Dietary glucarate as anti-promoter of 7, 12-dimethylbenz[a]anthracene-induced mammary tumorigenesis

Using as a criterion the inhibition of serum ß-glucuronidaseactivity, dietary calcium D-glucarate is shown to serve as anefficient slow-release source in vivo of D-glucaro-l, 4-lactone,the potent endogenous inhibitor of this enzyme. Using the 7,12-dimethylbenz[a]anthracene model of mammary tumor inductionin rats it is shown for the first time that feeding the ratscalcium D-glucarate-supplemented diet after treatment with thecarcinogen, inhibits tumor development by over 70%. Supportiveevidence is presented for the theory that calcium D-glucarateinhibits or delays the promotion phase of mammary carcinogenesisby lowering endogenous levels of estradiol and precursors of17-ketosteroids. Therefore, dietary glucarate can be used tolower blood and tissue levels of ß-glucuronidase,and in turn of those carcinogens and promoting agents whichare excreted, at least in part, as glucuronide conjugates.     

Serum haptoglobin levels in mice with 7, 12-dimethylbenz[a]anthracene-induced tumors.

Serum haptoglobin (Hp) levels were determined periodically in mice treated with the carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA). The magnitude and duration of the Hp response to tumors induced by DMBA were dependent on the tumor type; lymphocytic lymphomas elicted a minimal response, whereas mice bearing mammary carcinomas and stomach squamous cell carcinomas had high Hp levels which remained elevated throughout most of the period of tumor development. In the majority of mice with mammary carcinomas, the initial rise in serum Hp coincided fairly closely with the first appearance of palpable masses. Some variation in the magnitude of the Hp response was observed between individual mice bearing chemically-induced tumors of similar histological types.     

Sequential Hormone Therapy with Medroxyprogesterone Acetate for 7, 12--Dimethylbenz [{alpha}]Anthracene-induced Rat Mammary Tumors

Two types of sequential hormone therapy with medroxyprogesteroneacetate (MPA) were examined in female 93 Sprague—Dawley(SD) rats with 7,12-dimethylbenz [] anthracene (DMBA) inducedmammary tumors. Estradiol (E₂) priming induced progesteronereceptors (PgR) in most cases and, after induction of PgR, MPAoften showed an augmented antitumor effect. For this reason,E₂ priming + MPA had a more marked antitumor effect on DMBAtumors than MPA alone. Tamoxifen (TAM) (0.1 mg/kg) priming inducedPgR more frequently than TAM (0.4 mg/kg) priming. Furthermore,treatment with TAM priming (0.1 mg/kg) + MPA showed a more markedantitumor effect than with TAM priming (0.4mg/kg) + MPA or MPAalone. In the above two priming therapies with MPA, the latteris more practical for the clinical treatment of breast cancerthan the former because the mechanism of action of E₂ is usuallythought to show tumorigenic activity on breast cancer, whilethe mechanism of action of TAM is thought to show an antitumoreffect. It is suggested that the sequence of administrationof MPA after TAM priming may be favorable for the treatmentof breast cancer.     

Inhibition of N-methyl-N-nitrosourea- and 7,12-dimethylbenz[a] anthracene-induced rat mammary tumorigenesis by dietary cholesterol is independent of Ha-Ras mutations

Dietary cholesterol has previously been shown to inhibit rat mammary tumorigenesis but the mechanisms remain unclear. Uptake of serum low density lipoprotein cholesterol by tissues leads to down-regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis that catalyzes the formation of mevalonate. In addition to being a precursor of cholesterol, mevalonate is necessary for DNA synthesis and cell proliferation. Isoprenoids, also derived from mevalonate, are required for the post-translational modification of Ras proteins that are mutated in a number of carcinogen-induced rat mammary tumors. The purpose of this study, therefore, was to determine whether inhibition of tumorigenesis by cholesterol is dependent on the frequency of mutations in the Ha-ras gene. Female Sprague-Dawley rats (30/group) were given a single dose of either N-methyl-N-nitrosourea (MNU, 50 mg/kg i.p.) or 7, 12-dimethylbenz[a]anthracene (DMBA, 100 mg/kg intragastrally), carcinogens that produce tumors with either a high (MNU) or low (DMBA) frequency of Ha-ras mutations in codon 12 or 61, respectively. Rats were fed either a control AIN-93G diet or the control diet supplemented with 0.3% cholesterol for 14 weeks. Dietary cholesterol significantly decreased the final tumor incidence in rats given DMBA (83 versus 100%, P < 0.05) or MNU (53 versus 77%, P < 0.05). HMG-CoA reductase activity was higher in mammary tumors than in normal mammary glands, but the activity of this enzyme was reduced by cholesterol feeding only in mammary glands and not in tumors. Tumors induced by MNU had a high frequency of Ha-ras mutations in both the control (65%) and cholesterol-fed (68%) groups. Tumors induced by DMBA had a low frequency of Ha-ras mutations that also did not differ between the control (21%) and cholesterol-fed (18%) groups. These findings show that dietary cholesterol inhibits mammary tumorigenesis induced by either MNU or DMBA and that the inhibition is independent of the type or extent of mutations in the Ha-ras gene.     

