Vitamin E inhibition of normal mammary epithelial cell growth is associated with a reduction in protein kinase C(alpha) activation.

Tocopherols and tocotrienols represent the two subclasses within the vitamin E family of compounds. However, tocotrienols are significantly more potent than tocopherols in suppressing epidermal growth factor (EGF)-dependent normal mammary epithelial cell growth. EGF is a potent mitogen for normal mammary epithelial cells and an initial event in EGF-receptor mitogenic-signalling is protein kinase C (PKC) activation. Studies were conducted to determine if the antiproliferative effects of specific tocopherol and tocotrienol isoforms are associated with a reduction in EGF-receptor mitogenic signalling and/or PKC activation. Normal mammary epithelial cells isolated from midpregnant BALB/c mice were grown in primary culture, and maintained on serum-free media containing 10 ng/mL EGF as a mitogen, and treated with various doses (0-250 microm) of alpha-, gamma-, or delta-tocopherol or alpha-, gamma-, or delta-tocotrienol. Treatment with growth inhibitory doses of delta-tocopherol (100 microm), alpha-tocotrienol (50 microm), or gamma- or delta-tocotrienol (10 microm) did not affect EGF-receptor levels, EGF-induced EGF-receptor tyrosine kinase activity, or total intracellular levels of PKC(alpha). However, these treatments were found to inhibit EGF-induced PKC(alpha) activation as determined by its translocation from the cytosolic to membrane fraction. Treatment with 250 microm alpha- or gamma-tocopherol had no affect on EGF-receptor mitogenic signalling or cell growth. These findings demonstrate that the inhibitory effects of specific tocopherol and tocotrienol isoforms on EGF-dependent normal mammary epithelial cell mitogenesis occurs downstream from the EGF-receptor and appears to be mediated, at least in part, by a reduction in PKC(alpha) activation.

Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues.

Male offspring exposed in utero to antiandrogens often display alterations in androgen-dependent developmental markers (e.g., anogenital distance [AGD], nipple retention) together with clearly adverse responses such as genital malformations and reproductive tract lesions. The objectives of this study were to determine whether in utero exposure to flutamide results in permanent changes in male AGD and nipple retention, characterize the dose-response relationship between flutamide-mediated alterations in these landmarks and clearly adverse antiandrogenic effects, and establish the predictive value and relationship between AGD and nipple retention, and other adverse manifestations. Male offspring were exposed in utero to 0, 6.25, 12.5, 25, or 50 mg/kg/day (po) of flutamide from gestation days 12 to 21. Offspring were uniquely identified at birth, and various androgen-mediated end points (AGD, areola/nipple retention, cryptorchidism, reproductive tract weights, and malformation incidence) were examined throughout life. In utero flutamide exposure significantly decreased the AGD on postnatal day (PND) 1 and increased areola/nipple retention in male rats on PND 13. Flutamide-induced alterations in AGD and areolae/nipples in early postnatal life correlated with a reduction in AGD and retained nipples observed in the adult. Prenatal flutamide exposure resulted in dose-responsive increases in cryptorchidism. Hypospadias were observed in all flutamide-exposed offspring. In utero flutamide exposure induced partial or complete prostate agenesis and decreased the weights of the seminal vesicles, levator ani bulbocavernosus (LABC) muscle, testes, and epididymides in a dose-dependent manner. Epididymal malformations were observed mainly in the 50 mg/kg/day flutamide dose group. In general, flutamide-induced alterations in dihydrotestosterone (DHT)- and testosterone (T)-dependent development each had similar respective dose-response curves. DHT-mediated development was more sensitive to in utero flutamide exposure than T-dependent processes. However, the dose-response curves for flutamide-induced changes in cryptorchidism and seminal vesicle weight were intermediate between the dose-response curves for DHT- and T-mediated development, indicating that proper development of these tissues may require both androgens. The LABC also displayed a dose-dependent decrease in weight that was similar to dose-response observed with seminal vesicle weight and was the most sensitive T-dependent end point measured. Flutamide-induced decreases in AGD predicted subsequent malformations as evidenced by logistic regression and receiver operator characteristic analysis of malformations versus AGD. However, the AGD that would predict a 10% incidence of seminal vesicle malformations is equivalent to a female AGD. An almost fully feminized phenotype of 10-12 nipples was observed in animals that had malformations in T-dependent tissues, whereas 6 or more nipples were observed in animals with malformation in DHT-dependent tissues. These data suggest that flutamide-mediated changes in AGD and nipple retention are not sensitive predictors of altered T-mediated development.

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells.

