Increased expression of peripheral benzodiazepine receptor (PBR) in dimethylbenz[a]anthracene-induced mammary tumors in rats

Expression of peripheral benzodiazepine receptors (PBR) has been found in every tissue examined; however, it is most abundant in steroid-producing tissues. Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the nucleus suggests that there may be other roles for PBR. Our laboratory reported earlier a significant increase of PBR density in the nucleus of DMBA-induced malignant submandibular glands of rats, suggesting a role of PBR in nuclear events of peripheral tissues. Since then numerous studies have demonstrated the abundance of PBR in tumors. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. Based on studies with a battery of human breast cancer cell lines and several human tissue biopsies, Hardwick et al. suggested that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression. The purpose of the present study is to confirm this hypothesis by developing an animal breast cancer model and correlating the above events with the breast cancer. Weanling rats were maintained on a diet containing animal protein (casein) for 30 days and then a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage to the animals. Control animals received the vehicle only. After 122 days of DMBA administration, the animals were sacrificed. All tumors were detected by palpation. B_max of PBRs was 52.6% and 128.4% higher in the non-aggressive and aggressive cancer tissues, respectively, than that in normal tissues. Cholesterol uptake into isolated nuclei was found to be higher in both non-aggressive and aggressive tumor breast tissue than that in control tissue. There was also corresponding increase in B_max of PBRs in the nucleus of cancer tissues. Furthermore, the nuclear nucleoside triphosphatase (NTPase) activity was found to be higher in aggressive tumor tissues than that in non-aggressive tumor tissues. In conclusion, these data suggest that PBR ligand binding, and PBR-mediated cholesterol transport into the nucleus may be involved in the development of mammary gland adenocarcinoma, thus participating in the advancement of the disease.     

Beneficial effects of soy protein in the initiation and progression against dimethylbenz [a] anthracene-induced breast tumors in female rats

Objective: Although recent studies link altered cellular redox state to protein dysfunction in various disease-states, such associations are least studied in clinical diabetes. Therefore, this study assessed the levels of reduced glutathione (GSH) and Na⁺/K⁺ ATPase activities in type 2 diabetic patients with and without microangiopathy. Methods: The study group comprised of a total of 160 subjects, which included non-diabetic healthy controls (n = 40) and type 2 diabetic patients without (n = 60) and with microangiopathy (n = 60), defined as presence of retinopathy with or without nephropathy. Erythrocyte Na⁺/K⁺ ATPase activity and GSH levels were estimated spectrophotometrically and fluorometry was used to determine the plasma thiobarbituric acid reactive substances (TBARS) and serum advanced glycation end products (AGEs). Results: GSH levels in diabetic subjects without (4.8± 0.15 μmol/g Hb) and with microangiopathy (5.2± 0.14 μmol/g Hb) were significantly lower (p < 0.001) compared to control subjects (6.3± 0.14 μmol/g Hb). Erythrocyte Na⁺/K⁺ ATPase activity was significantly reduced (p < 0.001) in diabetes subjects with (272± 7 nmol Pi/mg protein/h) and without microangiopathy (304 ± 8) compared to control (374 ± 6) subjects. TBARS were significantly higher (p < 0.001) in diabetes subjects with (10.65± 0.81 nM/ml) and without microangiopathy (9.90± 0.5 nM/ml) compared to control subjects (5.18± 0.18 nM/ml). Advanced glycation end product levels were also significantly (p < 0.001) elevated in diabetic subjects with microangiopathy (8.2± 1.8 AU) when compared to diabetes subjects without microangiopathy (7.0± 2.0 AU) and control subjects (4.6± 1.9 AU). On multivariate regression analysis, GSH levels showed a positive association with the Na⁺/K⁺ ATPase activity and negative association with TBARS and AGE levels. Conclusion: Hypoglutathionemia and increased oxidative stress appears to be early biochemical aberrations in diabetes, and through protein alterations, oxidative stress and redox modifications may contribute to pathogenesis of diabetic microangiopathy.     

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.     

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.     

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.     

