Androgen-dependent Human Prostate Cancer Cell Research Articles

Hypoxia regulation of expression and angiogenic effects of vasoactive intestinal peptide (VIP) and VIP receptors in LNCaP prostate cancer cells

Vascular endothelial growth factor (VEGF) is a main factor promoting neovascularization (angiogenesis) of solid tumours as prostate carcinoma. Hypoxia stimulates VEGF gene expression by activating the hypoxia-inducible factor-1 (HIF-1α). In the present study, the hypoxia-mimicking agent Ni2+ induced vasoactive intestinal peptide (VIP) expression at both mRNA and peptide levels but it did not modify the expression of VIP receptors (VPAC1, VPAC2 and PAC1 receptors) in androgen-dependent human LNCaP prostate cancer cells. VIP increased the mRNA levels of VPAC1 and PAC1 receptors whereas it decreased VPAC2 receptor mRNA level. These features support that hypoxia up-regulation of VIP gene expression in prostatic carcinoma may lead to VIP regulation of the expression of its receptors by means of autocrine/paracrine mechanisms. Either VIP or hypoxia mimetics with Ni2+ increased VEGF expression whereas both conditions together resulted in an additive response. It suggests two independent mechanisms for the observed pro-angiogenic activities of VIP and hypoxia. VIP did not stimulate HIF-1α mRNA expression but increased the translocation of HIF-1α from the cytosolic compartment to the cell nucleus. Moreover, VIP was unable to modify the expression of the HIF-1α inhibitor FIH-1 discarding the possibility of an indirect effect of VIP on HIF-1 transactivation.

Alteration of gene expression in response to bone morphogenetic protein-2 in androgen-dependent human prostate cancer LNCaP cells

Bone morphogenetic protein (BMP)-2, a multifunctional member of the transforming growth factor (TGF)-beta superfamily with powerful osteoinductive effects, has various biological activities in a variety of cells. We observed that BMP-2 inhibits cell proliferation in the androgen-dependent human prostate cancer cell line, LNCaP. To investigate the mechanism of inhibition of androgen-dependent growth by BMP-2, we compared the gene expression in LNCaP cells treated with dihydrotestosterone (DHT) to that of LNCaP cells treated with DHT and BMP-2, using DNA microarray analysis. Of 8,400 human genes on the gene chip, 38 genes were up-regulated by >2.0-fold and 48 genes were down-regulated by <0.5-fold by treatment with BMP-2. These genes were involved in a variety of cellular functions, including signal transduction, transcription regulation, enzymes, transporters, structural molecules and translation. RT-PCR analysis showed that CH1CL and BMX were up-regulated and DACH1 and WNT5A were down-regulated by treatment with BMP-2. Furthermore, we detected an increase of WNT5A protein in the medium by DHT and inhibition of the increase by BMP-2. In the present study, we identified several BMP-2-responsive genes in LNCaP cells. Further studies of the roles of these genes may clarify the mechanisms underlying the inhibition of cell proliferation by BMP-2 and identify better approaches for the prevention and treatment of prostate cancer.

Doxazosin induces apoptosis in LNCaP prostate cancer cell line through DNA binding and DNA-dependent protein kinase down-regulation.

Doxazosin is a quinazoline-based compound acting as an alpha-1-adrenergic inhibitor shown to induce apoptosis in prostate cancer cell lines via an alpha-1-adrenergic receptor-independent mechanism. To better understand the mechanism of doxazosin-induced apoptosis in prostate cancer, we performed cDNA microarray to analyze gene expression changes produced by doxazosin in the androgen-dependent human prostate cancer cell line, LNCaP. We found that 70 and 92 genes were deregulated after 8 and 24 h of doxazosin treatment, respectively. These genes are involved in several cellular processes such as cell-cycle regulation, cell adhesion and signal transduction pathways. Strikingly, we found that doxazosin induces deregulation of genes implicated in DNA replication and repair, such as GADD45A, XRCC5 and PRKDC. These facts, together with the demonstration of the ability of doxazosin to bind DNA, allowed us to propose a novel mechanism of action for doxazosin in prostate cancer cells that implies DNA-damage mediated apoptosis by down-regulation of XRCC5 and PRKDC genes.

