Anti-tumor promoting potential of luteolin against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats

In this study, we evaluated the anti-tumor potential of luteolin (30mg/kg, p.o.), combined with cyclophosphamide (10mg/kg, i.p.) (LU+CYC) orally administered for 20 days; and CYC individually for 10 days against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in Wistar rats. Combination treatment (LU+CYC) inhibited the incidence rate of tumors and decreased tumor volume significantly without changing the total body weight of the animals. Long-term treatment did not show any apparent toxicity in rats. The CYC-treated group showed potential reduction of tumor volume (74%), severe toxicity, and loss of body weight. In order to elucidate the anticancer mechanism of luteolin, antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) generation in the liver, kidney and breast, as well as protein profiles, were also examined. Biochemical analysis of the combination-treated group showed significant (P<0.01; P<0.05) inhibition of lipid peroxide (LPx) formation (oxygen-free radicals), the level and the activity of SOD, CAT and GPx were found to be very high than the LU and CYC individually treated rats at a 30mg/kg dose. 2D gel electrophoresis analysis revealed that (56kDa) high molecular weight protein was detected in tumors of rats receiving combination treatment than the cancer controls. The biological significance of that protein involved for the dysfunction of cancer cells and induces apoptosis. Histopathological changes also confirmed the formation of tumor tubules and neovascularization after the treatment. Overall, these results suggest that the combination treatment provided antioxidant defense with strong chemopreventive activity against the genesis of DMBA-induced mammary tumors.     

Response of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells to tamoxifen, in vitro

Primary cell cultures from a density-defined cell subpopulation of the DMBA-induced rat mammary tumors were exposed to tamoxifen during their log phase of growth. Growth inhibition and the ultrastructure of surviving cells were examined along with the influence of this antiestrogen on the secreted proteins as determined by pulse labeling with [35S]methionine and fluorography. Cell growth was remarkably inhibited at clinically achievable concentrations. However, ultrastructural changes in the surviving cells were minimal, the most noteworthy being the accumulation of myelin bodies. Protein secretion was affected in the defined subpopulation of several tumors by the reduced production of a high-molecular-weight protein. These tumors may represent a population of estrogen-sensitive tumors within the DMBA-induced mammary tumor model.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

Consumption of a buckwheat protein extract retards 7,12-dimethylbenz[alpha]anthracene-induced mammary carcinogenesis in rats

Female rats were examined for the effects of feeding buckwheat protein extract (BWPE) on the development of mammary tumor caused by administration of 7,12-dimethylbenz[alpha]anthracene. The percentage of rats with palpable mammary tumors and serum estradiol were lower in the BWPE-fed animals than the casein-fed ones, implying that BWPE intake retarded the mammary carcinogenesis by lowering serum estradiol.     

Antioxidative effects of hesperetin against 7,12-dimethylbenz(a)anthracene-induced oxidative stress in mice

We investigated the effects of the chronic administration of hesperetin on the activation of the antioxidant defence system in mice in which oxidative stress had been induced by 7,12-dimethylbenz(a)anthracene (DMBA). Mice were divided randomly into three treatment groups. Hesperetin was administered orally to two of the three groups at 10 and 50 mg/kg body weight for 5 weeks. Subsequently, each group was subdivided randomly into DMBA-treated and untreated groups. The DMBA-treated groups were intragastrically administered a dose of 34 mg/kg BW in corn oil vehicle twice a week for 2 weeks. The TBARS value showed a tendency to decrease following hesperetin treatment; these decreases were significantly greater in the DMBA-treated than the untreated groups. Hesperetin significantly decreased the carbonyl content at the high dose in both DMBA-treated and untreated mice. Catalase and SOD activity were increased by hesperetin; this increase was more pronounced in DMBA-treated than untreated mice. Catalase, Mn-SOD, and CuZn-SOD expression analyses supported these results. Although the GSH-px and GR activity were little affected, hesperetin treatment significantly increased the GSH/GSSG ratio in the DMBA-treated group in a dose-dependent manner. These results suggest that hesperetin shows antioxidant activity and plays a protective role against DMBA-induced oxidative stress.     

