Weiping Ye

Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.

Liposomes Containing (-)-Gossypol-Enriched Cottonseed Oil Suppress Bcl-2 and Bcl-xL Expression in Breast Cancer Cells

ABSTRACTPurposeWe have demonstrated that (-)-gossypol-enriched cottonseed oil [(-)-GPCSO] can down-regulate Bcl-2 expression in MCF-7 and primary cultured human breast cancer epithelial cells (PCHBCECs). However, this agent has not been evaluated in vivo due to its limited solubility. We aimed to develop liposomes containing (-)-GPCSO to suppress Bcl-2/Bcl-xL expression.Methods(-)-GPCSO liposomes were prepared and evaluated for effects on breast cancer cell viability, MDA-MB-231 xenograft tumor growth, cellular Bcl-2 and Bcl-xL mRNA levels, and chemosensitivity to paclitaxel.Results(-)-GPCSO liposomes prepared had excellent stability. Cytotoxicity of (-)-GPCSO liposomes was significantly reduced compared to (-)-GPCSO in culture medium. Bcl-2 and Bcl-xL mRNA expression was down-regulated by (-)-GPCSO in culture medium or (-)-GPCSO liposomes in MDA-MB-231 cells. In PCHBCECs, Bcl-2 and Bcl-xL expression were down-regulated by (-)-GPCSO liposomes. (-)-GPCSO in culture medium induced only a mild reduction in Bcl-xL. In the MDA-MB-231 xenograft tumor model, (-)-GPCSO liposomes exhibited tumor-suppressive activity and significantly reduced intratumoral Bcl-2 and Bcl-xL expression. Cytotoxicity of paclitaxel was increased by pretreatment with (-)-GPCSO liposomes in MDA-MB-231 and PCHBCECs.ConclusionsFindings suggest that (-)-GPCSO liposomes warrant continued investigation as a chemosensitizer for breast cancers exhibiting Bcl-2-/Bcl-xL-mediated drug resistance.

Zeranol Down-Regulates p53 Expression in Primary Cultured Human Breast Cancer Epithelial Cells through Epigenetic Modification

Epidemiological studies have suggested that there are many risk factors associated with breast cancer. Silencing tumor suppressor genes through epigenetic alterations play critical roles in breast cancer initiation, promotion and progression. As a growth promoter, Zeranol (Z) has been approved by the FDA and is widely used to enhance the growth of beef cattle in the United States. However, the safety of Z use as a growth promoter is still under debate. In order to provide more evidence to clarify this critical health issue, the current study investigated the effect of Z on the proliferation of primary cultured human normal and cancerous breast epithelial cells (PCHNBECs and PCHBCECs, respectively) isolated from the same patient using MTS assay, RT-PCR and Western blot analysis. We also conducted an investigation regarding the mechanisms that might be involved. Our results show that Z is more potent to stimulate PCHBCEC growth than PCHNBEC growth. The stimulatory effects of Z on PCHBCECs and PCHBCECs may be mediated by its down-regulating expression of the tumor suppressor gene p53 at the mRNA and protein levels. Further investigation showed that the expression of DNA methylatransferase 1 mRNA and protein levels is up-regulated by treatment with Z in PCHBCECs as compared to PCHNBECs, which suggests a role of Z in epigenetic modification involved in the regulation of p53 gene expression in PCHBCECs. Our experimental results imply the potentially adverse health effect of Z in breast cancer development. Further study is continuing in our laboratory.

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.

In vitro transformation of MCF-10A cells by sera harvested from heifers two months post-Zeranol implantation

Among many risk factors of breast cancer, estrogens and non-estrogenic endocrine disruptors are considered to play critical roles in human breast carcinogenesis. Zeranol (Z) is a non-steroidal agent with potent estrogenic activity and has been widely used as an FDA approved beef growth promoter in the US. Recently, concerns have been raised about the potential adverse health risk by consumption of products containing biologically active Z and its metabolites. By utilizing cell proliferation assay, soft agar assay, quantitative real-time PCR and Western blotting analysis, we examined the potentially tumorigenic activity of bio-active Z containing sera harvested from heifers two months post Z-implantation and the underlying mechanisms. Our results showed that the growth of MCF-10A exposed to 0.2, 1 and 5% Z-containing serum (ZS) treatment for 3 weeks was 1.3, 1.75 and 1.8-fold faster compared to that of the control sera. After further investigation, we found that ZS increased cyclin D1 and decreased p53 expression at the mRNA and protein levels in MCF-10A compared to the controls. More importantly, treatment of 1% Z-containing sera for 21 days stimulated MCF-10A cells anchorage-independent colony formation in soft agar which illustrates its capability of inducing human normal breast epithelial cell neoplastic transformation. Our experimental results suggest that long-term exposure of low levels of Z and its metabolites contained in beef products might be a potential risk factor in human breast cancer initiation and development.

