gamma-Tocotrienol inhibits ErbB3-dependent PI3K/Akt mitogenic signalling in neoplastic mammary epithelial cells.

The antiproliferative effects of gamma-tocotrienol are associated with suppression in epidermal growth factor (EGF)-dependent phosphatidylinositol-3-kinase (PI3K)/PI3K-dependent kinase-1 (PDK-1)/Akt mitogenic signalling in neoplastic mammary epithelial cells. Studies were conducted to investigate the direct effects of gamma-tocotrienol treatment on specific components within the PI3K/PDK-1/Akt mitogenic pathway. +SA cells were grown in culture and maintained in serum-free media containing 10 ng/ml EGF as a mitogen. Treatment with 0-8 microm gamma-tocotrienol resulted in a dose-responsive decrease in the +SA cell growth and a corresponding decrease in phospho-Akt (active) levels. However, gamma-tocotrienol treatment had no direct inhibitory effect on Akt or PI3K enzymatic activity, suggesting that the inhibitory effects of gamma-tocotrienol occur upstream of PI3K, possibly at the level of the EGF-receptor (ErbB1). Additional studies were conducted to determine the effects of gamma-tocotrienol on ErbB receptor activation. Results showed that gamma-tocotrienol treatment had little or no effect on ErbB1 or ErbB2 receptor tyrosine phosphorylation, a prerequisite for substrate interaction and signal transduction, but did cause a significant and progressive decrease in the ErbB3 tyrosine phosphorylation. Because ErbB1 or ErbB2 receptors form heterodimers with the ErbB3 receptor, and ErbB3 heterodimers have been shown to be the most potent activators of PI3K, these findings strongly suggest that the antiproliferative effects of gamma-tocotrienol in neoplastic +SA mouse mammary epithelial cells are mediated by a suppression in ErbB3-receptor tyrosine phosphorylation and subsequent reduction in PI3K/PDK-1/Akt mitogenic signalling.

Antitumor efficacy in rats of CGP 19984, a thiazolidinedione derivative that inhibits luteinizing hormone secretion.

The antitumor efficacy and the hormonal effects of the thiazolidine-dione derivative (sodium methyl((-3-methyl-2- ([5-methyl-3-(2-methylallyl)-4-oxo-2 thiazolidinyliden]hydrazono)-4-oxo-5-thiazolidinyl)) phosphate, CGP 19984, have been studied in in vivo rat prostatic and mammary cancer models. CGP 19984 significantly inhibited growth of the androgen-dependent Dunning R3327 rat prostate adenocarcinoma. Concomitant with tumor inhibition, a significant decrease in circulating luteinizing hormone and testosterone levels was observed, suggesting that the antitumor effects of drug treatment resulted primarily from inhibition of luteinizing hormone release and subsequently decreased testosterone synthesis. Drug treatment had little effect on serum prolactin or corticosterone levels. Animals showed no adverse effects from CGP 19984 except for a modest loss of body weight. In female rats, growth of the estrogen-independent MTW-9B rat mammary tumor was also inhibited by CGP 19984 and uterine weight and tumor progesterone receptor levels were reduced. The latter suggests that CGP 19984 treatment decreases circulating estrogen in female rats. However, the inhibitory effect of CGP 19984 on the growth of the MTW-9B tumor does not appear to be mediated by the action of the drug to lower estrogen levels, since this tumor is not dependent on estrogen for growth, and lower doses of CGP 19984 were found to be equally effective in reducing uterine weight, but had no antitumor activity. The ability of CGP 19984 to suppress gonadal function and to inhibit tumor growth suggests that this drug may have potential clinical application in the treatment of both hormone-dependent and -independent prostate and breast cancers.

Relationship of hormones to inhibition of mammary tumor development by underfeeding during the "critical period" after carcinogen administration.

