Purpose: Development of prostate cancer prevention strategies is an important priority to overcome high incidence, morbidity, and mortality. Recently, we showed that Nexrutine, an herbal extract, inhibits prostate cancer cell proliferation through modulation of Akt and cAMP-responsive element binding protein (CREB)^mediated signaling pathways. However, it is unknown if Nexrutine can be developed as a dietary supplement for the prevention of prostate cancer. In this study, we used the transgenic adenocarcinoma of mouse prostate (TRAMP) model to examine the ability of Nexrutine to protectTRAMP mice from developing prostate cancer. Experimental Design: Eight-week-oldTRAMP mice were fed with pelleted diet containing 300 and 600 mg/kg Nexrutine for 20 weeks. Efficacy of Nexrutine was evaluated by magnetic resonance imaging at 18 and 28 weeks of progression and histologic analysis of prostate tumor or tissue at the termination of the experiment. Tumor tissue was analyzed for modulation of various signaling molecules. Results: We show that Nexrutine significantly suppressed palpable tumors and progression of cancer in the TRAMP model. Expression of total and phosphorylated Akt, CREB, and cyclin D1 was significantly reduced in prostate tissue from Nexrutine intervention group compared with tumors from control animals. Nexrutine also inhibited cyclin D1 transcriptional activity in androgen-independent PC-3 cells. Overexpression of kinase dead Akt mutant or phosphorylationdefective CREB inhibited cyclin D1transcriptional activity. Conclusions: The current study shows that Nexrutine-mediated targeting of Akt/CREBî nduced activation of cyclin D1prevents the progression of prostate cancer. Expression of CREB and phosphorylated CREB increased in human prostate tumors compared with normal tissue, suggesting their potential use as prognostic markers.Prostate cancer is the second leading cause of cancer-related deaths in men and expected to lead to f27,350 deaths in 2006 (1). African American men have the highest incidence of prostate cancer in the world, whereas Asian men native to their countries who consume a low-fat, high-fiber diet have the lowest risk (2). Epidemiologic studies suggest that a reduced risk of cancer is associated with the consumption of a phytochemical-rich diet that includes fruits and vegetables (3). Evidence suggests that prostate cancer progresses from normal epithelium to proliferative inflammatory atrophy, to low-grade prostatic intraepithelial neoplasia (PIN), and to high-grade PIN that eventually progresses to the more aggressive-metastatic and clinically evident prostate cancer (ref. 4 and references therein). Such preneoplastic lesions have been found in young men in their 20s and are common in men in their 50s (5). However, clinically detectable prostate cancer does not generally manifest itself until the 60s. In addition, the occurrence of precancerous lesions is more prevalent (about 1 in three men) than the incidence of carcinoma (about one in nine men; ref. 6). Therefore, the develop...

Purpose: The purpose of this study is to investigate whether Fas-associated death domain interleukin-1 converting enzyme like inhibitory protein (FLIP) inhibition is a therapeutic target associated with 2-methoxyestradiol (2-ME 2 )^mediated tumor regression. Experimental Design: Expression and levels of FLIP were analyzed using (a) real-time PCR and immunoblot analysis in androgen-independent PC-3 cells treated with the newly formulated 2-ME 2 and (b) immunohistochemistry in different Gleason pattern human prostate tumors. Transient transfections and chromatin immunoprecipitation (ChIP) assays were used to identify the transcription factors that regulate FLIP. Involvement of FLIP in 2-ME 2^i nduced tumor regression was evaluated in transgenic adenocarcinoma mouse prostate (TRAMP) mice. Results: High Gleason pattern (5+5) human prostate tumors exhibit significant increase in FLIP compared with low Gleason pattern 3+3 (P = <0.04). 2-ME 2 reduced the levels and promoter activity of FLIP (P = 0.001) in PC-3 cells. Transient expression assays show sequences between -503/+242 being sufficient for 2-ME 2^i nduced inhibition of FLIP promoter activity. Cotransfection experiments show that overexpression of Sp1 activated, whereas Sp3 inhibited, Sp1 transactivation of FLIP promoter activity (P = 0.0001). 2-ME 2 treatment reduced binding of Sp1 to the FLIP promoter as evidenced by ChIP. Further, levels of FLIP associated with Fas or FADD decreased, whereas cleavage of caspase-8, levels of Bid, and apoptosis increased in response to 2-ME 2 treatment in PC-3 cells. Administration of 2-ME 2 regressed established prostate tumors in TRAMP mice that were associated with reduced expression of FLIP and Sp1. Conclusion: Targeting Sp1-mediated FLIP signaling pathway may provide a novel approach for prostate cancer management.

