Zeranol induces deleterious effects on the testes and the prostate gland of mature rats.

BACKGROUND: Endocrine disrupters have been shown to affect the male and female reproductive systems and to alter potential fertility. OBJECTIVES: This study was conducted to evaluate the effect of a continuous-release pellet containing 12 mg of zeranol for 30 days on the testes and the prostate gland of mature male rats. RESULTS: Zeranol treatment induced significant decrease of the testes and the prostate gland weights which were associated with a remarkable atrophy of the testicular seminiferous tubules and prominent regression of the glandular compartment of the prostate gland. However, zeranol treatment increased the thickness of the periductal layer of stromal cells of the prostate gland from a thin layer that express intense immunostaining of SM-actin and mild vimentin to a thicker layer of cells that exhibited intense immunostaining for both SM-actin and vimentin. CONCLUSION: These findings suggest that zeranol-induced changes to the prostate gland could result from either a direct effect of zeranol on the prostate gland or an indirect effect by interfering with testosterone production through disruption of testicular function.

(-)-Gossypol-enriched cottonseed oil inhibits proliferation and adipogenesis of human breast pre-adipocytes.

BACKGROUND: Breast cancer is the most commonly diagnosed cancer in women. Obesity is an important risk factor for developing breast cancer and is one of few risk factors that women can modify to prevent cancer. (-)-Gossypol-enriched cottonseed oil [(-)-GPCSO] contains 65% (-)-gossypol and 35% (+)-gossypol. Previous studies have demonstrated that both (-)-gossypol and (-)-GPCSO have potent anticancer activity against multiple types of cancer, including breast cancer. In addition, (-)-GPCSO reduced body weight gain and food intake in young female rats. However, the role of (-)-GPCSO on adipogenesis in human breast pre-adipocytes remains unclear. MATERIALS AND METHODS: Primary human breast pre-adipocytes were induced to differentiate in adipogenic medium in the presence of (-)-GPCSO. The proliferation of pre-adipocytes was determined with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-H-tetrazolium (MTS) assay. Lipid accumulation and glycerol 3-phosphate dehydrogenase (GPDH) activity were measured during adipocyte differentiation. mRNA expression of cyclin-D1, B-cell lymphoma-2 (BCL-2), Peroxisome Proliferator-Activated Receptor-γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin was analyzed by real-time PCR. RESULTS: (-)-GPCSO inhibited proliferation of pre-adipocytes and down-regulated the expression of cyclin-D1 and BCL-2. (-)-GPCSO also significantly decreased adipogenesis, as determined by inhibition of GPDH activity, triglyceride content (TG), and down-regulation of the expression of PPARγ, C/EBPα and leptin. CONCLUSION: These findings suggest that (-)-GPCSO has the potential as a food supplement to inhibit adipogenesis, and therefore, reduce obesity.

Testosterone effect on the expression of genes that mediate testosterone metabolism and genes that mediate the effect of those metabolites on the prostate.

AIMS: The aim of this study was to investigate the effect of testosterone treatment on the proliferation index and the mRNA expression levels of 5α-reductase, CYP7B1, androgen receptor (AR), and estrogen receptor ß (ΕRß) in the canine prostate. MAIN METHODS: Immature dogs were treated with testosterone for one month, after which prostate gland growth was assessed by comparing the proliferation index in prostates from testosterone-treated dogs with that of untreated control dogs. The relative mRNA expression levels of the aforementioned genes in the prostate glands of testosterone-treated and untreated dogs were determined by real time PCR. KEY FINDINGS: Testosterone treatment induced a highly significant reduction in proliferation index in prostate gland. This inhibition of prostate gland growth was associated with differential mRNA expression of 5α-reductase, CYP7B1, AR, and ΕRß by the prostate gland of testosterone-treated dogs, as compared to that of untreated dogs. While the expression levels of 5α-reductase and CYP7B1 mRNA were significantly down-regulated by testosterone treatment, the expression level of ER-ß mRNA was highly up-regulated. In contrast, AR mRNA expression was not significantly altered. SIGNIFICANCE: Prostate gland proliferation appeared to be associated with the expression levels of genes that encode proteins that control intra-prostatic levels of testosterone metabolites and their respective receptors. Testosterone treatment may regulate gene expression in the prostate to generate a phenotype that suppresses growth-promoting signaling through AR and enhances anti-proliferative signaling through ERß. Therefore, targeting disturbances of this genetic machinery in benign prostate hyperplasia and prostate cancer is of a therapeutic potential.

