An unhydrolyzable analogue of N-(4-hydroxyphenyl)retinamide. synthesis and preliminary biological studies.

The synthesis of a nonhydrolyzable, carbon-linked analogue (4-HBR) of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) using Umpolung methods is described. Preliminary studies of biological activity show 4-HBR is similar to 4-HPR in its actions although a potentially relevant and desirable difference is its reduced suppression of plasma vitamin A levels. These results show that 4-HPR does not have to be hydrolyzed to retinoic acid to produce its chemotherapeutic 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.

Chemotherapeutic evaluation of 4-hydroxybenzylretinone (4-HBR), a nonhydrolyzable C-linked analog of N-(4-hydroxyphenyl) retinamide (4-HPR) against mammary carcinogenesis.

The antitumor effects of N-(4-hydroxyphenyl)retinamide (4-HPR), and its stable C-linked analog, 4-hydroxybenzylretinone (4-HBR) on the regression of established 7,12-dimethylbenz(a)anthracene(DMBA)-induced rat mammary tumors were compared. 4-HBR is a stable and nonhydroyzable derivative which cannot be converted in vivo to retinoic acid (RA). The results indicate that 4-HBR decreased mammary tumor volumes to the same extent as equimolar concentration (2 mmol/kg diet) of 4-HPR (-45% for 4-HBR vs. -42% for 4-HPR, p<0.01). Both 4-HPR and 4-HBR bind very poorly to nuclear retinoid receptors RARs and RXRs. The similarity of physicochemical properties of 4-HPR and 4-HBR as well as their equal antitumor potency suggests that 4-HPR like 4-HBR, is acting directly rather than through hydrolysis to free RA. Treatment with 4-HPR caused an almost 65% decrease in serum retinol levels. These results suggest that 4-HBR may have a significant chemotherapeutic advantage over 4-HPR, as the nonhydrolyzable analog may not cause night blindness which occurs as a significant side effect of 4-HPR usage.

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.

Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been observed to reduce the relative risk of breast cancer. This prompted our investigation of the chemopreventive potential of celecoxib, a specific cyclooxygenase 2 blocker, against mammary carcinogenesis induced by 7,12-dimethyl-benz(a)anthracene in female Sprague Dawley rats. Treatment with celecoxib was examined and compared to treatment with the general NSAID, ibuprofen, and to a control group receiving only dimethylbenz(a)anthracene. Dietary administration of celecoxib (1500 ppm) produced striking reductions in the incidence, multiplicity, and volume of breast tumors relative to the control group (68%, 86%, and 81%, respectively; P < 0.001). Ibuprofen also produced significant effects, but of lesser magnitude (40%, 52%, and 57%, respectively; P < 0.001). These results help confirm the chemopreventive activity of NSAIDs against breast cancer and provide the first evidence that a cyclooxygenase 2 blocking agent, celecoxib, possesses strong chemopreventive activity against mammary carcinogenesis.

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.

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.

Comparative chemopreventive activity of ibuprofen and N-(4-hydroxyphenyl) retinamide against the development and growth of rat mammary adenocarcinomas.

The chemopreventive effects of Ibuprofen on the development and growth of 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary tumors were examined. A well known breast cancer chemopreventive retinoid, N-(4-hydroxyphenyl) retinamide (4-HPR) was also included in this study for comparison. At 7 days prior to receiving a single intragastric dose of 15 mg DMBA, rats were fed a control chow diet, as well as diets containing either 1000 mg/kg diet of Ibuprofen or 1.5 mmol/kg diet of 4-HPR. Ibuprofen and 4-HPR markedly increased tumor latency. At 112 days post DMBA intubation, tumor incidence was 86% in control rats as compared to 74% and 62% in rats receiving Ibuprofen, and 4-HPR diets respectively (p < 0.05). Ibuprofen and 4-HPR reduced tumor burden and tumor volume almost to the same extent. The control rats had an average of 2.26 tumors/rat compared to 1.42 and 1.46 tumors/rat in the Ibuprofen or 4-HPR groups respectively (p < 0.05). Similarly, average tumor volume was 3.25 cm3 in the control rats compared to 0.86 cm3 and 0.83 cm3 in rats receiving Ibuprofen and 4-HPR diets respectively (p < 0.05). These results suggest that Ibuprofen may have potential in the chemoprevention and treatment of breast cancer.

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.

Chemotherapeutic evaluation of N-(4-hydroxyphenyl) retinamide-O-glucuronide in the rat mammary tumor model.

The growth inhibitory effects of N-(4- Hydroxyphenyl) retinamide (4-HPR) and its glucuronide derivative, N-(4-Hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) on established DMBA induced rat mammary tumors were compared. The results indicate that the glucuronide analog had a greater antitumor potency than equimolar concentration of the free retinoid. Tumor regression occurred in 75% of the rats fed 2 mmol/Kg diet of 4-HPROG. In a 6-week study, the maximum tolerated dietary dose (MTD) was found to be 3.5 mmol/Kg diet for 4-HPR and 5 mmol/Kg diet in the case of 4-HPROG. The higher potency and lower toxicity of the glucuronide suggests that this conjugate may have an in vivo chemotherapeutic advantage over the parent free retinoid.