Maintenance of milk protein gene expression in a subpopulation of 7,12-dimethylbenz (a) anthracene-induced rat mammary carcinoma cells grown on attached collagen gels

Summary Milk protein gene expression was studied in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma cells enriched or depleted for casein production grown on attached collagen gels. Culture of these cells in the presence of 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (10 μg/ml), and prolactin (5 μg/ml) maintained α-, β-, and γ-casein and whey acidic protein mRNAs at levels identical to cells isolated from perphenazine-treated rats. Whey acidic protein mRNA levels in the tumor cells relative to the 14-d lactating gland were greater than those of the casein mRNAs. Withdrawal of prolactin from the casein-producing cells resulted in the loss of all four milk protein mRNAs. Subsequent addition of prolactin to the withdrawn cells caused a rapid accumulation of these mRNAs to prewithdrawal levels. Milk protein gene expression in this tumor cell subpopulation is modulated by prolactin (in the presence of insulin and hydrocortisone) in a similar manner to that observed in the normal mammary gland when these tumor cells are cultured on attached collagen gels. This work was supported by National Institutes of Health grant CA 16303. M. L. Johnson was the recipient of NIH Fellowship, HD 06157.     

Effects of estradiol and prolactin on steroid receptor levels in 7,12-dimethylbenz(a)anthracene-induced mammary tumors and uterus in the rat.

In order to eliminate changes of pituitary hormone secretion secondary to treatment and be able to assess the direct effect of 17β-estradiol and prolactin at the level of hormone-dependent mammary tumors induced in the rat by 7,12-dimethylbenz(a)anthracene (DMBA), the effects of the two hormones on steroid receptor levels were investigated in hypophysectomized-ovariectomized animals. Treatment with prolactin alone led to tumor estrogen receptor levels higher than those found after estrogen administration. When the level of progesterone receptors was measured as parameter of estrogen action, it was found that combined treatment with prolactin and 17β-estradiol led to levels much higher than those found after individual hormone treatment. Tissue specificity of this interaction between prolactin and 17β-estradiol is indicated by the absence of effect of prolactin on the estrogen-induced stimulation of the progesterone receptor in uterine tissue. These data demonstrate the direct stimulatory effect of prolactin on estrogen receptor levels in DMBA-induced mammary carcinoma and indicate the central role of this pituitary hormone as modulator of estrogen action and growth of DMBA-induced mammary tumors.     

Scanning electron microscopy of 7,12-dimethylbenz(a)-anthracene-induced mammary carcinoma in the female Sprague-Dawley rat

The surface morphology of normal mammary glands and mammary carcinomas was examined under the scanning electron microscope after digestion of connective tissue and the basal lamina with collagenase, hyaluronidase and hydrochloric acid (HCl). Two types of cells were clearly identified in the acini of normal glands; granular epithelial cells and stellate myoepithelial cells. Spindle-shaped myoepithelial cells lying longitudinally along the mammary ducts were also recognized. 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas consisted of irregular masses of cells which had polypoid or columnar processes with rounded heads; the masses appeared to be composed of a single type of rhomboid cell. The tumors lacked the stellate or spindle-shaped myoepithelial cells found in normal acini and ducts.     

Specific stimulation by estradiol of tissue-type plasminogen activator production in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor cells.

The hormonal regulation of two plasminogen activators, tissue-type plasminogen activator (t-PA) and urokinase (u-PA), was studied both in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma and in DMBA-induced rat mammary dysplasia. t-PA activity in DMBA-mammary carcinoma was decreased markedly by oophorectomy and recovered upon estradiol administration to reach the maximum level at 12 hr. In contrast to its effect on DMBA-mammary carcinoma, estradiol had no effect on t-PA activity in DMBA-mammary dysplasia. Furthermore, DMBA-mammary carcinoma cells in primary culture displayed similar estrogen-dependency in production of t-PA, while t-PA production in DMBA-mammary dysplasia cells was not under the control of estradiol in vitro. Moreover, estrogen-stimulated production of u-PA activity was not observed in DMBA-mammary carcinoma cells or DMBA-mammary dysplasia cells both in vivo and in vitro. Taken together, these results suggest that estrogen stimulates the production of t-PA but not u-PA and that this estrogen dependency of t-PA is limited to malignant DMBA-mammary tumor cells.     