Seven days prior to 7,12-dimethylbenz(a)anthracene (DMBA) administration, virgin 50-day-old female Sprague-Dawley rats were placed on a food-restricted diet and continued on this regimen until 30 days after DMBA injection. One day prior to and 7 days after DMBA administration, animals were given daily 0.1-ml s.c. injections of 0.9% NaCl solution (controls), haloperidol (HAL; 0.5 mg/kg) to increase prolactin secretion, growth hormone (GH; 0.5 mg/kg), estradiol benzoate (EB; 1 microgram/rat), or a combination of HAL, EB, and GH. Drug and hormone treatments were terminated after 8 days, but underfeeding continued for 30 days after DMBA administration, after which time all animals were placed on ad libitum feeding for the remainder of the 26-week experiment. Food restriction for 7 days prior to and 30 days after DMBA administration resulted in a significant reduction in average tumor number and size by the end of the 26-week experiment. Treatment for 8 days with EB produced a significant increase in mammary tumor incidence despite underfeeding, whereas underfed rats given the combination of HAL EB, and GH showed development and growth of mammary tumors equal to that of full-fed controls. Both EB and HAL significantly raised blood prolactin levels. GH alone had no apparent effect on mammary tumor incidence. These results indicate that reduced food intake during the "critical period" for induction of mammary tumors in rats by DMBA can produce inhibition of mammary tumor development throughout the 6-month period of this experiment and that administration of EB or the combination of EB, HAL, and GH for only 8 days can counteract the inhibition by underfeeding.

Effects of high dietary fat on the growth and development of ovarian-independent carcinogen-induced mammary tumors in rats.

This study examined the influence of high dietary fat intake on the development of ovarian-independent mammary tumors in both vehicle-treated controls and rats made deficient in estrogen and prolactin during tumor induction. The majority of 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors in rats are dependent on estrogen and prolactin for growth, and suppression of prolactin and estrogen at the time of tumor initiation causes a reduction in tumor incidence and increase in tumor latency. However, the majority of mammary tumors which do develop in these animals exhibit ovarian-independent growth. Sprague-Dawley rats were given 7.5 mg DMBA p.o. at 57 days of age. Starting 1 day prior to and continuing for 7 days after DMBA administration, rats were given daily injection of vehicle or the combination of tamoxifen (20 micrograms/rat) plus bromocryptine (5 mg/kg). At the end of drug treatment, rats in each treatment group were equally divided and placed on normal fat (5% corn oil) or high fat (20% corn oil) diets for the duration of the experiment. Vehicle-treated rats were ovariectomized 27 wk and drug-treated rats 47 wk after DMBA administration to determine tumor ovarian dependency. Vehicle-treated rats fed high fat diets showed significant increases in mammary tumor incidence and number as compared to similarly treated rats fed a normal fat diet, with approximately 80% of the tumors in each group being ovarian dependent. Likewise, tamoxifen-bromocryptine-treated rats fed a high fat diet showed a significant enhancement in mammary tumor number, although not incidence, as compared to similarly treated rats fed a normal diet. Tumors in these drug-treated groups displayed essentially the same incidence of ovarian dependence (23%). Tamoxifen-bromocryptine-treated groups displayed a 2-fold increase in latency of tumor appearance as compared to vehicle-treated controls; however, this long latency was not reduced when these rats were fed a high fat diet. These results demonstrate that high dietary fat stimulates ovarian-dependent and -independent mammary tumorigenesis in rats but does not influence the hormonal responsiveness of these tumors.

Comparative effects of different animal and vegetable fats fed before and during carcinogen administration on mammary tumorigenesis, sexual maturation, and endocrine function in rats.

The purpose of this investigation was to determine whether diets high in animal or vegetable fat affected mammary tumorigenesis when fed to rats only prior to and during the initiation phase of carcinogenesis. Weanling 21-day-old female Sprague-Dawley rats were divided into different dietary treatment groups and were allowed to feed and libitum on one of the following diets: 5% (normal fat) corn oil; 20% (high fat) corn oil; 20% palm oil; 20% beef tallow; or 20% lard. At 52 days of age, all rats were given p.o. 7.5 mg 7,12-dimethylbenz(a)anthracene (DMBA). One week following DMBA administration, all rats were switched to the 5% corn oil control diet and were maintained on this diet for the duration of the experiment. Rats fed a 20% lard diet during the treatment period showed a significant increase in mammary tumor incidence and number 19 weeks after DMBA administration, when compared to all other dietary treatment groups. Rats fed a 20% beef tallow diet during this same time period also demonstrated enhanced mammary tumor development, during the 10- to 19-week time period after DMBA. Mammary tumor development in rats fed 20% corn oil or palm oil diets during this treatment period was similar to that of normal fat controls. Estrogens are potent stimulators of mammary tumor growth and development in rats. Because mammary tumorigenesis was enhanced in rats fed high animal, but not vegetable fat diets, it was possible that estrogens present in animal fat might be responsible for this stimulation. Further studies demonstrated however, that increased mammary tumorigenesis in rats fed diets high in animal fat could not be explained on the basis of endocrine stimulation. Average day of vaginal opening for all groups fed 20% fat diets was similar and occurred earlier than in normal fat controls. In addition, 50- to 65-day-old rats in the different dietary treatment groups showed no differences in basal or surge levels of serum prolactin, luteinizing hormone, or estradiol. Rat diestrus uterine weight also showed no significant differences among dietary treatment groups. Thus diets containing high levels of animal fat caused little if any increased estrogenic activity in rats. In conclusion, high dietary intake of lard and beef tallow, but not vegetable fat, fed from weaning until only 1 week after DMBA administration, significantly enhances mammary tumorigenesis in rats. The mechanism(s) by which animal fat induces this stimulation is not clear, but it does not appear to result from endogenous or exogenous endocrine stimulation.

