Estrogen receptors in TRAMP C2 cells

Abstract only availableAccording to a 2005 study done by the American Cancer Society, prostate cancer is the second most common type of cancer among American men. It has been shown that estrogen receptors alpha and beta play significant roles in the development and inhibition of prostate cancer. To further understand the roles ERs play in prostate cancer, a Transgenic Adenocarcinogenic of the Mouse Prostate (TRAMP) model was utilized. Simply put, these DNA engineered mice are highly likely to develop a prostate cancer similar to the type experienced by humans. Similar to humans, in TRAMP mice there are different stages of prostate cancer; well differentiated carcinoma (WDC) and poorly differentiated carcinoma (PDC) are the stages being we study extensively. It has been shown that double transgenic ER alpha knockout/ TRAMP mice have decreased incidence of PDC, while ER beta knockout/ TRAMP mice have increased incidence of PDC, which implies different roles for ER α and ER β in prostate cancer. The TRAMP C2 cell line is derived from TRAMP mice and potentially serve as a good model for in vitro studies of prostate cancer. This cell line would be useful for studying estrogen effects on prostate cancer, if it contained ER α and β. Our hypothesis is that TRAMP C2 cells are ER α and ER β positive. The goal of this research is to test for the presence of these proteins in the TRAMP C2 cell line. To test for the presence of ER alpha and ER beta, the Western blot method was used. Western Blot is a widely accepted and efficient method for detecting a specific protein among a mixture of many different ones. In conclusion, both estrogen receptor α and β are present in TRAMP C2 cells. With this confirmation, the cure to prostate cancer is one step closer because this TRAMP C2 cell line will be well suited for determining the benefits of ER alpha and ER beta manipulation.NSF-REU Program in Biological Sciences & Biochemistr

Genes targeted by the Hedgehog-signaling pathway can be regulated by Estrogen related receptor β

Additional file 1. Table S1: Known Hh-signaling pathway target genes. Table S2: Result of all pairwise comparisons of differentially expressed genes. Table S3: Hh-signaling differentially responsive genes

Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells

Additional file 2: Table S1.  Gene ontology analysis result. Table S2. Esrrb expression with DY131 treatment (control vs. Esrrb + DY131)

Studying DNA methylation changes of CpG islands in different stages of prostate cancer by pyrosequencing

Abstract only availableProstate cancer is one of the most common forms of cancer in men. Our lab is currently investigating changes in DNA methylation that occur during cancer progression, and in response to the soy phytoestrogen genistein treatment. We analyze genome-wide methylation differences by using the mouse DMH (mouse-Differential Methylation Hybridization) assay, a form of microarray. We are specifically looking at broad sets of CpG islands, areas rich in cytosine-guanine dinucleotides, that are subject to epigenetic modifications. The hypermethylation of CpG islands is correlated with the silencing of a gene while hypomethylation is correlated with a gene being actively transcribed. We were looking for potential new oncogenes or tumor suppressors. To study these genes we have a mouse model called TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate), which is a good model to study the progression of prostate cancer and metastasis because it is similar to human prostate cancer. We are using double transgenic mice that are WT or KO for the transcription factor estrogen receptor alpha, on a TRAMP background. The removal of ERα has been correlated with DNA methylation changes. These methylation changes showed up in our microarray screen that led us to find a set of genes that were differentially methylated across cancer progression. We selected one gene: Kinesin superfamily protein 9 (K3_E17) which has been shown on our microarrays to be methylated in well differentiated carcinoma and unmethylated in hyperplasia and poorly differentiated carcinoma. To confirm the methylation status we performed pyrosequencing, a new method to specifically study short sequences of DNA for methylation at specific CG sites. Our hypothesis is that in well differentiated carcinoma Kinesin 9 is hypermethylated, which will correlate with this gene being turned off. This would mean that Kinesin 9 might be acting as a tumor suppressor.Life Sciences Undergraduate Research Opportunity Progra

