Androgen-dependent Human Prostate Cancer Research Articles

The Mitogen-Activated Protein Kinase Phosphatase Vaccinia H1–Related Protein Inhibits Apoptosis in Prostate Cancer Cells and Is Overexpressed in Prostate Cancer

Androgen ablation during the initial stages of prostate cancer causes regression of the tumor due to an increase in apoptosis and reduced cellular proliferation. However, prostate cancer invariably progresses to an androgen-independent state for poorly understood reasons. Previous studies showed that c-Jun NH(2) terminal kinase (JNK) is required for 12-O-tetradecanoylphorbol-13-acetate (TPA)- and thapsigargin (TG)-induced apoptosis in the androgen-responsive prostate cancer cell line LNCaP. Androgens protect LNCaP cells from TPA-induced or TG-induced apoptosis via down-regulation of JNK activation. However, the molecular mechanisms of this inhibition are not clear. Here, we systematically investigated the possible regulation of mitogen-activated protein kinase phosphatases/dual-specificity phosphatases during apoptosis of LNCaP cells and found that Vaccinia H1-related protein (VHR/DUSP3) is up-regulated by androgens during inhibition of apoptosis in LNCaP cells, but not in androgen-independent DU145 cells. Ectopic expression of wild-type VHR, but not a catalytically inactive mutant, interfered with TPA- and TG-induced apoptosis. Consistently, small interfering RNA-mediated knockdown of endogenous VHR increased apoptosis in response to TPA or TG in the presence of androgens. Furthermore, COS7 cells stably expressing wild-type VHR, but not a mutant, had a decrease in JNK phosphorylation. In vivo, VHR expression decreased in the androgen-dependent human prostate cancer xenograft CWR22 upon androgen withdrawal and was inversely correlated to JNK phosphorylation. Expression analysis in human prostate cancer specimens showed that VHR is increased in prostate cancer compared with normal prostate. These data show that VHR has a direct role in the inhibition of JNK-dependent apoptosis in LNCaP cells and may therefore have a role in prostate cancer progression.

Relationship between Glucocorticoid Receptor Signal Pathway and Androgen-Independent Prostate Cancer

Objective: To study the role of the glucocorticoid receptor (GR) signal pathway and downstream cytokine interleukin-6 (IL-6) in androgen-independent growth of prostate cancer (PC). Methods: The human androgen-dependent PC (ADPC) cell line LNCaP and androgen-independent PC (AIPC) cell line DU145 were cultured in vitro. Immunocytochemistry was used to examine the expression of the androgen receptor (AR), GR, HSP90 and IL-6. The GR antagonist RU486 was used to treat cultured cells, and the effects of RU486 on the proliferation of both cell lines were analyzed by MTT assay. Expression of HSP90 and IL-6 mRNA and protein was assessed by RT-PCR and Western blots, respectively. Results: LNCaP cells were AR-positive and GR-negative, whereas DU145 cells were GR-positive and AR-negative. The expression of HSP90 and IL-6 in DU145 cells were significantly stronger than that in LNCaP cells (p < 0.01). RU486 had no obvious effects on the growth of LNCaP cells, but exerted a significant time- and dose-dependent growth inhibition on DU145 cells at doses as low as 0.1 µmol/l. RU486 treatment of DU145 cells also resulted in a dose-dependent decrease in the expression of HSP90 and IL-6 mRNA and protein. Conclusions: The GR signal pathway may be the main survival pathway for DU145 cells. Abnormal hyperactivation of the GR signal pathway and its promoting the expression of HSP90 and IL-6 contribute to the progression of ADPC to AIPC after androgen ablation.

