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1.  The EPI Bioassay identifies natural compounds with estrogenic activity that are potent inhibitors of androgenic pathways in human prostate stromal and epithelial cells 
The reactive stromal phenotype is an important factor for prostate cancer progression and may be a new target for treatment and prevention. A new high efficiency preclinical protocol, the EPI bioassay, reflects the interaction of endocrine, paracrine and immune, (EPI) factors on induced androgen metabolism in human prostate reactive stroma. The bioassay is based on co-culturing human primary prostate stromal cells and LAPC-4 prostatic adenocarcinoma cells in a downscaled format of 96-well-plates for testing multiple doses of multiple target compounds. Metabolism of dehydroepiandrosterone (DHEA) with or without TGFβ1–induced stimulation (D+T) of the reactive stroma phenotype was assessed by increased testosterone in the media and PSA production of the epithelial prostate cancer cells. By using the non-metabolizable androgen R1881, effects from direct androgen action were distinguished from stromal androgen production from DHEA. Stromal cell androgenic bioactivity was confirmed using conditioned media from D+T-treated stromal cell monocultures in an androgen-inducible AR screening assay. We further showed that both agonists to estrogen receptor (ER), DPN (ERβ) and PPT (ERα), as well as estrogenic natural compounds including soy isoflavones attenuated D+T-induced PSA production. Studies with the pure ER agonists showed that activating either ERα or ERβ could inhibit both D+T-mediated and R1881-mediated PSA production with the D+T effect being more pronounced. In conclusion, natural compounds with estrogenic activity and pure ER agonists are very potent inhibitors of stromal conversion of DHEA to androgenic metabolites. More studies are needed to characterize the mechanisms involved in estrogenic modulation of the endocrine-immune-paracrine balance of the prostate microenvironment.
doi:10.1016/j.jsbmb.2011.12.003
PMCID: PMC3311472  PMID: 22207083
2.  Transforming growth factor β1 increase of hydroxysteroid dehydrogenase proteins is partly suppressed by red clover isoflavones in human primary prostate cancer-derived stromal cells 
Carcinogenesis  2011;32(11):1648-1654.
Transforming growth factor β1 (TGF-β1) increases dehydro-epiandrosterone (DHEA) metabolism to androgens and prostate-specific antigen (PSA) in a prostate tissue model where stromal (6S) cells and epithelial (LAPC-4) cells are cocultured. Red clover (RC) isoflavones inhibits transforming growth factor (TGF)-β-induced androgenicity. Mechanisms controlling those activities were explored. Three hydroxysteroid dehydrogenases (HSDs), 3β-HSD, HSD-17β1 and HSD-17β5 involved in metabolizing DHEA to testosterone (TESTO) were investigated. Individual depletion of HSDs in 6S cells significantly reduced TGF-β1/DHEA-induced PSA in LAPC-4 cells in cocultures. Monomer amounts of 3β-HSD were similar without or with TGF-β1 in both cell types but aggregates of 3β-HSD in 6S cells were much higher than those in LAPC-4 cells and were upregulated by TGFβ in 6S cells. Basal and TGF-β1-treated levels of HSD-17β1 and HSD-17β5 in LAPC-4 cells were significantly lower than in 6S cells, whereas levels of HSD-17β1 but not HSD-17β5 were TGFβ inducible. 6S cell HSD genes expression induced by TGFβ or androgen signaling was insignificant to contribute TGF-β1/DHEA-upregulated protein levels of HSDs. RC decreased TGF-β1- upregulation of aggregates of 3β-HSD but not HSD-17β1. Depletion of TGFβ receptors (TGFβ Rs) reduced TGF-β1/DHEA-upregulated HSDs and TESTO. Immunoprecipitation studies demonstrated that TGF-β1 disrupted associations of TGFβ Rs/HSDs aggregates, whereas RC suppressed the dissociations of aggregates of 3β-HSD but not HSD-17β1 from the receptors. Given that TGFβ Rs are recycled with or without ligand, TGF-β1-induced disassociation of the HSDs from TGFβ Rs may increase stability and activity of the HSDs. These data suggest a pathway connecting overproduction of TGFβ with increased PSA in prostate cancer.
doi:10.1093/carcin/bgr206
PMCID: PMC3218644  PMID: 21914638
3.  Androgen-Induced PSA Expression Requires Not Only Activation of AR But Also Endogenous IGF-I or IGF-I/PI3K/Akt Signaling in Human Prostate Cancer Epithelial Cells 
The Prostate  2010;71(7):766-777.
BACKGROUND
Prostate cancer (PrCa) risk is positively associated with levels of insulin-like growth factor I (IGF-I) and prostate specific antigen (PSA), both androgen receptor (AR) signaling target genes in PrCa cells. Although activated AR is required for androgen-induction of expression of both genes, effects of the IGF-I signaling pathways on the androgen-induction of PSA have not been studied.
METHODS
Human prostate stromal and epithelial cancer cells were treated alone or in coculture with steroid hormone and/or inhibitors. Gene or protein expression was analyzed by real time RT-PCR or Western blotting of lysates, nuclear extracts, or immunoprecipitated products.
RESULTS
In PrCa epithelial cells, endogenous IGF-I, significantly induced by R1881, was required for R1881-induction of PSA. Increased IGF-I correlated with accumulation of cytoplasmic dephospho β-catenin (CPDP β-catenin), a co-activator of AR signaling. Exogenous IGF-I enhanced R1881-induced PSA and accumulation of CPDP β-catenin in LAPC-4 cells. Functional depletion of IGF-I or IGF-I receptor diminished PSA induction. Induction of IGF-I reached a plateau while PSA consecutively increased. Inhibiting PI3K abolished R1881-induced Akt phosphorylation, CPDP and nuclear β-catenin and nuclear association of AR/β-catenin, consequently abrogating R1881-induced expression of IGF-I and/or PSA.
