Clinton J. Grubbs

Potential Role of Dietary Isoflavones in the Prevention of Cancer

The death rate from cancer in the United States is unacceptably high. However, examination of time-related changes in death rates from individual cancers reveals that deaths from some cancers (e.g., stomach cancer) have fallen dramatically over the past 50 years, whereas others (principally lung cancer) have risen sharply.1 Since lung cancer is strongly associated with cigarette smoking, new public health policies with regard to smoking should lead to a reduction in the rate of this cancer.

Chemoprevention by Grape Seed Extract and Genistein in Carcinogen-induced Mammary Cancer in Rats Is Diet Dependent

Many popular dietary supplements are enriched in polyphenols such as the soy isoflavones, tea catechins, and resveratrol (from grape skins), each of which has been shown to have chemopreventive activity in cellular models of cancer. The proanthocyanidins, which are oligomers of the catechins, are enriched in grape seeds and form the basis of the dietary supplement grape seed extract (GSE). Evidence suggests that the proanthocyanidins may be metabolized to the monomeric catechins. This study was carried out to determine whether GSE added to rodent diets protected against carcinogen-induced mammary tumorigenesis in rats and whether this was affected by the composition of the whole diet. Female rats were begun on 5%, 1.25%, or 0% (control) GSE-supplemented diets at age 35 d. At age 50 d they were administered 7,12-dimethylbenz[a]anthracene (DMBA) in sesame oil at 80 mg/kg body weight. They were weighed and monitored weekly for tumor development until 120 d after DMBA administration. Administration of GSE in AIN-76A diet did not show any protective activity of GSE against DMBA-induced breast cancer. However, administration of GSE in a laboratory dry food diet (Teklad 4% rodent diet) resulted in a 50% reduction in tumor multiplicity. In similar experiments, genistein administered in AIN-76A diet also failed to show chemopreventive activity against the carcinogen N-methyl-N-nitrosourea; however, when administered at the same dose in the Teklad 4% rodent diet, genistein exhibited significant chemopreventive activity (44-61%). These results demonstrate that GSE is chemopreventive in an animal model of breast cancer; moreover, the diet dependency of the chemopreventive activity for both GSE and genistein suggests that whether or not a compound is chemopreventive may depend on the diet in which the agent is administered.

Chemopreventive efficacy of anethole trithione, N-acetyl-L-cysteine, miconazole and phenethylisothiocyanate in the DMBA-induced rat mammary cancer model

The chemopreventive efficacy of N‐acetyl‐L‐cysteine (NAC), anethole trithione, miconazole and phenethylisothiocyanate (PEITC), each of which would be expected to alter carcinogen metabolism, was examined in the dimethylbenzanthracene (DMBA) mammary carcinogenesis model. In this protocol, animals were exposed to non‐toxic doses of the chemopreventives in the diet beginning 7 days prior to DMBA administration and then continuously throughout the duration of the assay (100 days post carcinogen). Miconazole, an antifungal agent with relatively broad inhibitory activity toward a variety of cytochromes P450, increased mammary tumor latency, decreased tumor incidence at the highest dose and decreased tumor multiplicity up to 60%. Anethole trithione, a substituted dithiolthione and an analog of the relatively broad‐spectrum chemopreventive oltipraz, was administered in the diet and significantly inhibited mammary cancer multiplicity but not cancer incidence. NAC, an antimucolytic agent, failed to inhibit DMBA‐induced mammary tumorigenesis. Surprisingly, treatment with DMBA plus PEITC, a potent inhibitor of cytochrome P450 2E1, actually increased the multiplicity of tumors relative to that observed with DMBA alone. Int. J. Cancer 72:95–101, 1997.

Rosiglitazone, a PPAR gamma agonist: potent promoter of hydroxybutyl(butyl)nitrosamine-induced urinary bladder cancers.

In an initial study to determine if rosiglitazone had chemopreventive activity, Fischer‐344 female rats were administered twice weekly doses of hydroxybutyl(butyl)nitrosamine (OH‐BBN), a urinary bladder specific carcinogen, for 8 weeks. Two weeks following the last dose of OH‐BBN, rats were administered rosiglitazone (50 mg/kg BW) daily by gavage for the remainder of the study (7 months). Only 57% of OH‐BBN‐treated animals developed palpable urinary bladder cancers during the course of the study, while all of the OH‐BBN plus rosiglitazone treated rats developed large cancers (p < 0.01). Surprisingly, examination for PPAR gamma by immunohistochemistry in the urinary bladders of rats showed that while untreated bladder urothelium and preneoplastic lesions clearly expressed PPAR gamma, frank carcinomas exhibited significantly lower levels. This was confirmed by employing microarray studies of the same samples. In additional studies, lower doses of rosiglitazone (10, 2 and 0.4 mg/kg BW/day) were administered. The 10 mg/kg BW/day dose greatly enhanced bladder cancer incidence (p < 0.01). The dose of 2 mg/kg BW/day, which is roughly equivalent to a standard human dose, also significantly increased bladder cancer incidence (controls, 48%; rosiglitazone‐treated, 84%). The lowest dose did not significantly increase tumor incidence (rosiglitazone at 0.4 mg/kg BW/day, 64%) or tumor weight in the rats, although there was a trend in that direction. Rosiglitazone alone (10 mg/kg BW/day) given in the absence of OH‐BBN did not result in bladder cancer formation when given for 10 months. In summary, rosiglitazone over a wide dose range enhanced urinary bladder carcinogenesis in the OH‐BBN model in rats. Published 2008 Wiley‐Liss, Inc.