Liposomes containing (-)-gossypol-enriched cottonseed oil suppress Bcl-2 and Bcl-xL expression in breast cancer cells.

PURPOSE: We 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. CONCLUSIONS: Findings 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 induces cell proliferation and protein disulfide isomerase expression in mammary gland of ACI rat.

BACKGROUND: Zeranol is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used as a growth promoter in the US beef industry. Consumption of beef derived from Zeranol-implanted cattle may be a risk factor for breast cancer. Protein disulfide isomerase (PDI) has been studied extensively as a key enzyme involving in the formation of the correct pattern of disulfide bonds in newly synthesized proteins. The relationship between PDI expression and cancer development has attracted interest of cancer researchers in recent years. MATERIALS AND METHODS: We implanted ACI rats with 12 mg Zeranol pellet and harvested the mammary tissues and tumor at day 110 after implantation and investigated the effect of Zeranol-implantation on cell proliferation by histological examination and proliferation in vitro. We also evaluated PDI mRNA expression in primary epithelial cells isolated from normal mammary glands and primary tumor cells from tumor specimens using real-time RT-PCR. To further confirm, we also evaluated the effect of Zeranol on PDI mRNA expression in primary epithelial cells isolated from normal mammary gland of ACI rats. RESULTS: We observed a palpable mammary tumor in one of three Zeranol-implanted ACI rats at day-110 post Zeranol-implantation. Zeranol-implantation significantly promoted the cell proliferation of primary mammary epithelial and stromal cells isolated from the mammary gland of normal ACI rats. PDI mRNA is over-expressed in primary tumor cells isolated from the tumor specimen and in Zeranol-treated primary cultured epithelial cells from the mammary gland of normal ACI rats. CONCLUSION: These findings suggest that up-regulated expression of PDI may play a critical role in mammary tumorigenesis and cell proliferation in response to Zeranol. Our findings implicate PDI as a biomarker for mammary tumorigenesis.

Serum derived from zeranol-implanted ACI rats promotes the growth of human breast cancer cells in vitro.

BACKGROUND: Zeranol (Z) is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used as a growth promoter in the US beef industry. Consumption of beef derived from zeranol-implanted cattle may be a risk factor for breast cancer. MATERIALS AND METHODS: The effect of serum on the proliferation of human breast cancer MCF-7 cell line and primary cultured human breast epithelial cells (PCHBECs) was investigated. ACI rats were implanted with 12 mg zeranol pellet and the serum was harvested at day 110 after implantation. The effect of zeranol-serum on mRNA expression of cell cycle regulating gene (cyclin D1) and tumor suppressor genes (p53, and p21) was evaluated using real-time RT-PCR. RESULTS: The serum derived from ACI rats 110 days post-zeranol implantation significantly promoted the proliferation of MCF-7 cells and primary cultured human breast epithelial cells compared to control serum. Zeranol-serum up-regulated cyclin D1 and down-regulated p53 and p21 expression in PCHBECs compared with control serum. CONCLUSION: Bio-active zeranol metabolites contained in meat produced from cattle after zeranol implantation may be a risk factor for breast cancer.

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.

Antioxidant properties of peptide fractions from silver carp (Hypophthalmichthys molitrix) processing by-product protein hydrolysates evaluated by electron spin resonance spectrometry.