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on preneoplastic (CL-S1), neoplastic (-SA), and highly malignant (+SA) mouse mammary epithelial cell growth and viability in vitro. Over a 5-day culture period, treatment with 0-120 microM alpha- and gamma-tocopherol had no effect on cell proliferation, whereas growth was inhibited 50% (IC50) as compared with controls by treatment with the following: 13, 7, and 6 microM tocotrienol-rich-fraction of palm oil (TRF); 55, 47, and 23 microM delta-tocopherol; 12, 7, and 5 microM alpha-tocotrienol; 8, 5, and 4 microM gamma-tocotrienol; or 7, 4, and 3 microM delta-tocotrienol in CL-S1, -SA and +SA cells, respectively. Acute 24-hr exposure to 0-250 microM alpha- or gamma-tocopherol (CL-S1, -SA, and +SA) or 0-250 microM delta-tocopherol (CL-S1) had no effect on cell viability, whereas cell viability was reduced 50% (LD50) as compared with controls by treatment with 166 or 125 microM delta-tocopherol in -SA and +SA cells, respectively. Additional LD50 doses were determined as the following: 50, 43, and 38 microM TRF; 27, 28, and 23 microM alpha-tocotrienol; 19, 17, and 14 microM gamma-tocotrienol; or 16, 15, or 12 microM delta-tocotrienol in CL-S1, -SA, and +SA cells, respectively. Treatment-induced cell death resulted from activation of apoptosis, as indicated by DNA fragmentation. Results also showed that CL-S1, -SA, and +SA cells preferentially accumulate tocotrienols as compared with tocopherols, and this may partially explain why tocotrienols display greater biopotency than tocopherols. These data also showed that highly malignant +SA cells were the most sensitive, whereas the preneoplastic CL-S1 cells were the least sensitive to the antiproliferative and apoptotic effects of tocotrienols, and suggest that tocotrienols may have potential health benefits in preventing and/or reducing the risk of breast cancer in women.

Effects of in utero exposure to linuron on androgen-dependent reproductive development in the male Crl:CD(SD)BR rat.

Linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea) is a herbicide that blocks androgen action in the male rat. Studies were undertaken to characterize the ability of linuron to activate transcription through the human androgen receptor (AR) in vitro and to determine whether in utero linuron exposure induces dose-responsive alterations in androgen-dependent reproductive development in the male rat. In vitro, linuron competitively antagonized transcriptional activity of the AR induced by dihydrotestosterone (DHT) in a dose-responsive manner with an equilibrium dissociation constant (K(B)) of 75.8 x 10(-8) M. Pregnant rats were administered linuron by gavage at 0, 12.5, 25, or 50 mg/kg/day (n = 11/group) from gestation day 12 to 21. Anogenital distance of resulting offspring was unaffected, whereas male areola/nipple retention was increased in a dose-responsive manner. Hypoplastic testes in adult offspring were seen in 2/56 rats (2/10 litters), 8/69 rats (4/11 litters), and 5/44 rats (3/8 litters), while hypoplastic epididymides occurred in 1/56 rats (1/10 litters), 8/69 rats (4/11 litters), and 2/44 rats (1/8 litters) in the 12.5, 25, and 50 mg/kg/day dose groups, respectively. Partial agenesis of the epididymides was observed in 3/44 rats (2/8 litters) only in the 50 mg/kg/day group. These data indicate that in utero exposure to linuron preferentially impairs testosterone-mediated, rather than DHT-mediated, reproductive development. This effect is distinctly different from the effects induced by flutamide, an AR antagonist that shares structural similarities with linuron. Furthermore, these data suggest that dose-response studies utilizing late gestational exposure to endocrine-active compounds may be more robust than the traditional or EPA-modified multigeneration protocols in identifying adverse effects.

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells.

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on normal mammary epithelial cell growth and viability. Cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 0-120 microM alpha- and gamma-tocopherol had no effect, whereas 12.5-100m microM tocotrienol-rich fraction of palm oil (TRF), 100-120 microM delta-tocopherol, 50-60 microM alpha-tocotrienol, and 8-14 microM gamma- or delta-tocotrienol significantly inhibited cell growth in a dose-responsive manner. In acute studies, 24-h exposure to 0-250 microM alpha-, gamma-, and delta-tocopherol had no effect, whereas similar treatment with 100-250 microM TRF, 140-250 microM alpha-, 25-100 microM gamma- or delta-tocotrienol significantly reduced cell viability. Growth-inhibitory doses of TRF, delta-tocopherol, and alpha-, gamma-, and delta-tocotrienol were shown to induce apoptosis in these cells, as indicated by DNA fragmentation. Results also showed that mammary epithelial cells more easily or preferentially took up tocotrienols as compared to tocopherols, suggesting that at least part of the reason tocotrienols display greater biopotency than tocopherols is because of greater cellular accumulation. In summary, these findings suggest that the highly biopotent gamma- and delta-tocotrienol isoforms may play a physiological role in modulating normal mammary gland growth, function, and remodeling.