Promotion of mammary cancer development by tamoxifen in a mouse model of Brca1-mutation-related breast cancer

Loss of full-length Brca1 in mammary epithelial cells of the mouse mammary tumor virus (MMTV)-Cre Brca1 conditional exon 11 deletion mouse model results in the development of mammary adenocarcinomas with similar genetic changes to those found in human BRCA1-mutation-related breast cancers. We used this experimental model to evaluate the chemopreventive effect of tamoxifen on the development of mammary preneoplasia and adenocarcinoma. No protective effects of tamoxifen administration on mammary cancer development were found. Instead, tamoxifen treatment significantly increased rates of mammary epithelial cell proliferation and the prevalence of mammary hyperplasia at 6 months of age. Tamoxifen-exposed mice developed adenocarcinomas at younger ages than control mice and a higher percentage of mice developed adenocarcinomas by 12 months of age. Both whole mouse and tissue culture cell models were used to test if loss of full-length Brca1 was associated with a relative increase in the agonist activity of tamoxifen. Tamoxifen induced increased ductal growth in MMTV-Cre Brca1 conditional mice compared to wild type. Estrogen receptor alpha (ERα) expression was downregulated in the tamoxifen-induced hyperplasias. Reducing BRCA1 levels in MCF-7 cells using siRNA resulted in a relative increase in the agonist activity of tamoxifen. Results suggest a model of mammary cancer progression in which loss of full-length Brca1 altered the agonist/antagonist activity of tamoxifen, resulting in tamoxifen-induced mammary epithelial cell proliferation with subsequent loss of ERα expression and development of ERα-negative hyperplasias and adenocarcinomas.

Cancer chemopreventive activity of a mixture of Chinese herbs (antitumor B) in mouse lung tumor models

Antitumor B (ATB), also known as Zeng Sheng Ping, is a Chinese herbal mixture composed of six plants. Previously, clinical studies have shown a significant chemopreventive efficacy of ATB against human esophageal and lung cancers. In the present study, A/J mice harboring a dominant-negative p53 and/or heterozygous deletion of Ink4a/Arf and treated with benzo[a]pyrene were used to investigate the chemopreventive effects of ATB on chemically induced lung tumorigenesis. Mice with various genotypes treated with ATB displayed a significant reduction in lung tumor multiplicity and tumor load. Treatment with ATB resulted in an approximately 40% decrease in tumor multiplicity and a 70% decrease in tumor load in both wild-type mice and in mice with a loss of the Ink4a/Arf tumor suppressor genes. Interestingly, ATB decreased tumor multiplicity and volume by 50 and 90%, respectively, in mice with a dominant-negative p53 and in mice with both a p53 mutation and deletion of Ink4a/Arf. Kras2 mutation analysis of the lung tumors revealed that tumors harbored mutations in the 12th codon of Kras2. There were no differences in either the incidence or types of mutations between tumors treated with or without ATB. Oligonucleotide array analysis revealed 284 genes that were differentially expressed in mouse lung tumors as compared to the normal lung, and it was found that 114 out of these 284 genes changed their expression toward the normal levels in tumors treated with ATB. Most of the genes modulated by ATB belong to several cellular signaling pathways, including Notch (Notch homolog 2, manic fringe homolog), growth factor (FGF intracellular-binding protein, PDGFα), G protein-Ras-MAPK (MAPK3, MAP3K4, rab3A, Rap1, RSG5, PKCθ), ubiquitin-proteasome (CDC34, Cullin1, 26S proteasome), and apoptosis (BAD promoter, caspase 3). These results suggest that ATB is an effective chemopreventive against mouse lung tumorigenesis. Furthermore, ATB exhibited an enhanced inhibitory effect in animals harboring genetic alterations (Kras2, p53, and Ink4a/Arf), which are often seen in human lung adenocarcinomas.