Silver carp processing by-product protein is usually discarded as an industrial solid waste. In this study the protein was recovered using a pH-shift method, after which seven commercial proteases were separately employed to prepare antioxidative hydrolysates. Among the hydrolysates, pepsin hydrolysates, which had the highest free radical-scavenging activity, were further separated into five peptide fractions, SCPH-I (>10kDa), SCPH-II (5-10kDa), SCPH-III (3-5kDa), SCPH-IV (1-3kDa), and SCPH-V (<1kDa), by using ultrafiltration. The antioxidative properties of the peptide fractions were investigated, using a free radical-scavenging assay, by electron spin resonance. The results show that SCPH-V had the highest scavenging effects on DPPH (1,1-diphenyl-2-picrylhydrazyl), hydroxyl and superoxide anion radicals. SCPH-V had potent antioxidant activity in the prevention of the peroxidation of linoleic acid and alleviation of H2O2-induced oxidative stress in human intestinal epithelial caco-2 cells. The results indicated that the antioxidant capacity of silver carp by-product hydrolysates could be enhanced by ultrafiltration.

Zeranol may increase the risk of leptin-induced neoplasia in human breast.

Breast cancer and obesity are serious health problems and their relationship has been studied for many years. Leptin is mainly secreted by adipocytes and plays a key role in breast cancer development. Leptin expression is up-regulated in obese individuals and promotes breast cancer cell growth. On the other hand, exposure to environmental estrogens has been found to be directly related to breast cancer. Zeranol (Z) is a non-steroidal anabolic growth promoter used in the beef industry in the US. This study focused on the evaluation of Z and Z-containing sera (ZS) and its adverse health risk to human consumption of Z-containing meat produced from Z-implanted beef cattle. We hypothesized that Z increases the risk of breast neoplasia in women, particularly in obese women. A cell proliferation assay, ELISA analysis, RT-PCR and Western blot analysis were conducted. Our study demonstrated that Z and ZS collected from Z-implanted heifers stimulated the proliferation of primary cultured human normal breast epithelial cells (HNBECs) by up-regulating cyclin D1 expression. Leptin increased the sensitivity of HNBECs to Z, and Z increased the ability of HNBECs to secrete leptin. These results suggest an interaction between leptin and Z in HNBECs. Furthermore, Z may play a role in leptin-induced breast neoplasia.

Aromatase expression in leptin-pretreated human breast pre-adipocytes is enhanced by zeranol and suppressed by (-)-gossypol.

BACKGROUND: Obesity is associated with an increased risk of estrogen-dependent breast cancer. Recently, concerns have been raised regarding the role of zeranol (Z), a non-steroidal anabolic growth promoter with potent estrogenic activity widely used in the U.S.A. beef industry, as a possible contributor to an increased incidence of human breast cancer. This study hypothesized that obese individuals may be at greater risk of developing zeranol-induced breast cancer. MATERIALS AND METHODS: The aromatase mRNA expression level of three cell types isolated from adipose tissues were assayed by RT-PCR, and the cell proliferation of primary cultured human normal breast pre-adipocytes (HNBPADs) was investigated using the CellTiter96® non-radioactive method. The effects of Z and gossypol on aromatase expression of leptin-pretreated HNBPADs were evaluated by RT-PCR. RESULTS: HNBPADs expressed higher aromatase than primary cultured human breast epithelial cells and stromal cells. Z enhanced the mitogenic activity of leptin and increased aromatase expression in HNBPADs. Moreover, (-)-gossypol counteracted Z- and leptin-induced cell proliferation and aromatase expression. CONCLUSION: These results suggested that bioactive Z metabolites contained in meat produced from Z-implanted beef cattle may increase estrogen biosynthesis in obese individuals by increasing aromatase expression and estrogen production, which will promote cell sensitivity and increase breast cancer cell growth.

Function and regulatory mechanisms of the candidate tumor suppressor receptor protein tyrosine phosphatase gamma (PTPRG) in breast cancer cells.

