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.     

Androgen receptor-dependent regulation of Bcl-xL expression: Implication in prostate cancer progression

BACKGROUND: Recently we reported that silencing the androgen receptor (AR) gene reduced Bcl-xL expression that was associated with a profound apoptotic cell death in prostate cancer cells. In this study we further investigated AR-regulated Bcl-xL expression. METHODS: Prostate cancer cell line LNCaP and its sublines, LNCaP/PURO and LNCaP/Bclxl, were used for cell proliferation assay and xenograft experiments in nude mice. Luciferase gene reporters driven by mouse or human bcl-x gene promoter were used to determine androgen regulation of Bcl-xL expression. RT-PCR and Western blot assays were conducted to assess Bcl-xL gene expression. Chromatin immunoprecipitation assay was performed to determine AR interaction with Bcl-xL promoter. Bcl-xL-induced alteration of gene expression was examined using cDNA microarray assay. RESULTS: In cultured prostate cancer LNCaP cells, androgen treatment significantly increased Bcl-xL expression at mRNA and protein levels via an AR-dependent mechanism. Promoter analyses demonstrated that the AR mediated androgen-stimulated bcl-x promoter activation and that the AR interacted with bcl-x promoter. Enforced expression of Bcl-xL gene dramatically increased cell proliferation in vitro and promoted xenograft tumor growth in vivo. Genome-wide gene profiling analysis revealed that Bcl-xL expression was significantly higher in metastatic and castration-resistant diseases compared to normal prostate tissues or primary cancers. Bcl-xL overexpression significantly increased the expression of cyclin D2, which might be responsible for Bcl-xL-induced cell proliferation and tumor growth. CONCLUSIONS: Taken together, our data strongly suggest that androgen stimulates Bcl-xL expression via the AR and that increased Bcl-xL expression plays a versatile role in castration-resistant progression of prostate cancer.     

神经内分泌分化对前列腺癌细胞生长及雄激素受体表达影响的实验研究

目的探讨神经内分泌分化对前列腺癌细胞生长的作用及对雄激素受体表达的影响。方法建立神经内分泌分化的前列腺癌细胞模型PC3MNE和LNCaPNE;采用甲基噻唑基四唑(MTT)试验观察其调节前列腺癌细胞生长的作用[以吸光度值(A)表示];采用逆转录聚合酶链反应和Western杂交方法检测LNCaPNE对LNCaP细胞雄激素受体表达的影响。结果PC3MNE的培养上清液可促进PC3M细胞的生长(A值在培养24h为034±018与050±009,48h为038±016与057±009,72h为038±015与055±005,P均<005);在有雄激素时,LNCaPNE的培养上清液不促进LNCaP细胞的生长,对其雄激素受体的表达也没有显著影响;去除雄激素后,LNCaPNE的培养上清液可促进LNCaP细胞的生长,并能下调其雄激素受体的表达(P均<005)。结论在雄激素阻断后,神经内分泌分化的前列腺癌细胞能以旁分泌的方式支持其他前列腺癌细胞生长,降低其雄激素受体的表达。     

Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases

BACKGROUND: The present study was undertaken to evaluate anti-proliferative and -apoptotic activities of mahanine, a plant derived carbazole alkaloid, in prostate cancer cells and to determine its molecular mechanism by which it induces apoptotic cell death. METHODS: The growth inhibitory and apoptotic inductive effect of mahanine on prostate cancer cells were examined by measuring cell proliferation and BrdU labeling, caspase activity, DNA fragmentation, and Western blot analyses. RESULTS: Mahanine inhibited growth of PC3 and LNCaP prostate cancer cells in a dose and time-dependent manner. Mechanistically, mahanine inhibited cell-survival pathway by dephosphorylation of PIP3 dependent kinase 1 (PDK1) thereby deactivation of Akt and downregulation of Bcl-xL. In addition, mahanine activated caspase pathway (caspases 9 and 3) and eventually cleavage of DNA repair enzyme, PARP resulting DNA fragmentation and apoptosis. CONCLUSIONS: Mahanine inhibits growth and induces apoptosis in both androgen-responsive, LNCaP and androgen-independent, PC3 cells by targeting cell survival pathway.     