Hypoxia selects for androgen independent LNCaP cells with a more malignant geno‐ and phenotype

Hypoxia confers resistance to common cancer therapies, however, it has also has been shown to result in genetic alterations which may allow a survival advantage and increase the tumorigenic properties of cancer cells. Additionally, it may exert a selection pressure, allowing expansion of tumor cells with a more aggressive phenotype. To further assess the role of hypoxia in malignant progression in prostate cancer we exposed human androgen dependent prostate cancer cells (LNCaP) to cycles of chronic hypoxia and isolated a subline, LNCaP-H1. This article describes the partial characterization of this cell line. The LNCaP-H1 subline showed altered growth characteristics and exhibited androgen independent growth both in vitro and in vivo. Furthermore, these cells were resistant to mitochondrial-mediated apoptosis, probably since the endogenous levels of Bax was lower and Bcl-2 higher than in the parental LNCaP cells. Microarray analysis revealed that a complex array of pathways had differential gene expression between the 2 cell lines, with LNCaP-H1 cells exhibiting a genetic profile which suggests that they may be more likely metastasize to distant organs, especially bone. This was supported by an in vitro invasion assay, and an in vivo metastasis study. This study shows that hypoxia can select for androgen independent prostate cancer cells which have a survival advantage and are more likely to invade and metastasize.

Expression of PIAS3 an inhibitor of activated STAT3 protein in human prostate cancer

To compare the expressions of PIAS3, an inhibitor of activated STAT3 protein in human androgen-dependent prostate cancer (ADPC) and androgen-independent prostate cancer (AIPC) and to explore the relationship between PIAS3 and the development of AIPC. (1) cDNA microarray technique was used to detect the PIAS3 gene in 20 samples of ADPC and 5 samples of AIPC obtained during operation. (2) Carcinoma of prostate (CaP) cells of the lines LNCaP, DU145, and PC3 were cultured and underwent semi-quantitative RT-PCR and Western blotting to detect the mRNA and protein expression of PIAS3 gene. (3) Ten samples of ADPC tissue and 5 samples of AIPC tissue obtained during operation underwent immunohistochemistry to observe the expression of PIAS3 protein. (1) cDNA microarray technique showed that there were 17 genes significantly down-regulated and 22 were significantly up-regulated in the AIPC tissues in comparison with the AIPC tissues, (2) The mRNA and protein expressions of PIAS3 in the AIPC cells of the lines PC3 and DU145 were higher than those in the ADPC cells of the line LNCaP. (3) The expression of PIAS3 protein in the AIPC tissues was significantly higher than that in the ADPC tissues (P < 0.05). Our results suggested that PIAS3 gene may play an important role in the development of androgen independent prostate cancer so as to promote the continuous cellular proliferation without androgen.

A neutralizing anti‐fibroblast growth factor (FGF) 8 monoclonal antibody shows anti‐tumor activity against FGF8b‐expressing LNCaP xenografts in androgen‐dependent and ‐independent conditions

Fibroblast growth factor 8-isoform b (FGF8b) has been detected in human clinical sex-organ related cancers including hormone-refractory prostate cancer. There are, however, few relevant experimental models. A murine monoclonal anti-FGF8 antibody, KM1334, has been shown to neutralize FGF8b and inhibit the growth of androgen-dependent mouse mammary SC-3 cells in vitro and in vivo. In the present study, we evaluated the anti-tumor activity of KM1334 against androgen-dependent and -independent progression of FGF8b-expressing human prostate cancer xenografts. FGF8b cDNA was transfected into androgen-dependent human prostate cancer cell line LNCaP, and its xenograft tumors were established subcutaneously in SCID mice with or without castration. KM1334 at the dose of 400 microg/head was injected twice weekly. FGF8b-expressing LNCaP cells secreted FGF8b, showed enhanced level of Erk1/2 phosphorylation, and showed more potent growth properties than mock-expressing cells in vitro and in vivo. KM1334 reduced these properties in vitro, inhibited tumorigenecity in vivo (T/C=0.33), and showed anti-tumor activity against established tumors (T/C=0.47) of FGF8b-expressing cells. FGF8b-expressing LNCaP tumors were androgen-dependent. However, they recurred as androgen-independent FGF8b positive tumors after castration. KM1334 also inhibited the growth of established FGF8b-expressing tumors in the androgen-independent states (T/C=0.47). These results indicate that humanized monoclonal antibodies, conserving the paratope of KM1334, are a promising candidate for therapy of FGF8b-expressing clinical prostate cancers. Follow-up studies using xenograft models with clinical FGF8b-expressing tumors are required to validate these early findings.

