M. Margaret Juliana

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.

Papilloma Development Is Delayed in Osteopontin-Null Mice: Implicating an Antiapoptosis Role for Osteopontin

Osteopontin is a secreted, adhesive glycoprotein, whose expression is markedly elevated in several types of cancer and premalignant lesions, implicating its association with carcinogenesis. To test the hypothesis that induced osteopontin is involved in tumor promotion in vivo, osteopontin-null and wild-type (WT) mice were subjected to a two-stage skin chemical carcinogenesis protocol. Mice were initiated with 7,12-dimethylbenz(a)anthracene (DMBA) applied on to the dorsal skin followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate (TPA) for 27 weeks. Osteopontin-null mice showed a marked decrease both in tumor/papilloma incidence and multiplicity compared with WT mice. Osteopontin is minimally expressed in normal epidermis, but on treatment with TPA its expression is highly induced. To determine the possible mechanism(s) by which osteopontin regulates tumor development, we examined cell proliferation and cell survival. Epidermis from osteopontin-null and WT mice treated with TPA thrice or with DMBA followed by TPA for 11 weeks showed a similar increase in epidermal hyperplasia, suggesting that osteopontin does not mediate TPA-induced cell proliferation. Bromodeoxyuridine staining of papillomas and adjacent epidermis showed no difference in cell proliferation between groups. However, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analyses indicated a greater number of apoptotic cells in DMBA-treated skin and papillomas from osteopontin-null versus WT mice. These studies are the first to show that induction of the matricellular protein osteopontin facilitates DMBA/TPA-induced cutaneous carcinogenesis most likely through prevention of apoptosis.

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.

Mycoplasma pulmonis and lymphoma in bioassays in rats.

Lymphomas were reported to be induced in rats in bioassays of aspartame, methyl-tertiary-butyl ether (MTBE), and other chemicals conducted by a nonprofit cancer research organization. European regulatory authorities concluded that lymphomas in the aspartame study were caused by Mycoplasma pulmonis and suggested that this also was the case for the MTBE bioassay. To assess the role of M. pulmonis in these bioassays, we reviewed the tumor data for the aspartame and MTBE bioassays and, additionally, the organizations bioassay of methanol. For all 3 studies, the most frequently reported hematopoietic neoplasm was lympho-immunoblastic lymphoma, the most frequently affected organ was the lung, and, in almost half of the rats with this diagnosis, the lung was the only affected organ. Lesions diagnosed as lymphoma in published illustrations had pleomorphic cellular morphology and appeared to contain neutrophils. Information from these reports and other sources indicated that lesions typical of M. pulmonis disease were prevalent among the aspartame and MTBE study rats and that the rats were not specific-pathogen-free. Because the lymphoma type, cellular morphology, and organ distribution reported in these studies are atypical of lymphoma in rats, because lymphocyte and plasma cell accumulation in the lung is characteristic of M. pulmonis disease, and because M. pulmonis disease can be exacerbated by experimental manipulations, including chemical treatment, we suggest that a plausible alternative explanation for the reported results of these bioassays is that the studies were confounded by M. pulmonis disease and that lesions of the disease were interpreted as lymphoma.

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.

Preventive effects of Polyphenon E on urinary bladder and mammary cancers in rats and correlations with serum and urine levels of tea polyphenols

Polyphenon E, a standardized mixture of green tea polyphenols, was examined for its chemopreventive efficacy against chemically induced urinary bladder and mammary cancers. In the present study, Polyphenon E was administered after the last dose of 4-hydroxybutyl(butyl)nitrosamine, or roughly 30% of the way into the experiment. Polyphenon E (100 or 250 mg/kg body weight/d) caused a dose-dependent decrease in palpable urinary bladder tumors [low dose, 14 of 34; high dose, 6 of 35; controls, 20 of 34 (P < 0.01)]. In the mammary cancer model, Polyphenon E [333 or 1,000 mg/kg body weight (BW)/d] was administered beginning 5 days after a single dose of methylnitrosourea. In contrast to its significant efficacy in bladder tumor prevention, Polyphenon E had a minimal effect in the prevention of mammary cancers. Levels of polyphenols were determined in the urine and serum of rats. Relatively high levels of various polyphenols (and metabolites) were found in the urine. However, virtually no epigallocatechin-3-gallate was observed in the urine because of low systemic bioavailability; although it represents almost 65% of the polyphenols in Polyphenon E. Levels of polyphenols in serum were 50× to 1,000× less than were observed in urine. The bioavailability of these tea polyphenols to different organ sites may contribute to the differing preventive efficacy of Polyphenon E against urinary bladder and mammary cancers. [Mol Cancer Ther 2007;6(7):2022–8]