Chemopreventive Efficacy of Naproxen and Nitric Oxide–naproxen in Rodent Models of Colon, Urinary Bladder, and Mammary Cancers

Nonsteroidal anti-inflammatory drugs (NSAID) have been highly effective in preventing colon, urinary bladder, and skin cancer preclinically, and also in clinical trials of colon adenoma formation. However, certain NSAIDs cause gastrointestinal ulceration and may increase cardiovascular events. Naproxen seems to cause the lowest cardiovascular events of the common NSAIDs other than aspirin. Nitric oxide (NO)-naproxen was tested based on the finding that adding a NO group to NSAIDs may help alleviate GI toxicity. In the azoxymethane-induced rat colon aberrant crypt foci (ACF) model, naproxen administered at 200 and 400 ppm in the diet reduced mean ACFs in the colon by about 45% to 60%, respectively. NO-naproxen was likewise administered in the diet at roughly equimolar doses (300 and 600 ppm) and reduced total ACF by 20% to 40%, respectively. In the hydroxybutyl (butyl) nitrosamine rat urinary bladder cancer model, NO-naproxen was given at 183 or 550 ppm in the diet, and naproxen at 128 ppm. The NO-naproxen groups had 77% and 73% decreases, respectively, in the development of large urinary bladder tumors, whereas the 128 ppm naproxen group also showed a strong decrease (69%). If treatments were started 3 months after hydroxybutyl (butyl) nitrosamine, NO-naproxen (550 ppm) and naproxen (400 ppm) were also highly effective (86-94% decreases). In the methylnitrosourea-induced mammary cancer model in rats, NO-naproxen and naproxen showed nonsignificant inhibitions (12% and 24%) at 550 and 400 ppm, respectively. These data show that both naproxen and NO-naproxen are effective agents against urinary bladder and colon, but not mammary, carcinogenesis.

Short-term Modulation of Cell Proliferation and Apoptosis and Preventive/Therapeutic Efficacy of Various Agents in a Mammary Cancer Model

Purpose: The methylnitrosourea (MNU)-induced mammary cancer model in rats is similar to estrogen receptor–positive breast cancer in women. In prevention studies using this model, tumor incidence and multiplicity were typically primary end points. The ability of various agents administered for a short period to modulate cell proliferation [proliferation index (PI)] and apoptosis [apoptotic index (AI)] in mammary cancers was compared with their efficacy in long-term prevention and therapy studies. Experimental Design: Rats were injected with MNU to induce mammary cancers. For the prevention studies, agents were administered by gavage or in the diet beginning 5 days after MNU. For proliferation (PI) and apoptosis (AI) experiments, animals with a palpable mammary cancer were treated with the agents for only 4 to 7 days. PI was determined following 5-bromodeoxyuridine labeling whereas AI was determined using the terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay. Therapeutic efficacy was evaluated by measuring cancer size over a 6-week period. Results: Treatments with differing chemopreventive efficacy and mechanism(s) of action were examined: (a) hormonal treatments [tamoxifen, vorozole (an aromatase inhibitor), and ovariectomy]; (b) retinoid X receptor agonists (targretin, 9-cis retinoic acid, and UAB30); (c) inducers of drug-metabolizing enzymes (indole-3-carbinol, 5,6 benzoflavone, and diindoylmethane); (d) agents that alter signal transduction (R115777, a farnesyltransferase inhibitor); Iressa (an epidermal growth factor receptor inhibitor); sulindac and celecoxib (cyclooxygenase 1/2 and cyclooxygenase 2 inhibitors); and (e) diverse agents including meclizine, vitamin C, and sodium phenylbutyrate. Correlations between inhibition of PI, increase of AI, and chemopreventive efficacy were observed. Although most agents with moderate or low preventive efficacy suppressed PI, they minimally affected AI. Conclusions: The data confirmed that the short-term effects of various agents on cell proliferation and apoptosis in small mammary cancers can predict their preventive/therapeutic efficacy. Thus, these biomarkers can be used to help determine the efficacy of compounds in phase II clinical prevention trials.

Paradigms for primary prevention of ovarian carcinoma.

Objective: To provide the clinician with current concepts regarding prevention of ovarian cancer. Specifically, in this review, we provide a rationale for chemoprevention of ovarian cancer, a description of promising chemopreventive agents, and an overview of surgical strategies used in the prevention of ovarian cancer.

Effect of Canthaxanthin on Chemically Induced Mammary Carcinogenesis

Canthaxanthin, a carotenoid with no vitamin A activity, was evaluated for its efficacy in the prevention of chemically induced mammary cancers. Canthaxanthin was administered in the diet at two dose levels (3,390 or 1,130 mg/kg diet). In the dimethylbenzanthracene-induced mammary cancer model, diet supplementation with canthaxanthin for 3 weeks prior to the carcinogen resulted in a 65% reduction in the number of mammary cancers. The feeding of canthaxanthin after the administration of methylnitrosourea had no significant effect on mammary carcinogenesis. These data demonstrate that canthaxanthin, at least in these models of mammary cancer, is active in preventing cancer initiation and not promotion. Analysis of tissues by high-pressure liquid chromatography revealed that canthaxanthin levels in the liver are very high when compared to those in the mammary gland. The observation that canthaxanthin is highly effective in preventing cancer initiation without toxicity suggests that carotenoids not possessing vitamin A activity should be further evaluated as chemopreventive agents.