Effect of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis on the opioid peptide levels in the rat central nervous system.

Mammary tumors were induced in female Sprague-Dawley rats by giving a single oral dose of 20 mg 7,12-dimethylbenz[a]anthracene (DMBA). Animals were killed after full development of tumors 4 months after the ingestion of DMBA. Opioid peptides in various tissues were estimated by radioimmunoassay (RIA). Tumor-bearing rats (n = 5) had higher (P less than 0.05) contents of beta-endorphin in pituitary (+60%), striatum (+52%) and midbrain (+85%) compared to animals with no tumors. However, tumor-bearing rats showed a decrease of 35% in striatal met-enkephalin content. Dynorphin level decreased (P less than 0.05) in pituitary (-49%) and hypothalamus (-29%) of tumor-bearing rats. Thus for the first time, we report the alteration in the level of these neuropeptides during the process of chemical carcinogenesis.

Ibuprofen-induced inhibition of cyclooxygenase isoform gene expression and regression of rat mammary carcinomas.

A single dose of 75 mg/kg 7,12 dimethylbenz[a]anthracene was administered to 50-day-old virgin female Sprague-Dawley rats and 100 days later, animals were randomized and provided with Teklad rodent chow mixed with a dose of 25 mg/rat/day ibuprofen for 35 days. Ibuprofen treatment reduced tumor volume (P < 0.05) and significantly inhibited gene expression of both cyclooxygenase- and cyclooxygenase-2 (P < 0.02). These results indicate that ibuprofen induced significant regression of established mammary carcinomas which was associated with inhibition of expression of isoforms of the gene responsible for prostaglandin production.

Combined effects of interferon and steroid hormones on 2',5'-oligoadenylate synthetase activity in chronic lymphocytic leukemia cells.

2',5'-oligoadenylate synthetase (OAS) has been implicated in the effects of interferons (INF) and steroid hormones on cell growth and differentiation. We studied the combined effects in vitro of hormones and INF on OAS activity in cells from 10 patients with chronic lymphocytic leukemia. IFN enhanced OAS activity in all cells studied and also induced morphologic transformation. Diethylstilbestrol was also found to induce OAS activity, but not morphologic transformation. Progesterone, tamoxifen and dihydrotestosterone had no effect on enzyme activity nor on morphology. In five samples, all with estrogen receptor activity, DES and INF were synergistic in inducing OAS activity. TAM-INF synergism was observed in one sample. DES and TAM did not, however, significantly enhance IFN-induced morphologic transformations. Hydrocortisone reduced OAS activity, and antagonized INF-induced enzyme activity and morphologic transformations. We conclude that hormones can modulate OAS activity in CLL cells. These findings may be of importance in the design of INF-based therapies of CLL.

Chemotherapeutic evaluation of Celecoxib, a cyclooxygenase-2 inhibitor, in a rat mammary tumor model.

Epidemiological and experimental studies have shown that non-steroidal anti-inflammatory drugs (NSAIDs) reduce the relative risk of human cancer, including breast cancer. Recently, research studies in our laboratories have shown that the selective cyclooxygenase-2 (COX-2) blocker, Celecoxib, given daily in the diet, significantly inhibited the induction of rat mammary tumors by 7, 12-dimethylbenz(a)anthracene (DMBA). These studies were extended to evaluate Celecoxib for its effectiveness as an antineoplastic agent in this rat mammary tumor model. We examined the growth inhibitory effects of Celecoxib, given daily in the diet, on the volume and the number of established mammary tumors, vis-a-vis the cancer load (CL). Tumors continued to grow actively in control rats fed chow diet only. In contrast, the Celecoxib-supplemented diet (1500 mg/kg diet) significantly decreased the size of the mammary tumors in rats over the 6 week treatment period, resulting in an average reduction in tumor volume of approximately 32%, relative to the baseline volume (p<0.04). At the end of the 6 week treatment period, average tumor volume was 1.45 cm3 and 0.13 cm3 in the control and Celecoxib treated rats respectively. Tumor regression occurred in 90% of the rats. In addition, new tumors continued to emerge in the control group, in contrast to their significantly decreasing numbers in the Celecoxib treated group over the same time period (p<0.05). These results indicate that Celecoxib has significant antineoplastic activity, in addition to its anticarcinogenic effects.

