C. C. Kuo

Zeranol enhances leptin-induced proliferation in primary cultured human breast cancer epithelial cells

Breast cancer is the leading type of cancer in women in the United States. One of the known risk factors of breast cancer is obesity. Leptin is a product of the obese (ob) gene and plays an important role in breast cancer development. Its expression is up-regulated in obesity and it promotes breast cancer cell growth. Exposure to environmental estrogenic disruptors has been found to be directly related to the increase in the incidence of breast cancer. Zeranol (Z) is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used in the US beef industry. The objective of this study was to determine the mechanisms of Z- and leptin-induced proliferation of primary cultured human breast cancer epithelial cells (HBCECs). A cell proliferation assay was used to determine the extent to which Z is capable of enhancing the mitogenic activity of leptin in HBCECs. RT-PCR was used to explore the possible mechanisms by quantifying the transcription of cyclin D1 and ObR genes. Our results demonstrated that when the HBCECs were pre-treated with 3 nM leptin for 24 h, the sensitivity to Z exposure greatly enhanced the mitogenic action of leptin. The experimental data observed show that there is interaction between leptin and Z in HBCEC growth.

Abstract 816: Anti-inflammatory effects of black raspberries in ulcerative colitis are associated with demethylation of genes in the Wnt signaling and protective modulation of toll-like receptor pathway

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Ulcerative Colitis (UC) causes pervasive, chronic inflammation of the colon and is a potential precursor to colon cancer. Reduced Interleukin 10 (IL10), an anti-inflammatory cytokine, is associated with an increased risk of developing UC in rodents and humans. IL10 knockout (KO) mice develop UC spontaneously with subsequent development of colon tumors. Toll-like receptor and Wnt pathways are deregulated in UC patients and IL10 KO mice. Toll-like receptor pathway plays critical roles in the inflammatory response partly by mediating the Wnt pathway. We previously showed that 5-10% dietary freeze-dried black raspberries (BRBs) reduced chronic inflammation and tumor development in IL10 KO mice; aberrant promoter methylation of negative Wnt regulators, e.g., dkk2, dkk3, and sfrp1, were observed in colon from these animals. The goals of the current study were to determine if the anti-inflammatory effects of BRBs on IL10 KO mice are associated with modulation of the Wnt pathway through DNA demethylation, with alteration of enzymes regulating DNA methylation, and with modulation of toll-like receptor pathway. IL10 KO mice were fed control or 5% BRB diet for 4 wks. Cells from bone marrow and spleen as well as colon tissue were collected. Promoter methylation of dkk2, dkk3, and sfrp1 was quantified by Pyrosequencing. Protein expression of enzymes regulating DNA methylation, e.g., histone deacetylase 1, 2 (HDAC1, 2), methyl binding domain 2 (MBD2), and DNA methyltransferase 3B (DNMT3B), and β-catenin were assessed using quantitative immunohistochemistry. mRNA expression of genes associated with the Wnt and toll-like receptor pathways were quantified using real-time PCR based arrays, each of which contained 84 genes. Increased promoter methylation of dkk2, dkk3, and sfrp1 was observed in colon from KO mice when compared with colon from wild-type mice. The methylation levels of these genes in bone marrow and spleen were lower than those in colon. BRBs significantly decreased promoter methylation of dkk2 in colon and dkk3 in spleen. This was associated with decreased protein expression of HDAC1, HDAC2, MBD2, and DNMT3B. The expression of 84 genes in both pathways was different in colon of IL10 KO mice when compared to colon of wild-type mice. BRBs modulated 80% (67/84) and 95% (80/84) of differentially expressed genes toward normal levels of expression in the Wnt and toll-like receptor pathways, respectively. Protein expression of nuclear β-catenin was decreased by BRBs. In summary, berries are capable of reversing aberrant promoter methylation of genes in the Wnt pathway presumably through inhibition of proteins regulating DNA methylation. These changes could result in protective modulation of the Wnt pathway and/or its interaction with the toll-like receptor pathway which, in turn, reduce inflammation in ulcerative colitis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 816. doi:10.1158/1538-7445.AM2011-816

Abstract 3956: Differential miR-141 expression in primary cultured human breast cancer epithelium (PCHBCEs) and normal adjacent part epithelium (PCHBNEs) correlates with tumor suppressor protein tyrosine phosphataseγ (PTPγ)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL MicroRNAs (miR) have been shown to be extensively involved in tumorigenesis by post-transcriptional inhibition of oncogenes and/or tumor-suppressor genes. The purpose of our study is to investigate the difference in miRNA expression between cancer epithelium and epithelium from normal breast adjacent tissues. The clinical samples were procured from the Tissue Procurement Program at the Ohio State University Comprehensive Cancer Hospital from breast cancer patients who underwent partial or complete mastectomy. The miRNA profiling for one pair of PCHBCEC and PCHBNEC from the same patient was carried out, and the difference of miRNA expression and potential target genes were further verified by realtime qPCR in 7 pairs of clinical samples. The miRNA profiling showed that 24 miRNAs (let-7a, let-7c, let-7f, let-7g, miR-24, miR-28, miR-29a, miR-29c, miR-30a-5p, miR-30d, miR-92, miR-125a, miR-126*, miR-132, miR-135a, miR-135b, miR-137, miR-141, miR-182, miR-200c, miR-339, miR-365, miR-425-5p, miR-391, p<0.05) are statistically up-regulated in cancer while only 1 miRNA (miR-221) is down-regulated. Based on the magnitude of change and predicted target, we selected miR-141 for further validation. Compared with its normal adjacent counterpart, 4 PCHBCECs had lower miR-141 while 2 were up-regulated and 1 unaltered. Further validation on gene expression of the samples confirmed the negative correlation of miR-141 with its putative target PTPγ. Our comparison of PCHBCECs and PCHBNECs under the same genetic background demonstrated a distinct expression of miRNAs. The dysregulation of miR-141 was shown to result in modulation of the potential tumor-suppressor gene PTPγ which might have an impact on the etiological process of tumor lesion and discriminate cancer epithelial cells from their surrounding normal breast epithelial compartments. Our results implicate that miR-141 might serve as molecular biomarker for therapy of human breast cancer patients. (Supported by NIH R01 Grant ES 015212). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3956. doi:10.1158/1538-7445.AM2011-3956

Leptin and zeranol up-regulate cyclin D1 expression in primary cultured normal human breast pre-adipocytes.

