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.

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.

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 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.

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.

Conjugated linoleic acid induces apoptosis through estrogen receptor alpha in human breast tissue.

BACKGROUND: Conjugated linoleic acid (CLA), a naturally occurring fatty acid found in ruminant products such as milk and beef, has been shown to possess anti-cancer activities in in vivo animal models and in vitro cell culture systems. In human breast cancer, the overall duration of estrogen exposure is the most important risk factor for developing estrogen-responsive breast cancer. Accordingly, it has been suggested that estrogen exposure reduces apoptosis through the up-regulation of the anti-apoptosis protein, Bcl-2. Bcl-2, an anti-apoptotic protein, regulates apoptosis and plays a crucial role in the development and growth regulation of normal and cancerous cells. Our research interest is to examine the effects of CLA on the induction of apoptosis in human breast tissues. METHODS: The localization of Bcl-2 in both normal and cancerous human breast tissues was determined by immunohistochemical staining and the Bcl-2 protein expression was tested by western blot analysis. Co-culture of epithelial cells and stromal cells was carried out in the presence or absence of CLA to evaluate apoptosis in the context of a cell-cell interaction. RESULTS: The results showed that both normal and cancerous breast tissues were positive for Bcl-2 staining, which was higher overall in mammary ducts but very low in the surrounding stromal compartment. Interestingly, by quantifying the western blot data, basal Bcl-2 protein levels were higher in normal breast epithelial cells than in cancerous epithelial cells. Furthermore, treatment with 17beta-estradiol (E2) stimulated growth and up-regulated Bcl-2 expression in estrogen responsive breast epithelial cells; however, these carcinogenic effects were diminished by either CLA or 4-Hydroxytamoxifen (Tam) and were suppressed further by the combination of CLA and Tam. In both one cell type cultured and co-culture systems, CLA induced cell apoptosis in ERalpha transfected MDA-MB-231 cells but not in the wild type MDA-MB-231 cells. CONCLUSION: These data, therefore, demonstrate that ERalpha plays important roles in CLA induced apoptosis in human breast tissues.

A liposomal delivery vehicle for the anticancer agent gossypol.

BACKGROUND: Gossypol has recently been identified as a potential anticancer agent. In this study, a novel liposomal formulation is evaluated for delivery of gossypol. MATERIALS AND METHODS: Gossypol was incorporated into liposomes. The liposomes were characterized for physical and chemical properties and for in vitro cytotoxicity. RESULTS: Gossypol was stably encapsulated in these liposomes and exhibited cytotoxicity towards cancer cells. CONCLUSION: Liposomes can be used effectively as gossypol carriers.

Modulation of multidrug resistance gene expression in human breast cancer cells by (-)-gossypol-enriched cottonseed oil.

BACKGROUND: Multidrug resistance (MDR) is a major impediment to successful cancer chemotherapy. P-glycoprotein (P-gp), the product of the multidrug resistance 1 (MDR1) gene, acts as an efflux pump and prevents sufficient intracellular accumulation of several anticancer agents, thus, playing a major role in MDR. Tamoxifen (Tam), ICI 182 780 (ICI) and Adriamycin (Adr) alone or with (-)-gossypol-enriched cottonseed oil [(-)-GPCSO] possible effects on cell growth inhibition and regulation of MDR1, mRNA and P-gp expression were examined in both an MDR human breast cancer cell line, MCF-7/Adr cells, and primary cultured human breast cancer epithelial cells (PCHBCEC). MATERIALS AND METHODS: Cells were treated with 0.05% of (-)-GPCSO either in the absence or presence of either 0.1 microM Tam, ICI or Adr for 24 h. RESULTS: Using the non-radioactive cell proliferation MTS assay, none of these chemotherapeutic agents alone inhibited MCF-7/Adr cell and PCHBCEC proliferation; meanwhile, the combination of 0.1 microM Tam, ICI or Adr with 0.05% (-)-GPCSO significantly reduced MCF-7/Adr cell growth by approximately 34%, 32% and 23%, respectively, of that of the vehicle-treated cells. For PCHBCEC, the combination of 0.05% (-)-GPCSO with 0.1 microM of Tam, ICI and Adr reduced cell growth to about 94%, 90%, and 71% respectively, of the vehicle treated PCHBCEC. Furthermore, (-)-GPCSO inhibited MDR1/P-gp expression in both MCF- 7/Adr and PCHBCEC in a dose-dependent manner. Our results provide insight into the MDR-reversing potential of (-)-GPCSO in human breast cancer cells resistant to current chemotherapeutic agents.

