Ronald Nines

Effects of lyophilized black raspberries on azoxymethane-induced colon cancer and 8-hydroxy-2'-deoxyguanosine levels in the Fischer 344 rat

This study examined the effects of lyophilized black raspberries (BRB) on azoxymethane (AOM)-induced aberrant crypt foci (ACF), colon tumors, and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in male Fischer 344 rats. AOM was injected (15 mg/kg body wt ip) once per week for 2 wk. At 24 h after the final injection, AOM-treated rats began consuming diets containing 0%, 2.5%, 5%, or 10% (wt/wt) BRB. Vehicle controls received 5% BRB or diet only. Rats were sacrificed after 9 and 33 wk of BRB feeding for ACF enumeration and tumor analysis. ACF multiplicity decreased 36%, 24%, and 21% (P < 0.01 for all groups) in the 2.5%, 5%, and 10% BRB groups, respectively, relative to the AOM-only group. Total tumor multiplicity declined 42%, 45%, and 71% (P < 0.05 for all groups). Although not significant, a decrease in tumor burden (28%, 42%, and 75%) was observed in all BRB groups. Adenocarcinoma multiplicity decreased 28%, 35%, and 80% (P < 0.01) in the same treatment groups. Urinary 8-OHdG levels were reduced by 73%, 81%, and 83% (P < 0.01 for all groups). These results indicate that BRB inhibit several measures of AOM-induced colon carcinogenesis and modulate an important marker of oxidative stress in the Fischer 344 rat.

Chemopreventive Properties of Black Raspberries in N-Nitrosomethylbenzylamine-Induced Rat Esophageal Tumorigenesis: Down-regulation of Cyclooxygenase-2, Inducible Nitric Oxide Synthase, and c-Jun

Our laboratory has used a rodent model of human esophageal squamous cell carcinoma to identify putative chemopreventive agents for this disease and to determine their mechanisms of action. In the present study, we treated F344 rats with the esophageal carcinogen, N-nitrosomethylbenzylamine (NMBA), thrice per week for 5 weeks. Beginning 1 week later, they were fed a synthetic diet containing 5% black raspberries (BRB) for the duration of the bioassay (25 weeks). Rats were sacrificed at weeks 9, 15, and 25. Esophageal tissues were collected, and tumor data were recorded. The expression and enzymatic activities of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of c-Jun in the esophagi, were evaluated to investigate the mechanism(s) by which black raspberries modulate tumorigenesis. At week 25, BRB inhibited tumor multiplicity, the standard end point in this tumor model, from 3.78 +/- 0.41 tumors per rat in NMBA-treated animals to 2.23 +/- 0.21 tumors per rat in animals treated with NMBA plus BRB (P < 0.005). BRB reduced mRNA and protein expression levels of COX-2, iNOS, and c-Jun as well as the level of prostaglandin E(2) in preneoplastic lesions of the esophagus at week 25. The berries inhibited mRNA expression of iNOS and c-Jun, but not COX-2, in papillomatous lesions of the esophagus. Prostaglandin E(2) and total nitrite levels were also decreased by BRB in papillomas. These results suggest a novel tumor suppressive role of BRB through inhibition of COX-2, iNOS, and c-Jun.

Prospective grading of neoplastic change in rat esophagus epithelium using angle-resolved low-coherence interferometry

Angle-resolved low-coherence interferometry (a/LCI) is used to obtain quantitative, depth-resolved nuclear morphology measurements. We compare the average diameter and texture of cell nuclei in rat esophagus epithelial tissue to grading criteria established in a previous a/LCI study to prospectively grade neoplastic progression. We exploit the depth resolution of a/LCI to exclusively examine the basal layer of the epithelium, approximately 50 to 100 microm beneath the tissue surface, without the need for exogenous contrast agents, tissue sectioning, or fixation. The results of two studies are presented that compare the performance of two a/LCI modalities. Overall, the combined studies show 91% sensitivity and 97% specificity for detecting dysplasia, using histopathology as the standard. In addition, the studies enable the effects of dietary chemopreventive agents, difluoromethylornithine (DFMO) and curcumin, to be assessed by observing modulation in the incidence of neoplastic change. We demonstrate that a/LCI is highly effective for monitoring neoplastic change and can be applied to assessing the efficacy of chemopreventive agents in the rat esophagus.