Inhibition of lung tumorigenesis in A/J mice by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine and myo-inositol,individually and in combination

Isothiocyanates, their N-acetylcysteine conjugates, and myo-inositol (MI) are inhibitors of lung tumorigenesis in A/J mice. However, chemoprevention by combinations of these compounds in different temporal sequences has not been examined. This is important for developing practical approaches to lung cancer chemoprevention in smokers and ex-smokers. We used a tumor model in which A/J mice are treated with 8 weekly doses of benzo[a]pyrene (B[a]P) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and killed 19 weeks after the final treatment. In Experiment 1, isothiocyanates or their N-acetylcysteine conjugates were added to the diet (1 or 3 micro mol/g) from 1 week before until 1 week after carcinogen treatment. The compounds were 2-phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), N-acetyl-S-(N-benzyl-thiocarbamoyl)-L-cysteine (BITC-NAC), N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC), and N-acetyl-S-(N-3-phenylpropylthiocarbamoyl)-L-cysteine (PPITC-NAC). Significant reductions in lung tumor multiplicity were observed in mice treated with PEITC, PEITC-NAC, PPITC and PPITC-NAC. PEITC-NAC was chosen for combination studies with MI (Experiment 2). Mice were treated with B[a]P plus NNK without or with PEITC-NAC (3 micro mol/g diet), MI (55.5 micro mol/g diet), or PEITC-NAC plus MI (3 micro mol plus 55.5 micro mol/g diet). Different temporal sequences of dietary additions were investigated: carcinogen treatment phase; post-carcinogen treatment phase; entire experiment; 50% of carcinogen treatment phase until termination; and 75% of carcinogen treatment phase until termination. All treatments reduced lung tumor multiplicity except PEITC-NAC post-carcinogen or from 75% of the carcinogen treatment phase. Reduction of lung tumor multiplicity by PEITC-NAC plus MI was greater than that in the mice treated with the agents alone in all temporal sequences. When all results were combined, PEITC-NAC plus MI was significantly more effective than the agents alone. There was a significant trend for reduction in lung tumor multiplicity with increased duration of treatment by the chemopreventive agents. These results provide a basis for further development of mixtures of PEITC-NAC and MI for chemoprevention of lung cancer.     

Inhibition of 7,12-dimethylbenz[a]anthracene- and urethan-induced lung tumor formation in A/J mice by long-term treatment with dehydroepiandrosterone

Long-term treatment with the adrenal steroid dehydro-epiandrosteronereduces weight gain without suppressing appetite and inhibitsthe occurrence of 7,12-di-methylbenz[a]anthracene- and urethan-inducedlung tumors in A/J mice.     

Antitumor effects of a nonsteroidal aromatase inhibitor (CGS 16949A) on 7, 12-dimethylbenz[alpha]anthracene-induced mammary tumors in rats.

The effects of a nonsteroidal aromatase inhibitor, CGS 16949A, on female Sprague-Dawley (SD) rats with 7, 12-dimethylbenz[alpha]anthracene (DMBA)-induced mammary cancers were examined in relation to estrogen receptors (ER). Rat tumor sizes in each treated group were significantly smaller (P less than 0.05) and rat body weights in most treated groups were significantly increased (P less than 0.05) compared to those in the control group (no treatment) at all measurement points during treatment. Rat uterine weights in each treated group decreased significantly compared with those in the control group (P less than 0.05). There was no significant difference between ER-positive and ER-negative groups in tumor size, body weight or uterine weight. At increased doses of CGS 16949A in the experiment, further increases in testosterone levels and further decreases in estradiol levels were shown to occur. The results suggest the mechanisms of CGS 16949A action not to be influenced by the presence or absence of ER, but to be due to its potent aromatase inhibition of the conversion of androgens to estrogens.     