Prostate cancer cell type-specific involvement of the VDR and RXR in regulation of the human PTHrP gene via a negative VDRE

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDRE hPTHrP) within the human PTHrP gene and involves an interaction between nVDRE hPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRα forms part of the nuclear protein complex that interacts with nVDRE hPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9- cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9- cis-retinoic acid enhances the 1,25(OH) 2D 3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9- cis-retinoic acid. Promoter activity driven by nVDRE hPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH) 2D 3 and EB1089 in both cell lines. Co-treatment with these compounds and 9- cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH) 2D 3 in a cell type-specific manner in prostate cancer cells.

Role of androgen receptor in the progression of human prostate tumor cells to androgen independence and insensitivity

Various studies have implicated the androgen receptor (AR) in the progression of androgen-dependent human prostate cancer cells to androgen-independent and androgen-insensitive phenotypes, but the exact role of AR in progression is unclear. To mimic the clinical situation and test the role of AR in progression, we cultured androgen-dependent LNCaP 104-S prostate tumor cells in the presence of the antiandrogen Casodex (bicalutamide) to derive resistant (CDXR) clones. In a second step, we cultured CDXR cells in the presence of the androgen R1881, which generated androgen- and Casodex-insensitive (IS) cells. These cells were then characterized with regard to AR function and the effect of ectopic AR expression or AR knockdown on androgen sensitivity. CDXR cells showed increased AR expression and transcriptional activity. CDXR cell proliferation was unaffected by Casodex but was repressed by androgen in vitro and in vivo. IS cells, on the other hand, had greatly reduced AR expression and activity compared to CDXR cells. Knockdown of AR expression in CDXR cells produced cells that were insensitive to androgen. Conversely, re-expression of AR in IS cells regenerated cells that were repressed by androgen. Knockdown of AR expression in 104-S cells produced cells that remained stimulated by androgen, while overexpression of AR in 104-S cells generated an androgen-repressed phenotype but did not confer androgen-independent growth. Increased AR expression determines whether prostate cancer cells are repressed by androgen, but is not required for androgen independence. These results may have implications for anti-AR therapy for prostate cancer.

Expression profiling of androgen-dependent and -independent LNCaP cells: EGF versus androgen signalling

Prostate cancer development often includes a shift from androgen-dependent to androgen-independent growth. It is hypothesized that, during this transition, growth factors like the epidermal growth factor (EGF) gain importance as activators of tumour cell proliferation. To study this, androgen- and EGF-regulation of growth and gene-expression was analysed in the androgen-dependent human prostate cancer cell line LNCaP-FGC (FGC) and its androgen-independent derivative line LNCaP-LNO (LNO). It was observed that androgen-dependent FGC cells require exposure to either androgens or EGF to proliferate. This is in contrast to androgen-independent LNO cells that showed significant proliferation in medium depleted of androgens and growth factors. Gene expression data were obtained for the androgen-dependent FGC and androgen-independent LNO cells cultured in the presence or absence of androgens (synthetic R1881) or EGF for different time periods. Expression profiling showed that many cell cycle genes, including a number of androgen- and EGF-regulated genes, are constitutively activated in androgen-independent LNO cells. Furthermore, the overlap between changes in gene expression activated by androgen and EGF receptor signalling pathways was found to be very high (75%). These results partly explain why androgen-independent LNO cells can proliferate in the absence of androgenic stimulation. However, possibly other, so far unknown, signal transduction pathways that induce and maintain proliferation, have also been activated.

