The Effect of Phyllanthus debilis Methanolic Extract on DNA Methylation of TAC1 Gene in Colorectal Cancer Cell Line

Aim/Background: In colorectal cancer, TAC1 was shown to be highly methylated in early carcinogenesis. Using HT29 cell line as a model of colorectal cancer, we postulated that Phyllanthus debilis methanolic extract could regulate DNA methylation in TAC1 gene promoter region and thus could alter the progression of colorectal cancer. Methodology: Cell culture study was done using HT29 cells, which were treated with 0.117 mg/ml P. debilis methanolic extract or 0.5 mu M 5-Aza-2-Deoxycytidine (5-Aza) for 72 h. Cells were harvested at 72 h and were extracted for DNA. The DNA was bisulfite modified before been PCR and later was pyrosequenced. Results: The treatment with P. debilis significantly decreased the DNA methylation of TAC1 gene at site 1 (93.1% +/- 2.4% vs. 100% +/- 0.1%, P < 0.05), site 2 (86.5% +/- 1.3% vs. 91.7% +/- 0.2%, P < 0.05), and site 3 (96.7% +/- 0.9% vs. 100% +/- 0%, P < 0.05); but, no significant changes of DNA methylation were seen at site 4 (96.5% +/- 1.9% vs. 93.6% +/- 0.6%, P > 0.05). The average of all Cytosine nucleotide followed by Guanine nucleotide (CpG) sites methylation was reduced, but not statistically significant when compared to the untreated cells (mean methylation 93.2% +/- 2.4% vs. 96.3% +/- 2.2%, P > 0.05). For cells treated with 5-Aza, DNA methylation was significantly decreased only at site 2 (88.6% +/- 0.8% vs. 91.7% +/- 0.2%, P < 0.05), but there was no significant methylation changes at site 1 (94.8% +/- 2.4% vs. 100.0% +/- 0.06%, P > 0.05), site 3 (98.9% +/- 1.1% vs. 100% +/- 0%, P > 0.05), site 4 (90.6% +/- 1.6% vs. 93.6% +/- 0.6%, P > 0.05), and the average of all CpG sites (mean methylation 93.2% +/- 2.3% vs. 96.3% +/- 2.2%, P > 0.05). Conclusion: Treatment of methanolic extract of P. debilis reduces the methylation of promoter region of TAC1 gene, with better effect than low dose 5-Aza at 72 h of treatment. The anticancer effect of P. debilis may be partly been regulated through DNA methylation.