NUTRIGENOMICS EFFECTS OF PHYTOCHEMICAL INDICAXANTHIN IN in vitro CELL SYSTEMS

Nutrition plays a key role in many aspects of health, indeed epidemiological studies provide evidence that diet can play essential roles in reducing the risk of chronic diseases. Accumulating evidence indicates that dietary compounds (phytochemicals) from fruit and vegetables can modulate multiple cancer-inflammation pathways which are relevant for chemoprevention and are commonly deregulated by epigenetic mechanisms in cancer cells, including drug detoxification, cell cycle regulation, apoptosis induction. Epigenetic refers to heritable alterations in gene expression that do not involve modification of the underlying genetic DNA sequence. Although epigenetic changes are heritable in somatic cells, these modifications are also potentially reversible, which makes them attractive and promising avenues for tailoring cancer preventive and therapeutic strategies. The aim of this study was to investigate the antiproliferative potential of a Cactus pear pigment extract (Indicaxanthin) to assess the phenotypic effects on colorectal cancer cell lines and to investigate the biological function and/or epigenetic mechanisms for Indicaxanthin’s activity. The antiproliferative effects of Indicaxanthin (Ind), were investigated on a number of human cancer cell lines including hepatocarcinoma cells (HepG2, Ha22T, HUH 7), breast cancer cells (MCF7), cervix epithelial carcinoma (HeLa) and colorectal carcinoma cells (Caco2, LOVO1, DLD1, HT29, HCT116). Ind caused a clear dose-dependent decrease in the proliferation of Caco2, with minor effect on the other cell lines. Flow cytometric analysis showed a pro-apoptotic effect of the pigment at 48h in Caco2 cells. Incubation of proliferating Caco2 cells with Ind (10μl to 100μl ) remarkably reduced the global DNA 5-methyl cytosine methylation and caused a stable demethylation of the tumor suppressor p16INK4a gene promoter, with reactivation of the silenced mRNA expression and accumulation of p16INK4a protein. A decrease of the hyperphosphorylated retinoblastoma protein in favour of its hypophospohrylated status was observed, with unaltered level of the cycline-dependent kinase CDK4. Analysis of cell distribution in the cell cycle phases after Ind treatment showed arrest of Caco2 cells in the S- G2/M- phase. The effect of Ind on LINE-1 methylation levels in the other colorectal cancer cell lines was therefore explored as well as the effect of Ind on promoter methylation status of other genes implicated in colon carcinogenesis. Ind is able to alter the methylation status on global and specific gene level. To rationalize the mechanism of DNA methylation changes induced by Ind, the effect of the phytochemical on the activity and the level of DNA methyltransferase (DNMT) was evaluated. Ind induced a dose-dependent inhibition of DNMT activity on Caco2 cells, while did not affect DNMT1 and DNMT3b level. However a significant increment of DNMT3a was evident in Caco2 cell line. Aberrant expression of DNMTs and their isoforms has been found in many types of cancer and their contribution to aberrant DNA methylation has been proposed. Following this hypothesis, the effect of Ind on the expression of DNMT splice variant, was investigated. The expression of DNMT increased appreciably only in some cell lines with respect to only some DNMT genes after Ind treatment. DNA demethylation may take place as an active mechanism by the activity of specific enzymes. In order to assess whether Ind is able to induce an alteration in DNA demethylase expression, which may explain the effects of Ind on DNA methylation in the tested cell lines, the effect of Ind on the DNA demethylase expression was investigated. Ind induced an increased expression of some enzymes with demethylating activity (TET2, MBD4) relatively only in some cell lines. In this work it appears that the phytochemical Indicaxanthin induces different effects (both epigenetic and biochemical) on colorectal cancer cell lines tested (reduction of proliferation, induction of apoptosis, induction of cell cycle arrest, change in DNA methylation, alteration of gene expression). The effects brought by Ind, are often variable depending on the cell line used, such phenomenon can be attributed to the cell lines themselves. Furthermore from in silico molecular imaging tests, in this study it was supposed that epigenetic effects of Ind are not mediated by a direct alteration of DNMT expression, rather by an influence of Ind on their activities. Then, Ind binding the DNMT and altering their methyltransferase function could cause an imbalance of global DNA methylation.