Improving mouse-DMH screening capability by adding 2000 mouse CpG islands

Abstract only availableDNA methylation alteration, in correlation with gene expression, is involved in development and progression of many cancers. Using a microarray based method, mouse-DMH (Differential Methylation Hybridization), our lab is able to study DNA methylation changes during prostate cancer progression in the TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mouse model. Currently, there are about 3000 CpG islands on the microarray, which were used as probes to detect DNA methylation changes. In order to improve our ability to screen for a larger number of CpG island methylation changes, we are working on adding about 2000 more mouse CpG islands onto the array. In addition, we have successfully designed primers and PCR amplified CpG islands for tumor suppressor genes and proto-oncogenes which have been previously reported in literature to be differentially methylated during development of human prostate cancer. These genes include AR (Androgen Receptor), ER (Estrogen Receptor alpha), ER(Estrogen Receptor beta) and GSTP1 (Glutathione S-Transferase PI). Primer design and PCR amplification for other known tumor suppressors/oncogenes is still in process. The microarray-based mouse-DMH is a tool of great potential. It can easily be adapted to screen for DNA methylation changes in other mouse cancer models and generate valuable data leading to understanding of the molecular mechanism behind cancer development, which will in turn contribute to treatment of human cancers.NSF-REU Biology & Biochemistr

Spinach, a cyclops, and the search for a cure for prostate cancer

Abstract only availableOther than skin cancer, prostate cancer is the most prevalent form of cancer in men. The likelihood of developing prostate cancer increases with age; so that nearly every male will die with some form of prostate cancer though most likely not from the cancer itself. Prostate cancer is regulated by endocrine and dietary factors, as well as genetic predisposition. In the male reproductive tract, Sonic Hedgehog (Shh) signaling is necessary for the development of the prostate. It has also been found to be extremely important in the growth of a number of tumor types including prostate cancer, basal cell carcinoma (skin cancer), medulloblastoma, glioma, sarcoma, tumors of the digestive tract, small cell lung cancer and pancreatic carcinoma. The Lubahn lab has been studying the roles of Estrogen Receptors and estrogenic compounds on prostate cancer, and has recently used a series of phytoestrogens, natural estrogens found in plants, to inhibit the hedgehog-signaling pathway. One natural product, cyclopamine, is able to inhibit the pathway and has been shown to inhibit prostate cancer cell growth both in vitro and in vivo xenograft models. Additionally, some flavanoid compounds in spinach have been found to have a similar chemical structure to several other phytoestrogens that have been shown to have preventative effects on prostate cancer. A competitive binding assay was performed using various doses of cyclopamine and an unpurified spinach extract to determine Kd for both compounds to ER alpha and ER beta. The compounds were also tested in the NIH-3T3 Shh Light II cells, which have a stably transfected Gli-Luciferase reporter, to see if they downregulated the hedgehog signaling pathway.Food for the 21st Century Undergraduate Research Program in Nutritional Science

Can diet influence cancer cell growth? Effects of phytoestrogens on the hedgehog-signaling pathway in prostate cancer [abstract]

Abstract only availableProstate cancer continues to be one of the most common cancers affecting American men. By better understanding the mechanisms involved in prostate cancer cell proliferation, better treatments can be developed and more cases can be prevented. One mechanism that has been linked to prostate cancer is the Sonic hedgehog-signaling (Shh) pathway. Normally, the hedgehog pathway is active only during embryonic development. Several types of tumors, including those of the prostate, demonstrate inappropriate activation of the hedgehog pathway in the adult. Cyclopamine, a steroidal alkaloid isolated from , is able to inhibit the pathway and has been shown to inhibit prostate cancer cell growth both and using xenograft models, . Despite the promising initial results of cyclopamine treatment, the compound is a very potent teratogen, and its cost makes it an unrealistic answer as a widespread cancer cure. Botanical compounds may provide a cost effective and abundant alternative. We hypothesize that various botanicals including genistein, EGCG, curcumin, and quercetin will disrupt the Shh pathway and inhibit cell growth. TRAMP-C2 and PC3 prostate cancer cell lines were used as models for hedgehog pathway response to phytoestrogens. Protein assays measuring cell growth following treatment with each botanical were performed. Real-Time RT-PCR experiments to measure mRNA concentrations of hedgehog target genes were also performed. Initial results indicate that phytoestrogens are decreasing prostate cancer cell growth up to 70 percent with genistein being the strongest inhibitor. An approximate IC50 value of 30μM was found for genistein. The Shh pathway also responds to the presence of phytoestrogens with decreased hedgehog target mRNA concentration following phytoestrogen treatment

