Curcumin-mediated demethylation of the proximal promoter CpG island enhances the KLF4 recruitment that leads to increased expression of p21Cip1 in vitro

Curcumin, the active component of the spice turmeric, induce global DNA hypomethylation as it has been shown to inhibit DNA methyltransferases. It promotes cell death in cancer cells by arresting in the G1 phase. It was explained to cause increased expression of cell cycle regulator, p21 (WAF1/Cip1); however, the mechanism remains not clear. The p21 promoter harvests a CpG island (CGI) in the proximal region enriched with CG dinucleotide clusters with Kruppel-like factor 4 (KLF4) transcription factor binding site. We probed the p21 promoter CGI (spanning from -135 to +12, respective to the transcription start site) to detect alterations in cytosine methylation level in response to curcumin exposure in four different human cancer cell lines: A431, A549, MCF7, and HeLa. We observed curcumin (20 µM) treatment significantly increased the expression of p21, and the promoter CGI was demethylated in a dose-dependent manner. The curcumin significantly raised the level KLF4 and enhanced the p21 promoter occupancy by KLF4. From our results we hypothesize that curcumin-mediated demethylation of the p21 proximal promoter and increased KLF4 expression as well as its binding to its proximal promoter could serve as a mechanism that could be hypothesized to cause upregulation of p21 in presence of curcumin and thus its therapeutic implications could further be investigated.