Dietary fish oil associated with increased apoptosis and modulated expression of Bax and Bcl-2 during 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinogenesis in rats

The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on induction of apoptosis in mammary carcinogenesis model. Mammary carcinogenesis was initiated by a single, tail vein injection of 7,12-dimethylbenz(alpha)anthracene (DMBA) (0.5mg/0.2ml corn oil/100g body weight) at 7 weeks of animal age. Ninety female Sprague-Dawley rats were divided into two parts: part one was used for histology and immunohistochemical study and part two for morphological analysis. Each part consists of three experimental groups having 15 animals, i.e., Group A (DMBA control), Group B (DMBA+fish oil) and Group C (DMBA+corn oil). Rats were fed either fish oil or corn oil (0.5ml/day/rat) by oral gavage, 2 weeks prior to DMBA injection. Treatment was continued 25 weeks, studying histopathology, expression of Bax and Bcl-2 proteins by immunohistochemistry and apoptosis by TUNEL assay and morphological study at 36 weeks. Results showed that the fish oil-treated group exhibited a substantial increase in Bax (p<0.05) immunolabelling and a reduction of Bcl-2 immunopositivity (p<0.05), and increased TUNEL-positive apoptotic cells (p<0.05); however, corn oil treatment did not show these beneficial effects toward mammary preneoplasia. We conclude that fish oil has the potential to play a significant role in limiting mammary tumourigenesis in vivo.     

Metabolism of [4-14C]estradiol by 7,12-dimethylbenz(a)anthracene-induced mammary tumor peroxidase

The peroxidase and estradiol-metabolizing activities of mammary tumors induced by 7,12-dimethylbenz(a)anthracene were determined in fresh and stored tissue. In both cases, a wide variation in peroxidase activity was observed in 47 different tumors tested. The properties of the enzyme found in the tumors were similar to those of lactoperoxidase. It is suggested that the amount of peroxidase present might reflect the ability of tumor cells to differentiate in response to hormonal stimulation and be indicative of the degree of tumor progression.     

Chemopreventive efficacy of geraniol against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

Abstract

The status of lipid peroxidation, antioxidants, and detoxification enzymes were used as biochemical end points to assess the chemopreventive potential of geraniol, a monoterpene, in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Topical application of 0.5% DMBA in liquid paraffin, three times a week, for 14 weeks developed well-differentiated squamous cell carcinoma in the buccal pouch of golden Syrian hamsters. Although 100% tumor formation was noticed in hamsters treated with DMBA alone, intragastric administration of geraniol, at a dose of 250 mg/kg body weight (b.w.) to DMBA-treated hamster completely prevented the formation of oral tumors. Furthermore, geraniol significantly reduced lipid peroxidation by-products and improved the status of enzymatic and non-enzymatic antioxidants as well as modulated the status of phase I and phase II detoxification enzymes, favoring the excretion of carcinogenic metabolite, during DMBA-induced oral carcinogenesis. The present study concludes that the chemopreventive potential of geraniol relies on its anti-lipid peroxidative and antioxidant function as well as modulatory effects on phase I and II detoxification enzymes to excrete the carcinogenic metabolite, during DMBA-induced hamster buccal pouch carcinogenesis.     

Chemopreventive efficacy of geraniol against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

The status of lipid peroxidation, antioxidants, and detoxification enzymes were used as biochemical end points to assess the chemopreventive potential of geraniol, a monoterpene, in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Topical application of 0.5% DMBA in liquid paraffin, three times a week, for 14 weeks developed well-differentiated squamous cell carcinoma in the buccal pouch of golden Syrian hamsters. Although 100% tumor formation was noticed in hamsters treated with DMBA alone, intragastric administration of geraniol, at a dose of 250 mg/kg body weight (b.w.) to DMBA-treated hamster completely prevented the formation of oral tumors. Furthermore, geraniol significantly reduced lipid peroxidation by-products and improved the status of enzymatic and non-enzymatic antioxidants as well as modulated the status of phase I and phase II detoxification enzymes, favoring the excretion of carcinogenic metabolite, during DMBA-induced oral carcinogenesis. The present study concludes that the chemopreventive potential of geraniol relies on its anti-lipid peroxidative and antioxidant function as well as modulatory effects on phase I and II detoxification enzymes to excrete the carcinogenic metabolite, during DMBA-induced hamster buccal pouch carcinogenesis.     

Pineal gland in rats with 7,12-dimethylbenz(a)anthracene-induced mammary tumors subjected to manipulations known as enhancers of pineal actions

The ultrastructure of pinealocytes was studied in rats with 7,12-dimethylbenz(a)anthracene-induced mammary tumors which were subjected to experimental manipulations known as enhancers of pineal actions (anosmia, underfeeding or cold exposure). In these animals we found: (I)--more nuclei with deep nuclear invaginations; (II)--a large number of cytoplasmic organelles, including lipid droplets, myeloid bodies, synaptic ribbons and lysosomes; (III)--numerous degenerative changes. In general, we found an increase in structural features related to pineal photoneuroendocrine activity. Our results indicate that pineal-dependent inhibition of neoplastic growth induced by these experimental manipulations, previously reported, can be mediated through an increase in pineal metabolic activity.