Chemopreventive efficacies of aspirin and sulindac against lung tumorigenesis in A/J mice

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely prescribed drugs. In this study, we demonstrated the efficacy of aspirin to inhibit lung tumorigenesis in A/J mice. Lung tumors (9.9 tumors/mouse) were induced by the tobacco-specific nitrosamine, 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), administered in drinking water between week 0 and week +7. Groups of mice were fed sulindac (123 mg/kg diet), acetylsalicylic acid (ASA; 294 mg/kg), non- buffered Aspirin (294 mg/kg) or buffered Aspirin (294 mg/kg) in AIN-76A diet from week -2 to the end of the bioassay (week +23). These doses are comparable to the maximal doses recommended for humans. ASA and non- buffered Aspirin were the most effective inhibitors and reduced lung multiplicities by 60 and 62%, respectively. Sulindac inhibited lung tumor multiplicity by 52%. Inhibition by buffered Aspirin was not statistically significant. We evaluated the efficacies of NSAIDs to inhibit NNK activation by h1A2 v2 cells expressing human P-450 1A2. Salicylates, at doses of 500 microM and 1 mM, had no effect on NNK activation. Sulindac and its sulfide and sulfone metabolites (1 mM) inhibited NNK metabolism by 90, 92 and 65%, respectively. We observed a 76% inhibition with SKF 525A, a P-450 inhibitor. Taken together, these results indicate that salicylates and sulindac could be equally effective as chemopreventive agents, but they could differ in their mode of action.      

Dietary selenium fails to influence cigarette smoke-induced lung tumorigenesis in A/J mice

The goal of the study was to determine if dietary selenium inhibited the induction of lung tumorigenesis by cigarette smoke in A/J mice. Purified diets containing 0.15, 0.5, or 2.0 mg/kg selenium in the form of sodium selenite were fed to female A/J mice. Half of the mice in each dietary group were exposed to cigarette smoke 6 h/day, 5 days/week for five months followed by a four month recovery period in ambient air, while the other half were used as controls. After the recovery period, the mice were euthanized, and their lungs were removed for further analysis. Mice exposed to smoke had a higher tumor incidence and a higher tumor multiplicity, whereas dietary Se did not affect either the tumor incidence or tumor multiplicity. An increase in dietary selenium led to increased levels of selenium in the lung as well as GPx protein levels, but dietary Se did not affect lung SOD protein levels. In conclusion, these data confirm the carcinogenic activity of cigarette smoke in mice but show that dietary Se provided as sodium selenite does not affect smoke-induced carcinogenesis in this model.     

Mouse skin papilloma formation by chronic dermal application of 7, 12-dimethylbenz[a] anthracene is not reduced by diet restriction

Diet restriction has repeatedly been shown to reduce the incidenceof spontaneous and chemically induced tumors in rodents. However,no conclusive data are available to show whether carcinogenesisby chronic exposure to a genotoxic agent can also be retarded.In this study, diet restriction to 70% was investigated fora protective effect on the formation of skin papilloma in maleNMRI mice treated twice weekly with 20 nmol 7,12-dimethylbenz(a)anthracene(DMBA). Rather surprisingly, no protection was seen. Both timeof onset of papilloma formation (13 weeks in both groups) andtime of 50% cumulative incidence (t₅₀; 17.5 and 18 weeks) weresimilar in the unrestricted and the restricted group. In contrast,a clearly protective elTect was found in mice initiated with100 nmol DMBA and promoted twice weekly with 2.5 nmol 12-O-tetradecanoylphorbol-13-acetate:the onset of papilloma formation increased from 7 to 11.5 weeks,the t₅₀ was shifted from 8.5 to 19 weeks. Diet restriction,therefore, was not protective under conditions of chronic exposureto a genotoxic carcinogen. It cannot be considered a universalmeasure of cancer prevention.     