Effect of human chorionic gonadotrophin on 7,12-dimethylbenz[a]anthracene-induced DNA binding and repair synthesis by rat mammary epithelial cells

The administration of the placental hormone human chorionic gonadotrophin (HCG) to 50-day-old virgin Sprague--Dawley rats has been shown to reduce the incidence of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer. We now report from studies using rat mammary epithelial cells in culture that the anti-carcinogenic effect of HCG may be related to its effect on DNA binding of DMBA and on DNA repair. The results showed that the level of excision repair in cells derived from young virgin (YV) rats grown in the presence of HCG (10 units/ml) was 2.5-4.0 times higher than the level exhibited by control YV cells and 1.5-2.5 times over that obtained for cells from old virgin and parous rats. The effect of HCG on DMBA-DNA binding was also determined in YV cells cultured in the presence of HCG (10 units/ml). Results from this study indicated that DMBA-DNA binding was inhibited by 30-40% in HCG-treated cells as compared to control cells. DNA binding of DMBA was also determined with mammary epithelial cells from YV rats which were given subcutaneous injections of HCG (5 units/rat) 5 times per week for 4 weeks. Using this in vivo-in vitro protocol, DMBA-DNA binding was 17-51% lower in cells from HCG-treated rats than in cells derived from control saline-treated rats. These results suggest that the protective effect provided by HCG against DMBA-induced mammary tumorigenesis may be attributed to its ability to inhibit binding of the carcinogen to mammary cell DNA and to its ability to increase the level of excision repair in the cells.     

Relationship between immune state and tumor growth rate in rats bearing progressive and non-progressive mammary tumors

 Impaired immune responses occur frequently in cancer patients or in tumor-bearing animals, but the mechanisms of the tumor-induced immune defects remain poorly understood. The aim of the present study was to determine the relevance of the immune system in the control of tumor growth. We have developed a model of progressive and non-progressive mammary tumor, chemically induced in female Wistar rats. In this model we evaluated the development of an immune response after immunization of rats bearing progressive and non-progressive tumors with a non-related antigen, such as sheep red blood cells. We also studied the activation state of peritoneal macrophages from animals bearing tumors by evaluating the production of free radicals. Our findings indicated that the cell-mediated immunity in rats bearing progressive tumors fails to respond to heterologous antigen in vivo, as demonstrated by a negative delayed-type hypersensitivity reaction, and is accompanied by minor nitric oxide production by peritoneal exudate cells as well as a lower capacity for macrophage activation. The study of non-progressive tumor-bearing rats indicated that the cell-mediated immune response was intact and an activated state of macrophages was found in vivo. The results described in this paper should be taken into account when therapies based on cancer vaccines are chosen for the treatment of cancer. Received: 26 February 1998 / 9 April 1998     

Transformation of [14C]-pregnenolone and production of corticosteroids by adrenal glands from rats bearing a transplantable mammotropic pituitary tumor

Rats with transplants of Furth's mammotropic pituitary tumor (MtT-F4) have high plasma levels of ACTH, growth hormone and prolactin. Adrenals from normal rats and rats bearing the tumor for 2, 3 and 5 weeks were incubated with [¹⁴C]-pregnenolone in a time-course study. MtT-F4 rats showed an inhibition in the conversion to aldosterone and 18-hydroxy corticosteronc, with an increase in steroids produced mainly in the zona fasciculata-reticularis: corticosterone, deoxyeorticostcrone and 18-hydroxy deoxycorticosterone. Measurement of steroids in adrenal venous blood showed decreased aldosterone secretion (P < 0.001 vs. controls at the 3rd and 5th weeks of the tumor growth) and an increase in corticosterone and deoxyconicosterone secretion at the 5th week (P < 0.001 and P < 0.05 vs controls, respectively). A proportional higher increment in deoxycorticosterone secretion at the 5th week was found. Determination of mitochondrial 11β-hydroxylation of deoxycorticosterone showed higher basal activity in MtT-F4 adrenals than in controls (P < 0.001); however, Krebs cycle intermediates at 10mM stimulated hydroxylation better in normal than in MtT-F4 mitochondria. The reaction was stimulated better in MtT-F4 than in controls by β-hydroxybutyrate and α-glycerophosphate. Thus, rats bearing the MtT-F4 showed (1) functional inhibition of the zona glomerulosa with concomitant hypcrfunction of the fasciculata-reticularis, and (2) abnormal regulation of the mechanisms supporting 11β-hydroxylation. These changes may be due to the high levels of ACTH circulating in these animals.     

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.     

Mechanistic studies on the inhibitory action of dietary dibenzoylmethane, a beta-diketone analogue of curcumin, on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis

Dietary factors play important roles in the carcinogenic process. The results of epidemiological data and some laboratory animal studies indicate that certain naturally occurring and synthetic components are able to block the carcinogenic process and inhibit the development of certain cancers. Dibenzoylmethane (DBM), a curcumin-related beta-diketone analogue has been reported to exhibit a remarkable inhibitory effect on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in Sencar mice. The present study investigated the possible mechanisms of inhibitory action of DBM on DMBA-induced mammary tumorigenesis in mice. The summarized results indicate that: (1) in in-vitro studies. DBM inhibited DMBA metabolism and the formation of DMBA-DNA adducts in a dose-dependent manner; (2) in the assay of competitive binding to estrogen receptors with [3H]-estradiol in vitro, DBM showed weak binding affinity; (3) in vivo, feeding of 1% DBM in the diet of immature Sencar mice for 4 -5 weeks decreased the uterine and parametrial fat pad weights, and lowered the serum estrogen and triglyceride levels. This study provides insight into the mechanisms involved in the inhibitory action of DBM in mouse mammary tumorigenesis.     

