalateral regions of benign tissues. IGF-1R was expressed primarily in the basal cells of normal glands and highly expressed in prostate cancer. Inhibition of IGF-1R in PC-3N cells decreased MT1-MMP expression and treatment of LNCaP cells with a synthetic androgen and IGF-1 increased MT1-MMP expression. CONCLUSIONS: These data demonstrate that MT1-MMP is highly expressed in the apical cytoplasmic regions of the luminal cells in PIN and prostate cancer when compared to basalateral cytoplasmic membrane staining in benign glands. Additionally, we demonstrate that IGF-1R is highly expressed in human prostate carcinoma. These findings suggest that MT1-MMP localization and IGF-1R expression in prostate carcinoma could be predictive biomarkers for aggressive disease and support IGF-1R as a promising therapeutic target to decrease processes of prostate cancer metastasis.     

The androgen receptor pathway is by-passed in prostate cancer cells generated after prolonged treatment with bicalutamide

BACKGROUND: Experimental work in various prostate cancer models revealed that the androgen receptor is frequently upregulated and implicated in tumor progression. However, little attention has been paid to the androgen receptor-signaling pathway in the development of therapy resistance in patients who receive chronic treatment with a non-steroidal anti-androgen. METHODS: We have generated a novel subline, LNCaP-Bic, after prolonged treatment with androgen and bicalutamide in vitro. Proliferation of LNCaP-Bic cells in the absence or presence of androgen, tocopherol succinate, and/or bicalutamide was assessed by cell counting. Androgen receptor expression was determined by Western blot. Luciferase activity was measured in cells transfected with an androgen-responsive reporter. RESULTS: In basal conditions, proliferation of LNCaP-Bic cells increased more than threefold over that of control LNCaP cells. Neither synthetic androgen R1881 nor bicalutamide showed any effect on LNCaP-Bic growth in vitro. Androgen receptor expression did not differ between the cell subline generated in the presence of bicalutamide and parental LNCaP cells. The ability of R1881 to induce reporter gene activity in LNCaP-Bic cells was reduced by 56%. Tocopherol succinate caused inhibition of proliferation only in the parental cell line although the androgen receptor and prostate-specific antigen were down regulated by the vitamin E derivative in both parental LNCaP and LNCaP-Bic cells. CONCLUSIONS: Androgen receptor-mediated signal transaction is not enhanced in cells selected in the presence of bicalutamide. Our data may suggest that a more differentiated approach in targeting the androgen receptor is needed in prostate cancers that become resistant to classic endocrine treatment.     

Androgens stimulate both growth rate and epidermal growth factor receptor activity of the human prostate tumor cell LNCaP

LNCaP cells (derived from a lymph node carcinoma of the human prostate) contain androgen receptors and show androgen-responsive growth in vitro. Maximal effects on growth were seen at 0.1 nM of the synthetic androgen R1881 or 0.2 nM of epidermal growth factor (EGF); both compounds independently increased the growth rate 2-3 times. EGF-receptors were measured after 6 days culture in the presence or absence of 0.1 nM R1881. A 2.3-fold increase in receptor number/cell was found when binding was measured at 0 degrees C (from 12,500 to 28,900 sites/cell in stimulated cells). The kD value (0.45 nM) was not affected by androgen treatment. The increase of EGF-receptor activity was first observed between 6 and 12 h after exposure to androgen. It is concluded that LNCaP cells are sensitive to low concentrations of EGF (or EGF-like compounds) and that one of the mechanisms involved in androgen action on these cells is an increase of EGF-receptor expression at the cell surface.     

Dutasteride, the dual 5alpha-reductase inhibitor, inhibits androgen action and promotes cell death in the LNCaP prostate cancer cell line