Piper cubeba Targets Multiple Aspects of the Androgen-Signalling Pathway. A Potential Phytotherapy against Prostate Cancer Growth?

Despite the high prevalence of prostate cancer (PC) in the Western world, there is a dearth of effective medication. Since the androgen-signalling pathway is very much involved in PC growth and development, we investigated the potential of Piper cubeba L. extract, P9605, in targeting multiple events simultaneously within this pathway. This may be more effective compared to an antiandrogen monotherapy. Our results indicated that P9605 inhibited proliferation in androgen-dependent LNCaP human prostate cancer cells by reducing DNA synthesis and inducing apoptosis. This antigrowth effect was less pronounced in androgen-independent PC-3 prostate cancer cell lines. P9605 potently inhibited 5 alpha-reductase II activity, which is responsible for converting testosterone to its active form, dihydrotestosterone (DHT), in the prostate. It also acted as an antagonist at recombinant wild-type androgen receptors (AR). P9605 suppressed cell growth and prostate-specific antigen (PSA) secretion stimulated by physiological concentrations of DHT in LNCaP cells. Interestingly, it down-regulated AR levels. In conclusion, our findings suggest that P9605 may potentially retard the growth of androgen-dependent PC via several mechanisms.

Highly tumorigenic human androgen receptor‐positive prostate cancer cells overexpress angiogenin

We have recently established a highly tumorigenic cell line, LNCaP-CR, derived from human androgen-dependent prostate cancer LNCaP cells. In the present study, we examined the genes responsible for the high tumorigenicity of LNCaP-CR cells. The cDNA microarray analysis and protein array of secreted factors indicated angiogenin (ANG), an angiogenic factor, as a candidate gene. Reverse transcription-polymerase chain reaction and immunoassay confirmed that LNCaP-CR cells expressed high levels of ANG but not vascular endothelial growth factor (VEGF), compared with the parental LNCaP cells. We also proved that another tumorigenic androgen receptor-positive prostate cancer cell line, 22Rv1, secretes higher levels of ANG than VEGF. To assess the contribution of ANG to the highly tumorigenic phenotype, we transfected the ANG gene into LNCaP cells in order to overexpress ANG, and also transfected ANG small interfering RNA-expressing constructs into LNCaP-CR cells to downregulate ANG. Overexpression of ANG in LNCaP cells did not affect their growth in vitro, but it significantly enhanced tumorigenicity and angiogenesis in vivo. In contrast, knockdown of ANG expression in LNCaP-CR cells also did not affect the growth in vitro, but it led to a significant decrease in tumorigenicity and angiogenesis. Taken together, ANG is one of the genes responsible for the high tumorigenicity of LNCaP-CR cells. Thus, our results support the idea that ANG is an attractive target for cancer therapy and show that LNCaP-CR cells are useful for studying ANG action and experimental therapeutic approaches targeting ANG.

Inhibitory effect of snake venom toxin from Vipera lebetina turanica on hormone-refractory human prostate cancer cell growth: induction of apoptosis through inactivation of nuclear factor κB

