Integrating ayurvedic medicine into cancer research programs part 2: Ayurvedic herbs and research opportunities.

The aim of this two-part review in this issue is to provide some basic perspectives from Ayurveda, the traditional medicine of India, and to discuss how current research methodologies may be used to shed light on mechanisms of Ayurvedic treatments to support cancer care and prevention. It addresses some of the challenges for scientific validation of Ayurvedic herbal compounds, protocols, and modalities in four areas. Part 1 [1] has reviewed Ayurvedic theories and applications of body constitution (Prakriti), digestion (Agni and Ama) and mind-body-spirit health in relation to cancer. Here in Part 2, the focus is on preclinical and clinical research of Ayurvedic botanical herbs, with a review of pertinent literature on three selected herbs, Curcumin, Ashwagandha, and Triphala. A discussion of the challenges and possibilities of research in Ayurveda is offered to guide the development of translational research programs. Ayurvedic modalities are not intended as a substitute for allopathic treatments of cancer but as an integrative component for prevention and restoration of strength and immunity.

Integrating ayurvedic medicine into cancer research programs part 1: Ayurveda background and applications.

Integration of Ayurveda into our current health care research programs is critical to making progress in global wellness and in disease prevention and control, especially for cancer. Ayurveda promotes restoration of the innate healing mechanisms existing in the body for optimal immunity, resilience, and health. Ayurveda also has an abundant resource of botanical products containing diverse pharmaco-active ingredients and millennia of experience of clinical applications for health benefits. But there is a lack of evidence-based research to demonstrate its efficacy and potential. This 2-part review is written from the perspective of a western-trained biomedical scientist and student of Ayurveda. It aims to educate research scientist peers about the opportunities and challenges for scientific validation of Ayurvedic herbal compounds, protocols, and modalities and inspire more research in this area. Part 1 will review several aspects of Ayurveda including principles of body constitution (Prakriti), digestion (Agni and Ama) and mind-body health, in relation to cancer. Part 2 [1] will focus on Ayurvedic botanical resources used for cancer and research studies will be discussed on selected herbal compounds. Research gaps and opportunities will be identified to guide development of research programs to validate safety and efficacy of these therapies. Importantly, the use of Ayurvedic modalities is not intended to substitute for allopathic treatments for cancer but as an integrative component for prevention and restoration of strength and immunity post treatment.

TGFß1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

The inflammatory tissue microenvironment can be an active promoter in preneoplastic cancer lesions. Altered steroid hormone metabolism as induced by the inflammatory microenvironment may contribute to epithelial cancer progression. Dehydroepiandrosterone sulfate (DHEAS) is the most abundant endogenous steroid hormone present in human serum and can be metabolized to DHEA, androgens and/or estrogens in peripheral tissues. We have previously reported that TGFß1-induced reactive prostate stromal cells increase DHEA metabolism to active androgens and alter prostate cancer cell gene expression. While much of the focus on mechanisms of prostate cancer and steroid metabolism is in the epithelial cancer cells, this study focuses on TGFß1-induced effects on DHEA metabolic pathways and enzymes in human prostate stromal cells. In DHEA-treated primary prostate stromal cells, TGFß1 produced time- and dose-dependent increases in metabolism of DHEA to androstenedione and testosterone. Also TGFß1-treated prostate stromal cells exhibited changes in the gene expression of enzymes involved in steroid metabolism including up-regulation of 3ß hydroxysteroid dehydrogenase (HSD), and down-regulation of 17ßHSD5, and 17ßHSD2. These studies suggest that reactive prostate stroma and the inflammatory microenvironment may contribute to altered steroid metabolism and increased intratumoral androgens.

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 TGFß1-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. 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 (ERß) 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 ERß 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.

Transforming growth factor ß1 increase of hydroxysteroid dehydrogenase proteins is partly suppressed by red clover isoflavones in human primary prostate cancer-derived stromal cells.

