Development of Novel Dna Methyltransferases Inhibitors for Breast Cancer Treatment

Background and significance: Breast cancer is the most common non-cutaneous cancer diagnosed in women in the United States and is second only to lung cancer as the leading cause of cancer-related mortality. There is an urgent need to develop new anti-cancer drugs which have high tumor selectivity, high killing effect and low toxicity to normal cells. Cancer is caused by the accumulation of both genetic and epigenetic changes. Methylation of CpG islands, which are about 1-2 kb in length in or near the promoter and first exon regions of genes, is considered to be one of the major epigenetic aberrations that causes gene inactivation in cancer. Previous report has revealed that on an average there exist about 600 aberrantly methylated CpG islands in each tumor. Promoter hypermethylation may block the binding of transcription factors, or modify chromatin structure, which in turn blocks access by transcription factors. The potential reversibility of epigenetic states offers exciting opportunities for novel cancer drugs that can reactivate epigenetically silenced tumor suppressor genes (TSGs). Blocking 5’-cytosine-methyltransferase (DNMT), which is enzyme that methylates the cytosine residue of CpGs, could potentially inhibit or reverse the process of epigenetic silencing. Overexpression of DNMTs has been reported for various malignancies, including hepatomas and prostate, breast and lung tumors. Pharmacologic inhibitors of DNA methylation thus provide an attractive and rational approach to reversal of epigenetic silencing of TSGs, with the hope that they will reactivate TSGs genes in tumor cells and restore activity in critical cellular pathways. Study design: Natural compounds were collected from Department of Pharmaceutical Sciences of University of Kentucky and Graduate Institute of Pharmacognosy of Taipei Medical University. To develop potential DNMT inhibitors, high throughput screening of different drugs and natural compounds will be first performed by ELISA-based methods. In vivo DNA methylation assay will be serially examined in the cells. Highly potenticial DNMT inhibitor will be further treated in the breast cancer and normal cells to test whether novel inhibitors enable to induce cancer cytoxicity, inhibit metastasis, apoptosis induction, differenciation promotion. Identification of drug induced demethylation and TSGs re-expression also will be futher performed in the cells. Preliminary results: Using mithramycin A (MMA), and it analogs, which are known to be a GC and CG-rich DNA binding agent, we have found that MMA reduced CpG island methylation of anti-metastasis TSGs, including slit homolog 2 (SLIT2) and tissue inhibitor of metalloproteinase 3 (TIMP3) genes, and associated with the prevention of metastasis. MMA re-expressed mRNA levels for SLIT2 and TIMP3 genes and also inhibited the invasion phenotypes of CL1-5 cells as indicated by its inhibition of cancer cell migration. The mechanism may be mediated by interaction of MMA with DNMT1, leading to the depletion of DNMT1 protein. I am further testing MMA analog, MMA-SK. MMA-SK kills cancer cells without harming healthy cells and is a more tumor-specific cytotoxic drug than MMA in cell based test. I am now testing MMA-SK in the breast cancer (MCF7, MDA-MB-231, MDA-MB-361) and a non-cancerous human mammary epithelial cell line (H184B5F5/M10) to discovery tumor-specific DNMT inhibitors.