We investigated whether the snake venom toxin (SVT) from Vipera lebetina turanica inhibits cell growth of human prostate cancer cells by inducing apoptosis and also studied possible signaling pathways involved in this cell death. SVT inhibited growth of PC-3 and DU145 cells, androgen-independent prostate cancer cells, but not LNCaP cells, a human androgen-dependent prostate cancer cell. Cells were arrested in the G(2)-M phase by SVT with a concomitant decrease in the expression of the G(2)-M phase regulatory protein cyclin B1 and were also arrested in the G(1)-S phase with decreasing expression of cyclin-dependent kinase 4, cyclin D1 and cyclin E. In addition to the growth-inhibitory effect, SVT increased the induction of apoptotic cell death. Untreated PC-3 cells show high DNA binding activity of nuclear factor kappaB (NF-kappaB), an antiapoptotic transcriptional factor, but this was inhibited by SVT and accompanied by a significant inhibition of p50 translocation into the nucleus, as well as phosphorylation of inhibitory kappaB. Consistent with the induction of apoptosis and inhibition of NF-kappaB, this toxin increased the expression of proapoptotic proteins such as p53, Bax, caspase-3, and caspase-9, but down-regulated antiapoptotic protein Bcl-2. However, SVT did not show an inhibitory effect on cell growth and caspase-3 activity in cells carrying mutant p50 and inhibitory kappaB kinase plasmids. Confocal microscopy analysis showed that SVT is taken up into the nucleus of the cells. These findings suggest that a nanogram concentration range of SVT from V. lebetina turanica could inhibit hormone-refractory human prostate cancer cell growth, and the effect may be related to NF-kappaB signal-mediated induction of apoptosis.

Angiotensin II type 1 receptor antagonist as an angiogenic inhibitor in prostate cancer

Angiotensin II (AII) type 1 receptor (AT1R) antagonists are used widely as antihypertensive agents, and long-term AT1R blockade may have a protective effect against cancer. We previously demonstrated that specific AT1R blockade with candesartan, an AT1R antagonist, inhibited vascular endothelial growth factor (VEGF) production and dramatically decreased lung metastasis of renal cancer by inhibiting tumor angiogenesis. This study was then undertaken to investigate the effects of AT1R blockade using candesartan in prostate cancer (PCa). We first determined whether hormone-independence is associated with tumor angiogenesis and AT1R expression. Accordingly, we postulated that AT1R blockade may affect angiogenesis in androgen-independent PCa rather than in androgen-dependent PCa, and investigated the effects of AII and candesartan on PCa cell lines and a tumor xenograft model. A human hormone-refractory PCa (HRPC) and C4-2 androgen-independent PCa cell line showed significantly higher expression of VEGF, MVD, and AT1R than did human androgen-dependent PCa and an LNCaP androgen-dependent PCa cell line. In vitro, AII and candesartan did not directly affect the proliferation of LNCaP and C4-2 cells, but candesartan significantly suppressed VEGF production in C4-2 cells. In vivo, candesartan significantly suppressed VEGF expression, serum PSA concentration and tumor growth (1.1 +/- 0.2, 45.0 +/- 17.6 ng/ml, 235.8 +/- 37.4 mm(3)) in C4-2 xenografts in castrated mice, compared with the controls (2.4 +/- 0.6, 376.7 +/- 74.2 ng/ml, 830.8 +/- 147.6 mm(3)). Candesartan exerted preventive effects on HRPC, rather than on androgen-sensitive PCa, through the inhibition of tumor angiogenesis.

Repression of androgen receptor in prostate cancer cells by phenethyl isothiocyanate

Prostate cancer usually progresses to androgen refractory after an initial anti-androgen treatment. The androgen receptor (AR) is a pivotal factor for the androgen-mediated growth and maintenance of the prostate. Abnormality of the AR, such as overexpression has been postulated to be related to the hormone independent growth of the cancer. Although we previously demonstrated that the AR expression could be modulated by isothiocyanates, which are natural constituents of cruciferous vegetables, the mechanism, however, remained to be clarified. We have since investigated the mechanism of phenethyl isothiocyanate (PEITC) in AR regulation. A human androgen dependent prostate cancer cell line LNCaP (AD) and its sub-line LNCaP (AI), i.e. androgen independent but overexpressing AR, were exposed to PEITC. The effects of PEITC on cell growth and AR expression/transcription were analyzed with MTT assay, real-time PCR and western blotting. The AR promoter activity was analyzed with the reporter activity after transfection with pAR-luc. The effects on Sp1, the major transcription factor of the AR, were tested with Sp1-luc activity, western blotting and electrophoretic mobility shift assay. PEITC induced a significant growth inhibition, with equal IC(50), in both AD and AI cells. The AR present in both cells was repressed as demonstrated with real-time PCR and western blot. PEITC mediates dual effects at transcriptional and post-translational levels to regulate the AR. At transcriptional level the AR level was reduced via inhibition of the transcription factor Sp1, and at post-translational level by accelerating protein degradation. PEITC represses AR transcription and expression, and mediates growth arrest in androgen dependent and independent prostate cancer cells. With the AR modulation and growth attenuation, PEITC and possibly other isothiocyanates, may prevent and inhibit hormone sensitive and refractory prostate cancer.

