(T0930-04-27) DNA Hydroxymethylation and TET Enzymes Changes Induced by the Curcumin Analog Dimethoxycurcumin

Purpose: Dimethoxycurcumin (DMC) is a more soluble and stable analog of curcumin that is known to induce epigenetic and gene expression changes in tumor cells. Catalysis of DNA methylation (5mC) to hydroxymethylation (5hmC) is controlled by a family of enzymes known as Ten-Eleven Translocation (TET). 5hmC plays a role in regulating gene expression that is distinct from the closely related 5mC. In this study, we investigated the effect of the parent compound curcumin (Cur) and its analog DMC on global DNA hydroxymethylation and on the activity and expression of the TET isoforms (TET1, TET2 and TET3) enzymes.

Methods: Leukemia cell lines HL60 and U937 were treated with different concentrations of Cur and DMC for 48 hours. DNA extracts were used for global quantitation of the epigenetic mark 5hmC using a specific antibody against 5hmC and ELISA-based fluorometric assay. Nuclear extracts and RNA extractions were used to measure the activity of TET isoforms and their gene expression, respectively.

Results: Both Cur or DMC increased global 5hmC in U937 cells. On the other hand, HL60 cells demonstrated similar increase in global 5 hmC with Cur only but not DMC (Figure 1). The enzymatic activity of the TET enzymes was increased by both drugs in both HL60 and U937 cells (Figure 2). The three isoforms of TET showed an increase in gene expression with both Cur and DMC treatment, however, TET3 showed minimal fold increase compared to TET1 and TET2 (Figure 3).

Conclusion: We identified TET enzymes as a novel epigenetic target for Cur and DMC. The impact of increasing TET activity and expression on tumor growth and gene expression of tumor suppressor genes is not clear and will be investigated. Genome-wide analysis 5hmC level is also warranted to identify the gene loci with increase in 5hmC level.

Acknowledgements: Research reported in this abstract was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence (WV-INBRE). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Figure 1. Effect of Cur and DMC on global DNA hydroxymethylation.

Figure 2. Effect of Cur and DMC on the enzymatic activity of TET enzymes.

Figure 3. TET isoforms gene expression changes induced by Cur and DMC.