Inhibition of androgen-sensitive LNCaP prostate cancer growth in vivo by melatonin: association of antiproliferative action of the pineal hormone with mt1 receptor protein expression

BACKGROUND: Potential involvement of the mt1 receptor in the antiproliferative action of melatonin on androgen-sensitive LNCaP cells, and melatonin-induced modulation of androgen-insensitive PC-3 cell growth, have been reported in vitro. The effects of melatonin on prostate cancer cell proliferation and their association with mt1 receptor expression were investigated in athymic nude mice xenograft models of LNCaP and PC-3 cells. METHODS: Daily saline or melatonin (4 microg/g body weight) was given to nude mice before or after tumor cell inoculation. Tumor volume was measured periodically, and expression of PCNA, cyclin A, PSA, and mt1 receptor was assessed by immunohisto(cyto)chemistry and/or Western blotting. RESULTS: Melatonin inhibited the growth of LNCaP tumors, without affecting the growth of PC-3 xenografts, in nude mice. It induced significant decreases in the expression of PCNA, cyclin A, and PSA in LNCaP tumors. Expression of mt1 receptor protein was demonstrated in LNCaP cells, but not in PC-3 cells, both in vivo and in vitro. CONCLUSIONS: The antiproliferative action of melatonin on LNCaP tumor growth was demonstrated in vivo, and its association with mt1 receptor protein expression suggests the potential involvement of the receptor in the antitumor activity of the pineal gland hormone.     

Endothelin-1 production by prostate cancer cell lines is up-regulated by factors involved in cancer progression and down-regulated by androgens.

BACKGROUND: Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone. METHODS: We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1). RESULTS: Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression. CONCLUSIONS: In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.     

Effect of the neuropeptides bombesin and calcitonin on the growth of prostate cell lines, PC-3, DU 145, and LNCaP

Neuroendocrine cells are present in normal and tumoral prostate tissue, the neuropeptides secreted by this cells have a biological functions that have not been fully elucidated. The presence of neuroendocrine cells in prostatic carcinoma have been shown to increase tumor progression. We characterized the in vitro proliferative influence of bombesin and calcitonin in androgen-insensitive, PC-3 and DU-145, and androgen-sensitive, LNCaP, cell lines of human prostate cancers. The influence of these neuropeptides on proliferation were assessed using the colorimetric XTT assay and by cells counts with a hemocytometer. The growth of PC-3 and DU-145 cell lines is stimulated by bombesin and calcitonin but exerted any stimulatory effect on the proliferation of the LNCaP cell line. This indicate that bombesin and calcitonin can modulate proliferation of androgen-insensitive human prostate cell lines "in vitro" and may be potential paracrine growth promoters in stablished androgen irresponsive human prostatic carcinoma cells.     

Melatonin reduces prostate cancer cell growth leading to neuroendocrine differentiation via a receptor and PKA independent mechanism

BACKGROUND: Melatonin, the main secretory product of the pineal gland, inhibits the growth of several types of cancer cells. Melatonin limits human prostate cancer cell growth by a mechanism which involves the regulation of androgen receptor function but it is not clear whether other mechanisms may also be involved. METHODS: Time-course and dose-dependent studies were performed using androgen-dependent (LNCaP) and independent (PC3) prostate cancer cells. Cell number, cell viability, and cell cycle progression were studied. Neuroendocrine differentiation of these cells was evaluated by studying morphological and biochemical markers. Finally, molecular mechanisms including the participation of melatonin membrane receptors, intracellular cAMP levels, and the PKA signal transduction pathway were also analyzed. RESULTS: Melatonin treatment dramatically reduced the number of prostate cancer cells and stopped cell cycle progression in both LNCaP and PC3 cells. In addition, it induced cellular differentiation as indicated by obvious morphological changes and neuroendocrine biochemical parameters. The role of melatonin in cellular proliferation and differentiation of prostate cancer cells is not mediated by its membrane receptors nor related to PKA activation. CONCLUSIONS: The treatment of prostate cancer cells with pharmacological concentrations of melatonin influences not only androgen-sensitive but also androgen-insensitive epithelial prostate cancer cells. Cell differentiation promoted by melatonin is not mediated by PKA activation although it increases, in a transitory manner, intracellular cAMP levels. Melatonin markedly influences the proliferative status of prostate cancer cells. These effects should be evaluated thoroughly since melatonin levels are diminished in aged individuals when prostate cancer typically occurs.     

mRNA expression of the five membrane-type matrix metalloproteinases MT1-MT5 in human prostatic cell lines and their down-regulation in human malignant prostatic tissue