Relative efficacy of the opioid antagonist, naltrexone, on the initiation and promotion phases of rat mammary carcinogenesis.

We have shown earlier that naltrexone, a long acting opioid antagonist, inhibits the development and growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors. We extended these studies to determine the independent effects of naltrexone when fed at 75 mg/kg diet during the initiation (I), promotion (P), or initiation plus promotion (I+P) phases of DMBA-induced rat mammary carcinogenesis. The percentage of rats with palpable tumors as well as the number of tumors per rat were determined during the 130 day experiment. When fed during the 1, P, or I+P phases, mammary tumor incidence was significantly inhibited by 27%, 60% and 45% respectively, as compared to the control group. Similarly, tumor multiplicity was significantly reduced by naltrexone treatment. Tumor multiplicity was reduced by 40%, 73%, and 70% when fed naltrexone during I, P, and I+P respectively, as compared to the controls. These results indicate that naltrexone acts on the I and P phases, with the effects being maximal when fed during the P and I+P phases.

Chemopreventive activities of C-glucuronide/glycoside analogs of retinoid-O-glucuronides against breast cancer development and growth.

The O-glucuronide analog of N-(4-hydroxyphenyl)retinamide (4-HPROG) has shown a greater chemopreventive activity than the parent N-(4-hydroxyphenyl)retinamide (4-HPR). However, this compound is relatively unstable. In order to improve stability and efficacy, we have prepared a number of stable C-linked analogs of 4-HPROG (C-phenyl and C-benzyl glucuronosyl, glucosyl, and xylosyl analogs). These analogs are stable toward acid hydrolysis and the glucuronosyl analogs resist the actions of beta-glucuronidase. The analogs were prescreened for their antiproliferative potential in vitro using cultured human MCF-7 breast cancer cells. Selected analogs were then evaluated for their ability to inhibit the development and growth of tumors in the 7,12-dimethylbenzanthracene-induced rat mammary tumor model. Although the stable C-linked analogs bound poorly to the nuclear retinoic acid receptors, many showed more potency than the less stable 4-HPROG in inhibiting tumor incidence and multiplicity in vivo. The glucuronide/glucoside analogs are more potent than the xylosides, and the C-benzyl more effective than the C-phenyl analogs. The higher potency of at least two C-linked analogs (retinamidobenzyl glucuronide and retinamidobenzyl glucose) suggests that these analogs may have a chemopreventive advantage over the parent retinamide and its natural O-glucuronide.

Relative efficacy of glucarate on the initiation and promotion phases of rat mammary carcinogenesis.

The independent effects of the potential cancer chemopreventive agent calcium glucarate (CGT) when fed (128 mmol/kg diet) during the initiation (I), promotion (P) or (I+P) phases of 7,12-dimethylbenzanthracene-induced rat mammary carcinogenesis, was compared to that of the known chemopreventive agent N-(4-hydroxyphenyl) retinamide (4-HPR) fed (2.0 mmol/kg diet) during these same phases. CGT and especially 4-HPR both significantly increased tumor latency when fed during the P-phase. When fed during I, P or I+P phases mammary tumor incidence was reduced compared to the controls 33%, 42% and 67% by 4-HPR and 18%, 42% and 50% by CGT. Similarly, tumor multiplicity was significantly reduced by either agent. For example, as compared to the corresponding control, when fed during the I, P or I+P phases 4-HPR reduced tumor multiplicity 63, 34 and 63%, while CGT reduced tumor multiplicity 28, 42 and 63% respectively. CGT, like 4-HPR, acts on both the I and P phases with the effect being maximal when fed during P and I+P phases.

O(6)-alkylguanine DNA alkyltransferase activity levels in normal, benign and malignant human female breast.

The enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) is implicated in the repair of DNA damage by chemotherapeutic chloroethylnitrosoureas (CNUs) and plays a vital role in determining the sensitivity of cancer cells to this class of compounds. We have measured the AGT activity in 41 human female breast tissues including normal, benign and malignant specimens. The patients with breast cancer in advanced stages have shown a statistically significant 2.37-fold (p = 0.007) higher level of AGT activity compared to normal. An inter-individual variation of 1.8-fold in normal, 10.8-fold in benign and 7.6-fold in malignant tissues was observed. The average number of methyl group acceptor molecules per cell were estimated to be 19,053 +/- 903, 20,227 +/- 1,806 and 45,330 +/- 5,418 (SEM) in normal, benign and malignant tissue, respectively. These results for the first time emphasize the predictive role of AGT in selecting the responder patients by AGT determination in breast tumor biopsies and further encourage therapeutic trials based on biochemical modulation of CNU resistance against malignant cells.

Activity of D-glucarate analogues: synergistic antiproliferative effects with retinoid in cultured human mammary tumor cells appear to specifically require the D-glucarate structure.

