Peter S. Carlton

Interrelationships between dietary restriction, the IGF‐I axis, and expression of vascular endothelial growth factor by prostate adenocarcinoma in rats

Human studies suggest that excessive energy intake and obesity may influence prostate cancer progression. Rodent experiments demonstrate that diet restriction attenuates tumor growth in parallel with reduced vascular density. The present study examines changes in the insulin‐like growth factor I (IGF‐I) axis caused by dietary restriction and their association with the expression of vascular endothelial growth factor (VEGF) in prostate cancer. Weanling male Copenhagen rats were randomized into control or 40% dietary restricted groups (n = 5). After 8 wk, rats were implanted with rat AT6.3 prostate adenocarcinoma cells. Two weeks later, the animals were sacrificed and serum, normal prostate, liver, and prostate tumor samples were collected for analyses. Dietary restriction reduced serum concentrations of IGF‐I by 35% (P < 0.05) and increased IGF‐binding protein‐3 (IGFBP3) by sevenfold (P < 0.0001). Lower circulating IGF‐I concentrations were correlated with reduced IGF‐I mRNA expression in the liver, the primary source of circulating IGF‐I. Dietary restriction also lowered mRNA expression of IGF‐I (45%, P = 0.0242) and its receptor IGFIR (40%, P = 0.0083) in prostate tumors. Similarly, reduced VEGF mRNA (30%, P = 0.0176) and secreted VEGF protein (33%, P = 0.0003) were observed in prostate cancer of restricted rats. An in vitro study employing AT6.3 prostate cancer cells demonstrated dose‐ and time‐dependent stimulation of VEGF expression by IGF‐I. These results suggest that dietary restriction reduces endocrine and prostate tumor autocrine/paracrine IGF‐I expression, which contributes to reduced VEGF expression and signaling, to inhibit tumor angiogenesis associated with prostate tumorigenesis.

Incidence and effects of Ha‐ras codon 12 G→A transition mutations in preneoplastic lesions induced by N‐nitrosomethylbenzylamine in the rat esophagus

N‐nitrosomethylbenzylamine (NMBA)–induced rat esophageal tumorigenesis is an important model for squamous cell carcinoma of the human esophagus. In this model, previous studies have shown that the GGA→GAA Ha‐ras codon 12 mutation is present in the majority of papillomas. No other Ha‐ras mutation has been identified. Studies using other models of chemical carcinogenesis suggest that Ha‐ras activation has a critical role during tumor initiation. We have used laser‐capture microdissection and polymerase chain reaction–restriction fragment length polymorphism analysis to study the role of codon 12 Ha‐ras mutation at various stages of tumor development in the rat esophagus. Our results indicate that Ha‐ras mutation was present infrequently (4.3%) in premalignant lesions. The incidence of Ha‐ras mutation was high in papillomas (57.1%), however, and 50% of papillomas expressed mutant Ha‐ras RNA message. Additionally, there was a linear trend correlating increased incidence of Ha‐ras mutation with later papilloma stage. These data suggest the role of ras activation later in neoplastic development. To evaluate the potential mechanism of action by which Ha‐ras contributes to promotion and progression in this model, we compared mRNA expression of cyclin D1 and p27 in Ha‐ras mutant and Ha‐ras normal papillomas. We found no differences in mRNA expression of either cyclin D1 or p27 between these two papilloma populations. Our data suggest an important paradigm shift for the role of ras mutations in this model of chemical carcinogenesis, indicating a functional role of Ha‐ras activation in promotion/progression and not in the initiation phase of NMBA‐induced papillomagenesis.

Failure of dietary lyophilized strawberries to inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-and benzo[a]pyrene-induced lung tumorigenesis in strain A/J mice

In the present study, we examined the ability of lyophilized strawberries in the diet to inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and benzo[a]pyrene (B[a]P)-induced lung tumorigenesis in the A/J mouse. Groups of 20 mice were administered purified AIN-76A diet containing 10% lyophilized strawberries 1 week before carcinogen dosing, and for the duration of the study. NNK was administered by i.p. injection in five, 0.414 mg doses. B[a]P was administered by gavage in five, 0.2 mg doses. At 20 weeks, there were no significant differences in tumor incidence or tumor multiplicity between the NNK+10% lyophilized strawberry group and the NNK control group. Similarly, at 24 weeks, there were no differences in tumorigenesis between the B[a]P+10% lyophilized strawberry group and the B[a]P control group. Therefore, lyophilized strawberries at 10% in the diet failed to inhibit NNK- and B[a]P-induced mouse lung tumorigenesis.