BACKGROUND: The aim of this study was to assess the expression of membrane-type matrix metalloproteinases (MT-MMPs) 1-5 in the human prostatic cell lines BPH-1, LNCaP, DU 145, PC-3, in malignant and non-malignant prostatic tissue samples, and in epithelial cells cultured from these tissue samples. METHODS: Matched malignant and non-malignant tissue specimens were obtained from 12 men with untreated prostate carcinoma after radical prostatectomy. Expression of mRNA for the five MT-MMPs was quantified by real-time PCR technique and normalized to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The expression of the five MT-MMPs was distinctly different not only between the prostate cell lines but also varied in the same cell line. There was a general higher expression of all MT-MMPs except for MT3-MMP in the androgen-insensitive cells DU 145 and PC-3 compared with that in the androgen-sensitive LNCaP cells. Their relatively high expression in the benign prostatic cell line BPH-1 and also in the primary cell cultures from malignant and non-malignant tissue samples argues against a simple association between MT-MMP expression and invasiveness. In malignant tissue samples and their corresponding cell cultures, the expression of most MT-MMPs was down-regulated in comparison to the normal counterparts. There was no correlation between tumor classification data and the MT-MMP expression results. CONCLUSIONS: In contrast to other carcinoma, the down-regulation of most MT-MMPs is typical for prostate carcinoma. It seems to occur mainly in epithelial cells and has to be examined as special characteristic of this tumor entity in further studies.     

Growth inhibition of androgen-insensitive human prostate carcinoma cells by a 19-norsteroid derivative agent,mifepristone

Mifepristone, also known as RU 486, is a 19-norsteroid derivative. Currently, mifepristone is being tested in clinical trials on meningioma and breast cancer. In this study we analyzed whether mifepristone could inhibit the growth of human prostate cancer cells including androgen-insensitive (PC-3 and DU145) and androgen-sensitive (LNCaP) cell lines. At 1-nM concentration, mifepristone exhibited a marginal stimulatory action on LNCaP and PC-3 cells. Nevertheless, a dose-dependent growth inhibition on those same cell lines was observed at concentrations of 1 μM and 10 μM. Twenty-day exposure to the clinically achievable concentration of 1 μM mifepristone resulted in consistent inhibition of all three cell lines studied. Furthermore, this in vitro growth inhibition was reflected in an in vivo nude mouse system. Mifepristone at the dosage of 4 mg/100 g body weight completely suppressed the growth of PC-3 tumors for 21 days, although this was followed by a growth rate similar to that of the control tumor. To understand the possible mechanism of mifepristone inhibition, PC-3 cells were exposed to mifepristone in comparison with dexamethasone (Dex), progesterone, and 5 alpha-dihydrotestosterone (DHT), each at 1-μM concentration. The results demonstrated that while both DHT and Dex alone had essentially no effect on cell growth, progesterone alone resulted in a 20% growth inhibition, while mifepristone had more than 60% inhibition with a 16-day exposure. At an equal concentration, the degree of growth inhibition of PC-3 cells by mifepristone or progesterone was partially diminished by simultaneous exposure to Dex. In conclusion, our results demonstrated that the growth of androgen-insensitive prostate cancer cells can be directly inhibited by mifepristone in cultures. This in vitro inhibition is reflected in xenografted tumors.     

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.     

Regulation of lnvasive Potential of Human Prostate Cancer Cell Lines by Hepatocyte Growth Factor

Background: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro.
Methods: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgenindependent), and LNCaP (androgen-dependent). We studied the expression of the HCF receptor c-met proto-oncogene (c-met) by Western blotanalysis, and alsodetermined theeffectsof HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay.
Results: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells.
Conclusion: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.     

Downregulation of androgen receptor expression by luteolin causes inhibition of cell proliferation and induction of apoptosis in human prostate cancer cells and xenografts

BACKGROUND: Androgen receptor (ARs) play a crucial role in the development and progression of prostate cancer. Recent studies have suggested that prostate cancer cell proliferation is inhibited by AR downregulation. Our aim was to investigate how luteolin, a natural flavonoid, affects cell growth and AR expression in prostate cancer cells and xenografts. METHODS: We assessed prostate cancer cell (LNCaP, DU145, and PC-3) proliferation and apoptosis by MTT assay, flow cytometric analysis, and Western analysis. AR function was measured by evaluating the AR target molecule, prostate-specific antigen (PSA), by RT-PCR, Western blotting, and enzyme-linked immunosorbent assay. We determined the mechanism of AR downregulation with cycloheximide chase assays, proteasome inhibitor, and coimmunoprecipitation experiments. The effects of luteolin on growth inhibition in vivo were examined by LNCaP xenografts in SCID mice. RESULTS: Luteolin significantly repressed prostate cancer cell proliferation and induced apoptosis in LNCaP cells. PC-3 and DU145 cells were less susceptible to luteolin-mediated growth inhibition. Luteolin simultaneously suppressed intracellular and secreted PSA levels and repressed AR mRNA and protein expression in a dose- and time-dependent manner. Luteolin reduced the association between AR and heat-shock protein 90, causing AR degradation through a proteasome-mediated pathway in a ligand-independent manner. Luteolin also suppressed LNCaP xenograft tumor growth in SCID mice. CONCLUSION: Luteolin-mediated AR downregulation contributes to the inhibition of cell proliferation and the induction of apoptosis in LNCaP human prostate cancer cells, suggesting that AR is a molecular target for luteolin-mediated anticancer activity. Luteolin may act as a chemopreventive or chemotherapeutic agent for prostate cancer.     