Influence of underfeeding during the "critical period" or thereafter on carcinogen-induced mammary tumors in rats.

Normal cycling virgin female Sprague-Dawley rats were divided into different treatment groups and subjected to half ad libitum feed intake for 2 or 4 weeks at consecutive periods of time before and/or after administration of 7,12-dimethylbenz(a)anthracene (DMBA) and then were returned to full-feed. All rats at the end of their respective under-feeding periods showed reductions in serum prolactin and cessation of estrous cycles. However, only rats underfed 1 week prior to and 1 week after DMBA administration showed significant reductions in mammary tumorigenesis or the entire 21 weeks of the experiment when compared to full-feed controls. Rat groups underfed during subsequent weeks after DMBA administration showed no alterations in mammary tumor development as compared to full-fed controls. These results demonstrate that inhibition and perhaps permanent suppression of mammary tumorigenesis occurred only in rats underfed 1 week before and 1 week after DMBA administration. The inhibitory effects produced by this under-feeding regimen on mammary tumorigenesis may be mediated by suppression of prolactin and estrogen secretion.

Effects of alterations in early hormonal environment on development and hormone dependency of carcinogen-induced mammary tumors in rats.

The purpose of this study was to determine the early role of estrogen and prolactin on subsequent hormone dependency of carcinogen-induced mammary tumors. Virgin 57-day-old Sprague-Dawley rats were given i.v. injections of 7,12-dimethylbenz(a)anthracene (DMBA). One day prior to and 7 days after DMBA administration, the rats were divided into separate groups and given: a daily 0.1-ml s.c. injection of vehicle (controls); haloperidol (0.5 mg/kg) to increase prolactin secretion; estradiol benzoate (1 microgram/rat) to increase estrogen levels; bromocryptine (5 mg/kg) to inhibit prolactin release; tamoxifen (TAM, 20 micrograms/rat) to inhibit estrogen action; or the combination of TAM and bromocryptine. Drug and hormone treatments were terminated after 8 days. Sixteen weeks after DMBA administration, all animals were bilaterally ovariectomized, and 4 weeks later it was determined whether the mammary tumors were hormone dependent or independent. Treatment with TAM resulted in a significant reduction in incidence of mammary tumors, but also a 3-fold increase in the percentage of these tumors that showed hormone independence after ovariectomy as compared with that of control rats. Rats treated with the combination of TAM and bromocryptine also showed a significant reduction in tumor incidence and number, but a 5-fold greater percentage of hormone-independent tumors after ovariectomy. Rats given daily injections of haloperidol or estradiol benzoate showed only small differences in mammary tumor incidence or autonomy after ovariectomy, as compared with controls given injection vehicle alone. These results suggest that rats made deficient in estrogen and prolactin at the time of DMBA administration develop fewer tumors, but the tumors that develop are not dependent on these hormones for subsequent growth.

Role of estrogen and prolactin in stimulation of carcinogen-induced mammary tumor development by a high-fat diet.