Adipocytes account for more than 90% of human breast volume and secrete adipocytokines, which play a role in breast cancer development. Among the adipocytokines is leptin, which is secreted mainly by adipocytes and plays a key role in breast cancer development. Leptin expression is up-regulated in both obese and breast cancer patients, and promotes breast cancer cell growth. Exposure to environmental estrogens has also been found to be directly related to the development of breast cancer. Zeranol (Z) is a non-steroidal anabolic growth promoter with estrogenic activity that is widely used in the US beef industry due to its commercial benefits. Gossypol is a natural compound extracted from cottonseed that inhibits breast cancer growth, and is potentially a chemopreventive food component. This study focused on Z and bio-active Z-containing sera (ZS) collected from Z-implanted beef, and evaluated their adverse health risk to humans. We hypothesized that Z increases the risk of breast cancer in obese women. A cell proliferation assay, ELISA analysis, RT-PCR and Western blotting were performed to investigate the interaction of leptin, Z and (-)-gossypol in primary cultured normal human breast pre-adipocytes. The results indicated that Z and ZS stimulated the growth of pre-adipocytes isolated from normal human breast tissues by up-regulating cyclin D1 expression, while (-)-gossypol reversed this effect.

Abstract 5365: Zeranol-containing serum (Z-Sera) harvested from 30-day Z-implanted heifers induce transformation of human normal breast epithelial cells through down-regulation of the protein tyrosine phosphatase γ (PTPγ) tumor suppressor gene

The morbidity of breast cancer ranks first in the U.S. it remains the second leading cause of maligancy-related death in women, regardless of advances in novel therapeutic strategies. Many risk factors have been associated with breast cancer initiation, promotion and progression. Recently, Zeranol (Z), one of six growth promoters approved by FDA for use in the beef industry may cause health concerns due to the consumption of beef products containing bio-active Z metabolites. Our laboratory previously demonstrated that Z down-regulates the expression of estrogen-regulated PTPγ in primary cultured human normal breast epithelial cells and transforms the normal human breast epithelial cell line, MCF-10A, to neoplastic breast cancer cells. In the current study, we investigated the biologically active Z metabolites present in Z-Sera harvested from 30-day post Z implanted heifers (72 mg pellet) on MCF-7 cell growth and PTPγ mRNA expression in MCF-10A cells using non radioactive cell proliferation and real time PCR. Our results showed that Z-Sera at 0.5, 2.5 and 12.5% significantly increased the growth of MCF-7 cells by 42, 71, and 108%, respectively. While the sera harvested from the control heifers did not increase MCF-7 cell growth. Treatment with 2.5% Z-sera harvested 30, 60, 90 days post Z-implantation significantly down-regulated the expression of PTP γ mRNA in MCF-7 cells to 18.2, 36.5 and 51%, respectively. Furthermore, neoplastic transformation of MCF-10A cells resulted after exposure of 2.5% Z-Sera in culture medium to MCF-10A for 21-day. The expression of PTPγ mRNA, in transformed MCF-10A cells, was significantly reduced by 83, 96 and 97%, as compared to the controls. We hypothesize that the transformation of MCF-10A cells by bio-active Z metabolites contained in 2.5% Z-sera may be mediated through the down-regulation of estrogen-regulated PTPγ in MCF-10A cells. We have reported that the growth rate of pre-adipocytes isolated from the beef cattle 30-day post Z-implantation was 12 fold faster than the pre-adipocytes isolated from of the control beef cattle. In summary, our in vitro data suggest that potential adverse health risk may result from consuming beef products containing bio-active Z metabolites. (Supported by NIH grant R01 ES 015212). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5365.

Abstract 3072: Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer

Refractory to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in aberrant expression of downstream target genes in the disease. We hypothesized that aberrant expression of TGF-β/SMAD4 targets are mediated through epigenetic mechanism and also contribute to resistance to TGF-β meditated growth inhibition. Our previous report using chromatin immunoprecipitation microarray (ChIP-chip) identified FBXO32 as one of SMAD4 targets in immortalized ovarian surface epithelial cell (IOSE) (Qin et al., BMC Syst Biol, 17: 73, 2009). In the present study, we investigated the mechanism conferring FBXO32 down-regulation, its clinical significance, and its function in ovarian cancer. Our result showed that expression of FBXO32 was observed in normal ovarian surface epithelium but not in ovarian cancer cell lines (HeyC2, SKOV3, CP70, A2708, MCP2, MCP3) using real time RT-PCR. Promoter methylation of FBXO32 was seen in ovarian cancer cell lines, HeyC2 and SKOV3, that display constitutive TGF-β/SMAD4 signaling. Moreover, our finding that epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggested that epigenetic regulation of FBXO32 in ovarian cancer may be a common event. Re-expression of FBXO32 markedly impeded proliferation of a platinum-resistant ovarian cancer cell lines, HeyC2 and CP70 (colony number: HeyC2: 19.33 ± 3.06 vs 1 ± 0, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3072.