Chemopreventive Effects of a Selective Nitric Oxide Synthase Inhibitor on Carcinogen-Induced Rat Esophageal Tumorigenesis

The inducible nitric oxide synthase (iNOS) generates a high concentration of nitric oxide (NO) in tissues. Increased NO production is associated with many disorders including esophageal cancer. Previous studies in our laboratory demonstrated an association between increased iNOS expression and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. On the basis of these observations, we initiated a bioassay to evaluate the ability of S,S′-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), a selective iNOS inhibitor, to prevent the progression of esophageal tumors in rats preinitiated with NMBA. Rats were given s.c. injections of NMBA (0.25 mg/kg body weight) three times per week for 5 weeks. One week later, they were fed a synthetic diet containing either 50 or 100 ppm PBIT until the end of the bioassay (25 weeks). PBIT reduced the incidence of esophageal cancer from 96% in NMBA-treated rats to 83% and 77% (P < 0.05) in rats treated with 50 and 100 ppm PBIT, respectively. Tumor multiplicity was reduced from 3.64 ± 0.42 tumors per esophagus in NMBA-treated rats to 1.79 ± 0.25 (P < 0.001) and 1.50 ± 0.24 (P < 0.0001) in rats treated with 50 and 100 ppm PBIT, respectively. PBIT reduced the production of NO in NMBA-induced preneoplastic and papillomatous esophageal lesions when compared with comparable lesions in rats treated with NMBA only. iNOS mRNA expression was not modulated by PBIT. These observations suggest that iNOS plays a role in tumor development and that its selective inhibitor, PBIT, significantly inhibits esophageal tumor progression presumably through reducing the production of NO.

Prevention of mouse lung tumors by targretin

Targretin has indicated chemotherapeutic activity against nonsmall‐cell lung cancer and chemoprevention in rat mammary gland. Therefore, targretin was evaluated for the prevention of 4‐(methylnitrosoamino)‐1‐(3‐pyridyl)‐1‐butanol (NNK) and vinyl carbamate‐induced lung tumors in female strain A mice. Three experiments were performed: (i) a dose‐response study with vinyl carbamate‐induced tumors; (ii) a limited treatment study also with vinyl carbamate and (iii) prevention of NNK‐induced tumors. In the dose‐response study, 0, 10, 30, 100 and 300 mg/kg targretin were administered after vinyl carbamate. Dose levels of 30 mg/kg and greater significantly decreased tumor multiplicity by >19%. However, the efficacy of 30 and 300 mg/kg was not significantly different demonstrating a shallow dose‐response relationship. In the limited treatment study, 200 mg/kg targretin was administered to the mice from 4–13, 4–19, 4–25 and 23–25 weeks after vinyl carbamate. Administering targretin from weeks 4–19 and 4–25 decreased the multiplicity of tumors from 35.3 ± 1.43 to 29.1 ± 1.51 and 25.0 ± 0.93, respectively, and along with administering it from weeks 23–25 decreased tumor size. In the third study, when targretin (100 and 300 mg/kg) was administered for 3 weeks after NNK followed by a 20 weeks holding period, tumor multiplicity was reduced from 10.6 ± 1.13 to 6.38 ± 0.75 and 4.60 ± 0.70, respectively. Hence, targretin demonstrated both preventive and therapeutic activity with respect to mouse lung tumors supporting its further development as a preventive and therapeutic agent for lung cancer.

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.

In situ assessment of intraepithelial neoplasia in hamster trachea epithelium using angle-resolved low-coherence interferometry