The role of estrogen and prolactin in high-fat (HF) dietary stimulation of carcinogen-induced mammary tumors was examined in female Sprague-Dawley rats. At 55 days of age, the rats were given injections i.v. of 5 mg of dimethylbenz(a)anthracene and, 5 days later, rats were sham- or bilaterally ovariectomized. Ten days after dimethylbenz(a)anthracene administration, the rats were placed on either a 20.0% HF diet or a 4.5% control fat (CF) diet and were then subjected to various drug and endocrine treatments to maintain uniform levels of circulating estrogen and prolactin. Sham-operated intact rats and bilaterally ovariectomized rats were given daily injections of haloperidol to increase prolactin secretion, bromocryptine to decrease prolactin secretion, and/or estradiol benzoate (EB). The intact rats fed the HF diet showed significant stimulation of all parameters of mammary tumor development when compared to similarly treated rats fed the CF diet. In ovariectomized rats fed either the HF or CF diet, there was nearly complete inhibition of mammary tumor development. When the HF diet was given to ovariectomized rats treated daily with either haloperidol or EB, or EB and bromocryptine, some parameters of mammary tumor development were enhanced by the HF diet. However, in all cases, mammary tumorigenesis was reduced when compared to sham-operated control rats. Ovariectomized rats fed the HF diet and given both EB and haloperidol exhibited significantly greater tumor number per rat, increased average tumor size, and reduced tumor latency period when compared to similarly treated rats fed the CF diet. However, these parameters of mammary tumorigenesis were still reduced when compared to those of sham-control rats fed the HF diet. These results indicate that a HF diet requires adequate circulating levels of estrogen and prolactin to maximally promote increased mammary tumorigenesis in dimethylbenz(a)anthracene-treated rats. Moreover, the enhancing effects of a HF diet on mammary tumorigenesis can be achieved in the presence of similar circulating levels of estrogen and/or prolactin, whether decreased or increased. These results suggest, therefore, that mechanisms independent of altered secretion of estrogens and/or prolactin are involved in promotion of mammary tumorigenesis by high levels of dietary fat.

Inhibition of mammary tumor growth by dexamethasone in rats in the presence of high serum prolactin levels.

Female Sprague-Dawley rats with established 7, 12-dimethylbenz(a)anthracene-induced mammary tumors were given daily s.c. injections of 50 microgram dexamethasone per rat, 0.5 mg haloperidol per kg, or both for 3 weeks. Control rats received the injection vehicles only. Mammary tumor growth was measured at weekly intervals for 21 days, and blood was collected on Days 10 and 21 of treatment for assay of prolactin. Dexamethasone produced significant regression of mammary tumors and reduced serum prolactin levels, whereas haloperidol significantly increased mammary tumor growth and greatly elevated serum prolactin levels. When dexamethasone and haloperidol were injected together, there was significant regression of mammary tumors despite markedly elevated serum prolactin levels. No significant differences in specific prolactin binding to membrane preparations of mammary tumors from these animals were observed in any treatment group. These results indicate that dexamethasone, a synthetic glucocorticoid, can directly inhibit mammary tumor growth in the presence of elevated serum prolactin levels produced by haloperidol, and this inhibition is not due to a reduction of prolactin binding sites in the tumor tissue.

γ-Tocotrienol reversal of epithelial-to-mesenchymal transition in human breast cancer cells is associated with inhibition of canonical Wnt signalling.

OBJECTIVES: Frizzled-7 (FZD7) receptor-dependent activation of the canonical Wnt/ß-catenin pathway plays a crucial role in epithelial-to-mesenchymal transition (EMT) and breast cancer metastasis. FZD7 and its co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), are highly expressed in MDA-MB-231 and T-47D breast cancer cells, and endogenous ligands for FZD7 include Wnt3a and Wnt5a/b. γ-Tocotrienol, a natural isoform of vitamin E, inhibits human breast cancer cell proliferation and EMT. Here, studies have been conducted to investigate the role of the canonical Wnt pathway in mediating inhibitory effects of γ-tocotrienol on EMT in human breast cancer cells. MATERIALS AND METHODS: MDA-MB-231, T-47D and MCF-10A cells were maintained in serum-free defined media containing selected doses of γ-tocotrienol. Cell viability was determined using the MTT colorimetric assay, Western blot analysis was used to measure protein expression and the wound-healing assay was employed to study cell mobility and migration. Immunohistochemical fluorescence staining visualized expression and localization of EMT cell markers. RESULTS: γ-Tocotrienol was found to induce dose-responsive inhibition of MDA-MB-231 and T-47D cell growth at doses that had no effect on immortalized normal MCF-10A mammary epithelial cells. These growth inhibitory effects were associated with suppression in canonical Wnt signalling, reversal of EMT and significant reduction in breast cancer cell motility. CONCLUSIONS: γ-Tocotrienol suppression of metastatic breast cancer cell proliferation and EMT was associated with suppression of the canonical Wnt/ß-catenin signalling pathway.

Effects of ultra-wideband electromagnetic pulses on pre-neoplastic mammary epithelial cell proliferation.