Zanthoxyli Fructus induces growth arrest and apoptosis of LNCaP human prostate cancer cells in vitro and in vivo in association with blockade of the AKT and AR signal pathways

Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR-target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types.

Expression and Cellular Localization of Follicle-Stimulating Hormone Receptor in Normal Human Prostate, Benign Prostatic Hyperplasia and Prostate Cancer

FSH, identified as an endogenous product of the prostate, is a glycoprotein with proliferative activity. Increasing evidence of autocrine/paracrine activities of gonadotropins at extragonadal sites led us to investigate the gene expression and cellular localization of FSH-R in normal and diseased human prostates. Prostate specimens, including normal gland, BPH, PCa and human androgen refractory (PC3) and androgen dependent (LNCaP) prostate cancer cell lines (European Collection of Cell Cultures, Salisbury, United Kingdom), were analyzed for FSH-R expression by semiquantitative and real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. We also evaluated cyclic adenosine monophosphate production by cultured PC3 and LNCaP stimulated with human FSH. Little FSH-R expression was seen in 9 of 13 normal and 8 of 15 BPH specimens. Of 30 PCa samples 21 were FSH-R positive with generally higher expression compared to normal prostate and BPH samples. Real-time reverse transcriptase-polymerase chain reaction of matched normal/tumor pairs confirmed higher FSH-R mRNA expression in PCa. PC3 cells expressed FSH-R, while LNCaP cells were FSH-R negative. FSH-R protein was mainly localized in the glandular epithelium and in some stromal cells in normal prostate, BPH and PCa specimens. PC3 cells expressed FSH-R protein and their treatment with FSH induced a significant increase in cyclic adenosine monophosphate production. These results indicate that a subset of PCa expresses FSH-R mRNA and protein at levels higher than those of normal and hyperplastic tissues that express FSH-R. This suggests that FSH might contribute to some cases of PCa via a receptor mediated mechanism.

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.