Investigation of the enhancement of N-nitrosomethylbenzylamine-induced esophageal tumorigenesis by 6-phenylhexyl isothiocyanate.

Previous studies in our laboratory have shown that 6-phenylhexyl isothiocyanate (PHITC), enhances N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 rats while the shorter chain analogs, phenylethyl isothiocyanate (PEITC), and 3-phenylpropyl isothiocyanate (PPITC), inhibit NMBA-induced esophageal tumorigenesis. To test the hypothesis that PHITC influences the promotional stage of esophageal tumorigenesis, groups of 22-27 rats were dosed with vehicle or NMBA three times a week for 5 week, and fed a modified AIN-76A diet containing PHITC at concentrations of 0.0, 1.0, and 2.5 micromol/g. At the 25th week, the rats were killed, esophagi harvested and tumors counted. In the groups that received NMBA+PHITC, apparent but statistically insignificant increases in tumor multiplicity of 32 and 42% were found in comparison to rats treated with NMBA alone. A higher frequency of dysplastic lesions was found in rats treated with NMBA+2.5 micromol/g PHITC (71%) when compared to rats treated with NMBA only (12%). To test whether PHITC increased cellular proliferation, we evaluated proliferating cell nuclear antigen (PCNA) expression by immunohistochemistry. While there were no significant increases in PCNA staining in rats treated with NMBA+PHITC compared to rats treated with NMBA only, rats treated with PHITC only had a significantly higher PCNA index compared to untreated controls. Expression of cyclin D1, another biomarker of proliferation, was analyzed by semi-quantitative reverse transcription-polymerase chain reaction. There were no significant increases in cyclin D1 expression in groups treated with NMBA+PHITC compared to the group treated with NMBA only. Thus, while the data suggest a promotional effect by PHITC as manifested by a significant increase in dysplastic leukoplakia by the high dose of PHITC and an increase in the PCNA index by PHITC alone, PHITC does not appear to have a significant effect on esophageal cell proliferation.

FUNCTIONAL ROLE OF CYTOCHROME P-450 2A3 IN N -NITROSOMETHYLBENZYLAMINE METABOLISM IN RAT ESOPHAGUS

Previous in vitro studies demonstrated that the rat esophageal carcinogen N -nitrosomethylbenzylamine (NMBA) is metabolically activated by cytochrome P-450s (CYP) 2A3 and 2E1. However, the in vivo role of these P-450s in the metabolism of NMBA has not been fully evaluated. In this study, the effects of single and multiple doses of NMBA were investigated on CYP2A3 and CYP2E1 mRNA expression in the rat esophagus and lung. Seven- to 8-wk old male Fischer 344 rats were administered a single subcutaneous dose of NMBA at either 0.5 mg/kg or 2 mg/kg body weight, after which the rats were sacrificed at 1, 3, 6, 12, 24, 48, and 72 h. In the multiple-dose experiment, 2 groups of rats were dosed with 0.5 mg/kg body weight NMBA 3 times per week for 1 wk or 3 wk. The animals were sacrificed 24 h following the last treatment. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis demonstrated a reduction of CYP2A3 mRNA expression in lung and esophagus from NMBA-treated animals compared to dimethyl sulfoxide (DMSO)-treated vehicle controls. This reduction in CYP2A3 mRNA was significant at 48 h in the esophagus and at 24 and 48 h in the lung following a single dose of 2 mg/kg body weight NMBA. In contrast, CYP2E1 mRNA expression remained unchanged in rat lung following NMBA treatment and no consistent pattern of expression could be observed in the esophagus. In the multiple-dose study, a 32% and 25% reduction in esophageal CYP2A3 mRNA expression was observed at 1 and 3 wk, respectively. Similar reductions in CYP2A3 mRNA expression were also observed in the lung. Further, esophageal explants derived from animals pretreated with NMBA in vivo demonstrated a reduced ability to metabolize the carcinogen in vitro as compared to explants from vehicle control animals. Taken together, these data provide further support for a potential role of CYP2A3 in NMBA metabolism in the rat esophagus. Data suggest that CYP2A3 levels in the rat esophagus can be a determinant of its ability to metabolize this carcinogen in vivo.