Nano-Diamino-Tetrac (Ndat) Enhances Resveratrol-Induced Anti-Proliferation by Action on the Rrm2 Pathway in Colorectal Cancers

Drug resistance or low efficacy is a major concern in cancer therapy. Overexpression of ribonucleotide reductase M2 subunit (RRM2) has been associated with aggressive cancer behavior and chemoresistance. Resveratrol is a stilbenoid phytoalexin which binds to a specific site on cell surface integrin αvβ3 and induces apoptosis in cancer cells via nuclear translocation of COX-2. However, resveratrol activated RRM2 gene expression and protein translation in colon cancer cells; this paradoxical effect inhibits resveratrol-induced COX-2 nuclear translocation and apoptosis. Nano-diamino tetrac (NDAT) is a nano-particulate derivative of tetraiodothyroacetic acid (tetrac), and exerts anti-cancer properties via several mechanisms in different type of cancers. In the current project, we will investigate the effects of NDAT on anti-cancer activity of resveratrol in human colorectal cancers. The hypothesis is that treatment of colorectal cancer cells with resveratrol will induce RRM2 accumulation in these cells and the low effect can be blocked by NDAT treatment. NDAT will restore and potentiate the anti-proliferative actions of resveratrol in colorectal cancers. The SPECIFIC AIMS are:1. To define the mechanisms involved in the induction by resveratrol of RRM2 in colorectal cancer cells;2. To define the mechanisms by which NDAT restores/potentiates the effect of resveratrol in resveratrol-treated colorectal cancer cells;3. To conduct PK/PD modeling of the resveratrol-NDAT interaction in a perfusion bellows cell culture system and in in vivo xenograft model as required components of preclinical evaluation of the drug combination.The Methods used will include: (i) suppression by shRNA of expression of integrin αvβ3, and miR-21 or overexpression of mutant K-ras to define their contribution to resveratrol-induced resistance in colorectal cancer cells; phosphorylation status of several signaling pathway proteins will also be determined (Aims 1 and 2); (ii) qPCR and immunoblotting of integrin αvβ3 and signaling proteins in cells exposed to NDAT, to resveratrol and to the combination, as well as MTT and TUNEL assay (Aims 1 and 2); (iii) detection by CytoF of signal transduction pathways involved in resveratrol-induced resistance or the potentiating effect of NDAT (Aims 1 and 2); (iv) reporter assays to identify expression levels/interactions of expression of β-catenin/HMGA2/RRM2 related genes, such as HIF-1α (Aim 2); (v) Affymetrix arrays to identify miRNA signatures of resveratrol-treated resveratrol-resistant primary colorectal cancer cells (Aim 2); (vi) definition of PD of NDAT, resveratrol and the combination in a perfusion cell culture system (Aim 3) in preparation for (vii) xenograft studies to confirm the effects of NDAT on anti-proliferation (Aim 3).By understanding the mechanisms behind the low efficacy of resveratrol treatment in colorectal cancer cells, we will be enabled to examine clinical tissue samples and analyze anti-proliferative actions of NDAT in combination with resveratrol; this will support the development of new treatment approaches for RRM2-mediated cancer chemo-resistance/low efficacy such as gemcitabine in pancreatic cancer and resveratrol in colorectal cancers.