Electromagnetic ultra-wideband pulses (UWB) or nanopulses, are generated by a wide range of electronic devices used in communications and radar technology. However, the specific effects of nanopulse exposure on cell growth and function have not been extensively investigated. Here, studies have been conducted to determine the effects of prolonged exposure to non-ionizing, low to moderate intensity nanopulses on the growth of pre-neoplastic CL-S1 mammary epithelial cells in vitro. Cells were grown in culture and maintained in serum-free defined medium containing 10 ng/ml EGF and 10 microg/ml insulin as comitogens. Studies showed that 0.25-3.0 h exposure to nanopulses of 18 kV/m field intensity, 1 kHz repetition rate and 10 ns pulse width had no effect on CL-S1 cell growth or viability during the subsequent 72-h culture period. However, exposure to similar nanopulses for prolonged periods of time (4-6 h) resulted in a significant increase in cell proliferation, as compared to untreated controls. Additional studies showed that nanopulse exposure enhanced CL-S1 cell growth when cells were maintained in media containing only EGF, but had no effect on cells maintained in defined media that were mitogen-free or containing only insulin. Studies also showed that the growth-promoting effects of nanopulse exposure were associated with a relatively large increase in intracellular levels of phospho-MEK1 (active) and phospho-ERK1/2 (active) in these cells. These findings demonstrate that prolonged exposure to moderate levels of UWB enhanced EGF-dependent mitogenesis, and that this growth-promoting effect appears to be mediated by enhanced activation of the mitogen-activated protein kinase (MAPK) signalling pathway in pre-neoplastic CL-S1 mammary epithelial cells.

Anti-proliferative effects of γ-tocotrienol are associated with suppression of c-Myc expression in mammary tumour cells.

OBJECTIVES: Aberrant c-Myc activity plays a central role in cancer transformation. γ-tocotrienol is a member of the vitamin E family that displays potent anti-cancer activity. Here, studies were conducted to determine the role of c-Myc in mediating anti-proliferative effects of γ-tocotrienol in mammary cancer cells. MATERIALS AND METHODS: Treatment effects on mouse +SA and human MCF-7 mammary cancer cell proliferation were determined by MTT assay and Ki-67 staining. Protein expression was determined by western blot analysis. Immunofluorescence staining and qRT-PCR were used to characterize cellular c-Myc and MYC levels respectively. RESULTS: Anti-proliferative effects of γ-tocotrienol were associated with reduction in total c-Myc and phosphorylated-c-Myc-serine 62, and increase in phosphorylated-c-Myc-threonine 58 levels. γ-tocotrienol also reduced PI3K/Akt/mTOR and Ras/MEK/Erk mitogenic signalling, cyclin D1 and cyclin-dependent kinase 4 levels, and increased p27 levels. However, γ-tocotrienol had no effect on MYC mRNA levels. γ-tocotrienol also increased levels of FBW7 (E3 ligase that initiates ubiquitination of c-Myc), but had no effect on serine/threonine phosphatase PP2A or isomerase Pin 1 levels. Combined treatment with GSK3α/ß inhibitor LiCl or proteasome inhibitor MG132 blocked γ-tocotrienol-induced reductions in c-Myc. CONCLUSIONS: These findings indicate that anti-proliferative effects of γ-tocotrienol are associated with reduction in c-Myc that results from increase in GSK-3α/ß-dependent ubiquitination and degradation, rather than from reduction in c-Myc synthesis in +SA and MCF-7 mammary cancer cells.

Diminished bioactivity of circulating prolactin despite increased immunoreactive hormone release in old versus young male rats.

The Nb2 rat lymphoma PRL bioassay and standard RIA techniques were used to compare the impact of aging on immunoreactive (ir) and bioactive (bio) plasma PRL levels in 3- to 5- and 22- to 24-month-old male Copenhagen-Fischer rats. Basal plasma irPRL levels were significantly higher, while bioPRL were significantly lower in old compared to young rats. Administration of 10 mg/kg morphine similarly increased plasma irPRL levels in both age groups, but elevations in bioPRL were significantly lower in the older animals. Administration of 1 mg/kg haloperidol significantly increased circulating levels of ir- and bioPRL in both age groups, but these responses were significantly attenuated in the older rats. Treatment with 10 mg/kg bromocryptine resulted in significantly greater decreases in plasma irPRL levels in young rats, but induced similar decreases in bioPRL levels in both age groups. Administration of 25 micrograms/kg TRH induced similar increases in both ir- and bioPRL levels in young and old male rats. These results demonstrate that basal plasma bioPRL levels are significantly lower in old compared to young male rats, and specific drug treatments differentially alter the ratio of plasma bio- vs. irPRL levels in these age groups. These findings suggest that during the aging process not only quantitative changes but also qualitative changes occur in PRL secretion, which result in an overall decline in the biopotency of PRL released into the circulation.