(-)-Epigallocatechin gallate can prevent cisplatin-induced lung tumorigenesis in A/J mice

Risks of secondary lung cancer in patients with non-small cell lung cancer and small cell lung cancer are estimated to be 1-2% and 2-10% per patient per year, respectively. Cisplatin is widely used in the treatment of lung cancer and is also known as a carcinogen in experimental animals. In this study, the effect of (-)-epigallocatechin gallate (EGCG) on cisplatin-induced lung tumors in A/J mice was investigated. Female A/J mice (4 weeks old) were divided into four groups: group 1, control without treatment; group 2, EGCG treatment (1 mg/ml in tap water); group 3, weekly cisplatin treatment (1.62 mg/kg body wt, i.p.) for 10 weeks; group 4, cisplatin plus EGCG treatment (EGCG was started 2 weeks before cisplatin treatment). Four groups of mice were killed at week 30 after treatment. Tumor incidence was 26.3% (5/19) in group 1, 30% (6/20) in group 2, 100% (19/19) in group 3 and 94.4% (17/18) in group 4. Tumor multiplicity (the number of tumors per mouse, mean +/- SD) was 0.4 +/- 0.8 in group 1, 0.4 +/- 0.8 in group 2, 5.1 +/- 2.1 in group 3 and 2.8 +/- 2.3 in group 4. Tumor multiplicity was significantly reduced by adding EGCG to cisplatin-treated mice (P < 0.01). Furthermore, EGCG significantly reduced cisplatin-induced weight loss from 24.7-26.3% (cisplatin treatment) to 10.8-11.6% (cisplatin plus EGCG treatment) (P < 0.01). These findings suggest that EGCG can inhibit cisplatin-induced weight loss and lung tumorigenesis in A/J mice.     

Protection against 7, 12-dimethylbenz[a]anthracene-induced rat mammary carcinoma by infection with mouse xenotropic type C virus.

A single ip inoculation of female, outbred Sprague-Dawley rats with a viable mouse xenotropic type C virus significantly reduced the incidence and/or retarded the development of mammary carcinoma induced by 7, 12-dimethylbenz[a]anthracene administered orally 7 days after virus. Although infectious virus could not be isolated from organs of infected rats, high titers of circulating and tumor-associated antibodies were detected against the viral internal core protein p30, and a low-grade antibody response to intact virus or envelope glycoprotein was found. Moreover, a cell-mediated immune response, measured by lymphocyte transformation, was detected with the use of intact virus but not with p30 antigen. No immunity developed after a single inoculation of UV-inactivated virus. These data indicated that inoculation of adult individuals of heterologous species with viable xenotropic mouse type C virus resulted in the rapid disappearance of infectious virus from the recipient, followed by the development of both humoral and cellular immunity to virion constituents. These events led, by unknown mechanisms, to the effective retardation of chemical carcinogenesis when infection preceded carcinogen administration.     

Predominant role of prolactin in stimulating the growth of 7, 12-dimethylbenz(a)anthracene-induced rat mammary tumor.

The effect of prolactin in supporting the growth of 7, 12-dimethylbenz(a)anthracene-induced mammary tumor in adult female Sprague-Dawley rats was investigated when estrogen receptors were blocked by the nonsteroidal antiestrogen, Tamoxifen, ICI 46,474. Following an oophorectomy-induced remission, perphenazine, which stimulates endogenous prolactin release, was able to restore tumor growth whether or not Tamoxifen was added. A second course of perphenazine treatment, instituted after the tumors were allowed to shrink, was again effective in stimulating tumor growth. After a regression in tumor size induced by oophorectomy and daily administration of Tamoxifen, perphenazine was able to restore original tumor size despite continued treatment with Tamoxifen. In intact rats, after regression was obtained by daily administration of Tamoxifen and the prolactin inhibitor, lergotrile mesylate, perphenazine induced tumor growth when the latter was discontinued, even though Tamoxifen was continued for 50 days. Estrogen receptors measured at the time of maximum stimulation by perphenazine were undetectable. On the other hand, estradiol did not stimulate tumor growth when serum prolactin was depressed to undetectable levels by lergotrile. These results indicate that prolactin supports the growth of 7, 12-dimethylbenz(a)anthracene-induced rat mammary tumor and that estrogen receptors are not required under these conditions.     