Primary cultured human breast epithelial cells up-regulate protein disulfide isomerase in response to zeranol.

BACKGROUND: Experimental data at the molecular, cellular and organismal levels have implicated dietary components in cancer. Exposure to numerous growth factors, hormones and environmental agents, that can regulate signaling events, is involved in carcinogenesis. Research targets on gene-nutrient interactions may give useful information for the development of a novel diet-based intervention for the at-risk population. MATERIALS AND METHODS: To examine the proteomic effect of a low level dietary compound on potential breast cancer, primary human breast epithelial cells were isolated and cultured in media with or without zeranol, an anabolic, non-estrogenic growth promoter with estrogenic activity, used in beef cattle and naturally found in some fungus in grain. The cells then underwent proteomic analysis. RESULTS: 2-D electrophoresis showed that protein disulfide isomerase (PDI) was up-regulated 5-fold in the breast epithelial cells exposed to zeranol. PDI has been shown to be up-regulated in a variety of cancerous tissues, although this is the first reported up-regulation of PDI in breast tissue. CONCLUSION: PDI may be a useful marker of dietary exposure to zeranol.

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.

Conjugated linoleic acid (CLA) up-regulates the estrogen-regulated cancer suppressor gene, protein tyrosine phosphatase gamma (PTPgama), in human breast cells.

BACKGROUND: Conjugated linoleic acid (CLA), a naturally occurring compound found in ruminants products, has been shown to possess anticancer properties in vivo and in vitro. There are several CLA isomers in ruminant-produced foods, among which t10, c12-CLA and c9, t11-CLA are the most potent. Protein tyrosine phosphatase gamma (PTPgamma) has been implicated as a tumor suppressor gene in kidney and lung cancers. Our previous results indicated that estradiol-17beta (E2)-induced suppression of PTPgamma may play a role in mammary tumorigenesis. MATERIALS AND METHODS: The effects of t10, c12-CLA and c9, t11-CLA on PTPgamma mRNA expression in human breast epithelial cells and stromal cells, isolated from surgical specimens of mammoplasty and breast cancer patients, were detected and quantified by RT-PCR RESULTS: The PTPgamma mRNA expression was lower in cancer than in normal breast cells. Both t10, c12-CLA and c9, t11-CLA significantly (p < 0.05) increased the PTPgamma mRNA levels in primary cultured normal breast epithelial cells, normal breast stromal cells and breast cancer epithelial cells, but not in breast cancer stromal cells. t10, c12-CLA appeared to be the most active isomer in estrogen receptor a (ERalpha)-positive human breast cancer epithelial cells. CONCLUSION: The results indicate that dietary CLA might serve as a chemo-preventive and chemo-therapeutic agent in human breast cancers by up-regulating the estrogen-regulated tumor suppressor gene, PTPgamma expression.

Effects of serum on (-)-gossypol-suppressed growth in human prostate cancer cells.

BACKGROUND: Gossypol, a natural polyphenolic compound present in cottonseeds, possesses antiproliferative and pro-apoptotic effects in in vivo and in vitro models. There are two enantiomers, (+)-gossypol and (-)-gossypol, the latter being a more potent inhibitor of cancer cell growth. Here, the effect of bovine serum albumin (BSA) and dextran-coated charcoal-treated fetal bovine serum (DCC-FBS)-containing medium on the ability of (-)-gossypol to inhibit the growth of human prostate cancer cells was studied. MATERIALS AND METHODS: BSA- and DCC-FBS-supplemented medium were used to examine the influence of serum proteins on the antiproliferative effects of (-)-gossypol in DU-145 cells, a human prostate cancer cell line. The viability of the DU-145 cells was determined by CellTiter 96 Aqueous assay. The expressions of mRNA and protein for the cell cycle regulators, cyclin-D1, Rb, CDK, p21 and TGF-beta, were determined by RT-PCR and Western blot analyses, respectively. RESULTS: (-)-Gossypol caused growth suppression of the DU-145 cells. In comparison with BSA-supplemented medium, DCC-FBS blocked the antiproliferative effects of (-)-gossypol at 1 and 2.5 microM, but not at 5 microM. Furthermore, (-)-gossypol treatment down-regulated cyclin-D1, Rb, CDK4 and CDK6, and up-regulated p21 and TGF-beta1 at the mRNA and/or protein levels. CONCLUSION: The data suggested that (-)-gossypol-suppressed prostate cancer cell growth may be influenced through cell cycle regulators, which may lead to better prognosis. We further speculate that (-)-gossypol might serve as a chemotherapeutic agent for human prostate cancer patients.