Bone morphogenetic protein-2 induces hypophosphorylation of Rb protein and repression of E2F in androgen-treated LNCaP human prostate cancer cells

Bone morphogenetic protein-2 (BMP-2), a multifunctional member of the transforming growth factor (TGF)-beta, superfamily, has powerful osteoinductive effects and causes cell cycle arrest in a variety of transformed cell lines. We have observed BMP-2-induced inhibition of cell proliferation in an androgen-dependent human prostate cancer cell line (LNCaP). To investigate the mechanism of inhibition of androgen-dependent growth by BMP-2, we examined the effect of dihydrotestosterone (DHT) and/or BMP-2 on cell cycle-related proteins in LNCaP cells. BMP-2 decreased the phosphorylation of retinoblastoma (Rb) protein induced by treatment with DHT. DHT-induced expression of cyclin A and cyclin-dependent kinase 2 (CDK2) protein was also inhibited by co-treatment with BMP-2. Furthermore, BMP-2 induced expression of p21(WAF1/CIP1), a CDK inhibitor. These results indicate that changes in expression of these proteins lead to modulation of the phosphorylation state of Rb. DHT-induced E2F-1 protein and mRNA expressions was also inhibited by BMP-2, suggesting that BMP-2 inhibits DHT-induced growth of LNCaP cells through a decrease in E2F protein expression and suppression of E2F activity by hypophosphorylation of Rb.

Transition of an androgen-dependent human prostate cancer cell line into an androgen-independent subline is associated with increased angiogenesis.

Androgen-independent prostate cancer is today an incurable disease, but increased understanding of the mechanisms for the transition into an androgen-independent state may increase the possibilities for more efficient strategies in the future. An androgen-independent subline, LNCaP-19, to the androgen-dependent prostate cancer cell line LNCaP was developed in vitro under standard culture conditions. The characteristics of LNCaP-19 regarding androgen responsiveness, PSA, and VEGF secretion was studied in vitro. The growth in vivo and the microvessel density (MVD) of the tumors were studied after inoculation in nude mice. LNCaP-19 grows equally well in dextran-charcoal stripped FBS (DCC-FBS) as in normal FBS, and rapidly gives rise to tumors in both intact and castrated mice, indicating a true androgen-independent growth. The PSA secretion from LNCaP-19 cells was lower than from LNCaP cells, while the VEGF level was comparable to the secretion from LNCaP cells without androgen stimulation. The MVD was increased in the LNCaP-19 tumors, and the vessels also displayed a changed morphology with exclusively small microvessels without lumen. LNCaP-19 shows characteristics resembling those of androgen-independent prostate cancer. An increased MVD and changed vessel morphology in the tumor, makes it an interesting model system for studies regarding angiogenesis in the context of the acquisition of androgen independence.

Protection of androgen-dependent human prostate cancer cells from oxidative stress-induced DNA damage by overexpression of clusterin and its modulation by androgen.

Recent studies reported that oxidative stress is one of the major factors associated with the progression of prostate cancer through the accumulation of DNA damage. In the present study, we investigated the effect of oxidative stress on cell injury using androgen-dependent human prostate cancer LNCaP cells overexpressing clusterin, which has been shown to play crucial roles in the acquisition of resistance to several apoptotic stimuli. We introduced clusterin cDNA into LNCaP cells which do not express a detectable level of clusterin expression, and generated a clusterin-overexpressing cell line (LNCaP/Cl) and a control vector only-transfected cell line (LNCaP/Co). The effects of hydrogen peroxide (H2O2) treatment on the LNCaP sublines with and without the addition of dihydrotestosterone (DHT) were analyzed using the in vitro mitogenic assay and lipid peroxidation assay, and morphological changes in the LNCaP sublines after H2O2 treatment were examined by staining with Hoechst 33258. The degrees of DNA damage induced by H2O2 into the LNCaP sublines were evaluated by the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. H2O2-induced apoptosis in LNCaP/Cl was significantly suppressed compared with that in LNCaP/Co through the inhibition of membrane damage; however, the measurement of 8-OHdG level demonstrated that DNA damage was more intensively accumulated in LNCaP/Cl cells than LNCaP/Co cells. Furthermore, DHT suppressed the incidence of apoptotic cell death and enhanced the formation of 8-OHdG in both LNCaP/Cl and LNCaP/Co cells after H2O2 treatment in a dose-dependent manner. These findings suggest that clusterin may contribute to conferring resistance to oxidative stress-mediated cellular injury on prostate cancer cells, especially in the presence of androgen.