307. Prostate Targeted TSTA Oncolytic Adenovirus

Top of pageAbstract Due to the limited treatment options currently available for recurrent hormone refractory prostate cancer (HRPC), gene therapy can provide a promising tool to fight this challenging disease. We employed a prostate-targeted gene expression system to develop a safe and efficient vector-based gene delivery approach. The combination of a modified PSA promoter/enhancer and the two- step transcriptional amplification (TSTA) system enabled us to achieve robust expression of transgenes while maintaining prostate specificity. Furthermore, integration of an efficient adenovirus- mediated gene delivery approach and noninvasive imaging enabled real-time assessment of in vivo gene expression by optical CCD imaging (Zhang 2003), as well as clinically-translatable PET radionuclide imaging, applicable to both early stage androgen dependent and late stage HRPC human prostate cancer xenograft models (Sato 2005). In applying PET to monitor HSV-tk-mediated suicide gene therapy, the TSTA system exhibited effective tumor cytoxicity and remarkably lowered liver toxicity compared to constitutive CMV promoter driven system (Johnson 2005). Given the utility and flexibility of the TSTA gene expression system, we further explored the application of this method to develop a robust, prostate-restricted oncolytic adenoviral vector to treat HRPC. This oncolytic vector is designed based on our previous findings that two distinct genes can be transcribed in opposite orientations from centrally located Gal4 binding sites (ASGT 2005; Ray 2004). Hence, in our oncolytic vector, the two key viral regulatory E1A and E1B gene are driven by the Gal4VP16 activator, which in turn is controlled by an augmented prostate-specific PSA promoter. To establish the feasibility of this system, we first developed a set of bi-directional E1 vectors with centrally located Gal4 sites, ranging from 0 (negative control), 2, to 4 tandem repeats. The prostate-specific Gal4VP16 was delivered in trans by a second vector. We discovered that the level of E1 protein products, viral DNA amplification and infectious virus production in PSA- and AR-positive LNCaP cells was non-existent in the virus with 0 Gal4 repeat (G0) and proportionally increased in the G2 (2 sites) and G4 (4 sites) virus. These proof-of-concept findings led us to advance this prostate specific oncolytic technology for in vivo application. Towards this end, we generated all-in-one adenoviral constructs that carry both bi-directional E1 (G0, G2, G4) and the activator expression cassettes (inserted in E3 region) in individual viruses. Infection study showed that all-in-one TSTA oncolytic adenovirus (G2 and G4) can specifically replicate in AR positive prostate cancer cells (LAPC-4 and LNCaP) but not in non-prostate cells (e.g. HeLa and A549). Real-time PCR analysis illustrated a robust increase in the copy number of oncolytic viral genome in prostate cancer cells, and this method is applied to establish the time course and efficiency of replication in comparison to wild-type adenovirus. We foresee that this TSTA oncolytic approach to be a promising gene therapy strategy for the advanced stages of prostate cancer.

Establishment of a highly tumorigenic LNCaP cell line having inflammatory cytokine resistance

Human androgen-dependent prostate cancer LNCaP cells are low tumorigenic even in immunodeficient mice and were killed by the synergistic effect of inflammatory cytokines, IL-β and IL-6. To establish a highly tumorigenic LNCaP cell line, we isolated the cytokine-resistant LNCaP-CR cell line and examined the phenotypes. The parental LNCaP cells were induced to commit apoptosis by the addition of IL-1β and IL-6, but LNCaP-CR cells showed strong resistance against the cytokine action. However, LNCaP-CR cells did not exhibit any resistance to various antitumor drugs investigated. While LNCaP cells formed only palpable tumors in SCID mice, LNCaP-CR cells readily made tumors and their growth was significantly higher than that of LNCaP cells. Moreover, LNCaP tumor-bearing mice gained the weight gradually, but LNCaP-CR tumor-bearing mice significantly lost their body weight. LNCaP-CR cells still responded to androgen action and expressed AR, erbB2, IL-1R, IL-6R, gp130, STAT3, p21, Bcl-2 and caspase-3 as well as LNCaP cells. These results indicate that LNCaP-CR cell line is a new type of tumorigenic LNCaP cell lines and should be useful for identifying responsible genes of tumorigenicity, cytokine resistance, and also cachexia.

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.

BM18: A novel androgen-dependent human prostate cancer xenograft model derived from a bone metastasis

Androgen-dependent prostate cancer (PrCa) xenograft models are required to study PrCa biology in the clinically relevant in vivo environment. Human PrCa tissue from a femoral bone metastasis biopsy (BM18) was grown and passaged subcutaneously through male severe combined immune-deficient (SCID) mice. Human mitochondria (hMt), prostate specific antigen (PSA), androgen receptor (AR), cytokeratin-18 (CK-18), pan-cytokeratin, and high molecular weight-cytokeratin (HMW-CK) were assessed using immunohistochemistry (IHC). Surgical castration was performed to examine androgen dependence. Serum was collected pre- and post-castration for monitoring of PSA levels. BM18 stained positively for hMt, PSA, AR, CK-18, pan keratin, and negatively for HMW-CK, consistent with the staining observed in the original patient material. Androgen-deprivation induced tumor regression in 10/10 castrated male SCID mice. Serum PSA levels positively correlated with BM18 tumor size. BM18 expresses PSA and AR, and rapidly regresses in response to androgen withdrawal. This provides a new clinically significant PrCa model for the study of androgen-dependent growth.