Keratinocyte growth factor (KGF) induces tamoxifen (Tam) resistance in human breast cancer MCF-7 cells.

BACKGROUND: Both estrogen receptor-alpha (ER-alpha) and progesterone receptor (PR) are good prognostic factors and indicators of benefit from endocrine therapy in breast cancer patients. The relationship of the ER-alpha and PR status and the difference in clinical benefit from endocrine therapy in breast cancer is currently unclear. It has been suggested that keratinocyte growth factors (KGFs) are important regulatory factors in breast cancer. Our laboratory has demonstrated that KGF can act as an estromedin for the stimulation of breast cancer cell growth. Also, KGF stimulates aromatase activity in primary cultured human breast cells. This enzyme is a key to the conversion of androgens to estrogens. In the present study, ER-alpha, two estrogen-regulated genes, PR and PTPgamma, KGF and their relationship with endocrine resistance in human breast cancer cells were investigated. MATERIALS AND METHODS: MCF-7 cells were treated with KGF (1, 5, 10, 20 ng/ml), KGF-13 (0.1, 1, 10 microM) or vehicles as control for 24 hours. KGF-13 is a potential receptor-binding pentapeptide constructed using the KGF peptide residues 101-105 (RTVAV) as a template, located within the beta 4--beta 5 loop. Total RNA were isolated and real-time PCR was employed to identify ER-alpha, PR and PTPgamma gene expressions in response to KGF and KGF-13. Western blot analysis was used to verify the levels of ER-alpha and PR protein, whereas immunohistochemical staining was used to detect PTPgamma expression in MCF-7 cells. To determine the response of MCF-7 cells to endocrine therapy, MCF-7 was treated with either 20 ng/ml KGF or 10 microM KGF-13 combined with 1, 3 and 5 microM of 4-hydroxytamoxifen (4OH-Tam). A non-radioactive cell proliferation assay was applied to determine the growth rate of MCF-7 cells. The results of real-time PCR and the cell proliferation assay were analyzed by Student's t-test and p-values of less than 0.05 were considered statistically significant. RESULTS: Our data showed that KGF significantly suppressed ER-alpha, PR and PTPgamma expression in MCF-7 cells. KGF suppressed ER-alpha, PR and PTPgamma mRNA to a maximal inhibition at 20 ng/ml by 88%, 57% and 61%, respectively. Western blot analysis and immunohistochemical staining confirmed the down-regulation of ER-alpha, PR and PTPgamma by KGF in protein levels. Ten microM KGF-13 also decreased ER-alpha expression. Ten microM KGF-13 significantly decreased ER-alpha, PR and PTPgamma mRNA expressions by 51%, 57% and 67%, respectively. These KGF-13-mediated mRNA down-regulations were also observed in protein levels. In a cell proliferation assay, 4OH-Tam (3, 5 microM) induced MCF-7 cell death. KGF and KGF-13 alone did not stimulate MCF-7 cell growth. KGF significantly disrupted 4OH-Tam cell killing effects by 1.2- and 1.3-fold at 4OH-Tam concentrations of 3 microM and 5 microM, respectively. KGF-13 significantly disrupted 4OH-Tam cell killing effects by 1.2- and 1.7-fold at 4OH-Tam concentrations of 3 microM and 5 microM, respectively. CONCLUSION: Our results suggested that not only ER-alpha and PR but also PTPgamma could be potential bio-makers for growth factor-induced endocrine resistant in breast cancer. KGF might increase the endocrine resistance via decreasing ER-alpha, PR and PTPgamma as well. Moreover, the functional analysis of KGF-13 implied possible applications of using short receptor-binding peptides derived from intact KGF as breast cancer therapeutic agents. Thus, our experimental data provided evidence of KGF-induced anti-hormone resistance in human breast cancer and suggested novel strategies for breast cancer via interference with KGF signaling.

Molecular mechanisms of (-)-gossypol-induced apoptosis in human prostate cancer cells.