Prostate stromal cell-derived hepatocyte growth factor induces invasion of prostate cancer cell line DU145 through tumor-stromal interaction.

BACKGROUND: In prostate cancer, several growth factors derived from stromal cells regulate tumor cell growth. Hepatocyte growth factor (HGF) possesses biological activities that promote cancer proliferation and invasion through tumor-stromal interaction. We examined how prostate stromal cell-derived HGF affects invasion of prostate cancer cells through this interaction. METHODS: The effects of HGF, various growth factors (transforming growth factor (TGF)-alpha, TGF-beta1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor), and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3, and DU145) were determined by collagen gel invasion assay. DU145 cells and PrSC were cocultured for Matrigel invasion chamber assay. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by the ELISA method and Western blotting. RESULTS: LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or cocultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta1. Native-type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. CONCLUSIONS: PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction, wherein DU145 cells secreted some HGF-inducer(s) for PrSC.     

Mitogenic signaling in androgen sensitive and insensitive prostate cancer cell lines

PURPOSE: To investigate the role of a specific mitogen activated protein kinase, extracellular signal-regulated kinase (ERK), in regulating cell proliferation induced by three potentially important prostate cancer mitogens that signal via different classes of receptors. MATERIALS AND METHODS: Androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cell lines were used in these studies. Epidermal growth factor (EGF), lysophosphatidic acid (LPA), and dihydrotestosterone (DHT) were the mitogenic stimulants and AG1478, a receptor tyrosine kinase inhibitor, and PD98059, an inhibitor of MEK, were the chemical inhibitors used in this study. Cell proliferation was measured using the WST-1 assay and ERK expression and activation was determined by immunoblotting for phospho- and total ERK. RESULTS: In androgen-sensitive LNCaP cells, epidermal growth factor (EGF) and dihydrotestosterone (DHT) both enhanced cell proliferation. EGF-stimulation dramatically increased ERK phosphorylation while DHT did not. In the androgen-insensitive cell line, PC-3, EGF- and LPA-induced ERK phosphorylation and cell proliferation. Inhibition of EGF- and LPA- induced ERK activation with the EGF receptor inhibitor, AG1478, or the MEK inhibitor, PD98059, attenuated their proliferative effects. Neither inhibitor had an effect on DHT stimulated cell proliferation. CONCLUSIONS: These data demonstrate heterogeneity of mitogenic signaling in prostate cancer cells, and support the hypothesis that androgens and growth factors utilize divergent signaling pathways in prostate cancer to induce proliferation.     

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 hepatocyte growth factor in invasion of prostate cancer cell lines through tumor-stromal interaction

We examined how prostate stromal cell-derived hepatocyte growth factor (HGF) affects invasion of prostate cancer cells through tumor-stromal interaction. The effects of HGF, various growth factors [transforming growth factor (TGF)-alpha, TGF-beta 1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor], and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3 and DU145) were determined by collagen gel invesion assay. DU145 cells and PrSC were co-cultured for matrigel invasion chamber assay. LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta 1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or co-cultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta 1. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by ELISA method and Western blotting. Native type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. In summary, PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction wherein DU145 cells secreted some HGF-inducer(s) for PrSC.     

Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells

BACKGROUND: Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro. METHODS: We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively. RESULTS: Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin. CONCLUSIONS: Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells.     

PC-SPES: a unique inhibitor of proliferation of prostate cancer cells in vitro and in vivo