Transforming growth factor ß1 (TGF-ß1) 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)-ß-induced androgenicity. Mechanisms controlling those activities were explored. Three hydroxysteroid dehydrogenases (HSDs), 3ß-HSD, HSD-17ß1 and HSD-17ß5 involved in metabolizing DHEA to testosterone (TESTO) were investigated. Individual depletion of HSDs in 6S cells significantly reduced TGF-ß1/DHEA-induced PSA in LAPC-4 cells in cocultures. Monomer amounts of 3ß-HSD were similar without or with TGF-ß1 in both cell types but aggregates of 3ß-HSD in 6S cells were much higher than those in LAPC-4 cells and were upregulated by TGFß in 6S cells. Basal and TGF-ß1-treated levels of HSD-17ß1 and HSD-17ß5 in LAPC-4 cells were significantly lower than in 6S cells, whereas levels of HSD-17ß1 but not HSD-17ß5 were TGFß inducible. 6S cell HSD genes expression induced by TGFß or androgen signaling was insignificant to contribute TGF-ß1/DHEA-upregulated protein levels of HSDs. RC decreased TGF-ß1- upregulation of aggregates of 3ß-HSD but not HSD-17ß1. Depletion of TGFß receptors (TGFß Rs) reduced TGF-ß1/DHEA-upregulated HSDs and TESTO. Immunoprecipitation studies demonstrated that TGF-ß1 disrupted associations of TGFß Rs/HSDs aggregates, whereas RC suppressed the dissociations of aggregates of 3ß-HSD but not HSD-17ß1 from the receptors. Given that TGFß Rs are recycled with or without ligand, TGF-ß1-induced disassociation of the HSDs from TGFß Rs may increase stability and activity of the HSDs. These data suggest a pathway connecting overproduction of TGFß with increased PSA in prostate cancer.

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.

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 ß-catenin (CPDP ß-catenin), a co-activator of AR signaling. Exogenous IGF-I enhanced R1881-induced PSA and accumulation of CPDP ß-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 ß-catenin and nuclear association of AR/ß-catenin, consequently abrogating R1881-induced expression of IGF-I and/or PSA. CONCLUSIONS: By integrating androgen, IGF-I and ß-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.

Dehydroepiandrosterone administration or G{alpha}q overexpression induces {beta}-catenin/T-Cell factor signaling and growth via increasing association of estrogen receptor-{beta}/Dishevelled2 in androgen-independent prostate cancer cells.

beta-Catenin/T-cell factor signaling (beta-CTS) plays multiple critical roles in carcinogenesis and is blocked by androgens in androgen receptor (AR)-responsive prostate cancer (PrCa) cells, primarily via AR sequestration of beta-catenin from T-cell factor. Dehydroepiandrosterone (DHEA), often used as an over-the-counter nutritional supplement, is metabolized to androgens and estrogens in humans. The efficacy and safety of unregulated use of DHEA are unclear. We now report that DHEA induces beta-CTS via increasing association of estrogen receptor (ER)-beta with Dishevelled2 (Dvl2) in AR nonresponsive human PrCa DU145 cells, a line of androgen-independent PrCa (AiPC) cells. The induction is temporal, as assessed by measuring kinetics of the association of ERbeta/Dvl2, protein expression of the beta-CTS targeted genes, c-Myc and cyclin D1, and cell growth. However, in PC-3 cells, another human AiPC cell line, DHEA exerts no detectible effects, partly due to their lower expression of Galpha-subunits and DHEA down-regulation of ERbeta/Dvl2 association. When Galphaq is overexpressed in PC-3 cells, beta-CTS is constitutively induced, including increasing c-Myc and cyclin D1 protein expression. This effect involved increasing associations of Galphaq/Dvl2 and ERbeta/Dvl2 and promoted cell growth. These activities require ERbeta in DU-145 and PC-3 cells because they are blocked by ICI 182-780 treatment inactivating ERbeta, small interfering RNA administration depleting ERbeta, or AR overexpression arresting ERbeta. These data suggest that novel pathways activating beta-CTS play roles in the progression of AiPC. Although DHEA may enhance PrCa cell growth via androgenic or estrogenic pathways, the effects of DHEA administration on clinical prostate function remain to be determined.

Endocrine-immune-paracrine interactions in prostate cells as targeted by 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 proinflammatory cytokines such as transforming growth factor beta1 (TGFbeta1), 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 +/- TGFbeta1 or interleukin-6. Prostate-specific antigen (PSA) expression and testosterone secretion in LAPC-4/6S cocultures were compared with those in monocultured epithelial and stromal cells by real-time PCR and/or ELISA. Combined administration of TGFbeta1 + DHEA to cocultures increased PSA protein secretion two to four times, and PSA gene expression up to 50-fold. DHEA + TGFbeta1 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 testosterone metabolism induced by TGFbeta1 + DHEA in prostate LAPC-4/6S cocultures. In this coculture model of endocrine-immune-paracrine interactions in the prostate, TGFbeta1 greatly increased stromal-mediated DHEA effects on testosterone production and epithelial cell PSA production, whereas red clover isoflavones reversed these effects.

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 tuoitiuot 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 17beta-estradiol (E(2)) 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.

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 dihydrotestosterone (DHT), T, or R1881), and E2 and assayed for prostate specific antigen (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.