Comparison of acute effects of dynorphin and beta-endorphin on prolactin release in the rat.

The ability of dynorphin and beta h-endorphin (beta h-EP) to stimulate prolactin (PRL) release in male rats was examined. Rats were injected intraventricularly with either 1 or 10 microgram dynorphin, 1 or 10 microgram beta h-EP, 10 microgram dynorphin together with 20 microgram naloxone (NAL), or an equivalent volume (13 microliter) of saline. Dynorphin caused a dose-dependent release of PRL which was significant 10 min after injection but not at later sampling times. beta h-EP also increased serum PRL levels and maintained elevated PRL levels for at least 60 min. NAL, a specific opiate antagonist, completely blocked dynorphin-induced PRL release. These results indicate that dynorphin is a potent stimulator of PRL release, but its stimulatory activity is transient relative to the activity exhibited by beta h-EP. Furthermore, dynorphin's action is specific since NAL completely blocked dynorphin induced PRL release.

Effect of naltrexone on stress-induced bioactive prolactin release in aging male rats.

Effects of the opiate receptor antagonist naltrexone (NALT, 10 mg/kg) on basal and novel environment stress-induced prolactin (PRL) release were examined in young (3-5 month) and old (22-24 month) male Copenhagen-Fischer 344 rats. Radioimmunoassay and Nb2 lymphoma bioassay were used to determine plasma immunoreactive (ir-) and bioactive (bio-) PRL levels, respectively. Although basal plasma irPRL levels were greater, bioPRL levels were significantly lower in old as compared to young rats. NALT induced significant decreases in basal plasma ir- and bioPRL concentrations in young rats, but had little or no effect on irPRL or bioPRL levels in old rats. Stress-induced elevations in irPRL levels were similar, but increases in bioPRL levels were attenuated in vehicle-treated old as compared to young rats. NALT pretreatment blocked stress-induced ir- and bioPRL release in both age groups. These results indicate that the role of endogenous opioid peptides (EOP) in regulating basal PRL secretion appears to be diminished, whereas EOP continue to be involved in stress-induced PRL release in the aging animal.

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.

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.

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.

Effects of Schistosoma mansoni on androgen regulated gene expression in the mouse.

Two-dimensional gel electrophoresis of liver mRNA translation products and dot-blot hybridization revealed that the levels of mRNA encoding major urinary proteins were greatly reduced in mice infected with Schistosoma mansoni. Major urinary protein mRNA levels are known to be androgen regulated. Dot-blot hybridization analysis of RNAs from various mouse tissues with a variety of cDNA probes indicated that all androgen-regulated mRNAs tested were reduced in infected mice. Administration of testosterone to infected animals restored urinary major urinary protein levels. Direct measurement of serum testosterone levels and seminal vesicle weights confirmed that chronic schistosome infection reduces testosterone to castration levels in male mice.

Effects of repetitive daily acute stress on pituitary LH and prolactin release during exposure to the same stressor or a second novel stress.

The present study was carried out to examine the effect of repetitive acute stress on pituitary secretion of prolactin (PRL) and luteinizing hormone (LH) during subsequent exposure to the same stimulus or to a second, novel stress. Intact adult male rats were subjected to either a single or 10 daily acute episodes of a psychological stress, transfer to a novel environment, or a physical stressor, restraint. A single acute exposure to either stress caused a temporary but significant increase in circulating concentrations of LH and PRL, and repetitive daily exposure to these stressors resulted in the habituation of stress-stimulated release of both hormones by the 10th consecutive day. When rats previously exposed to daily novel environment stress were subjected to a single episode of restraint stress, they showed an attenuation of both the LH and PRL secretory responses to this type of stress, compared to rats exposed to only one episode of restraint. These results indicate that repeated daily exposure to specific acute stress stimuli can result in the eventual habituation of both the LH and PRL hormonal responses to stress. At least with regard to the paired stressors examined in this study, adaptation to one type of acute stress stimulation may result in altered hormonal responsiveness to a second, unfamiliar stressor.