BACKGROUND: Protein phosphorylation is one of the essential steps in cell signaling, and aberrant phosphorylation is a common event in human cancer. The expression of receptor type protein tyrosine phosphatase gamma (PTPRG) in normal breast is found to be approximately 50-60% higher than that of breast tumor tissue. Overexpression of PTPRG inhibits anchorage-independent growth and proliferation of breast cancer cells. To understand the tumor suppression characteristics of PTPRG, we studied its tumor suppressive function in an athymic mouse model and evaluated factors that can potentially regulate its expression in breast cancer cells. MATERIALS AND METHODS: To investigate the function of PTPRG in vivo, athymic nude mice were implanted with MCF-7 cells overexpressing PTPRG. For in vitro study, protein levels of cell cycle regulators, cell cycle re-entry, and the phosphorylation levels of extracellular signal-regulated protein kinases 1/2 (ERK1/2) were examined. In addition, methylation assays were conducted to investigate the epigenetic modification on the promoter of PTPRG. RESULTS: Athymic nude mice bearing MCF-7 cells overexpressing PTPRG showed a reduction in tumor burden in comparison to animals implanted with MCF-7 cells transfected with vector alone. When these two cell lines were studied in an in vitro system, elevated mRNA and protein levels of cell cycle regulators, p21(cip) and p27(kip) were detected in MCF-7 cells overexpressing PTPRG compared to cells transfected with vector alone. Similarly, overexpression of PTPRG also delayed the re-entry of breast cancer cells into the cell cycle after serum starvation, and reduced the phosphorylation levels ERK1/2 in MCF-7 cells. In addition, methylation assays in PTPRG promoter in breast cancer cell lines (including SK-Br-3) revealed an aberrant methylation pattern. When SK-Br-3 and MCF-7 cells were treated with deoxy-5-azacytidine (DAC) and trichostatin A (TSA), these compounds reactivated the expression of PTPRG, suggesting an epigenetic control on its expression. CONCLUSION: Our results indicated that PTPRG inhibited breast tumor formation in vivo; PTPRG may up-regulate p21(cip) and p27(kip) proteins through the ERK1/2 pathway. This study also showed methylation-mediated silencing of PTPRG in breast cancer cell lines. These data indicate that PTPRG exhibits the characteristics of a breast tumor suppressor.

Induction of apoptosis by (-)-gossypol-enriched cottonseed oil in human breast cancer cells.

Induction of apoptosis is one of the mechanisms of chemotherapeutic agents against breast cancer. In addition, recent studies have shown that diets containing polyphenolic components possess anticancer activities either in vitro or in vivo by inhibiting cell proliferation and inducing apoptosis. The aim of our study was to explore the effects of (-)-gossypol-enriched cottonseed oil [(-)-GPCSO], a polyphenolic compound, on the proliferation of the breast cancer cell line MCF-7 as well as primary cultured human breast cancer epithelial cells (PCHBCEC). We investigated whether the mechanism of the effects of (-)-GPCSO was mediated via the induction of cell apoptosis and the regulation of Bcl-2 gene expression at both the mRNA and protein levels. Our results showed that (-)-GPCSO inhibited the proliferation of MCF-7 and PCHBCEC in a dose-dependent manner. (-)-GPCSO (0.1 and 0.2%) induced DNA fragmentation in both MCF-7 cells and PCHBCEC. (-)-GPCSO suppressed the expression of Bcl-2 at both the mRNA and protein levels in MCF-7 cells and PCHBCEC in a dose-dependent fashion. Our results suggest that the growth inhibitory effect of (-)-GPCSO on MCF-7 and PCHBCEC is due, at least partially, to the induction of cell apoptosis, which is mediated by down-regulation of Bcl-2 expression at both the mRNA and protein levels. It might be possible for (-)-GPCSO to be developed as a novel chemotherapeutic agent for breast cancer patients.

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.

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.

Serum harvested from heifers one month post-zeranol implantation stimulates MCF-7 breast cancer cell growth.