BACKGROUND: Reduction of T to DHT by 5alphaR in the prostate enhances androgenic activity for most targets. Inhibition of 5alphaR activity with finasteride attenuates androgen action in men and animal models. The objective of this study was to compare and contrast the effects of a potent new 5alphaR inhibitor, dutasteride, with finasteride in the LNCaP prostate cancer cell line. METHODS: LNCaP cells were incubated for varying times with T or DHT in steroid-free medium in the absence or presence of increasing doses of dutasteride or finasteride and the effects on 5alphaR activity, PSA accumulation in the medium, and on cell proliferation were determined. Drug effects on apoptosis were investigated using Annexin V staining and a cell death ELISA assay. Effects of the drugs on AR ligand-binding activity and on AR protein levels were determined. RESULTS: Dutasteride inhibited (3)H-T conversion to (3)H-DHT and, as anticipated, inhibited T-induced secretion of PSA and proliferation. However the drug also inhibited DHT-induced PSA secretion and cell proliferation (IC(50) approximately 1 microM). Finasteride also inhibited DHT action but was less potent than dutasteride. Dutasteride competed for binding the LNCaP cell AR with an IC(50) approximately 1.5 microM. High concentrations of dutasteride (10-50 microM), but not finasteride, in steroid-free medium, resulted in enhanced cell death, possibly by apoptosis. This was accompanied by loss of AR protein and decreased AR ligand-binding activity. Occupation of AR by R1881 partly protected against cell death and loss of AR protein. PC-3 prostate cancer cells, which do not contain AR, also were killed by high concentrations of dutasteride, as well as by 50 microM finasteride. CONCLUSIONS: Dutasteride exhibited some inhibitory actions in LNCaP cells possibly related to 5alphaR inhibition but also had antiandrogenic effects at relatively low concentrations and cell death-promoting effects at higher concentrations. Finasteride also was antiandrogenic, but less than dutasteride. The antiandrogenic effects may be mediated by the mutant LNCaP cell AR. Promotion of cell death by dutasteride can be blocked, but only in part, by androgens.     

Amplification of MMP-2 and MMP-9 production by prostate cancer cell lines via activation of protease-activated receptors

BACKGROUND: The matrix metalloproteinases (MMP) are a family of proteolytic enzymes involved in facilitating cancer metastasis. Protease-activated receptors (PARs) have previously been shown to be involved in pathways of MMP upregulation by tumor cells. METHODS: Two androgen independent prostate cancer cell lines, PC3 and DU-145, and one androgen dependent prostate cancer line LNCaP, were investigated. PAR expression was detected using RT-PCR and immunofluorochemistry (IFC) techniques. MMP activity assays were used to quantify the levels of MMP-2 and -9 on all three prostate cell lines after PAR activation. RESULTS: RT-PCR and IFC showed the presence of PAR-1 and PAR-2 in all cell lines investigated, only LNCaP showed PAR-3 and PAR-4 expression. Increased levels of MMP-2 and MMP-9 activity, up to sevenfold depending on prostate cancer cell line, following PAR activation by specific PAR peptides was shown. CONCLUSION: Preliminary studies show the activation of PAR-1 or PAR-2 produced increased levels of MMP-2 and MMP-9 activity in prostate cancer cell lines, indicating their potential role in the metastasis of prostate cancer cells.     

Androgens induce CD-9 in human prostate tissue

Based on microarray analyses of LNCaP and LNCaP-r prostatic cell-lines we tentatively identified CD-9 as an androgen sensitive protein. This prompted us to characterize the androgen sensitivity and the correlation to malignancy of CD-9 at the protein level. Using Western blot, RT-PCR and immunohistochemistry the expression of CD-9 was analysed in LNCaP cells stimulated during increasing time by the synthetic androgen R1881 and also in 88 specimens of human prostate cancer tissues. Expression of CD-9 was induced by R1881 in LNCaP. CD-9 was immunolocalized in human prostate tissue sections representing non-malignant tissue as well as tumour areas. In non-malignant glands CD-9 immunoreactivity was observed at the apical and lateral cell borders of luminal epithelial cells. Basal epithelial cells were largely unstained. In tumour areas CD-9 staining intensity was variable and apparently not related to primary Gleason grade. In prostate tissue from a patient under androgen ablation therapy no staining was observed in luminal epithelial cells or in the tumour areas, but some staining was observed in basal epithelial cells. CD-9 is regulated by androgens in LNCaP and present in human prostate specimens. The expression is variable and changes in expression levels. These and earlier studies using other tissues indicate that CD-9 and its cellular localization could have an important role in prostate cancer cell development.     

