Uncovering Molecular Events Associated with Chemosuppressant Effects of Flaxseed: A Microarray Analysis in Laying Hen Model of Ovarian Cancer.

Abstract

Despite considerable efforts to improve early detection and advances in therapy, ovarian cancer remains a major challenge. Significant lack of early-stage malignancy symptoms and evidence of disease after metastasis; contribute to very low survival rate in patients. Resistance to available chemotherapeutic drug regimes and tumor relapse added much to the lethality of the disease. These limitations encouraged us to search for other strategies for management of the disease. The activity of Omega-3 fatty acid reported against cancer, therefore it may be a safe agent to reduce the progression of ovarian cancer. A study was conducted to evaluate the efficacy of flaxseed (richest plant source of Omega-3 fatty acid) on laying hen an excellent animal model for ovarian cancer. Three hundred eighty-seven single-comb 2.5-year-old hens were randomly divided into Control and Flax groups. Control groups hens were subdivided into Control Normal and Control Cancer, whereas Flax group in Flax Normal and Flax Cancer. Control hens were fed with standard diet, while Flax was with diet supplemented with 10% flaxseed. Results show a significant reduction in the severity of the disease and increased survival of the laying hens fed with flaxseed. Moving further, we tried to elucidate the molecular mechanism underlying the tumor suppressive property of flaxseed. We used Agilent custom 4x44K chicken long oligo microarray, designed by Dr. Zhou of Texas A&M University. Routine RNA extraction and precipitation were performed from ovary tissue of 4 groups of hens. Samples were sent to the WM Keck center for Comparative & Functional Genomics, University of Illinois Urbana-Champaign for cDNA synthesis, labeling, hybridization, scanning and preliminary analysis. Microarray profiling demonstrated around 43,803 genes which have higher intensity of fluorescence after normalization against background. We applied 1.5 fold change cutoffs i.e. all genes expressing intensity of high or lower than 1.5 fold changes were only selected, this effort generated a reasonable numbers of genes for further analysis. We performed 6 way pair analysis of the gene list between the groups i.e. 1.Control Cancer to Control Norm; 2.Control Cancer to Flax Normal; 3. Flax Normal to Flax Cancer;4. Flax Cancer to Control Normal;5. Control Normal to Flax Normal and 6.Flax Cancer to Control Cancer which suggested significant alteration in expression pattern of genes between the sample groups. We applied various bioinformatics tools such as Multiple Experimental Viewer, Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Software Tools for analysis. Microarray analysis identified genes were associated with key molecular and cellular functions such as- cell survival, cell Cycle, molecular mechanisms of cancer, cell-to-cell signaling and cellular growth & proliferation. The top signaling pathways appeared on analyses were p53 signaling, FAK signaling, Fructose metabolism and EGF signaling. Representative important molecules were evaluated by RT-PCR and their expression profile matched with the microarray result. Altogether these data suggested that activated cell survival, cell cycle and p53 signaling may contribute to the reduction in the severity of disease and increased cell survival in hens. This study is an increment towards understanding the molecular mechanism underlying the effect of flaxseed rich diet in laying hen model of ovarian cancer. Accordingly, natural products such as flaxseed may be promising agent for dietary intervention in ovarian cancer-therapy. [Supported by NIH/NCCAM AT004085 and NCI CA133915] (poster)