Optical spectroscopy was used to evaluate the transformation of nuclear morphology associated with intraepithelial neoplasia in an animal model of carcinogenesis. In this pilot study, we have assessed the capability of angle-resolved low-coherence interferometry (a/LCI) to monitor in situ the neoplastic progression of hamster trachea epithelial tissue. By using the depth resolution made possible by coherence gating, the a/LCI system has been adapted to the unique geometry of the hamster trachea to allow us to extract useful nuclear morphometric information from cells in the epithelial layer without the need for exogenous staining or tissue fixation. Analysis of a/LCI nuclear morphology measurements has identified two important biomarkers of neoplastic transformation in hamster trachea epithelium, the size and the refractive index of epithelial cell nuclei. By comparing the a/LCI measurements of these two biomarkers to pathologic classification, we distinguished nuclear morphology changes for normal tissue, low-grade dysplasia, and high-grade dysplasia. Given its previous usefulness for tracking neoplastic change through nuclear morphometry measurements, the a/LCI technique may prove to be a useful tool in evaluating chemopreventive agents in future studies of hamster trachea epithelium. (Cancer Epidemiol Biomarkers Prev 2007;16(2):223–7)

The effects of a binary mixture of benzo(a)pyrene and 7H-dibenzo(c,g)carbazole on lung tumors and K-ras oncogene mutations in strain A/J mice.

Polycyclic aromatic hydrocarbons (PAH) and N-heterocyclic aromatic hydrocarbons (NHA) are environmental pollutants formed during the incomplete combustion of organic materials. Benzo(a)pyrene (BaP) and 7H-dibenzo(c,g)carbazole (DBC) are well-characterized representatives of the PAH and NHA classes of compunds, respectively. Both are demonstrated carcinogens that frequently co-occur in environmental mixtures. This preliminary study was conducted to investigate the effects of a binary mixture of BaP and DBC on lung carcinogenicity in the strain A/J mouse as manifested by tumor development and mutations in the K-ras gene. Male A/J mice were administered the following single intraperitoneal dose (mg/kg) combinations of BaP and DBC dissolved in a 0.2-mL volume of tricaprylin--10 DBC:10 BaP; 2 DBC:10 BaP; 2 DBC:100 BaP; and 10 DBC: 100 BaP, and each of the compounds alone at the same doses. Mice were sacrificed 8 months after carcinogen treatment and lung tumor multiplicity and K-ras mutations determined (high-dose combination). The combination of DBC and BaP produced fewer tumors than the sum of all tumors produced by each compound acting alone. The frequency of tumors with K-ras mutations was also less in a sample of the 10 DBC:100 BaP treatment group than in the same-dose, single compound-treated animals. The dominant mutations produced by BaP and DBC were expressed in tumors from animals treated with the mixture.Polycyclic aromatic hydrocarbons (PAH) and N -heterocyclic aromatic hydrocarbons (NHA) are environmental pollutants formed during the incomplete combustion of organic materials. Benzo(a)pyrene (BaP) and 7H-dibenzo(c,g)carbazole (DBC) are well-characterized representatives of the PAH and NHA classes of compounds, respectively. Both are demonstrated carcinogens that frequently co-occur in environmental mixtures. This preliminary study was conducted to investigate the effects of a binary mixture of BaP and DBC on lung carcinogenicity in the strain A/Jmouse as manifested by tumor development and mutations in the K-ras gene. Male A/Jmice were administered the following single intraperitoneal dose (mg/kg) combinations of BaP and DBC dissolved in a 0.2-mL volume of tricaprylin - 10 DBC:10 BaP; 2 DBC:10 BaP; 2 DBC:100 BaP; and 10 DBC: 100 BaP, and each of the compounds alone at the same doses. Mice were sacrificed 8 months after carcinogen treatment and lung tumor multiplicity and K-ras mutations determined (high-dose combination). The combination of DBC and BaP produced fewer tumors than the sum of all tumors produced by each compound acting alone. The frequency of tumors with K-ras mutations was also less in a sample of the 10 DBC:100 BaP treatment group than in the same-dose, single compound-treated animals. The dominant mutations produced by BaP and DBC were expressed in tumors from animals treated with the mixture.