Increased expression of tumor-associated trypsin inhibitor, TATI, in prostate cancer and in androgen-independent 22Rv1 cells

OBJECTIVES: Tumor-associated-trypsin inhibitor (TATI) is frequently coexpressed with trypsinogen in tumors. Recently, we found expression of trypsinogens in prostate cancer. We have now studied whether TATI is also expressed in prostate cancer and if TATI expression is associated with Gleason grade, proliferation, and neuroendocrine differentiation. METHODS: Expression of TATI and prostate-specific antigen (PSA) was studied by immunohistochemistry and in situ hybridization, and that of chromogranin A (CgA) and Ki-67 by immunohistochemistry. Immunofluorometric assays were used to quantify TATI and PSA in serum from prostate cancer patients and in medium of 22Rv1 prostate cancer cells. RESULTS: TATI expression was weak in benign prostatic epithelium and moderate to strong in prostate cancer and high-grade prostatic intraepithelial neoplasia. There was no correlation between TATI and Ki-67 immunostaining in a tissue microarray of 115 prostate cancer cores, but strong expression of TATI was associated with higher Gleason grade (p=0.002) and CgA immunostaining intensity (p=0.012). Serum TATI was elevated in 44% (29 of 66) of patients with prostate cancer, and the levels correlated with serum PSA (p<0.0001, r=0.306). DU145, PC-3, LNCaP, and 22Rv1 cells contained TATI mRNA as determined by RT-PCR, but only 22Rv1 cells produced detectable TATI protein. The synthetic androgen R1881 decreased secretion of TATI from 22Rv1 cells. CONCLUSIONS: We demonstrate for the first time that TATI is expressed in the benign and malignant prostate. Increased TATI protein expression is found in high-grade tumors and in 22Rv1 cells in which it is regulated by androgens.     

Melatonin and prostate cancer cell proliferation: interplay with castration,epidermal growth factor,and androgen sensitivity

BACKGROUND: Potential modulatory effects of melatonin on the proliferation of androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells were reported recently. In this study, we investigated the effects of combined melatonin and castration on LNCaP tumor growth in vivo, the interactions between melatonin and epidermal growth factor (EGF) on LNCaP cell proliferation, and melatonin actions on the proliferation of PC-3 and DU 145 cells. METHODS: Tumor development and growth in castrated nude mice inoculated with LNCaP cells or in intact animals inoculated with DU 145 cells, with or without daily melatonin treatment, were monitored by observation and caliper measurement. MT(1) receptor expression in native or transfected prostate cancer cell lines was examined by immunocytochemistry or 2-[(125)I]iodomelatonin binding. Cyclin D1 expression in LNCaP cells was assessed by Western blotting, and cell proliferation was measured by thymidine incorporation and/or cell count. RESULTS: Melatonin treatment was associated with further decreases in LNCaP tumor incidence and growth rate in castrated nude mice. Melatonin and 2-iodomelatonin (a melatonin receptor agonist) attenuated EGF-stimulated increases in LNCaP cell proliferation and cyclin D1 levels. Melatonin had no effect on the proliferation or growth of MT(1) receptor-expressing DU 145 cells, and of PC-3 cells in which MT(1) receptor protein was undetectable. The proliferation of transfected PC-3 cells expressing MT(1) receptor was unaffected by 2-iodomelatonin. CONCLUSION: Together with previous data, the present results indicate synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor. Androgen-sensitive prostate cancer cell proliferation may be modulated by opposite changes in cyclin D1 levels induced by activated MT(1) and EGF receptors. In androgen-insensitive prostate cancer cells, MT(1) receptor-mediated signal transduction may become defective not only through changes in membrane receptor protein expression and/or functions, but also by means of alterations in downstream postreceptor signaling events.     

5alpha-dihydrotestosterone inhibits 1alpha,25-dihydroxyvitamin D3-induced expression of CYP24 in human prostate cancer cells

BACKGROUND: A cross-talk between 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] and 5alpha-dihydrotestosterone (DHT) in the growth inhibition has been demonstrated, but the mechanism is unknown. METHODS: The expression of 25-hydroxyvitamin D(3) 24-hydroxylase (24-hydroxylase) was measured using a real-time quantitative RT-PCR assay and the catabolism of 1alpha,25-(OH)(2)D(3) was measured using a radioreceptor assay. RESULTS: Real-time RT-PCR showed that DHT at 1-100 nM significantly inhibited 1alpha,25-(OH)(2)D(3)-induced expression of 24-hydroxylase in LNCaP cells. Furthermore, the catabolism of 1alpha,25-(OH)(2)D(3) was decreased by 10 nM DHT. An androgen receptor (AR) antagonist, Casodex antagonized the DHT effect, whereas an AR agonist (due to the mutant AR in LNCaP cells) hydroxyflutamide did not. CONCLUSIONS: We demonstrated, for the first time, that DHT reduces the ability of 1alpha,25-(OH)(2)D(3) to induce 24-hydroxylase expression. Our results not only support the earlier finding of a cross-talk between androgen and vitamin D in human prostate cancer cells but also provide a possible mechanism how androgen and vitamin D signaling pathways may interact.     