Lycopene inhibits IGF-I signal transduction and growth in normal prostate epithelial cells by decreasing DHT-modulated IGF-I production in co-cultured reactive stromal cells.

Prostate stromal and epithelial cell communication is important in prostate functioning and cancer development. Primary human stromal cells from normal prostate stromal cells (PRSC) maintain a smooth muscle phenotype, whereas those from prostate cancer (6S) display reactive and fibroblastic characteristics. Dihydrotestosterone (DHT) stimulates insulin-like growth factor-I (IGF-I) production by 6S but not PSRC cells. Effects of reactive versus normal stroma on normal human prostate epithelial (NPE or PREC) cells are poorly understood. We co-cultured NPE plus 6S or PRSC cells to compare influences of different stromal cells on normal epithelium. Because NPE and PREC cells lose androgen receptor (AR) expression in culture, DHT effects must be modulated by associated stromal cells. When treated with camptothecin (CM), NPE cells, alone and in stromal co-cultures, displayed a dose-dependent increase in DNA fragmentation. NPE/6S co-cultures exhibited reduced CM-induced cell death with exposure to DHT, whereas NPE/PRSC co-cultures exhibited CM-induced cell death regardless of DHT treatment. DHT blocked CM-induced, IGF-I-mediated, NPE death in co-cultured NPE/6S cells without, but not with, added anti-IGF-I and anti-IGF-R antibodies. Lycopene consumption is inversely related to human prostate cancer risk and inhibits IGF-I and androgen signaling in rat prostate cancer. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on NPE cell death, decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization and inhibited IGF-I-stimulated NPE and PREC growth, perhaps by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3beta and tyrosine phosphorylation of GSK3. This study expands the understanding of the preventive mechanisms of lycopene in prostate cancer.

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

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

Comparative effects of DHEA and DHT on gene expression in human LNCaP prostate cancer cells.

BACKGROUND: DHEA is widely used as a dietary supplement in older men. Because DHEA can be converted to androgens or estrogens, such use may promote prostate cancer. In this study, the effects of DHEA were compared with those of DHT using gene expression array profiles in human LNCaP prostate cancer cells. MATERIALS AND METHODS: LNCaP cells were exposed to DHEA (300 nM), DHT (300 nM), or vehicle for 48 h, and mRNA expression was measured using Affymetrix HU-95 gene chips. Gene expression values were sorted in ascending order on the p-values corresponding to the extent of differential RNA expression between control and either hormone treatment. RESULTS: S100 calcium binding protein, neurotensin, 24-dehydrocholesterol reductase, and anterior-gradient 2 homologue were the four most differentially expressed genes (p-values all < 3 x 10(-5)). Nested tests of differential expression revealed lesser effects of DHEA versus DHT treatment (p < 0.01) for the S100 calcium binding protein and neurotensin genes. Microarray findings were confirmed by QRT-PCR. The top 83 genes exhibiting differential expression after DHEA or DHT were used for pathway analysis. DHT decreased expression of more genes involved in intercellular communication, signal transduction, nucleic acid binding and transport, and in structural components, such as myosin and golgin, than DHEA. CONCLUSION: These data revealed consistent, measurable changes in gene expression patterns following treatment of LNCaP prostate cancer cells with DHEA and DHT. Understanding the mechanisms of DHEA versus DHT actions in the prostate may help clarify the separate and interactive effects of androgenic and estrogenic actions in prostate cancer progression.

DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells.

Prostate cancer is one of the four most common cancers in the United States, affecting one of six men. Increased serum levels of androgens and IGF-I are associated with an augmented risk of prostate cancer. Dihydrotestosterone (DHT) and testosterone (T) stimulate prostate cancer cell growth, development, and function, whereas the effects of DHT and T in prostate stromal cells, and of dehydroepiandrosterone (DHEA) in prostate cancer or stromal cells, are uncertain. We investigated the actions of DHT, T, DHEA, and estradiol (E2) on insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor (R), IGF-binding protein (IGFBP)-2, IGFBP-3, and IGFBP-5 in primary cultures of human prostatic stromal cells by assessing cell proliferation, mRNA expression, and protein secretion by MTT growth assay, quantitative real-time PCR, and ELISA, respectively. DHT and T each increased IGF-I (7-fold) and decreased IGFBP-3 (2-fold) mRNA expression and protein secretion in a dose- and time-dependent manner and increased IGFBP-2 (2-fold) mRNA in a dose- and time-dependent manner. DHEA and E2 did not significantly alter these measures. Flutamide abolished the DHT-modulated increases in IGF-I and IGFBP-2, suggesting that the influences of DHT and T on these measures were androgen receptor mediated. None of the four steroids significantly affected IGF-IR, IGF-II, or IGFBP-5 mRNA levels or stromal cell proliferation. The effects of DHT on IGF-I, IGFBP-2, and IGFBP-3 were more pronounced in stromal cultures that did not express desmin. These data suggest that DHT and T promote prostate growth partly via modulation of the stromal cell IGF axis, with potential paracrine effects on prostate epithelial cells.