BACKGROUND: Management of prostate cancer that has spread beyond the capsule is a difficult problem. Innovative and nontoxic approaches to the disease are urgently required. Recently, a commercially available herbal mixture called PC-SPES showed potent antitumor activities on a variety of malignant cells in vitro. METHODS: PC-SPES was evaluated for its ability to inhibit clonal growth, and to induce cell cycle arrest of three human prostate cancer cell lines (LNCaP, PC-3, and DU 145). Western blot analysis examined the effect of PC-SPES on levels of p21(waf1), p27(kip1), Bcl-2, and E-cadherin in the three cell lines; and telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay. Furthermore, the effect of oral PC-SPES (250 mg/kg/day) on growth of PC-3 and DU 145 tumors present in male BNX nu/nu triple immunodeficient mice was studied. LNCaP cells were not analyzed in mice because they grow only with difficulty in these immunodeficient mice. RESULTS: PC-SPES markedly inhibited clonal growth of LNCaP, PC-3, and DU 145 prostate cancer cells, with a 50% inhibition (ED50) at approximately 2 microl/ml. Pulse-exposure studies showed that a 5-day pulse-exposure to PC-SPES (2 microl/ml) in liquid culture achieved a 50% inhibition of PC-3 clonal growth in soft agar, suggesting that the growth inhibition mediated by the extracts remained after removal of PC-SPES. Cell cycle analysis using the prostate cancer cell lines found that PC-SPES induced a significant increase in the number of cells in G0-G1 and G2/M, with a concomitant decrease in the number of cells in S phase. PC-SPES (2 microl/ml, 4 days) increased slightly the levels of p21(waf1) in the three cell lines, decreased by 40% the levels of Bcl-2 in PC-3, and the levels of p27(kip1) and E-cadherin and telomerase were unchanged in each of the lines. In vivo treatment with oral PC-SPES of male BNX mice having DU 145 tumors produced significant inhibition of their growth (P < 0.001), with no objective side effects including blood chemistries, weights, or autopsy analysis. The PC-SPES showed no statistical effect on the in vivo growth of PC-3 cells. CONCLUSIONS: PC-SPES inhibits clonal proliferation of human prostate cancer cells both in vitro and in vivo, using a murine model.     

The effect of papaverine on morphologic differentiation, proliferation and invasive potential of human prostatic cancer LNCaP cells

BACKGROUND: Intracellular cyclic adenosine monophosphate (AMP) level changes are thought to play an important role in inhibiting cell proliferation and inducing differentiation in several types of cells. It has been reported that cyclic AMP analogs induce terminal differentiation in human prostate cancer cells. Consequently, phosphodiesterase inhibitors may be useful in delineating the role of cyclic AMP in the differentiation of these cells. Therefore, the effect of phosphodiesterase inhibitors on morphologic differentiation, proliferation and invasive potential of human prostate cancer cells was investigated. METHODS: Three human prostate cancer cell lines PC-3, DU145 and LNCaP were treated with one of the phosphodiesterase inhibitors, papaverine, 3-isobutyl-1-methylxanthine (IBMX) or theophylline, for 6 days. Morphologic changes of these cells induced by phosphodiesterase inhibitors were observed by microscopy. Intracellular cyclic AMP levels in LNCaP cells were measured by radioimmunoassay using a cyclic AMP assay kit. The effect of papaverine on the proliferation and invasive potential of LNCaP cells were measured by cell counting and the Matrigel invasion chamber assay. RESULTS: Of the three agents, examined papaverine (10(-5) mol/L) is the most effective inducer of morphologic change and also raised intracellular cyclic AMP levels in LNCaP cells. However, unlike LNCaP cells, PC-3 and DU145 cells treated with phosphodiesterase inhibitors, including papaverine, showed little change in morphology. Additionally, proliferation and invasive potential of LNCaP cells were significantly inhibited by papaverine. CONCLUSION: The results suggest that papaverine induces terminal differentiation in LNCaP cells, which is correlated with an intracellular cyclic AMP-mediated pathway.     

Inhibition of human chorionic gonadotropin beta-subunit modulates the mitogenic effect of c-myc in human prostate cancer cells

BACKGROUND: Amplification of the proto-oncogene c-myc has been identified as one of the most common genetic alterations in prostate cancer, thus making it an attractive therapeutic target. However, certain prostate cancer cells are unresponsive to c-Myc inhibition. The purpose of this study was to test the hypothesis that effective growth inhibition in the refractory cancer cells can be achieved by blocking c-myc along with a growth factor using a novel phosphorodiamidate morpholino antisense oligomer-based approach. Human chorionic gonadotropin, a growth factor implicated in neoplasm, causes activation of c-myc through a G-protein-coupled pathway of signal transduction. METHODS: In this study, the effect of inhibition of beta-hCG and c-myc singly or in combination was evaluated in DU145 (RB -/-, p53-/-, androgen-independent) and LNCaP (Rb+/+, p53 +/+, androgen-sensitive) human prostate cancer cell lines and in a DU145 subcutaneous xenograft murine model. RESULTS: Antisense phosphorodiamidate morpholino oligomers directed against beta-hCG and c-myc caused a specific decrease of the target protein levels. Unlike LNCaP cells, DU145 cell growth was refractory to c-Myc inhibition. Unresponsiveness to c-myc inhibition in DU145 cells was overcome by targeting both beta-hCG and c-myc genes, resulting in potentiation of the antiproliferative effect seen with inhibition of beta-hCG alone. CONCLUSIONS: The inhibition of beta-hCG sensitizes prostate cancer cells to the antiproliferative effects of c-Myc inhibition, including tumors that are refractory to c-Myc decrease alone.