BACKGROUND: Gossypol, a natural compound present in cottonseeds, displays antiproliferative and pro-apoptotic effects against various cancer cells. The (-)-gossypol enantiomer is a more potent inhibitor of cancer cell growth. Here, the molecular mechanisms of apoptosis induced by (-)-gossypol were studied in human prostate cancer cells. MATERIALS AND METHODS: After the prostate cancer cell DU-145 had been treated with (-)-gossypol, the trypan blue exclusion assay and DNA fragment end-labeling assay were used to stain the dead cells and to detect DNA laddering, respectively. The effects of (-)-gossypol on the expression of apoptotic-regulated gene markers in both death receptor- and mitochondria-mediated apoptotic pathways, such as the Bcl-2 family and caspase, etc., were detected by RT-PCR and Western blot analysis. To further investigate the apoptotic pathways induced by (-)-gossypol, different caspase inhibitors were used to block caspase activities and cell viability was detected by the CellTiter 96 AQueous assay in DU-145 cells. RESULTS: At a 5-10 microM dose-level, (-)-gossypol significantly enhanced apoptosis measured by DNA fragmentation. (-)-Gossypol caused apoptosis in DU-145 cells through the down-regulation of Bcl-2 and Bcl-xL and the up-regulation of Bax at the mRNA and protein levels. (-)-Gossypol also activated caspases-3, -8 and -9 and increased PARP [poly (ADP-ribose) polymerase] cleavage. Furthermore, (-)-gossypol-induced apoptosis might be due to an increase in CAD (caspase-activated deoxyribonuclease) proteins and a decrease in ICAD (inhibitor of CAD) proteins. By using caspase inhibitors, (-)-gossypol caused apoptosis via the caspase-dependent pathways. CONCLUSION: Our results indicated that the apoptotic processes caused by (-)-gossypol are mediated by the regulation of the Bcl-2 and caspase families in human prostate cancer cells. Our data also suggested that (-)-gossypol may have chemotheraputic benefits for prostate cancer patients.

Conjugated linoleic acid (CLA) modulates prostaglandin E2 (PGE2) signaling in canine mammary cells.

BACKGROUND: Conjugated linoleic acid (CLA), a naturally occurring linoleic acid isomer found in ruminant-produced foods, has the potential to serve as an effective chemopreventive nutriceutical factor for breast cancer prevention based upon previous published studies. There are several CLA isomers in ruminant-produced food products, among which t10,c12-CLA and c9,t11-CLA are more potent. Expression of cyclooxygenase 2 (COX-2) in mammary tumors has been correlated with poor prognosis. Prostaglandin E2 (PGE2) is a major COX-2 product in various cancers and, as in humans, PGE2 concentrations in canine tumor tissues were frequently elevated. Moreover, a PGE2 receptor subtype, EP2, is highly expressed in mammary tumors. Thus, various studies have implicated the important role of PGE2 and EP2 in COX-2-regulated tumor development. MATERIALS AND METHODS: Mammary tumor and normal mammary tissues were both collected from a female dog with mammary tumor. Both malignant and normal mammary tissues were subjected to isolation of epithelial and stromal cells. The effects of t10,c12-CLA and c9,t11-CLA on proliferation, as well as COX-2 and EP2 protein expression in canine mammary normal and cancerous cells, were detected by CellTiter 96 AQueous assay and Western blot assay, respectively. RESULTS: Both t10,c12-CLA and c9,t11-CLA not only suppressed malignant mammary cell growth, but also exerted inhibitory effects on tumor-associated non-malignant mammary cells. Similarly, both t10,c12-CLA and c9,t11-CLA suppressed EP2 protein expression in both normal and malignant mammary cells. t10,c12-CLA was more effective in decreasing COX-2 protein expression in malignant mammary cells, while, in contrast, c9,t11-CLA down-regulated COX-2 protein expression in both normal and malignant mammary cells. CONCLUSION: The results indicate that the dietary component CLA regulates COX-2 and EP2 protein expression in both malignant mammary cells and cells from the tumor-associated stromal compartment. In turn, this may suppress PGE2 signaling, leading to better prognosis. We further speculate that the knowledge obtained from canine studies may also be beneficial to study human breast cancer.

Function analysis of estrogenically regulated protein tyrosine phosphatase gamma (PTPgamma) in human breast cancer cell line MCF-7.

Protein tyrosine phosphatase gamma (PTPgamma) is a member of the receptor-like family of tyrosine phosphatases and has been implicated as a tumor suppressor gene in kidney and lung cancers. Based on our previous findings, we hypothesize that PTPgamma is a potential estrogen-regulated tumor suppressor gene in human breast cancer. To examine the effects of PTPgamma on growth of MCF-7 human breast cancer cells and compare the estrogenic responses of human breast cells with different PTPgamma expression levels, we established several stably transfected MCF-7 cell lines expressing different levels of PTPgamma, which were confirmed by RT-PCR and immunostaining. In our work, we used the antisense construct to breakdown endogenous PTPgamma level in MCF-7 cells. The results from doubling time assay suggested that PTPgamma is capable of inhibiting MCF-7 breast cancer cell growth. We further demonstrated that PTPgamma is able to inhibit anchorage-independent growth of breast cancer cells in soft agar and reduce the estrogenic responses of MCF-7 cell proliferation to estradiol-17beta (E(2)) and zeranol (Z, a nonsteroidal growth promoter with estrogenic activity). Our data suggest that PTPgamma may function as an important modulator in regulating the process of tumorigenesis in human breast.