Effects of Phenylethyl Isothiocyanate on Early Molecular Events in N-Nitrosomethylbenzylamine–Induced Cytotoxicity in Rat Esophagus

There is little information on early molecular events in the development of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis and of the effects of chemopreventive agents on these events. In this study, we identified genes in rat esophagus that were differentially expressed in response to short-term NMBA treatment and modulated by cotreatment with phenylethyl isothiocyanate (PEITC). Rats were fed AIN-76A diet or AIN-76A diet containing PEITC for 3 weeks. During the 3rd week of dietary treatment, they were administered three s.c. doses of NMBA (0.5 mg/kg body weight). Rats were sacrificed 24 h after the last treatment; esophagi were excised and processed for histologic grading, microarray and real-time PCR analysis. Histopathologic analysis showed that treatment of rats with PEITC had a protective effect on NMBA-induced preneoplastic lesions in the rat esophagus. We identified 2,261 genes that were differentially expressed in the NMBA-treated versus control esophagi and 1,936 genes in the PEITC + NMBA versus NMBA-treated esophagi. The intersection of these two sets resulted in the identification of 1,323 genes in NMBA-treated esophagus, the vast majority of which were modulated by PEITC to near-normal levels of expression. Measured changes in the expression levels of eight selected genes were validated using real-time PCR. Results from 12 microarrays indicated that PEITC treatment had a genome-wide modulating effect on NMBA-induced gene expression. Samples obtained from animals treated with PEITC alone or cotreated with PEITC + NMBA were more similar to controls than to samples treated with NMBA alone.

Abstract 3686: Microarray analysis of dynamic regulation of genes in early and late stages of chemically induced esophageal carcinogenesis in rats.

Esophageal carcinogenesis is a multistage process characterized by morphological changes from normal esophagus to basal cell hyperplasia, dysplasia, carcinoma in situ and squamous cell carcinoma (SCC). Our laboratory has used the model of N-nitrosomethylbenzylamine (NMBA)-induced esophageal SCC in rats to investigate carcinogenesis and identify putative chemopreventive agents for this disease. The current study is part of large scale investigations on carcinogenesis of esophageal SCC. F344 rats were treated with NMBA (0.30 mg/kg b.w.) or a solution of 20% dimethyl sulfoxide (DMSO) in water (the solvent for NMBA) three times per week for five weeks. The esophageal tissues were collected at 6 and 29 weeks. The RNA pools from 3 individual RNA samples each were prepared for microarray analysis using Affymetrix Rat Genome 230 2.0 Array. Triplicate microarrays were completed for total 27 rats/9 microarrays. A P-value cutoff of 0.001 with a minimum 2.0-fold change was used to define the differently expressed genes. Ingenuity Pathway Analysis (IPA) software was used to identify cancer-related pathways and cellular functions. At 6 weeks (tumor incidence = 20%), 173 genes were modulated by NMBA when compared to control animals. Among which, 20 genes were associated with cancer, cellular development, cellular assembly and organization network; 34 genes were associated with inflammatory disease. The top five cellular functions altered by NMBA were cellular development, carbohydrate metabolism, cell death and survival, cellular growth and proliferation, and cellular function and maintenance. At 29 weeks (tumor incidence = 100%), we identified 1628 genes which were significantly up- or down-regulated by NMBA treatment when compared to normal animals and among which, 466 genes were involved in tumor development. In addition, we identified four cancer-related pathways (nuclear factor erythroid 2-related factor 2 mediated oxidative stress response, protein kinase A signaling, mitochondrial dysfunction, and AMP-activated kinase signaling) and five cellular functions (cellular development, cell morphology, cell death and survival, carbohydrate metabolism and molecular transport). Among genes altered during NMBA-induced esophageal carcinogenesis, 16 genes differently expressed only at 6 weeks including Gbp2, Psmb, Psmb9, Lgals3bp, Id1, Igfbp3, Agpat4, rCG_35099, Tes, RGD1563091, Cyp26b1, Itga1, Scarb1, LOC682861, Serpinb2 and Slfn3. The fold changes of Plcd4, Hbb, Bmper, Hba-a2, Defb4, LOC100134871 and LOC364653 increased from 6 weeks to 29 weeks. In conclusion, we identified differently expressed genes in early and late stages of NMBA-induced esophageal SCC by microarray analysis. Further investigations on biomarkers and cellular/molecular events during multistage of carcinogenesis in NMBA-rat preclinical model of esophageal SCC are underway. Citation Format: Ni Shi, Ronald G. Nines, Lianbo Yu, Michael J. Nicholl, Tong Chen. Microarray analysis of dynamic regulation of genes in early and late stages of chemically induced esophageal carcinogenesis in rats. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3686. doi:10.1158/1538-7445.AM2013-3686