Mutually antagonistic effects of androgen and activin in the regulation of prostate cancer cell growth.

Activin, a member of the TGFbeta superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.

The atypical GATA protein TRPS1 represses androgen-induced prostate-specific antigen expression in LNCaP prostate cancer cells

Prostate-specific antigen (PSA) is considered as an important marker for prostate cancer. Regulation of PSA gene expression is mediated by androgens bound to androgen receptors via androgen response elements (AREs) in its promoter and far upstream enhancer regions. In addition, GATA proteins contribute to PSA gene transcription by interacting with GATA motifs present in the PSA enhancer sequence. The TRPS1 gene contains a single GATA zinc finger domain and not only binds to forward consensus GATA motifs but also to an inverse GATA motif overlapping the ARE III in the far upstream enhancer of the PSA gene. Overexpression of TRPS1 in androgen-dependent human LNCaP prostate cancer cells inhibited the transcription of a transiently transfected PSA enhancer/promoter-driven luciferase reporter construct. Furthermore, overexpression of TRPS1 reduced the androgen-induced endogenous PSA levels secreted in culture medium of LNCaP cells. Our results suggest a role of TRPS1 in androgen regulation of PSA gene expression.

PC-SPES: Molecular mechanism to induce apoptosis and down-regulate expression of PSA in LNCaP human prostate cancer cells

PC-SPES is an eight-herbal mixture which has activity against prostate cancer cells and can reduce the serum level of prostate specific antigen (PSA) in more than 80% of individuals with prostate cancer. We conducted this study to begin to clarify the molecular mechanism by which PC-SPES inhibited the growth of prostate cancer cells and down-regulated expression of PSA. Western blot analysis, luciferase reporter assay using a variety of promoters of the PSA gene and the isolated androgen receptor response elements (ARE), as well as electrophoretic mobility shift assay (EMSA) were employed to study the effect of PC-SPES on DHT-induced expression of PSA in LNCaP androgen-dependent human prostate cancer cells. Also, Western blot analysis and luciferase reporter assay using 12-0-tetradecanoylphorbol-13-acetate response elements were employed to study the ability of PC-SPES to activate the c-Jun NH2-terminal kinase (JNK)/c-Jun/AP-1 signal pathway in these cells. Reporter studies showed that PC-SPES inhibited DHT-induced PSA promoter/enhancer-luciferase activity via inhibition of ARE transcriptional activity. Western blot analysis showed that PC-SPES down-regulated DHT-induced expression of PSA without decreasing DHT-induced nuclear level of AR. EMSA demonstrated that PC-SPES inhibited the binding of DHT-activated AR to ARE. Moreover, we found that PC-SPES phosphorylated JNK, increased levels of phosphorylated and unphosphorylated forms of c-Jun, and enhanced AP-1 transcriptional activity in LNCaP cells. Interestingly, when LNCaP cells were stably tranfected with the dominant negative JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), these cells no longer underwent apoptosis and growth inhibition in the presence of PC-SPES. But, PC-SPES still decreased levels of PSA in the LNCaP-JIP-1 cells. Taken together, PC-SPES inhibited binding of DHT-activated AR to AREs of PSA gene resulting in down-regulation of ARE transcriptional activity and expression of PSA, and this occurred independently of the JNK/c-Jun/AP-1 signal pathway. Also, PC-SPES activated the JNK/c-Jun/AP-1 signal pathway resulting in growth arrest and apoptosis of prostate cancer cells.