Dose-response effects of the COX-2 inhibitor, celecoxib, on the chemoprevention of mammary carcinogenesis.

Recent chemopreventive studies in our laboratories showed that the COX-2 inhibitor, celecoxib, inhibited the induction of mammary cancer by 7,12-dimethylbenz(a)anthracene (DMBA). In this study, we examined the relative chemopreventive effect of varying doses of celecoxib on the development and growth of DMBA-induced rat mammary tumors. At 10 days prior to receiving a single intragastric dose of 15 mg DMBA/rat, female Sprague-Dawley rats were fed a control chow diet or diets containing 250, 500, 1000 or 1500 ppm celecoxib until termination of the experiment. Administration of increasing doses of celecoxib inhibited mammary tumor incidence and multiplicity as well as tumor volume in a dose-dependent manner. At 122 days post DMBA-intubation, mammary tumor incidence was 100% in the control rats compared to 80%, 50%, 45% and 25% in rats receiving 250, 500, 1000 or 1500 ppm celecoxib, respectively (p<0.001). Similarly, tumor multiplicity and tumor volume were significantly reduced by increasing the dose of celecoxib from 250 to 1500 ppm in the diet. The control rats had an average of 3.46 tumors/rat compared to 1.80, 1.00, 0.75 and 0.50 tumors/rat in animals receiving 250, 500, 1000 or 1500 ppm celecoxib, respectively (p<0.001). Average tumor volumes in rats fed 250, 500, 1000 or 1500 ppm celecoxib were 0.42, 0.34, 0.31 and 0.16 cm3 compared to 1.29 cm3 in the control rats (p<0.001). There was a concomitant increase in the steady-state serum concentration of celecoxib with the dose. These results indicate that, in this rat model, the chemopreventive effect of celecoxib against breast cancer is dose-dependent and that celecoxib is effective even at lower dose levels.

Chemopreventive activity of a C-glucuronide analog of N-(4-hydroxyphenyl) retinamide-O-glucuronide against mammary tumor development and growth.

The long term chemopreventive effects of the N-(4-hydroxyphenyl) retinamide-O-glucuronide (4-HPROG), and its stable C-linked benzyl glucuronide analog, retinamidobenzyl glucuronide (4-HPRCG) on the growth and development of 7,12-dimethylbenz[a]anthracene-induced mammary tumors were compared. The retinamidobenzyl glucuronide is stable toward acid hydrolysis and resists the actions of beta-glucuronidase. The results indicate that the C-linked glucuronide analog, 4-HPRCG has a greater chemopreventive potency than an equimolar concentration of 4-HPROG. Tumor latency was 15% longer in rats fed 2 mmol/kg diet of 4-HPRCG as compared to 4-HPROG. At 80 days post DMBA-intubation, tumor incidence was 57% and 27% in the 4-HPROG and 4-HPRCG treated rats, respectively. Tumor multiplicity was also markedly decreased in the 4-HPRCG treated rats. At 80 days post DMBA intubation the control rats had an average of 1.43 tumors/rat compared to 0.71 and 0.36 tumors/rat in the 4-HPROG and 4-HPRCG respectively. The higher potency and low toxicity of 4-HPRCG suggest that this stable analog may have an in vivo chemopreventive advantage over its analog, 4-HPROG. The results also demonstrated that these glucuronide analogs do not bind effectively in vitro either to the nuclear retinoid receptors or to the cellular retinoid binding proteins. Regardless of the mode of action of these retinoids, they are clearly effective chemopreventive agents.