Role of notch-1 and E-cadherin in the differential response to calcium in culturing normal versus malignant prostate cells.

A panel of expression markers was validated and used to document that, when radical prostatectomy specimens are cultured in low (i.e., <260 micromol/L)-calcium (Ca2+)-serum-free, growth factor-defined (SFD) medium, what grows out are not prostatic cancer cells but basally derived normal transit-amplifying prostatic epithelial cells. The selective outgrowth of the normal transit-amplifying versus prostatic cancer cells is due to the differential effect of low-Ca2+ medium on the structure of Notch-1 and E-cadherin signaling molecules. In low-Ca2+ medium, Notch-1 receptor is conformationally in a constitutively active, cell autonomous form not requiring reciprocal cell-cell (i.e., ligand) interaction for signaling. Such signaling is required for survival of transit-amplifying cells as shown by the death of transit-amplifying cells induced by treatment with a series of chemically distinct gamma-secretase inhibitors to prevent Notch-1 signaling. Conversely, in low-Ca2+ medium, E-cadherin is conformationally inactive preventing cell-cell homotypic interaction, but low cell density nonaggregated transit-amplifying cells still survived because Notch-1 is able to signal cell autonomously. In contrast, when medium Ca2+ is raised to >400 micromol/L, Notch-1 conformationally is no longer constitutively active but requires cell-cell contact for reciprocal binding of Jagged-1 ligands and Notch-1 receptors between adjacent transit-amplifying cells to activate their survival signaling. Such cell-cell contact is enhanced by the elevated Ca2+ inducing an E-cadherin conformation allowing homotypic interaction between transit-amplifying cells. Such Ca(2+)-dependent, E-cadherin-mediated interaction, however, results in cell aggregation, stratification, and inhibition of proliferation of transit-amplifying cells via contact inhibition-induced up-regulation of p27/kip1 protein. In addition, transit-amplifying cells not contacting other cells undergo squamous differentiation into cornified (i.e., 1% SDS insoluble) envelopes and death in the elevated Ca2+ medium. Stratification and contact inhibition induced by elevated Ca2+ are dependent on E-cadherin-mediated homotypic interaction between transit-amplifying cells as shown by their prevention in the presence of a cell-impermanent, E-cadherin neutralizing antibody. In contrast to growth inhibition of normal transit-amplifying cells, supplementation of low-Ca(2+)-SFD medium with 10% FCS and raising the Ca2+ to >600 micromol/L stimulates the growth of all prostate cancer cell lines tested. Additional results document that, at physiologic levels of Ca2+ (i.e., >600 micromol/L), prostatic cancer cells are not contact inhibited by E-cadherin interactions and Notch-1 signaling is no longer required for survival but instead becomes one of multiple signaling pathways for proliferation of prostatic cancer cells. These characteristic changes are consistent with prostate cancer cells' ability to metastasize to bone, a site of high-Ca2+ levels.

Comparative effects of DHEA vs. testosterone, dihydrotestosterone, and estradiol on proliferation and gene expression in human LNCaP prostate cancer cells.

Serum levels of the adrenal androgen dehydroepiandrosterone (DHEA) peak in men and women in the third decade of life and decrease progressively with age. Increasing numbers of middle-aged and older individuals consume over-the-counter preparations of DHEA, hoping it will retard aging by increasing muscle and bone mass and strength, decreasing fat, and improving immunologic and neurobehavioral functions. Because DHEA can serve as a precursor to more potent androgens and estrogens, like testosterone (T), dihydrotestosterone (DHT), and 17beta-estradiol (E2), supplemental DHEA use may pose a cancer risk in patients with nascent or occult prostate cancer. The steroid-responsive human LNCaP prostate cancer cells, containing a functional but mutated androgen receptor (AR), were used to compare effects of DHEA with those of T, DHT, and E2 on cell proliferation and protein and/or gene expression of AR, prostate-specific antigen (PSA), IGF-I, IGF-I receptor (IGF-IR), IGF-II, IGF-binding proteins-2, -3, and -5, (IGFBPs-2, -3, and -5), and estrogen receptor-beta (ERbeta). Cell proliferation assays revealed significant stimulation by all four steroids. DHEA- and E2-induced responses were similar but delayed and reduced compared with that of T and DHT. All four hormones increased gene and/or protein expression of PSA, IGF-IR, IGF-I, and IGFBP-2 and decreased that of AR, ERbeta, IGF-II, and IGFBP-3. There were no significant effects of hormone treatment on IGFBP-5 mRNA. DHEA and E2 responses were similar, and distinct from those of DHT and T, in time- and dose-dependent studies. Further studies of the mechanisms of DHEA effects on prostate cancer epithelial cells of varying AR status, as well as on prostate stromal cells, will be required to discern the implications of DHEA supplementation on prostatic health.