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Endometriosis is a hormone-sensitive gynecological disorder characterized by the benign growth of endometrial-like tissue in the pelvic cavity. Endometriotic lesions composed of endometrial stromal cells (ESC) and glandular epithelial cells (EEC) are thought to arise from menstrual endometrial tissue reaching the pelvic cavity via retrograde menstruation. The cause of endometriotic lesion formation is still not clear. Recent evidence suggest that cytokines may play a role in the early development of endometriosis lesions. Because cytokines and growth factors signal via the v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase pathway, we have examined the role of Raf-1 in early steps of endometriosis lesion formation, specifically attachment of endometrial cells to peritoneal mesothelial cells (PMC) and invasion of endometrial cells through PMC (trans-mesothelial invasion). Raf-1 antagonist GW5074 decreased attachment to PMC and trans-mesothelial invasion by primary EEC and ESC. Raf-1 also mediated TGFβ-induced trans-mesothelial invasion by the established, low-invasive EEC line EM42. TGFβ treatment of EEC resulted in Raf-1 phosphorylation at S338 and phosphorylation of ERK, suggesting that TGFβ activates Raf-1 signaling in these cells. GW5074 had little effect on ESC proliferation but inhibited EEC growth significantly under reduced serum conditions. Antagonizing Raf-1 activity and expression via GW5074 and specific Raf-1 small interfering RNA, respectively, did not alter EEC resistance to growth inhibition by TGFβ. Raf-1 inhibition blocked induction of EEC growth by epidermal growth factor. Our data suggest that Raf-1 may mediate pathologic steps involved in early endometriosis lesion formation and may be a mediator of TGFβ and epidermal growth factor actions in endometriosis.

Breast cancers amplified for the tyrosine kinase receptor Her-2/neu constitute ~30% of advanced breast cancer cases, and are characterized by hormone independence and aggressive growth, implicating this pathway in breast oncogenesis. The induction of Her-2/neu leads to tumor development in 60% of transgenic mice. We have previously examined the effects of estrogen in the MMTV-Her-2/neu background by generating the MMTV-Her-2/neu x aromatase double transgenic mouse strain. MMTV-Her-2/neu x aromatase mice developed fewer mammary tumors than the Her-2/neu parental strain. Our present data show the induction of several estrogen-related genes, including the tumor suppressors BRCA1 and p53, and a decrease in several angiogenic factors. The phosphorylated forms of MAPK p42/44 and AKT were lower in the MMTV-Her-2/neu x aromatase double transgenic mice compared to the MMTV-Her-2/neu parental strain; conversely, phospho-p38 levels were higher in the double transgenic strain. The ERβ-selective antagonist THC reversed these changes. The regulation of these factors by ERβ was confirmed in clones of MCF7 breast cancer cells overexpressing Her-2/neu in combination with ERβ, suggesting that ERβ may play a direct role in regulating MAPK and AKT pathways. In summary, the data suggest that ERβ may play a major role in decreasing tumorigenesis and that it may affect breast cancer cell proliferation and survival by altering MAPK and AKT activation as well as modulation of tumor suppressor and angiogenesis factors. Treatment with selective ERβ agonist may provide therapeutic advantages for the treatment and prevention of breast cancer.

Nontoxic naturally occurring metabolite of estrogen namely 2-methoxyestradial (2ME 2 ) found in serum and urine has been shown to be antitumorigenic in various tumor models including the prostate. A recent study conducted in breast cancer cells showed growth stimulatory effect of 2ME 2 when used at low concentrations (10-750 nM). Studies from our laboratory has demonstrated prostate tumor preventive ability of 50 mg/kg 2-ME 2 . In this study we show that concentrations of 2-ME 2 as low as 1 µM is sufficient to inhibit proliferation and induce apoptosis in androgen responsive LNCaP cells. In addition oral administration of doses lower than 50 mg/kg prevented prostate tumor development in LNCaP xenograft model. The observed tumor growth inhibition was associated with induction of apoptosis, increased expression of Wee1 kinase and p34cdc2. In addition administration of 25 mg/kg 2-ME 2 prevented tumor development significantly that is associated with reduction in serum PSA levels.

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