D-Glucarate has shown modest chemopreventive and synergistic chemopreventive effects with retinoids in a number of tumor models as well as a similar antiproliferative effect in MCF-7 human tumor cells in culture. It has been postulated that D-glucarate exerts some of its effects by equilibrium conversion to D-glucarolactone, a potent beta-glucuronidase inhibitor. In the present study, D-glucarate and a number of its analogues, including D-glucarolactone, were evaluated as antiproliferatives in the MCF-7 model with and without added retinoid. Results suggest that the effects of glucarate are reasonably specific for its structure and may not require conversion to glucarolactone.

Mechanism of growth inhibition of mammary carcinomas by glucarate and the glucarate: retinoid combination.

In synergistic combination 0.75 mmol/kg diet of N-(4-hydroxyphenyl) retinamide and 32 mmol/kg diet of glucarate inhibits the growth of primary rat mammary tumors, but are equally effective as single agents at 1.5 and 128 mmol/kg diet, respectively. Dose-response studies suggest that like retinoids, glucarate acts directly on tumor cells, rather than having an adjuvant effect. Although synergism is maintained down to at least 0.38 mmol/kg diet of the retinoid, experiments using Vitamin A-deficient diets indicates 128 mmol/kg glucarate acts independent of retinoid. Both alone and in combination, glucarate and retinoid inhibited the growth of human mammary tumor cells grown in the athymic mouse, the growth of rat mammary tumors in germfree rats and the hormone-independent MTW 9a/R rat mammary tumor. Like retinoids, glucarate suppresses protein kinase C and induces transforming growth factor-beta, in the mammary tumor cells.

In vivo use of N-(4-hydroxyphenyl retinamide)-0-glucuronide as a breast cancer chemopreventive agent.

The inhibitory effects of N-(4-hydroxyphenyl) retinamide and its glucuronide derivative on the development and growth of 7,12-dimethylbenz (a) anthracene - induced rat mammary tumors in vivo were compared. The results indicate that the glucuronide had a greater chemopreventive potency than equimolar concentration of the free retinoid by all the criteria measured, mainly the inhibition of tumor incidence, multiplicity and tumor growth. HPLC analysis of the blood of the rats showed no hydrolysis of the glucuronide during its chronic consumption, indicating that the retinoid glucuronide is probably acting in vivo per se rather than through hydrolysis to the free retinoid. The higher potency and lower toxicity of the glucuronide suggests, for the first time, that the conjugate may have an in vivo chemopreventive advantage over the parent retinamide.

Basis for the anti-tumor and chemopreventive activities of glucarate and the glucarate:retinoid combination.

The biochemical basis for the cancer chemopreventive and anti-cancer activities of glucarate, retinoids (13-cis-retinoic acid, hydroxyphenyl retinamide) and their synergistic combination, has been evaluated. Neither alone nor in combination did these agents affect the level in the rat, of enzymes which are (a) known to correlate with reduced risk of carcinogenesis (detoxification enzyme, catalase, glutathione reductase) nor (b) enzymes which correlate with increased risk of carcinogenesis (beta-glucuronidase, xanthine oxidase, glucose-6-phosphate dehydrogenase). Retinoids, but neither glucarate nor its lactone inhibited free radical-induced lipid peroxidation. Both agents alone and synergistically in combination, raise cellular cAMP levels, repress protein kinase C and more generally inhibited DNA synthesis.

Effect of dietary soybean and licorice on the male F344 rat: an integrated study of some parameters relevant to cancer chemoprevention.

The individual and combined effects of dietary toasted soybean meal (3.13-25%) and dietary licorice root extract (0.38-3.0%) on selected liver and intestinal enzyme levels and on clinical chemistry and histopathological parameters were evaluated on male F344 rats. All parameters were measured one and three months after the 50-day-old rats were started on the diets. By use of newly developed high-performance liquid chromatography-based analytic methods, measurable levels of daidzein (2.67 micrograms/ml) and glycyrrhetinic acid (7.87 micrograms/ml) were detected in the sera of rats on the 25% soybean and 3% licorice diets, respectively. Histopathological evaluations of organs and tissues yielded only nonsignificant strain-related changes. At all dosages, there were no significant soybean- or licorice-related anatomic lesions or hematologic changes. In the clinical biochemistry profile, soybean meal caused moderate but significant dose-dependent decreases in serum cholesterol and increases in alkaline phosphatase, blood urea nitrogen, and phosphorus, which remained within the normal range. Liver glutathione transferase, catalase, and protein kinase C showed significant inductions (up to 50%) in response to increasing doses of soybean meal and licorice extract, with evidence for only marginal interaction between the two additives. Their effects on the intestinal mucosa were not significant. Ornithine decarboxylase levels, an indicator of promotional activity, were unchanged or repressed by the additives. The favorable effects of up to 25% toasted soybean meal and 3% licorice root extract on the levels of the four enzymes, without unfavorable changes in clinical parameters, might account in part for the chemopreventive activities of these additives. These effects would be in addition to direct inhibitory effects of known components in these additives on these or other enzymes or modulation of hormone activity that is not evaluated in this study.