Development of Dna Methyltransferases Inhibitors from Natural Products for Target Therapy of Lung Cancer

Background and significance: 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 tumor suppressor genes (TSGs) inactivation in tumor development. DNMTs are the enzymes which are responsible for DNA methylation through transfer of methyl group to cytosine residue of CpGs. Previous study reported that overexpression of DNMTs in lung cancers also creates novel therapeutic targets and encourages the search for inhibitors of DNMTs as anticancer treatments. We therefore hypothesized that DNMT may be a good target for lung cancer therapy. Blocking 5’-cytosine-methyltransferase (DNMT) could potentially reverse the process of epigenetic silencing and reactivate tumor suppressor genes (TSGs). 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 induce promoters de-methylation and reactivation of TSGs genes in tumor cells and restore activity of TSGs in critical cellular pathways. Study design: The natural products have been provided with a rich source of anti-cancer drugs. We have collected the natural products and their synthetic derivatives 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 natural compounds for DNMT-catalyzed DNA methylation will be first performed by ELISA-based methods. In vivo DNA methylation assay will be serially examined in the cells. High potential DNMT inhibitor will be further treated in the lung cancer and normal cells to demonstrate whether novel inhibitors enable to induce cancer cytoxicity, inhibit metastasis, activate apoptosis or promote differentiation. Identification of drug induced demethylation and TSGs re-expression also will be further performed in the cells. In vivo antitumor activity and toxicity of novel DNMT inhibitors will be also evaluated in xenograft model. Preliminary results: Using mithramycin A (MMA), 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-SK, an MMA derivative, which could kill cancer cells without harming healthy cells and is a more tumor-specific cytotoxic drug than MMA in cell based test. The animal model tests for MMA-derivatives will also be investigated.