Triiodothyronine modulates cell proliferation of human prostatic carcinoma cells by downregulation of the B-cell translocation gene 2

BACKGROUND: Studies suggest that triiodothyronine (T3) and cognate nuclear receptors (hTR) are involved in regulation of prostatic cell growth and differentiation. To probe mechanisms for T3 effects, we studied prostate carcinoma cells, investigating the effect of T3 on expression of the B-cell translocation gene 2 (BTG2), which regulates the G1/S transition of the cell cycle. METHODS: Effects of T3 on cell proliferation were determined by (3)H-thymidine incorporation. T3 modulation of BTG2 expression was investigated using immunoblots, Northern blots, and transient gene expression assays. The putative T3 response element was determined by electrophoretic mobility shift assay. RESULTS: T3 (0.1-1,000 nM) enhanced threefold the proliferation of prostate carcinoma cells and human androgen-dependent prostate carcinoma cells (LNCaP), but not PC-3 cells. T3 also inhibited BTG2 gene expression in LNCaP cells. Reporter assays showed that T3 downregulates by 50% promoter activity of the BTG2 gene in LNCaP cells but not PC-3 cells or thyroid-hormone receptor (TRbeta1)-overexpression PC-3 cells. Deleting the putative thyroid hormone response element (TRE; AGCGATGACCTCAGCG) blocked the inhibitory effect of T3 on BTG2 promoter activity. Electrophoretic mobility shift assays with purified TRbeta1 from in vitro translation, or with nuclear extracts from LNCaP cells and PC-3 cells, demonstrated the presence of T3 receptor binding sites in the TRE region. CONCLUSIONS: These results suggested that the T3 upregulates proliferation of LNCaP cells by downregulating BTG2 gene expression through the consensus TRE pathway.     

熊果酸对前列腺癌细胞雄激素非依赖性的逆转作用

目的 探讨中药成分熊果酸对雄激素非依赖性前列腺癌(AIPC)的治疗作用及其机制.方法 应用熊果酸处理体外培养的人雄激素依赖性前列腺癌(ADPC)细胞株LNCaP和AIPC细胞株DU145,噻唑蓝(MTT)比色法检测细胞活性及对人工合成雄激素R1881的反应性,免疫细胞化学检测熊果酸对雄激素受体(AR)、糖皮质激素受体(GR)、前列腺特异性抗原(PSA)及成活因子HSP90和白细胞介素(IL)-6表达的影响,逆转录.聚合酶链反应(RT-PCR)检测熊果酸对DU145细胞AR mRNA表达的影响.结果 熊果酸对不同浓度雄激素下的LNCaP细胞均呈浓度和时间依赖性生长抑制,20 mg/L的熊果酸作用96 h对LNCaP细胞的抑制率近50%.0.1 nmoL/L的R1881为最适生长浓度,熊果酸作用后,LNCaP细胞生长的最适雄激素浓度上升了10倍;熊果酸对DU145细胞的生长有浓度和时间依赖性抑制效应,DU145细胞对AR阻断剂羟氟他胺缺乏反应,熊果酸作用同时再应用氟他胺比单纯熊果酸的作用更明显,对细胞抑制率明显上升.熊果酸作用后,LNCaP和DUl45细胞IL-6、HSF90表达均明显下降(P＜0.05),DU145细胞GR表达明显降低(P＜0.01),AR和PSA蛋白及AR mRNA出现再表达.结论 熊果酸能改善前列腺癌细胞对雄激素的反应性,使LNCaP细胞对雄激素的依赖性加强,并诱发了DU145细胞对雄激素的反应性,其部分机制是降低了GR、HSP90、IL-6的表达并促进AR再表达.