Gene Expression Signature of DMBA-Induced Hamster Buccal Pouch Carcinomas: Modulation by Chlorophyllin and Ellagic Acid

Chlorophyllin (CHL), a water-soluble, semi-synthetic derivative of chlorophyll and ellagic acid (EA), a naturally occurring polyphenolic compound in berries, grapes, and nuts have been reported to exert anticancer effects in various human cancer cell lines and in animal tumour models. The present study was undertaken to examine the mechanism underlying chemoprevention and changes in gene expression pattern induced by dietary supplementation of chlorophyllin and ellagic acid in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model by whole genome profiling using pangenomic microarrays. In hamsters painted with DMBA, the expression of 1,700 genes was found to be altered significantly relative to control. Dietary supplementation of chlorophyllin and ellagic acid modulated the expression profiles of 104 and 37 genes respectively. Microarray analysis also revealed changes in the expression of TGFβ receptors, NF-κB, cyclin D1, and matrix metalloproteinases (MMPs) that may play a crucial role in the transformation of the normal buccal pouch to a malignant phenotype. This gene expression signature was altered on treatment with chlorophyllin and ellagic acid. Our study has also revealed patterns of gene expression signature specific for chlorophyllin and ellagic acid exposure. Thus dietary chlorophyllin and ellagic acid that can reverse gene expression signature associated with carcinogenesis are novel candidates for cancer prevention and therapy

Gross appearance (A) and photomicrographs of histopathological changes (B) in the buccal pouch mucosa of control and experimental animals (×20).

<p>Gross appearance (A) and photomicrographs of histopathological changes (B) in the buccal pouch mucosa of control and experimental animals (×20).</p

Transcript expression levels of TGFβRI, TGFβRII, Smad7, NF-κB p50, and NF-κB p65 in various experimental groups as determined by RT-qPCR.

<p>cDNA individually generated from the buccal pouch RNA of biological replicates was subjected to RT-qPCR analysis. Relative mRNA expression for each gene was determined and normalized to the average transcript expression of GAPDH internal control. The fold change in transcript expression for each gene was determined using the 2^−ΔΔCt method. Data are the mean ± SD of two separate experiments. Statistical significance was determined by Mann-Whitney test (P<0.05). a Significantly different from control. b Significantly different from DMBA.</p

Molecular pathways related to the genes differentially expressed in DMBA painted animals.

<p>Gene enrichment analysis for the up- and down-regulated genes was done using GSEA Pre Ranked Tool. The selection criteria were set at Benjamin-Hochberg P = 0.05. Only statistically significant pathways showing a permuted P-value = 0.05 and a positive (enrichment) z-score >2 were selected.</p

Gene expression profiles in various experimental groups.

<p>(A) Venn diagram analysis of the differentially expressed genes with P value = 0.05 and two fold change cut off in various experimental groups. (B) Heat maps depicting the gene expression profiles of hamsters treated with DMBA (B1), DMBA+chlorophyllin (B2) and DMBA+ellagic acid (B3). The heat map represents unsupervised clustering of differentially-expressed genes (P value = 0.05, fold change cut off of 2) using Imfit and eBayes (Empirical Bayes) method. Heat map values are log₂-transformed, normalized fluorescence ratios of control versus treatment groups. Red represents an expression level above the mean expression of a gene across all samples; black represents mean expression; green indicates expression lower than the mean.</p

Molecular pathways related to the genes commonly modulated by chlorophyllin and ellagic acid supplementation.

<p>Genes commonly modulated by chlorophyllin and ellagic acid in the enriched pathways (z-score >2) are listed. Gene enrichment analysis was done using GSEA Pre Ranked Tool. P value correction was done using Benjamini and Hochberg method.</p

Relative mRNA expression levels of MMP-2, MMP-9, TIMP-2 and Cyclin D1 in various experimental groups as determined by RT-qPCR.

<p>cDNA individually generated from the buccal pouch RNA of biological replicates was subjected to RT-qPCR analysis. Relative mRNA expression for each gene was determined and normalized to the average transcript expression of GAPDH internal control. The fold change in transcript expression for each gene was determined using the 2^−ΔΔCt method. Data are the mean ± SD of two separate experiments. Statistical significance was determined by Mann-Whitney test (P<0.05). a Significantly different from control. b Significantly different from DMBA.</p

Western blot analysis of the differentially expressed genes.

<p>(A) Representative immunoblots showing the protein levels of the differentially expressed genes in the buccal pouch total cell lysate. Protein samples (50 µg/lane) resolved on SDS-PAGE were probed with corresponding antibodies. Quantification was done by normalising the band density to that of β-actin. (B) Densitometric analysis. Each box plot represents the protein expression from control lysates for two determinations. a Significantly different from control by Mann-Whitney test (P<0.05). b Significantly different from DMBA.</p