Plant-derived 3,3′-Diindolylmethane Is a Strong Androgen Antagonist in Human Prostate Cancer Cells

3,3'-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a potential anticancer component of cruciferous vegetables. Our results indicate that DIM exhibits potent antiproliferative and antiandrogenic properties in androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA synthesis. These activities were not produced in androgen-independent PC-3 cells. Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM inhibited, in a concentration-dependent manner, the DHT-induced expression of a prostate-specific antigen promoter-regulated reporter gene construct in transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3 cells only when these cells were co-transfected with a wild-type androgen receptor expression plasmid. Using fluorescence imaging with green fluorescent protein androgen receptor and Western blot analysis, we demonstrated that DIM inhibited androgen-induced androgen receptor (AR) translocation into the nucleus. Results of receptor binding assays indicated further that DIM is a strong competitive inhibitor of DHT binding to the AR. Results of structural modeling studies showed that DIM is remarkably similar in conformational geometry and surface charge distribution to an established synthetic AR antagonist, although the atomic compositions of the two substances are quite different. Taken together with our published reports of the estrogen agonist activities of DIM, the present results establish DIM as a unique bifunctional hormone disrupter. To our knowledge, DIM is the first example of a pure androgen receptor antagonist from plants.

Inhibition of steryl sulfatase activity in LNCaP human prostate cancer cells

The enzyme steryl sulfatase may help support the growth of hormone-dependent tumors, including prostate cancers, by facilitating the conversion of circulating precursor steroids to active hormones. We sought to determine the presence of steryl sulfatase activity in the androgen-dependent human prostate cancer cell line LNCaP, and to determine if this activity was inhibited by known steryl sulfatase inhibitors. Intact LNCaP cultures had steryl sulfatase activity, as determined by conversion of [3H]estrone sulfate (E(1)S) to unconjugated steroids. The level of steryl sulfatase activity was relatively low (4.6 pmol/18 h/million cells) compared to MDA-MB-231 breast cancer cells (284.0 pmol/18 h/million cells). The observed activity in both cell lines was blocked by addition of 1 microM estrone sulfamate (EMATE), an active-site-directed, steroidal inhibitor of steryl sulfatase. Steryl sulfatase activity was also inhibited by Danazol, and by (p-O-sulfamoyl)-tetradecanoyl tyramine (C2-14), a non-steroidal inhibitor. Microsomes prepared from LNCaP cultures also showed steryl sulfatase activity, as determined by hydrolysis of [3H]E(1)S and [3H]dehydroepiandrosterone sulfate (DHEAS) to unconjugated forms. LNCaP and MDA-MB-231 microsomes both hydrolyzed E(1)S about two times faster than DHEAS. Hydrolysis of E(1)S in LNCaP and MDA-MB-231 microsomes was blocked by steryl sulfatase inhibitors with the following relative potencies: EMATE>C2-14>Danazol. These data demonstrate that LNCaP prostate cancer cells contain a steryl sulfatase with properties similar to that found in human breast cancer cells, and that the activity of this enzyme can be blocked by known steryl sulfatase inhibitors. Steryl sulfatase inhibitors may be useful as an adjuvant to androgen deprivation therapy for prostate cancer.

Neuroendocrine differentiation of the LNCaP prostate cancer cell line maintains the expression and function of VIP and PACAP receptors

The molecular mechanisms involved in differentiation of prostate cancer cells to a neuroendocrine (NE) cell phenotype are not well understood. Here we used the androgen-dependent human prostate cancer cell line LNCaP to perform a systematic and broad analysis of the expression, pharmacology, and functionality of vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptors. Reverse transcription polymerase chain reaction experiments, together with pharmacological approaches with a set of specific agonists and antagonists, demonstrated the presence of the three VIP/PACAP receptor subtypes (PAC 1, VPAC 1, and VPAC 2) with a major role for VPAC 1, acting through adenylate cyclase (AC) stimulation. An essentially similar pattern was observed by NE differentiated cells (4 days after serum deprivation) in spite of the important morphological changes observed. However, the expression of the prostate-specific antigen (PSA) decreased in NE cells (and increased again by dihydrotestosterone, DHT, treatment). The present demonstration of the induction of NE transdifferentiation in LNCaP cells by increasing concentrations of VIP adds value to previous observations on the role of cAMP in this process, an interesting topic in the comprehension of the molecular changes that are involved in the progression of prostate cancer to androgen independence.