Prostate cancer cells generated during intermittent androgen ablation acquire a growth advantage and exhibit changes in epidermal growth factor receptor expression

BACKGROUND: Intermittent androgen ablation is a palliative treatment option for advanced prostate cancer which is associated with less side effects, improved quality of life of patients, and reduced costs. Regulation of growth and survival of prostate cancer cells during intermittent androgen withdrawal has not been studied in appropriate models yet. METHODS: Two cycles of androgen withdrawal and supplementation were performed in human prostate cancer cells LNCaP in vitro. Proliferation of prostate cancer cell sublines established after intermittent androgen withdrawal was assessed in the absence or presence of epidermal growth factor (EGF) by protein determination. Cell cycle was analyzed with a flow cytometer. EGF was measured in the supernatants of LNCaP sublines with a commercial ELISA. EGF receptor mRNA and protein were determined by real-time PCR and Western blot, respectively. RESULTS: Basal proliferation rate of all newly generated LNCaP sublines increased over that of the parental LNCaP cell line. The highest stimulation of proliferation by exogenous EGF was observed in parental LNCaP cells. In each LNCaP derivative established during intermittent androgen withdrawal, the percentage of cells in the S phase of cell cycle was higher than that in parental LNCaP cells. EGF levels did not increase during intermittent androgen ablation. The expression of EGF receptor protein decreased following each cycle of androgen ablation and increased subsequently after androgen supplementation. EGF receptor (EGFR) mRNA was regulated in a similar manner in LNCaP derivatives established during the second cycle of intermittent withdrawal. CONCLUSIONS: Changes in the expression of the EGF receptor occur during intermittent androgen ablation but they cannot be solely responsible for increased basal proliferation. Alternatively, other ligands and receptors of the EGF system may become overexpressed during prolonged withdrawal and supplementation of androgenic hormones in prostate cancer therapy.     

Long-term exposure of tumor necrosis factor alpha causes hypersensitivity to androgen and anti-androgen withdrawal phenomenon in LNCaP prostate cancer cells

BACKGROUND: One of the mechanisms through which prostate cancers relapse during anti-androgen therapy may involve adaptation to low concentrations of androgen induced by anti-androgen therapies. Recent studies from our laboratory have reported that tumor necrosis factor-alpha (TNFalpha) is secreted from prostate cancer epithelial cells and LNCaP cells. We hypothesized that TNFalpha changes androgen-sensitivity in LNCaP cells. METHODS: We cultured LNCaP cells for more than 3 months in the presence of 50 ng/ml TNFalpha and established TNFalpha-resistant LNCaP cells (LN-TR2). Sensitivity to androgen was examined by the cell proliferation assay. We also transfected LNCaP and LN-TR2 cells with a luciferase reporter plasmid driven by prostate-specific antigen (PSA) promoter and compared PSA promoter activity. Nuclear localization of AR protein that binds to target genes was also examined by Western blotting. RESULTS: LN-TR2 cells had increased sensitivity to dihydrotestosterone (DHT) (i.e., proliferation and PSA promoter activation) than LNCaP cells. Total AR mRNA and AR protein levels were decreased in LN-TR2 cells. However, LN-TR2 cells demonstrated increased levels of nuclear AR compared to LNCaP cells. At 1 nM DHT, the anti-androgen bicalutamide stimulated LN-TR2 and inhibited LNCaP proliferation. CONCLUSIONS: Long-term exposure of TNFalpha causes hypersensitivity to DHT in LNCaP and this was associated with increased nuclear AR protein. Furthermore, hypersensitivity to androgen caused anti-androgen withdrawal phenomenon in the presence of DHT although bicalutamide itself did not stimulate LNCaP proliferation without androgen. This result may be one possible mechanism for the anti-androgen withdrawal phenomenon.     