Abstract 33: Microarray analysis of zeranol induced gene expression in primary cultured human breast cancer epithelial cells

Zeranol (Z) is a nonsteroidal mycotoxin with potent estrogenic activity. Z is one of six growth promoters approved by the FDA for use in the U.S. beef industry to accelerate weight gain, improve feed efficiency and increase the lean meat-to-fat ratio. Recently, the safety meat products from Z-implanted beef has been questioned on the basis that increased exposure of bioactive Z residues may have adverse health implications. Our previous study showed that serum, meat and meat extracts derived from beef cattle implanted with Z significantly stimulated the proliferation of primary cultured human normal and cancerous breast epithelial cells, primary cultured human breast pre-adipocytes, and human breast cancer cell lines. However, little is known about the mechanisms of Z stimulation on the growth of different types of breast cells. To systematically investigate gene expression patterns in primary cultured human breast cancer epithelial cells (PCHBCECs) after Z treatment, we performed DNA microarray analysis. After PCHBCECs were isolated from breast cancer tissue, they were treated with 30 nM Z or 0.1% DMSO, as a vehicle control, for 48 hr, and then mRNA was isolated and purified for gene expression analysis using the Human Genome U133 Plus 2.0 Array TM (Affymetrix Co. Ltd) in the Microarray Shared Resources at The Ohio State University Comprehensive Cancer Center. DNA microarray data were generated on the basis of the criteria of signal intensity and signal ratio. Our results showed that 10 genes were down-regulated and 25 genes were up-regulated in PCHBCECs treated with 30 nM Z as compared to the controls. Among the genes regulated by Z were, limb-bud and heart (LBH) and annexin-1 (ANXA1) which were decreased 2.27 and 2 fold, respectively and human HMG-CoA synthase 1 (HMGCS1) and methyltransferase like 7A (METTL7A) which were up-regulated to 3.8 and 3.25 fold, respectively compared with the controls. Our preliminary data also showed that Z decreased the expression of several tumor suppressor genes, such as protein tyrosine phosphatase γ (PTPγ), p16, p21, and increased DNMT1 expression in PCHBCECs which suggests epigenetic modification of Z in their promoter regions. Our results indicate that long term consumption of beef products with low level of Z or its metabolites might have adverse health effects on human breast. Further validation of our DNA microarray results is in progress in our laboratory (Supported by NIH R01Grant 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 33. doi:10.1158/1538-7445.AM2011-33

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 3984: Effect of zeranol on beef skeletal muscle growth by differential image gel electrophoresis (DIGE)

Zeranol is a resorcylic acid lactone produced by fungi of the genus Fusarium that grows on corn, wheat, barley, oats and sorghum. As such Zeranol is a virtually unavoidable contaminant of crops used to feed animals that are consumed by humans. Zeranol has been shown to have a positive impact on muscle growth during the finishing phase of beef cattle production, was first approved and licensed for use as a growth promotant in cattle and sheep in the USA by FDA in 1969. However, the mechanism of this interaction is unknown but is likely related to changes in muscle specific proteins observable during the increased growth phase during finishing. To determine the affect of Zeranol on beef cattle muscle growth, we performed four DIGE experiments. We compared protein expression level changes between beef cattle treated with Zeranol and untreated beef cattle. On day zero, 10 cattle were implanted with 72 mg of Zeranol pellets, the other half (n = 10) were implanted with vehicle. The beef cattle were raised at the Ohio State University Department of Animal Sciences Beef Barn in accordance with industry standards for 120 days. At 60 days, half the implanted beef cattle and half of the control beef cattle were harvested and the remaining beef cattle were harvested at day 120. Muscle samples were analyzed by DIGE and bioinformatics Seven proteins associated with energy metabolism (creatine kinase, glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase I, aconitase 2, enolase I, and triosephophate isomerase), 5 proteins with regulation of muscle contraction (slow skeletal muscle troponin T, calsequestrin I, myosin light chains 2 and 3, tropomyosin 3), 3 structural proteins (alpha actin, myosin binding protein C and kelch repeat and BTB domain), a protein associated with myotube formation, dihydrolipoamide dehydrogenase, and HSP70, a protein associated with meat quality, and albumin were identified. The differential regulation of these proteins indicates that muscle growth in cattle implanted with Z is due to fast skeletal muscle. (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 3984.

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.