Effects of a black raspberry diet on gene expression in the rat esophagus.

A diet containing 5% freeze-dried black raspberries (BRB) markedly inhibits esophageal cancer in rats treated with the carcinogen, N-Nitrosomethylbenzylamine (NMBA). We previously identified esophageal genes that become dysregulated after short-term treatment of rats with NMBA and determined which genes are maintained at near-normal levels of expression if the animals were fed 5% BRB prior to and during NMBA treatment. In this study, we report the effects of the BRB diet on gene expression in esophagi from untreated (control) animals. After 3 wk on a 5% BRB diet, control esophagi were excised, stripped of the submucosal and muscularis layers, and processed for histology and microarray profiling. RNA microarrays revealed that the BRB altered the expression levels of 36 genes; 24 were upregulated, and 12 were downregulated. Among the upregulated genes are genes associated with cellular matrix, signaling cascades, transcription regulation, apoptosis, metabolism, and intriguingly, contraction. Most of the downregulated transcripts are involved in cell regulation, signal transduction, and metabolism. Histopathological analyses revealed that the BRB have little or no effect on esophageal morphology. In conclusion, histological and molecular studies indicate that a 5% BRB diet produces only modest effects on the esophagus, the target tissue for NMBA carcinogenesis in the rat.

Global gene expression profiling unveils S100A8/A9 as candidate markers in H-ras-mediated human breast epithelial cell invasion.

The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model genetically engineered to constitutively activate the H-ras or N-ras signaling pathway. We previously showed that H-Ras, but not N-Ras, induces MCF10A cell invasion/migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. Thus, these cell lines provide an experimental system to separate the gene expression profile associated with cell invasion apart from cell proliferation/transformation. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N-Ras MCF10A, and H-Ras MCF10A cells and hierarchical clustering separated 412 genes into four clusters. We then tested whether S100A8 and S100A9, two of the genes which are most highly up-regulated in an H-Ras-specific manner, play a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, small interfering RNA-mediated knockdown of S100A8/A9 expression significantly reduced H-Ras-induced invasion/migration. Conversely, the induction of S100A8/A9 expression conferred the invasive/migratory phenotype to parental MCF10A cells. Furthermore, we provided evidence of signaling cross-talk between S100A8/A9 and the mitogen-activated protein kinase signaling pathways essential for H-Ras-mediated cell invasion and migration. Taken together, this study revealed S100A8/A9 genes as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide useful information which may lead to the identification of additional potential targets for the prognosis and/or therapy of metastatic breast cancer.

Carcinogen-altered genes in rat esophagus positively modulated to normal levels of expression by both black raspberries and phenylethyl isothiocyanate.

Our recent study identified 2,261 dysregulated genes in the esophagi of rats that received a 1-week exposure to the carcinogen N-nitrosomethylbenzylamine (NMBA). We further reported that 1,323 of these genes were positively modulated to near-normal levels of expression in NMBA-treated animals that consumed dietary phenylethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables. Herein, we report our results with companion animals that were fed a diet containing 5% freeze-dried black raspberries (BRB) instead of PEITC. We found that 462 of the 2,261 NMBA-dysregulated genes in rat esophagus were restored to near-normal levels of expression by BRB. Further, we have identified 53 NMBA-dysregulated genes that are positively modulated by both PEITC and BRB. These 53 common genes include genes involved in phase I and II metabolism, oxidative damage, and oncogenes and tumor suppressor genes that regulate apoptosis, cell cycling, and angiogenesis. Because both PEITC and BRB maintain near-normal levels of expression of these 53 genes, their dysregulation during the early phase of NMBA-induced esophageal cancer may be especially important in the genesis of the disease.

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.