Lack of Efficacy of the Statins Atorvastatin and Lovastatin in Rodent Mammary Carcinogenesis

The statins are highly effective in lowering cholesterol by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase. Recently, there has been conflicting epidemiologic data indicating that statins decrease the incidence of certain types of cancer, including breast cancer. Atorvastatin and lovastatin, statins with different lipophicilities, were administered in diet either as single agents or in combination with suboptimal doses of tamoxifen or the retinoid X receptor agonist bexarotene were evaluated for prevention of estrogen receptor–positive mammary cancers induced in the rat with methylnitrosourea. Atorvastatin (125 or 500 mg/kg diet) alone did not significantly alter cancer incidence or multiplicity. Suboptimal doses of tamoxifen (0.4 mg/kg diet) or bexarotene (80 mg/kg diet) reduced cancer multiplicity from 3.8 (control) to 2.9 and 0.9, respectively. Combining atorvastatin (500 mg/kg diet) with either of these effective agents minimally altered their efficacy. Although this dose of atorvastatin did not decrease serum triglyceride levels in control rats, it significantly decreased triglyceride levels that had been increased in bexarotene-treated rats. Experiments done with a second statin, lovastatin (100 and 400 mg/kg diet), yielded similar results: (a) limited activity when administered alone, (b) no obvious synergy with bexarotene, and (c) an ability to decrease bexarotene-induced increases in serum triglycerides. Thus, the statins had minimal activity in this model of mammary cancer in which approximately half of the cancers are mutated in the Ha Ras oncogene. Similarly, atorvastatin failed to alter the development of estrogen receptor–negative mammary carcinomas in a new animal model using bitransgenic mice (MMTV-Neu+/−/p53KO+/−), whereas bexarotene (250 mg/kg diet) was effective.

Screening Agents for Preventive Efficacy in a Bladder Cancer Model: Study Design, End Points, and Gefitinib and Naproxen Efficacy

PURPOSE We optimized agent testing in an in vivo bladder cancer model and determined the most sensitive, relevant protocol to test efficacy in clinical prevention trials. MATERIALS AND METHODS Female Fischer-344 rats (Harlan) were treated with the bladder carcinogen OH-BBN (TCI America, Portland, Oregon) for 8 weeks. Rats were treated with naproxen (400 mg/kg diet), aspirin (Sigma(R)) (300 or 3,000 mg/kg diet), Iressa(R) (10 mg/kg gefitinib body weight daily) or resveratrol (1,000 mg/kg diet) using 1 of 3 protocols, including treatment beginning 1) 1 week after OH-BBN and continuing for 7 months, 2) 3 months after OH-BBN after microscopic lesions already existed and continuing for 3 months, and 3) 1 week after OH-BBN and continuing for 4 months. In protocols 1 and 2 bladder lesion weight and large tumors were primary end points, and in protocol 3 microscopic cancer was the end point. RESULTS Using protocol 1 naproxen, Iressa, resveratrol, and low and high dose aspirin altered the formation of large bladder tumors by 87% (decreased), 90% (decreased), 3% (increased), 6% (decreased) and 60% (decreased), respectively. Using protocol 2 Iressa and naproxen were also highly effective. Protocol 3 evaluation revealed that only Iressa caused a significant decrease in microscopic bladder cancers (63%). CONCLUSIONS Initiating treatment after OH-BBN or when bladder lesions already existed showed naproxen and Iressa to be effective in preventing formation of large cancers. Low dose aspirin and resveratrol were ineffective. In protocol 3, in which microscopic lesions were the end point, only Iressa was effective. Thus, an established cancer end point appears preferable. Naproxen, which has an excellent cardiovascular profile, or epidermal growth factor receptor inhibitors may be effective in an adjuvant setting.

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM) on chemically-induced mammary carcinogenesis: Is DIM a substitute for I3C?

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid), indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a condensation product of I3C) to alter the induction of mammary cancers induced by the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea (MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon receptor (AhR) agonists, while DIM does not. The agents were initially examined for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were administered for 14 days starting 7 days prior to a single dose of the carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%, respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction of DMBA tumors. The effect of these agents on the promotion/progression phase of carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF (1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after the administration of MNU, and continually thereafter. 5,6-BF decreased MNU- induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at the high dose, while DIM at either dose had minimal effects on tumor multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM had minimal effects in either model; arguing that administration of DIM is not analogous to administration of I3C.