Estrogen Up-regulates Apolipoprotein E (ApoE) Gene Expression by Increasing ApoE mRNA in the Translating Pool via the Estrogen Receptor α-Mediated Pathway

The antiatherogenic property of estrogens is mediated via at least two mechanisms: first by affecting plasma lipoprotein profiles, and second by affecting the components of the vessel wall. Raising plasma apolipoprotein E (apoE) in mice protects them against diet-induced atherosclerosis (Shimano, H., Yamada, N., Katsuki, M., Gotoda, T., Harada, K., Murase, T., Fukuzawa, C., Takaku, F., and Yazaka, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1750-1754). It is possible that estrogen may be antiatherogenic at least in part by increasing plasma apoE levels. Therefore, we studied the regulation of apoE by estrogen. A survey of 15 inbred strains of mice showed that some mouse strains responded to injections or subcutaneously implanted pellets of estradiol by raising their apoB and apoE levels and some did not. We performed detailed studies in two "responder" strains, C57L and C57BL, and two "non-responder" strains, C3H and BALBc. Responders increased their plasma apoE levels 2.5-fold. Non-responders' levels were altered +/-10%. In the responders the distribution of apoE among the plasma lipoproteins shifted from high density lipoprotein toward the apoB-containing lipoprotein fractions. In nonresponders the shift was toward high density lipoprotein. Hepatic apoE mRNA levels and relative rates of apoE mRNA transcription were unchanged in all strains, suggesting that apoE regulation occurred at posttranscriptional loci. Therefore, we measured apoE synthesis in fresh liver slices and on isolated hepatic polysomes. Two-fold increases were noted but only in responders accompanied by selective 1.5-fold increases in polysomal apoE mRNA levels. Similar increases in apoE synthesis were also observed in castrated C57BL mice given either physiological or pharmacological replacement doses of estradiol, but not testosterone, suggesting that the effect of estradiol was specific on the distribution of apoE mRNA in the translationally active polysomal pool. Next, we examined whether the effects of estrogen on apoE translation were mediated by estrogen receptors (ER). ER-alpha knock-out mice and their wild-type littermates were administered estradiol. As expected, apoE levels and hepatic apoE synthesis increased more than 2-fold in the wild-type littermates, but only 20% increases in the plasma apoE and hepatic synthesis were observed in the ER knock-out mice. Hepatic apoE mRNA levels did not change in either the wild-type or the ER knock-out mice. Thus, estradiol up-regulates apoE gene expression by increasing levels of apoE mRNA in the polysomal translating pool. Furthermore, the increased polysomal recruitment of apoE mRNA is largely mediated by estrogen receptors

Antiproliferative and Antiestrogenic Activities of Bonediol an Alkyl Catechol from Bonellia macrocarpa

The purpose of this study was to investigate antiproliferative activity of bonediol, an alkyl catechol isolated from the Mayan medicinal plant Bonellia macrocarpa. Bonediol was assessed for growth inhibition of androgen-sensitive (LNCaP), androgen-insensitive (PC-3), and metastatic androgen-insensitive (PC-3M) human prostate tumor cells; toxicity on normal cell line (HEK 293) was also evaluated. Hedgehog pathway was evaluated and competitive 3H-estradiol ligand binding assay was performed. Additionally, antioxidant activity on Nrf2-ARE pathway was evaluated. Bonediol induced a growth inhibition on prostate cancer cell lines (IC50 from 8.5 to 20.6 µM). Interestingly, bonediol binds to both estrogen receptors (ERα (2.5 µM) and ERβ (2.1 µM)) and displaces the native ligand E2 (17β-estradiol). No significant activity was found in the Hedgehog pathway. Additionally, activity of bonediol on Nrf2-ARE pathway suggested that bonediol could induce oxidative stress and activation of detoxification enzymes at 1 µM (3.8-fold). We propose that the compound bonediol may serve as a potential chemopreventive treatment with therapeutic potential against prostate cancer