Down-regulation of macrophage inhibitory cytokine-1/prostate derived factor in benign prostatic hyperplasia

BACKGROUND: Macrophage inhibitory cytokine-1 (MIC-1) is a member of transforming growth factor-beta/bone morphogenetic protein (BMP) superfamily. Despite its potential role in prostatic regulation, little is known about its biological activity. METHODS: Expression profiling using 42K Affymetrix HuGeneFL array was conducted to compare symptomatic benign prostatatic hyperplasia (BPH), histological BPH without symptoms, and normal prostate samples from donors. MIC-1 gene expression was analyzed by RT-PCR in pure culture of prostate epithelial and stromal cells, and prostate cancer cells, LNCaP, PC-3, DU-145. Influence of androgens on MIC-1 expression in LNCaP cells was analyzed by Northern blot. Enhancement of promoter activity of MIC-1 by androgens was examined using reporter assays. RESULTS: In contrast to normal prostates, MIC-1 gene was down-regulated in BPH samples with symptoms and histological BPH obtained from cystoprostatectomy specimens (P < 0.005 and P < 0.01, respectively). Expression level of MIC-1 in androgen-sensitive LNCaP cells was high and enhanced by androgens, whereas in the androgen-insensitive PC-3 and DU-145 cells the expression level was low. An 11 kb promoter region of MIC-1 gene was identified to be 6- to 12-fold activated by androgens. CONCLUSIONS: Down-regulation of MIC-1 may play a role in the development of BPH. MIC-1 is positively regulated by androgens, but other regulatory factors remain unclear.     

Role of coordinated molecular alterations in the development of androgen-independent prostate cancer: an in vitro model that corroborates clinical observations

OBJECTIVE: To investigate the role of potential downstream targets of HER-2/neu, including the cell-cycle regulator p27, proliferation-associated protein Ki-67, apoptosis inhibitor Bcl-2, and signal-transduction molecule Akt (which is associated with cell survival), as the development of androgen-independent prostate cancer (AIPC) in patients who are initially responsive to androgen-ablation therapy (AAT) is a significant clinical problem. PATIENTS AND METHODS: Earlier studies showed that high levels of HER-2/neu tyrosine kinase receptor expression as assessed by immunohistochemistry were significantly associated with the development of AIPC, and we hypothesised that HER-2/neu overexpression provides an alternative proliferative stimulus upon androgen depletion. We established a unique clinical model system, comprising patients who received no AAT, or who had preoperative AAT, or those with advanced tumours resistant to AAT. To test our hypothesis in vitro, we stably transfected full-length HER-2/neu cDNA in androgen-responsive LNCaP cells and examined the effects of HER-2/neu overexpression on cell proliferation, apoptosis, androgen-receptor activation, and Akt phosphorylation upon androgen deprivation by using immunohistochemistry and Western blot technique. RESULTS: p27 expression was initially induced on exposure to AAT, and significantly decreased in AIPC (P < 0.001). There was also a significant increase in the Ki-67 index in AIPC (P = 0.001). Elevated Bcl-2 expression was closely associated with AAT (P = 0.002), suggesting that Bcl-2 expression is induced on initial exposure to AAT. Further, Bcl-2 expression was highest in hormone-resistant cancers (P < 0.001). Using the HER-2/neu transfected cell-line model, we confirmed the mechanistic basis of the clinical observations which elucidate the pathway leading to HER-2/neu-mediated androgen independence. On androgen deprivation, the HER-2/neu transfected cells had higher proliferation rates, lower G1 arrest, inhibited p27 up-regulation, a lower apoptotic index, and higher Bcl-2, prostate-specific antigen and phosphorylated Akt expression than the mock-transfected LNCaP cells. CONCLUSION: This study suggests that prostate cancer cells undergo a series of coordinated changes after exposure to AAT, which eventually result in the development of androgen independence. Further, in support of previous results, it appears that a major factor in this process is the induction of HER-2/neu overexpression, which occurs after initial exposure to AAT. HER-2/neu may contribute to the development of androgen independence through: (i) maintaining cell proliferation; (ii) inhibiting apoptosis; and/or (iii) inducing AR activation in a ligand-independent fashion. These effects may be mediated, at least in part, through activation of the PI3K/Akt pathway.     