Therapeutic effect of propolis and paclitaxel on hepatic phase I and II enzymes and marker enzymes in dimethylbenz(a)anthracene-induced breast cancer in female rats

Propolis, a natural beehive product has been known for centuries for a variety of beneficial traditional medicinal properties. The present study was conducted to ascertain the antineoplastic potential of propolis along with paclitaxel against experimental mammary carcinogenesis. Female Sprague Dawley rats at 55 days of age were treated with dimethylbenz(a)anthracene to induce breast cancer. Paclitaxel at a dose of 33 mg/kg body mass intraperitoneally and propolis 50 mg/kg body weight orally was administered to the experimental animals, immediately after the carcinogen treatment and continued until the termination of the study. At the end of the treatment activities of phase I and II xenobiotic metabolizing enzymes and liver marker enzymes were measured. A significant increase in carcinogen activating enzymes, cytochrome P(450), cytochrome b(5) and NADPH cytochrome C reductase with concomitant decrease in phase II enzymes, glutathione transferase and UDP-glucuronyl transferase were observed in animals with mammary cancer. Furthermore there was a significant decrease in alanine aminotransferase, aspartate aminotransferase with a sharp increase in alkaline phosphatase, acid phosphatase and 5' nucleotidase. Propolis treatment caused the activity of these enzymes return to almost normal control levels, indicating the protective effect of propolis against dimethyl benz(a) anthracene induced carcinogenesis. On the basis of the observed results propolis can be considered a promising chemotherapeutic agent and can be administered as an adjuvant with paclitaxel chemotherapy.     

Effects of dietary fat and alpha-tocopherol on gamma-glutamyltranspeptidase activity of 7, 12-dimethylbenz(alpha)anthracene-induced mammary gland adenocarcinomas

The levels of gamma-glutamyltranspeptidase (GGTP) (EC 2.3.2.2) were measured in 7, 12-dimethylbenz(alpha)anthracene-induced mammary adenocarcinomas, in control mammary gland tissue and in sera from tumor-bearing and control rats. The carcinogenic process was modulated by diets differing in the type and amount of fat with and without alpha-tocopherol supplementation. Rat mammary adenocarcinomas showed significantly elevated (up to 25-fold) levels of GGTP when compared with adjacent histologically-uninvolved tissue or with mammary tissue of control rats. GGTP activity in sera from tumor-bearing rats was also elevated up to 4-fold than in the corresponding controls. Histochemical studies of the frozen section of mammary adenocarcinomas indicated that GGTP was localized in neoplastic ductal epithelial cells. In tumor rats on alpha-tocopherol supplemented diets, GGTP activity in the adenocarcinomas was mainly in the particulate (membrane-bound) fraction. In contrast, the tumor rats receiving alpha-tocopherol-deficient diets, the total GGTP activity was distributed in both particulate and cytosolic fractions, suggesting an altered membrane-GGTP interaction. The levels of GGTP in control mammary gland and sera of control rats from the low fat dietary groups were up to 7-fold higher than the corresponding control values in either of two high-fat groups. These high levels of GGTP in the serum and tissues of animals from the low fat dietary group are consistent with lower tumor incidence through enhanced carcinogen detoxification.     

Effects of dietary linseed,evening primrose or fish oils on fatty acid and prostaglandin E2 contents in the rat livers and 7,12-dimethylbenz[a]anthracene-induced tumours

We examined the influence of diets supplemented with fish and vegetable oils on fatty acid and prostaglandin E2 (PGE2) contents in livers of non-7,12-dimethylbenz[a]anthracene (DMBA)- and DMBA-treated rats, and in DMBA-induced tumours. Decreased concentrations of saturated fatty acids and increased unsaturated fatty acid levels were observed in liver phospholipids of rats fed these oils. There was a marked difference in the concentrations of fatty acids found in the tumours and those present in liver lipids. Oleic acid was the main unsaturated fatty acid found in the tumour tissue. Both liver and tumour PGE2 contents were clearly correlated to the diet. The PGE2 concentrations were decreased in livers and tumours of rats fed fish (FO) and linseed oils (LO).     

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.