CONCLUSIONS
By integrating androgen, IGF-I and β-catenin signaling pathways, these data reveal that androgen-induced PSA expression requires activation of AR and endogenous IGF-I or IGF-I/PI3K/Akt signaling, suggesting a positive feedback cycle for increased production of PSA associated with PrCa.
doi:10.1002/pros.21293
PMCID: PMC3125406  PMID: 21031436
Akt; AR; β-catenin; IGF-I; prostate cancer; PSA; R1881
4.  DHEA metabolism in prostate: For better or worse? 
Dehydroepiandrosterone (DHEA) is commonly used in the USA as a nutritional supplement for antiaging, metabolic support or other uses. Investigations into understanding the effects of DHEA on human prostate cancer progression have posed more questions than answers and highlight the importance of communications between stromal and epithelial elements within the prostate that contribute to the regulation of DHEA metabolism. Intracrine metabolism of DHEA to androgens (A) and/or estrogens (E) may occur in one cell compartment (stromal) which may release paracrine hormones or growth/inhibitory factors to the epithelial cells. Alternatively no metabolism of DHEA may occur, resulting in no harmful consequences of high levels of DHEA in prostate tissues. We herein review the tissue components involved and interactions with the prohormone, DHEA and/or resulting metabolites, including dihydrotestosterone (DHT) or 17β-Estradiol (E2) in an in-vitro model of endocrine-immune-paracrine interactions within the prostate. This work raises questions and hypotheses concerning the role of DHEA in prostate in normal tissues, vs. preneoplastic tissues.
doi:10.1016/j.mce.2008.10.019
PMCID: PMC2667103  PMID: 19013497
DHEA; TGF β1; Androgen Receptor; Estrogen Receptor; stromal; epithelial prostate; PSA; testosterone; coculture; red clover isoflavones
5.  Endocrine-Immune-Paracrine Interactions In Prostate Cells: A Model For Mechanistic Studies Of Phytomedicines 
Dehydroepiandrosterone (DHEA) is used as a dietary supplement and can be metabolized to androgens and/or estrogens in the prostate. We investigated the hypothesis that DHEA metabolism may be increased in a reactive prostate stroma environment in the presence of pro-inflammatory cytokines such as TGFβ1 and further, whether red clover extract, which contains a variety of compounds including isoflavones, can reverse this effect. LAPC-4 prostate cancer cells were grown in coculture with prostate stromal cells (6S), and treated with DHEA +/- TGFβ1 or IL-6. PSA expression and testosterone (T) secretion in LAPC4/6S cocultures were compared with those in monocultured epithelial and stromal cells using real time PCR and/or ELISA. Combined administration of TGFβ1+DHEA to cocultures increased PSA protein secretion 2-4 times, and PSA gene expression up to 50-fold. DHEA + TGFβ1 also increased coculture production of testosterone over DHEA treatment alone. Red clover isoflavone treatment led to a dose-dependent decrease in PSA protein and gene expression and T metabolism induced by TGFβ1+DHEA in prostate LAPC-4/6S cocultures. In this coculture model of endocrine-immune-paracrine interactions in the prostate, TGFβ1 greatly increased stromal-mediated DHEA effects on T production and epithelial cell PSA production, whereas red clover isoflavones reversed these effects.
doi:10.1158/1940-6207.CAPR-08-0062
PMCID: PMC2757651  PMID: 19141600
DHEA; TGFβ1; stromal; prostate; PSA; testosterone; coculture; red clover isoflavones
6.  Human Prostate Stromal Cells Stimulate Increased PSA Production in DHEA-treated Prostate Cancer Epithelial Cells 
Dehydroepiandrosterone (DHEA) is commonly used as a dietary supplement and may affect prostate pathophysiology when metabolized to androgens and/or estrogens. Human prostate LAPC-4 cancer cells with a wild type androgen receptor (AR), were treated with DHEA, androgens (DHT, T, or R1881), and E2 and assayed for PSA protein and gene expression. In LAPC-4 monocultures, DHEA and E2 induced little or no increase in PSA protein or mRNA expression compared to androgen-treated cells. When prostate cancer-associated (6S) stromal cells were added in coculture, DHEA stimulated LAPC-4 cell PSA protein secretion to levels approaching induction by DHT. Also, DHEA induced 15-fold more PSA mRNA in LAPC-4 cocultures than in monocultures. LAPC-4 proliferation was increased 2–3 fold when cocultured with 6S stromal cells regardless of hormone treatment. DHEA-treated 6S stromal cells exhibited a dose- and time-dependent increase in T secretion, demonstrating stromal cell metabolism of DHEA to T. Coculture with non-cancerous stroma did not induce LAPC-4 PSA production, suggesting a differential modulation of DHEA effect in a cancer-associated prostate stromal environment. This coculture model provides a research approach to reveal detailed endocrine, intracrine, and paracrine signaling between stromal and epithelial cells that regulate tissue homeostasis within the prostate, and the role of the tumor microenvironment in cancer progression.
doi:10.1016/j.jsbmb.2008.06.008
PMCID: PMC2570207  PMID: 18621129
DHEA; stromal; prostate; PSA; coculture

Results 1-6 (6)