Cell growth and gene modulatory activities of Yunzhi (Windsor Wunxi) from mushroom Trametes versicolor in androgen-dependent and androgen-insensitive human prostate cancer cells.

The incidence of prostate cancer varies greatly throughout the world; it is highest in African-Americans and lowest in the Asian populations of China, India, and Japan. Geographical differences in both prevalence of latent prostate cancer and mortality have been postulated to be influenced by diverse tumor-promoting and protective factors, both environmental and dietary. Prostate cancer is a tumor with an extremely long latency; the pattern of prostate tumorigenesis, in terms of the display and sequence of appearance of particular molecular or biochemical features, or morphological changes, characterizing different stages of the carcinogenic process, is expected to be heterogeneous. Some insights into tumor heterogeneity and progression can be obtained from studies using cell lines, particularly those derived from different anatomical sites. The present study aims to investigate whether hormone-responsive LNCaP and androgen-refractory JCA-1, PC-3, and DU-145 prostate cancer cells are responsive to Yunzhi (YZ), a proprietary dietary supplement prepared from extracts of Trametes versicolor, also known as Coriolus versicolor (a mushroom consumed by Chinese for its purported health benefits), and to elucidate its mechanism of action. Ethanolic extracts (70%) of YZ significantly reduced LNCaP cell growth, down-regulated the levels of secreted PSA, but had less effects on the expression of intracellular PSA and did not affect levels of the androgen receptor. In androgen-unresponsive prostate cancer cells, YZ had a much less pronounced suppressive effect on proliferation of PC-3 and DU-145 cells, compared to LNCaP, and was inactive against JCA-1 cells. Western blot analyses show that the expression of Rb, a key regulatory protein in G1/S transition, and PCNA, integrally involved in mammalian cell DNA replication, were significantly reduced by treatment with YZ in PC-3 and DU-145 cells, respectively. In contradiction, none of these biochemical parameters were affected in JCA-1 cells under identical treatment conditions. Further analysis shows that YZ increased the levels of signal transducer and activator family of transcription factors STAT 1 and STAT 3 in JCA-1 and not LNCaP cells. The greater sensitivity of LNCaP cells to this polysaccharopeptide raises the possibility that YZ may be considered as an adjuvant therapy in the treatment of hormone responsive prostate cancer; additionally, it may have chemopreventive potential to restrict prostate tumorigenic progression from the hormone-dependent to the hormone-refractory state.

Development of human prostate cancer cells to neuroendocrine-like cells by interleukin-1.

Growth and development of some prostate epithelial cells are androgen-dependent. Non-androgenic hormones and growth factors may also influence prostate cells and the effect of interleukin 1beta (IL-1beta) has been investigated with an androgen-dependent human prostate cancer cell line LNCaP. Exposure of LNCaP cells to IL-1beta at picomole ranges resulted in a dose-dependent and progressive differentiation to neuroendocrine-like cells evidenced by dendrite formation and the development of specific neuroendocrine cell markers. Quantification by computer-based image analysis after immunostaining revealed a two-fold increase of chromogranin A in 90% of the cells and a ten-fold increase in the remaining 10%. Additionally, serotonin was developed in all the cells with the staining intensity increased by five-fold. Significant increase in cytokeratin 8 and reduction of prostate specific antigen was also noted. Proliferation was reduced in parallel to the cellular development. The IL-1beta effect was irreversible after several days of IL-1beta incubation. IL-1beta is produced constitutively and its secreted level has an inversed relation during the exponential and plateau phases of cell growth. An IL-1 autocrine regulation in the growth and differentiation of prostate cells is discussed.

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

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. 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. 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. 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.

Effect of vitamin C on prostate cancer cells in vitro: effect on cell number, viability, and DNA synthesis.