Trk receptor inhibition induces apoptosis of proliferating but not quiescent human osteoblasts.

Prostate cancer frequently metastasizes to the skeleton, producing painful osteoblastic lesions, which are associated with significant morbidity and mortality. This bone tropism involves the bidirectional paracrine interactions between prostate cancer cells and osteoblasts. These interactions enhance prostate cancer cell survival and proliferation of osteoblasts. Therefore, agents that can induce apoptosis of prostate cancer cells and proliferating osteoblasts would be highly advantageous. Previously, we have documented that the unique survival pathway for prostate cancer cells involves a neurotrophin/Trk receptor autocrine pathway. The indocarbazole compounds, CEP-701 and CEP-751, are potent inhibitor of this Trk receptor survival signaling and thus selectively induces apoptosis of prostate cancer cells in various in vitro and in vivo models. In this study, we documented the effects of CEP-751 on the conditionally immortalized osteoblastic cell line, hFOB, in vitro. At the permissive temperature of 34 degrees C, these cells express large T antigen, inducing their continuous proliferation, whereas at 39 degrees C, T antigen is degraded and the cells stop proliferating without undergoing apoptosis. Trk receptors are expressed in hFOB cells, as determined both by reverse transcription-PCR and Western blots. These osteoblasts were shown to produce nerve growth factor and brain-derived neurotrophic factor but not neurotrophin-3, as measured by ELISA. hFOB osteoblasts, cultured at 34 degrees C, secreted significantly (P < 0.01) more brain-derived neurotrophic factor and nerve growth factor into the medium than hFOB cells cultured at 39 degrees C. Because the Trk/neurotrophin axis is present in both proliferating and quiescent (i.e., nonproliferating) osteoblasts, the effects of 48 h of exposure to various doses of CEP-751 on cell viability and apoptosis of hFOB cells were assessed by trypan blue exclusion assays and 4',6-diamidino-2-phenylindole nuclear staining. Cell viability and apoptosis of hFOB cells at 34 degrees C were significantly and dose-dependently decreased compared with untreated proliferating cells. In contrast, even the highest concentration of CEP-751 (200 nM) did not affect cell viability and apoptosis of quiescent hFOB cells cultured at 39 degrees C. This trk inhibition-induced cytotoxicity was confirmed using early-passage, proliferating normal (i.e., non-SV40-transformed) human osteoblasts, which also express Trk receptor protein. These combined results demonstrate that proliferating osteoblasts acquire a sensitivity to trk inhibition- induced apoptosis not shared with normally quiescent osteoblasts.

Effect of normal endometrial stroma on growth and differentiation in Ishikawa endometrial adenocarcinoma cells.

Endometrial cancer is characterized by alterations in the stromal cells and the supporting extracellular matrix in addition to the intrinsic alterations of the malignant epithelial cells. We have developed a cell culture model that demonstrates the role of stromal cells in the regulation of proliferation, hormone responsiveness, and differentiation of an endometrial adenocarcinoma cell line (Ishikawa). Conditioned medium (CM) was collected from normal primary human endometrial stromal cells grown on plastic or within the basement membrane extract, Matrigel. The CM produced by stromal cells cultured in contact with Matrigel markedly inhibited Ishikawa cell proliferation compared with CM from stromal cells cultured on plastic. Ishikawa cell proliferation varied with steroid hormone treatment in the presence of CM from stromal cells embedded in Matrigel. When the Ishikawa cells were placed in coculture in contact with stromal cells in Matrigel, production of a differentiated epithelial secretory product, glycodelin, was induced. Gene expression of stromal cell hormone receptors, growth factors, and integrins was analyzed by reverse transcription-PCR in the presence of Matrigel to determine the potential factors involved in stromal regulatory function. These combined studies imply that the phenotype of the Ishikawa cells can be induced to differentiate to more closely resemble normal endometrial epithelium by reintroduction of stromal factors and appropriate extracellular matrix. Additionally, the study shows that basement membrane proteins influence the regulatory function of stromal cells as they mediate epithelial cell growth.