Androgen receptor or estrogen receptor-beta blockade alters DHEA-, DHT-, and E(2)-induced proliferation and PSA production in human prostate cancer cells

BACKGROUND: Dehydroepiandrosterone (DHEA) is an endogenous steroid that is metabolized to androgens and/or estrogens in the human prostate. DHEA levels decline with age, and use of DHEA supplements to retard the aging process is of unproved effectiveness and safety. LNCaP and LAPC-4 prostate cancer cells were used to determine whether DHEA-modulated proliferation and prostate specific antigen (PSA) production were mediated via the androgen receptor (AR) and/or ERbeta. METHODS: Cells were treated with DHEA, DHT, or E(2) and antagonists to AR (Casodex-bicalutamide) or ER (ICI 182,780) or siRNA to the respective receptors. Proliferation was assessed by MTT assay and PSA mRNA and protein secretion were measured by quantitative real-time PCR and ELISA. Associations of AR and ERbeta were analyzed by co-immunoprecipitation studies and fluorescent confocal microscopy. RESULTS: DHEA-, T-, and E(2)-induced proliferation of LNCaP cells was blunted by Casodex but not by ICI treatment. In LNCaP cells, Casodex and ICI suppressed hormone-induced PSA production. In LAPC-4 cells, DHT-stimulated PSA mRNA was inhibited by Casodex and ICI, and the minimal stimulation by DHEA was inhibited by ICI. Use of siRNAs confirmed involvement of AR and ERbeta in hormone-induced PSA production while AR-ERbeta co-association was suggested by immunoprecipitation and nuclear co-localization. CONCLUSIONS: These findings support involvement of both AR and ERbeta in mediating DHEA-, DHT-, and E(2)-induced PSA expression in prostate cancer cells.     

Cyclooxygenase-2 promotes prostate cancer progression

BACKGROUND: Cyclooxygenase (COX) -2, an inducible isoform of COX, has been observed to be expressed in prostate cancer. Several studies have reported that COX-2 overexpression is associated with carcinogenesis, cell growth, angiogenesis, apoptosis, and invasiveness in a variety of tumor types. METHODS: To investigate the function of COX-2 in prostate cancer directly, we stably transfected human full-length COX-2 cDNA into LNCaP cells (LNCaP-COX-2), which express low levels of endogenous COX-2. RESULTS: The level of COX-2 mRNA and protein and the COX activity in COX-2 LNCaP-COX-2 cells was significantly increased compared with parent and control-transfected cells. Overexpression of COX-2 increased both proliferation in vitro and tumor growth rate in vivo. However, the pro-tumor effect was neither associated with changes of androgen receptor (AR) expression level nor AR activity. Furthermore, addition of the major metabolites of COX-2-mediated arachidonic acid metabolism did not alter the proliferation of LNCaP-COX-2 cells in vitro. LNCaP-COX-2 cells had increased secretion of vascular endothelial growth factor (VEGF) protein, suggesting that angiogenesis induced by COX-2 stimulates tumor growth in vivo. CONCLUSION: These data demonstrate that COX-2 contributes to prostate cancer progression and suggest that it mediates this effect, in part, through increased VEGF.     

Inhibitory effects of nordihydroguaiaretic acid (NDGA) on the IGF-1 receptor and androgen dependent growth of LAPC-4 prostate cancer cells