Breast cancer is a serious disease in the US. Numerous risk factors have been linked to this disease. The safety of using growth promoters, such as zeranol, remains under debate due to the lack of sufficient in vitro and in vivo evidence. Using CellTiter 96(™) Aqueous Non-Radioactive Cell Proliferation assay, real-time PCR and Western blot analysis, we evaluated the effects of sera harvested from experimental and control heifers before and after one month of zeranol implantation on the growth of human breast cancer cell line MCF-7 as well as the involved mechanisms. We found that sera harvested from the heifers following one month of zeranol implantation were more mitogenically potent in stimulating the proliferation of MCF-7 cells when compared to sera harvested from the same heifers before zeranol implantation and the control heifers. Further investigation found that dextran-coated charcoal suppressed the stimulating effect of the sera on MCF-7 cell growth. The mechanisms involved in the MCF-7 cell proliferation stimulated by zeranol-containing sera may include up-regulation of cyclin D1 and down-regulation of p53 and p21 expression at the mRNA and protein levels in the cells. The results suggest that the consumption of beef products containing biologically active residues of zeranol or its metabolites is a risk linked to breast cancer development. Further investigation is required in order to clarify this critical issue.

Mitogenic activity of zeranol in human breast cancer cells is enhanced by leptin and suppressed by gossypol.

BACKGROUND: The molecular links between breast cancer and obesity have been studied for many years. Obesity significantly increases the incidence rate and chance of morbidity of breast cancer. Leptin, mainly secreted by adipocytes, plays an important role in breast cancer development. Leptin expression is up-regulated in obesity and it can promote breast cancer cell growth. Zeranol is used as an anabolic growth promoter to stimulate cattle growth in the U.S. beef industry. (-)-Gossypol, a natural polyphenolic compound extracted from cottonseed, is an anticancer chemopreventive agent. MATERIALS AND METHODS: Zeranol, leptin and (-)-gossypol were used to investigate MCF-7 Adr cell growth. RESULTS: Leptin enhanced the sensitivity of MCF-7 Adr cells to zeranol and increased cell growth. Exposure to zeranol may lead to initiation of transformation of normal breast cells to breast preneoplastic cells. CONCLUSION: It is suggested that obese individuals may be at greater risk of developing zeranol-induced breast cancer.

Keratinocyte growth factor (KGF) regulates estrogen receptor-alpha (ER-alpha) expression and cell apoptosis via phosphatidylinositol 3-kinase (PI3K)/Akt pathway in human breast cancer cells.

BACKGROUND: It is suggested that the phosphatidylinositol 3-kinase (PI3K/Akt) pathway may lead to tamoxifen (Tam) resistance in the estrogen receptor-alpha (ER-alpha)-positive breast cancer cell line, MCF-7. Our previous results demonstrated that keratinocyte growth factor (KGF) down-regulates ER-alpha expression and increases Tam resistance in MCF-7 cells. Therefore, we hypothesized that a possible mechanism for developing Tam resistance could be the regulation of ER-alpha and Bcl-2 family proteins through modulation of Akt activity. MATERIALS AND METHODS: MCF-7 cells were treated with KGF, LY294002 (LY), a PI3 kinase inhibitor, 4-OH-Tam, KGF with LY, KGF with LY and 4-OH-Tam, or vehicles as control for 24 hours. Total RNA was extracted from MCF-7 cells and real-time PCR was employed to identify the ER-alpha expression in response to KGF. To determine that the resistance to 4-OH-Tam-inducing cell killing after the KGF treatment was due to the inactivation of the apoptotic pathway, low molecular weight DNA was isolated from cells of different treatments and inter-nucleosomal DNA fragmentation was investigated. The phosphorylation of signaling intermediates Akt, Bad, the activation of caspase-9, and the expression of ER-alpha, Bcl-2, Bcl-xL, and Bax were evaluated by immunoblot analysis for the study of KGF signaling effects. To determine the involvement of PI3K/Akt pathway in the survival effect of KGF, the growth rate of MCF-7 cell was measured by non-radioactive cell proliferation assay after treatments of KGF, LY, 4-OH-Tam, KGF with LY, KGF with LY and 4-OH-Tam, or vehicles as control for 3 days. The results of real-time PCR and cell proliferation assay were analyzed by Student's t-test and p-values of less than 0.05 were considered statistically significant. RESULTS: Our results showed that in MCF-7 cells KGF increased Akt phosphorylation and induced ER-alpha mRNA expression which could be blocked by a PI3K/Akt pathway inhibitor, LY. KGF treatment also induced apoptosis based on the observation of the suppression of DNA fragmentation, variable increase in the expression of the Bcl-2 and Bcl-xL proteins and the decrease of the active form of caspase-9 protein, whereas LY blocked the anti-apoptotic effects of KGF. In the cell proliferation assay, KGF maintained MCF-7 cell survival in the presence of 4-OH-Tam which could be blocked by LY. CONCLUSION: We confirmed the regulation of ER-alpha by KGF in human breast cancer cells at both mRNA and protein levels. We further demonstrated that KGF may play an inhibitory role in the induction of breast cancer cell apoptosis, conferring resistance against anticancer drugs on breast cancer cells.