Translational studies on aromatase, cyclooxygenases, and enzyme inhibitors in breast cancer.

Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Regulation of aromatase expression in human tissues is quite complex, involving alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Our hypothesis is that higher levels of COX expression result in higher levels of prostaglandin E2 (PGE2), which in turn increases CYP19 expression through increases in intracellular cyclic AMP levels. This biochemical mechanism may explain the beneficial effects of non-steroidal anti-inflammatory drugs (NSAIDs) on reducing the risks of breast cancer. The effects of NSAIDs (ibuprofen, piroxicam, and indomethacin), a COX-1 selective inhibitor (SC-560), and COX-2 selective inhibitors (celecoxib, niflumic acid, nimesulide, NS-398, and SC-58125) on aromatase activity and CYP19 expression were investigated in breast cancer cell culture systems. Dose-dependent decreases in aromatase activity were observed following treatment with an NSAID or COX inhibitor, with the most effective agents being COX selective inhibitors. Real time PCR analysis of aromatase gene expression showed a significant decrease in mRNA levels in treated cells when compared to vehicle control. These results suggest that the effect of COX inhibitors on aromatase occurs at the transcriptional level. To further probe these interactions, short interfering RNAs (siRNA) were designed against either human CYP19 mRNA or human COX-2 mRNA. Treatment of breast cancer cells with aromatase siRNAs suppressed CYP19 mRNA and aromatase enzyme activity. Finally, treatment with COX-2 siRNAs downregulated the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated suppression of COX-2 also resulted in suppression of aromatase mRNA. In summary, pharmacological regulation of aromatase and cyclooxygenases can act locally in an autocrine fashion to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer.

Effects of age on growth and ERbeta mRNA expression of canine prostatic cells.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an age-dependent prostatic disease in human males and dogs. The prostatic stromal estrogen level of health control and BPH patients increases significantly with age, while the dihydrotestosterone (DHT) level is not connected with age. Moreover, experimentally estrogens have induced BPH in the presence of androgens. Our aim was to investigate the effects of age on the proliferation and estrogen receptor beta (ER/3) mRNA of canine prostatic epithelial and stromal cells. MATERIALS AND METHODS: Epithelial and stromal cells were isolated from canine prostatic tissues. The proliferation of these cell types from dogs of different ages was assessed by thymidine incorporation assay, while the expression and identification of ERbeta mRNA were performed by RT-PCR and DNA sequence. RESULTS: Prostatic epithelial cells isolated from 1-year-old dogs exhibited a greater proliferative activity than those of 4-year-old dogs. In contrast, the prostatic stromal cells from 4-year-old dogs proliferated more rapidly than the cells from 1-year-old dogs. ERbeta mRNA expression was detected in the canine prostatic epithelial and stromal cells, decreasing with age. The partial DNA sequence showed that the canine ERbeta sequence shares 90.0%, 87.0% and 83.0% of its nucleotide homology with human, rat and mouse ERbeta, respectively. CONCLUSION: The decrease in the expression of ERbeta in prostatic cells with age reduces its negative control over the androgen receptor, associated with the overgrowth of canine prostatic stromal cells, which further induces the development of canine BPH.

Decreased MCF-7 breast cancer cell proliferation by serum from a selected line of beef cattle.

One way to combat or treat breast cancer, the most common cancer among women, is to decrease or prevent proliferation of cancerous cells. Many animal species of agricultural importance have been selected for various growth traits, typically through altered proliferative capacity of some cell types. Previous studies in two lines of cattle divergently selected for serum IGF-I concentration have shown that low IGF-I cattle had increased growth rates and high IGF-I cattle had decreased growth rates. Serum from either the high or low IGF-I lines of cattle were administered to MCF-7 breast cancer cell lines and doubling times were determined. The MCF-7 cells treated with serum from cattle with high serum IGF-I concentrations took 26% longer to double than MCF-7 cells treated with serum from cattle with low serum IGF-I concentrations. In conclusion, model systems employing agricultural animals may provide novel insight into mechanisms of cell proliferation.