Immunosuppressant inhibition of P-glycoprotein function is independent of drug-induced suppression of peptide-prolyl isomerase and calcineurin activity.

PURPOSE: P-glycoprotein is a 170-kDa plasma membrane multidrug transporter that actively exports cytotoxic substances from cells. Overexpression of P-glycoprotein by tumor cells is associated with a multidrug-resistant phenotype. Immunosuppressive agents such as cyclosporins and macrolides, have been shown to attenuate P-glycoprotein activity. However, the mechanism by which some immunosuppressants inhibit P-glycoprotein function has not been determined. Since cyclosporin and macrolide immunosuppressants inhibit calcineurin (CaN) phosphatase and FKBP12 peptideprolyl isomerase (FKBP12 PPI) activity, studies were conducted to determine if these effects are directly related to the inhibitory effects these immunosuppressants have on P-glycoprotein function. METHODS: Western blot analysis was performed to assess CaN and FKBP12 protein levels in P-glycoprotein-negative (MCF-7) and -positive (MCF-7/Adr) breast cancer cell lines. P-glycoprotein function was determined by intracellular doxorubicin accumulation and/or cytotoxicity assays before and after CaN and FKBP12 were independently inhibited by pharmacological antagonists. RESULTS: CaN and FKBP12 levels were similar in MCF-7 and MCF-7/Adr cells. P-glycoprotein function was not affected by treatment of P-glycoprotein-expressing MCF-7/Adr cells with CaN and FKBP12 antagonists. CONCLUSIONS: These results demonstrate that the inhibitory effects of immunosuppressive agents on P-glycoprotein function are independent of CaN or FKBP12 PPI activity.

Genistein and erbstatin inhibition of normal mammary epithelial cell proliferation is associated with EGF-receptor down-regulation.

Epidermal growth factor (EGF) is a potent mitogen for normal mouse mammary epithelial cells grown in primary culture. EGF activation of the EGF-receptor (EGF-R) induces intrinsic tyrosine kinase activity which results in EGF-R autophosphorylation and tyrosine phosphorylation of other intracellular substrates involved in EGF-R signal transduction. Genistein and erbstatin are anticancer agents which have been shown to be potent tyrosine kinase inhibitors. However, the effects of these compounds in modulating EGF-dependent normal mammary epithelial cell proliferation is presently unknown. Therefore, studies were conducted to determine the effects of genistein and erbstatin on EGF-dependent proliferation, and EGF-R levels and autophosphorylation in normal mouse mammary epithelial cells grown in primary culture and maintained in serum-free media. Chronic treatment with 6.25-100 microM genistein or 1-16 microM erbstatin significantly decreased EGF-dependent mammary epithelial cell proliferation in a dose-responsive manner. However, the highest doses of genistein (100 microM) and erbstatin (16 microM) were found to be cytotoxic. Additional studies showed that acute treatment with 6.25-400 microM genistein did not affect EGF-R levels or EGF-induced EGF-R autophosphorylation, while acute treatment with 1-64 microM erbstatin caused a slight reduction in EGF-R levels, but had no effect on EGF-dependent EGF-R autophosphorylation in these cells. In contrast, chronic treatment with similar doses of genistein or erbstatin resulted in a large dose-responsive decrease in EGF-R levels, and a corresponding decrease in total cellular EGF-R autophosphorylation intensity. These results demonstrate that the inhibitory effects of chronic genistein and erbstatin treatment on EGF-dependent mammary epithelial cell proliferation is not due to a direct inhibition of EGF-R tyrosine kinase activity, but results primarily from a down-regulation in EGF-R levels and subsequent decrease in mammary epithelial cell mitogenic-responsiveness to EGF stimulation.

Effects of protein tyrosine phosphatase inhibitors on EGF- and insulin-dependent mammary epithelial cell growth.