Gossypol inhibits the growth of MAT-LyLu prostate cancer cells by modulation of TGFbeta/Akt signaling.

Gossypol (GP), a male contraceptive compound naturally present in cottonseed products, possesses anti-proliferative and anti-metastatic effects in vitro and in vivo. However, the detailed mechanisms responsible for the effects of GP on the cell cycle of prostate cancer cells remain to be elucidated. In the present study, we investigated the effects of GP on the regulation of the cell cycle of rodent prostate cancer MAT-LyLu cells and the mechanisms of GP-induced growth inhibition. Our results showed that GP inhibited the cell proliferation and colony formation in a dose-dependent manner by the up-regulation of expression and secretion of transforming growth factor beta1 (TGFbeta1) and down-regulation of expression of Akt and phospho-Akt protein. The inhibition of cell growth was also demonstrated by cell cycle arrest at G0/G1 phase. Furthermore, GP decreased the expression of cyclin D1, Cdk4 and phospho-Rb in MAT-LyLu cells. Thus, the inhibitory effects of GP on the proliferation of MAT-LyLu prostate cancer cells are associated with modulation of TGFbeta1 and Akt signaling, which influence the expression of regulatory proteins such as cyclin D1, Cdk4 and phospho-Rb which regulate cell cycle progression of prostate cancer cells.

(-)-Gossypol reduces invasiveness in metastatic prostate cancer cells.

BACKGROUND: Acquisition of metastatic ability by prostatic cancer cells is the most lethal aspect of prostatic cancer progression. (-)-Gossypol, a polyphenolic compound present in cottonseeds, possesses anti-proliferative and proapoptotic effects in various cancer cells. MATERIALS AND METHODS: In this study, the differences between MAT-LyLu, rat prostate cancer cells, with a novel isolated subline from metastasized tumors in the lungs of MAT-LyLu-bearing Copenhagen rats (MLL cells) were compared with respect to cell growth and invasion. The effects of (-)-gossypol on cell viability, colony formation, invasive ability and cell migration in MAT-LyLu and MLL cells were also evaluated. RESULTS: Results showed that MLL cells displayed higher growth ability, colony formation and aggressive penetration than those of MAT-LyLu cells. MLL cells possess lower protein expression of Bcl-xL and nm23-H1 than those of MAT-LyLu cells, implying differences in invasive ability. Moreover, (-)-gossypol treatment induced a dose-dependent inhibition of invasive activity and cell viability and reduced Bcl-2 and Bcl-xL proteins but induced nm23-H1 protein in both cell lines. CONCLUSION: These findings illustrated that (-)-gossypol reduced in vitro invasion of both the parental MAT-LyLu cells and the isolated MLL cells, suggesting that (-)-gossypol might serve as a chemotherapeutic and/or chemopreventive agent.