Comparative ability of ibuprofen and N-(4-hydroxyphenyl)retinamide to inhibit development of rat mammary adenocarcinomas associated with differential inhibition of gene expression of cyclooxygenase isoforms.

A rodent model of carcinogen-induced mammary tumorigenesis was used to determine the comparative growth inhibitory effects of dietary administration of either 1000 mg/kg of the non-steroidal antiinflammatory drug (NSAID) ibuprofen or 1.5 mmol/kg of the synthetic retinoid N-(4-hydroxyphenyl)-retinamide (4-HPR). In addition, the effects of these compounds on gene expression and protein production of the two isoforms of the cyclooxygenase (COX) gene which are responsible for prostaglandin production were examined. Experimental diets were provided to rats beginning at 7 days prior to administration of a single intragastric dose of 15 mg dimethylbenz[a]anthracene (DMBA) and diets were provided ad libitum until the study was terminated at 16 weeks later. Ibuprofen significantly decreased levels of gene expression of both COX-1 and COX-2 (p < 0.01). Although dietary 4-HPR did significantly diminish levels of COX-1 gene expression (p < 0.01) in rat mammary adenocarcinomas, this synthetic retinoid did not significantly inhibit COX-2 gene expression. COX-1 protein was localized to endothelial cells, infiltrating inflammatory cells, and tumor cells, while COX-2 protein was detected primarily within tumor cells. Although ibuprofen was more effective in inhibiting COX-2 gene expression than 4-HPR, ibuprofen and 4-HPR were equally effective in inhibiting development of carcinogen-induced mammary adenocarcinomas.

Genetic induction and upregulation of cyclooxygenase (COX) and aromatase (CYP19): an extension of the dietary fat hypothesis of breast cancer.

A novel model of mammary carcinogenesis is proposed involving sequential induction and upregulation of cyclooxygenase and aromatase genes by essential fatty acids prominent in the US diet. The basic carcinogenic processes are: (1) constitutive prostaglandin biosynthesis and formation of mutagenic oxygen and nitrogen free radicals responsible for tumor initiation; (2) PGE-2-induced expression of aromatase and constitutive estrogen biosynthesis which sustains mitogenesis and tumor promotion; and (3) PGE-2-induced expression of vascular endothelial growth factor which stimulates angiogenesis and tumor metastasis.

Putative metabolites derived from dietary combinations of calcium glucarate and N-(4-hydroxyphenyl)retinamide act synergistically to inhibit the induction of rat mammary tumors by 7,12-dimethylbenz[a]anthracene.

Calcium glucarate and N-(4-hydroxyphenyl)retinamide were evaluated individually and in combination in the diet as preventative chemical agents, by using the induction of rat mammary tumors by 7,12-dimethylbenz[a]anthracene as the test system. When tested separately over 18 weeks, optimal doses of calcium glucarate (128 mmol/kg of diet) or N-(4-hydroxyphenyl)retinamide (1.5 mmol/kg of diet) administered daily inhibited tumor incidence by 50% or 57% and tumor multiplicity by 50% or 65%, respectively. Suboptimal doses of calcium glucarate (32 mmol/kg) and of N-(4-hydroxyphenyl)retinamide (0.75 mmol/kg) inhibited tumor incidence by 15% and 5% but had no inhibitory effect on tumor multiplicity. In contrast, the combination of calcium glucarate (32 mmol/kg) and N-(4-hydroxyphenyl)retinamide (0.75 mmol/kg) inhibited tumor incidence and tumor multiplicity by 50%. Similar synergism was observed with the combination of calcium glucarate (64 mmol/kg) and N-(4-hydroxyphenyl)retinamide (0.75 mmol/kg), the inhibition being 55-60%. HPLC analysis of the bile of female rats injected intraperitoneally with a single dose of the retinamide [60 mg/kg (body weight)] showed that the excretion of the retinamide and its glucuronide were markedly suppressed by pretreatment with an oral dose of calcium glucarate [4.5 mmol/kg (body weight)].