Purpose: Estrogen receptor (ER) and the Piwi-Like Protein 1 (PIWIL1) oncogene alter the expression and methylation of oncogenes and tumor suppressor genes. Potential cross-talk between ER and the methylated-DNA binding protein, Kaiso, and its downstream genes c-Myc and CDKN2A remains unexplored. Dual targeting of ER and PIWIL1 might be an excellent therapeutic approach. We aimed to explore natural drug that exerted anti-carcinogenicity in MCF-7 and MDA-MBA-231 with high efficacy and its mode of action.
Methods: Initially, we used in silico molecular docking to identify potential blockers for ER and PIWIL1 among seven natural drugs previously reported anticancer compounds. The best candidate was tested in vitro for its influence on KAISO expression by RT-PCR, promoter methylation of c-Myc and CDKN2A by methylation-specific PCR, cell cycle and apoptosis by flowcytometry, and cytotoxicity by MTT assay in the ER(+) MCF-7, and ER(−) MDA-231-MB breast cancer cell lines.
Results: Betulinic acid (BA) docked ER-α and PIWIL1 perfectly at critical amino acids with the lowest binding energy. Moreover, ER and PIWIL1 docked Kaiso with root mean square deviation = 5.05 Å and 4.75 Å, respectively, at the critical amino acid Y537. Experimentally, BA caused a dose-dependent cytotoxicity in both MCF-7 and MDA-MB-231 cells (IC50= 21.3 ± 0.05 and 100 ± 0.34 µM), respectively. BA caused a significant dose-dependent G0/G1 cell cycle arrest, apoptosis induction, and upregulated Kaiso expression coincided with increased methylation of c-Myc and demethylation of CDKN2A promoter regions.
Conclusion: We are proposing a novel anticancer mechanism of action for BA via potential inhibition of Estrogen-ER-α and PIWIL1 signaling, modulation of KAISO expression and selective alterations in methylation state of c-Myc and CDKN2A key cancer-associated genes.