Epidermal growth factor (EGF)- and insulin-dependent mammary epithelial cell mitogenesis is mediated by specific tyrosine kinase receptors. Receptor tyrosine kinase activity is highly regulated in normal cells, whereas amplification of intracellular protein tyrosine phosphorylation is associated with abnormal growth and/or neoplastic transformation. Since protein tyrosine phosphatases (PTPs) are involved in regulating receptor tyrosine kinase signaling, studies were conducted to determine the effects of the PTP inhibitors, vanadate and pervanadate, on mitogen-receptor signal transduction and cell growth. Mammary epithelial cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 2-8 microM vanadate or pervanadate greatly increased intracellular protein tyrosine phosphorylation. However, in the presence of optimal mitogenic stimulation (10 ng/ml EGF and 10 microg/ml insulin), these treatments induced a slight, but significant decrease in cell growth. In contrast, these treatments significantly increased mammary epithelial cell growth, albeit less than optimally, under submitogenic culture conditions (500 pg/ml EGF and 10 microg/ml insulin). Neither vanadate nor pervanadate was found to mimic the mitogenic actions of EGF and/or insulin in these cells. The growth-stimulatory effects of PTP inhibitors in submitogenic conditions appear to result from enhanced receptor tyrosine kinase mitogenic signaling, whereas PTP inhibitor attenuation of optimal cell growth may be due to the suppression of PTP activity associated with cell cycle progression. In addition, treatment with PTP inhibitors was not found to stimulate anchorage-independent growth, as determined by the inability of single cells to form colonies in soft agarose. In conclusion, these data demonstrate that optimal mitogen-dependent mammary epithelial cell growth requires both receptor tyrosine kinase and PTP activity. Furthermore, PTP inhibitor-induced amplification of receptor tyrosine kinase mitogenic signaling is not in itself sufficient to induce enhanced cell growth or phenotypic expression of neoplastic transformation.

Protein kinase C isoenzyme expression in normal mouse mammary epithelial cells grown in primary culture.

Mammary epithelial cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained in serum-free media containing 10 ng/ml epidermal growth factor (EGF) for an 8-day culture period. Western blot and scanning densitometric image analysis showed the presence of protein kinase C(PKC)alpha (82 kDa), delta(75 kDa), eta(90 and 78 kDa), and zeta(82, 74, and 65 kDa), whereas PKCbeta, gamma, and theta were not detected in either the cytosolic or membrane fractions in these cells. Cytosolic and membrane levels of PKCalpha and 82 kDa PKCzeta band progressively increased throughout the 8-day culture period. During this same time, cytosolic PKCdelta levels decreased, while membrane levels of PKCdelta showed no change. Cytosolic and membrane levels of PKCeta and the 74- and 65-kDa PKCzeta bands displayed some fluctuations but remained relatively constant during the 8-day culture period. Other studies showed that 24-hr treatment with 100 nM of phorbol 12-myristate 13-acetate (PMA), resulted in the downregulation of PKCalpha, delta, and eta, and the 82-kDa PKCzeta band. However, PMA treatment had no effect on cytosolic and membrane levels of the 74- and 65-kDa PKCzeta bands. Since PKC activation is associated with hormone- and growth factor-dependent mammary epithelial cell proliferation, these findings suggest that increases and/or decreases in the relative levels of the different PKC isoenzymes in proliferating cells may indicate their possible role in mediating or regulating EGF-dependent mitogenesis.

Relationship between epidermal growth factor receptor levels, autophosphorylation and mitogenic-responsiveness in normal mouse mammary epithelial cells in vitro.

Mammary epithelial cells were isolated from mid-pregnant BALB/c mice, grown within collagen gels and maintained on DME/F12 (1:1) media containing 10% bovine calf serum and 10 micrograms/ml insulin. Initial time-course and dose-response studies showed that epidermal growth factor (EGF)-induced autophosphorylation of the EGF-receptor (EGF-R) in these cells was maximal 5 min after exposure to 75 ng/ml EGF. Mammary epithelial cells displaying little or no growth during their first 2 days in primary culture cells were found to contain low levels of EGF-R. However, EGF-induced autophosphorylation of the EGF-R in these cells was extremely intense. Subsequent studies demonstrated that during the proliferative and plateau phases of growth, EGF-R levels progressively increased, while conversely EGF-induced autophosphorylation of the EGF-R decreased over time in primary culture. These results demonstrate that EGF-R levels and autophosphorylation do not show a direct correlation with mammary epithelial cell mitogen-responsiveness. Intense EGF-R autophosphorylation appears to be required for initiating growth, but sustained mammary epithelial cell proliferation occurs when EGF-R autophosphorylation is low. This inverse relationship between EGF-R levels and autophosphorylation may reflect changes in receptor affinity and function during the various phases of mammary epithelial cell growth in primary culture.