BACKGROUND: Nordihydroguaiaretic acid (NDGA) is an inhibitor of the IGF-1 receptor (IGF-1R) in breast and other cancers, and concomitantly inhibits tumor growth both in cultured cells and animals. The current study evaluates the effect of NDGA on the androgen-stimulated growth of human prostate cancer cells. METHODS: LAPC-4 prostate cancer cells in tissue culture were androgen starved for 3 days, 1 nM dihydrotestosterone (DHT) and other androgens were then added for up to 7 days, and cell proliferation measured. IGF-1R protein expression was measured by Western blot, and IGF-1R mRNA expression by quantitative PCR. IGF-1R receptor kinase activation was measured by ELISA. RESULTS: After 7 days, LAPC-4 growth was doubled by 1 nM DHT. NDGA had a rapid effect to inhibit IGF-1R autophosphorylation induced by IGF-1. DHT increased the expression of IGF-1R protein and mRNA levels. Maximal IGF-1R protein levels were observed 3 days after the addition of androgen. In addition, NDGA, at 10 microM or less, inhibited DHT-induced proliferation in both cells grown in plates and cells grown in soft agar. Androgen receptor (AR) studies by FRET revealed that NDGA had no conformational effects on the AR in response to ligand. CONCLUSIONS: NDGA blocks the DHT-induced growth of LAPC-4 prostate cancer cells by several mechanisms including rapid inhibition of the IGF-1R kinase, and a dose-dependent inhibition of androgen stimulation of IGF-1R expression. Clinical studies of this agent will determine its efficacy in the setting of androgen-dependent prostate cancer.     

Regulation of Bcl-2 during androgen-unresponsive progression of prostate cancer

The progression of prostate cancer from androgen-responsive to an androgen-unresponsive state remains the greatest obstacle in the treatment of this disease. Androgen-unresponsive prostate cancer is highly resistant to chemotherapy and radiation treatment that kill cells by the induction of apoptosis. Elucidating the molecular mechanisms of apoptosis regulation in prostate cancer can be useful in the development of new strategies for effective therapy of androgen-unresponsive cancer. We analyzed the Bcl-2 family of apoptosis regulators using various passages of the LNCaP prostate cancer cell line, which serve as an in vitro model for the progression of prostate cancer from androgen-responsive to androgen-unresponsive. In our model, progressively higher passages of LNCaP cells represent the progression to androgen-unresponsiveness. We examined the basal mRNA expression of the Bcl-2 family of apoptosis regulators. Under normal growth conditions, both androgen-responsive and androgen-unresponsive LNCaP cells express the Bcl-2 family of genes at similar levels. Western blot analysis showed the presence of Bcl-2 protein in androgen-responsive cells but not in androgen-unresponsive cells. Both androgen-responsive and androgen-unresponsive cells expressed Bax protein at similar levels. When exposed to oxidative stress, androgen-responsive cells underwent apoptosis but androgen-unresponsive cells exhibited resistance suggesting that the progression to androgen-unresponsiveness was associated with altered regulation of apoptosis. Treatment with paclitaxel or sodium butyrate induced apoptosis in both androgen-responsive and androgen-unresponsive cells suggesting that the apoptotic machinery is still intact in androgen-unresponsive LNCaP cells.     

Prosaposin upregulates AR and PSA expression and activity in prostate cancer cells (LNCaP)

BACKGROUND: Prosaposin overexpression and/or genomic amplification have been demonstrated in androgen-independent (AI) prostate cancer cell lines and tissues. Here, we explored the possibility for a functional relationship between prosaposin and androgen receptor (AR) in LNCaP cells. METHODS: The effect of prosaposin or its active molecular derivatives (e.g., saposin C) on expression and activity of androgen receptor (AR) and prostate-specific antigen (PSA) was examined by using immunoblotting, RT-PCR, transfection, and reporter gene assays, immunofluorescence staining, and inhibitors of signal transduction pathways. RESULTS: Prosaposin or saposin C, in an AI-manner, (a) increased AR mRNA and protein expression and nuclear AR content and its phosphorylation state; (b) increased PSA mRNA and protein expression; and (c) upregulated PSA- and an androgen-inducible probasin (PB)-reporter gene activity in LNCaP and AR-transfected PC-3 cells. Induction of PSA expression and reporter activity was substantially blocked or prevented with the antiandrogen bicalutamide, pertussis toxin, or inhibitors of MAPK- and PI3K/Akt-signaling pathways, indicating an androgen-agonistic effect for saposin C that involves AR and multiple signaling pathways. CONCLUSIONS: The results for the first time introduce prosaposin as an androgen-agonist in prostate cancer cells. This finding, together with the growth-promoting effect and overexpression of prosaposin, may support a growth advantage to AI prostate cancer cells.