Efficacy of deguelin and silibinin on benzo(a)pyrene-induced lung tumorigenesis in A/J mice

We evaluated deguelin and silibinin in A/J mice treated with the tobacco-specific carcinogen benzo(a)pyrene (BP) for their ability to inhibit pulmonary adenoma formation and growth. Animals were treated with either deguelin (5.0 or 10.0 mg/kg body weight, by gavage) or silibinin at doses of 0.05% and 0.1% in the diet, approximately 10 days before a single intraperitoneal dose of BP. We found that oral administration of deguelin reduced tumor multiplicity by 56% and tumor load by 78%, whereas silibinin treatment at doses of 0.05% and 0.1% in the diet did not show any significant efficacy on either tumor multiplicity or tumor load. The result indicates that deguelin significantly inhibits pulmonary adenoma formation and growth in A/J mice. Finding new and effective agents that can prevent lung cancer is urgently needed because cancer of the lungs remains the principal cause of cancer deaths in the United States and because effective chemoprevention of this cancer type remains elusive. Thus, deguelin appears to be a promising new preventive agent for lung cancer and may be considered for further studies in other animal models and in clinical trials.     

Dose dependent inhibitory effects of dietary 8-methoxypsoralen on NNK-induced lung tumorigenesis in female A/J mice

We have reported that pretreatment by stomach tube with 8-methoxypsoralen (methoxsalen; 8-MOP), a potent human CYP2A6 inhibitor, strongly suppresses lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice (Cancer Res. 2003). Here, we examined inhibitory effects with administration in the diet. When the mice were 7 weeks of age, they received dietary supplementation with 8-MOP at concentrations of 1, 10 or 100 ppm for 3 days prior to a single dose of NNK (2mg/0.1 ml saline/mouse, i.p.) or an equal volume of saline (vehicle control). The experiment was terminated 16 weeks after the first 8-MOP treatment and lung proliferative lesions were analyzed. The incidences and multiplicities in the 8-MOP 100 ppm-treated group were significantly reduced as compared with values for the NNK alone group (P<0.001). Multiplicities of NNK-induced lung proliferative lesions were also reduced in a dose dependent manner (Spearman rank correlation coefficient; rho=-0.806, correction P<0.0001). Mouse CYP2A4 and CYP2A5 differ from each other only 11 amino acids, and are closely related to the human CYP2A6. One hour after the last of three daily doses of 8-MOP (0.5, 5 or 50mg/kg body weight in 0.2 ml corn oil, given by stomach tube) or an equal volume of corn oil (vehicle control), given to the mice at 7 weeks of age, isolation of lung and liver RNAs demonstrated no effects on CYP2A4 and CYP2A5 mRNA levels with 8-MOP. In conclusion, the results of this study showed that clear dose response inhibitory effects of 8-MOP on NNK-induced lung tumorigenesis in female A/J mice fed diets containing 8-MOP, due to inhibition of enzyme activity of CYP2A4 and CYP2A5, rather than their gene expression.     

Tobacco smoke-induced lung tumorigenesis in mutant A/J mice with alterations in K-ras, p53, or Ink4a/Arf

A/J mice with genetic alterations in K-ras, p53, or Ink4a/Arf were employed to investigate whether mice carrying these germline mutations would be susceptible to tobacco smoke-induced lung tumorigenesis. Transgenic mice of both genders and their wild-type littermates were exposed to environmental cigarette smoke for 6 months, followed by recovery in air for 5 months. A significant increase of lung tumor multiplicity was observed in K-ras, p53, or Ink4a/Arf mutant mice when compared with wild-type mice. Furthermore, an additive effect was observed between the mice with a mutant p53 transgene and an Ink4A/Arf deletion during tobacco smoke-induced lung tumorigenesis. Sequence analysis of the K-ras gene indicated that the mutations had occurred at either codon 12/13 or 61 in both spontaneously occurring (air control) and tobacco smoke-induced lung tumors. K-ras mutations were found in 62% of the tumors from air-control animals and 83% in those exposed to tobacco smoke. The mutation spectrum found in tumors from mice exposed to tobacco smoke is somewhat similar to that in tumors from air-control mice. In addition, we identified three novel mutations at codon 12: GGT (Gly) --> TTT (Phe), ATT (Ile), and CTT (Leu). These findings provide evidence that K-ras, p53, and Ink4a/Arf mutations play a role in tobacco smoke-related lung carcinogenesis. The similarity of the mutation spectra in the K-ras oncogene observed in tobacco smoke-induced tumors, as compared to spontaneous tumors, suggests that tobacco smoke enhances lung tumorigenesis primarily through promoting spontaneously occurring K-ras mutations.