Many studies describe the protective role of vitamin C (ascorbic acid) against cancer development and in treatment of established cancer. The present study investigated whether ascorbic acid demonstrates a therapeutic benefit for prostate cancer. Androgen-independent (DU145) and androgen-dependent (LNCaP) human prostate cancer cell lines were both treated in vitro with vitamin C (0-10 mM). Cell counts, cell viability, and thymidine incorporation into DNA were determined. Treatment of DU145 and LNCaP cells with vitamin C resulted in a dose- and time-dependent decrease in cell viability and thymidine incorporation into DNA. Vitamin C induced these changes through the production of hydrogen peroxide; addition of catalase (100-300 units/ml), an enzyme that degrades hydrogen peroxide, inhibited the effects of ascorbic acid. Superoxide dismutase, an enzyme that dismutates superoxide and generates hydrogen peroxide, did not prevent decreases in cell number and DNA synthesis, suggesting further the involvement of hydrogen peroxide in vitamin C-induced changes. These results clearly indicate that reactive oxygen species (ROS) are involved in vitamin C-induced cell damage. However, that singlet oxygen scavengers such as sodium azide and hydroquinone and hydroxyl radical scavengers such as D-mannitol and DL-alpha-tocopherol did not counteract the effects of ascorbic acid on thymidine incorporation suggests that vitamin C-induced changes do not occur through the generation of these ROS. Vitamin C inhibits cell division and growth through production of hydrogen peroxide, which damages the cells probably through an as yet unidentified free radical(s) generation/mechanism. Our results also suggest that ascorbic acid is a potent anticancer agent for prostate cancer cells.

Characterization of high‐affinity receptors for bombesin/gastrin releasing peptide on the human prostate cancer cell lines PC‐3 and DU‐145: Internalization of receptor bound 125I‐(Tyr4) bombesin by tumor cells

Specific receptors for bombesin/gastrin releasing peptide (GRP) on the androgen-independent human prostate cancer cell lines PC-3 and DU-145 were characterized. No specific binding of 125I-[Tyr4]-bombesin to the androgen-dependent human prostate cancer cell line LNCaP was detectable. The binding of 125I-[Tyr4]-bombesin to PC-3 and DU-145 cells was found to be time- and temperature-dependent, saturable, and reversible. Scatchard analysis revealed a single class of binding sites with high affinity (Kd 9.8 x 10(-11) M for PC-3, and 9.1 x 10(-11) M for DU-145 cells at 25 degrees C) and with a binding capacity of 44,000 binding sites/cell and 19,000 binding sites/cell, respectively. Bound 125I-[Tyr4]-bombesin was rapidly internalized by PC-3 cells. The nonhydrolyzable GTP analog GTP-gamma-S caused a dose-dependent inhibition of 125I-[Tyr4]-bombesin binding to PC-3 and DU-145 cells, indicating that a G-protein (guanine nucleotide-binding protein) couples the bombesin receptor to intracellular effector systems. Bombesin and GRP(14-27) inhibited the binding of 125I-[Tyr4]-bombesin to both cell lines in a dose-dependent manner with inhibition constants (Ki) of 0.5 nM and 0.4 nM, respectively. Both cell lines express the bombesin/GRP preferring bombesin receptor subtype, since, in displacement studies, neuromedin B was more than 200 times less potent than bombesin and GRP(14-27) in inhibiting the binding of 125I-[Tyr4]-bombesin. Two synthetic bombesin/GRP antagonists, RC-3095 and RC-3110, powerfully inhibited the specific binding of 125I-[Tyr4]-bombesin with Ki 0.92 nM and 0.26 nM on PC-3 cells, and 3.3 nM and 0.89 nM on DU-145 cells, respectively. These findings indicate that the PC-3 and DU-145 human prostate cancer cell lines possess specific high-affinity receptors for bombesin/GRP, and are suitable models for the evaluation of the antineoplastic activity of new bombesin/GRP antagonists in the treatment of androgen-independent prostate cancer.