Phyllanthus debilis Methanolic Extract Reduces the Viability of Human Colorectal Adenocarcinoma (HT-29) Cells and Increases LINE-1 and Alu DNA Methylation

Abstract

Phyllanthus debilis was shown to have a strong anti-proliferative effect on cancer cells with less effect in normal cells. However, its mechanism on the epigenetic mechanism at repeat sequences is unknown. This study was carried out to determine the effect of P. debilis extract on long interspersed nuclear element-1 (LINE-1) and Alu DNA methylation. The anti-proliferative effect of P. debilis methanolic extract on human colorectal adenocarcinoma (HT-29) at 24 hours was done using trypan blue assay. LINE-1 and Alu methylation measurement on the HT-29 cell line was done after 72 hours of treatment using Pyrosequencing. The effect of P. debilis methanolic extract at 24 hours on the viability of HT-29 cells was dose-dependent with the half-maximal inhibitory concentration (IC50) concentration of 0.1 mg/mL. Treatment with P. debilis methanolic extract showed significantly higher Alu DNA methylation when compared with the untreated HT-29 cells (37.0 ± 2.5% vs 32.3 ± 4.3%, p<0.05). Similarly, treatment with 5-aza-2-deoxycytidine also significantly increased the Alu DNA methylation compared with the untreated HT-29 cells (46.0 ± 2.3% vs 37.0 ± 2.5%, p<0.05). For LINE-1, there was a significant increase of LINE-1 methylation when treated with P. debilis extract (80.3 ± 1.3% vs 76.3 ± 2.1%, p<0.05) and with 5-aza-2-deoxycytidine (81.8 ± 4.3% vs 76.3 ± 2.1%, p<0.05) when compared with untreated cells. In conclusion, treatment of P. debilis methanolic extract on HT-29 cell line reduces the viability of HT-29 cells and increases the methylation of Alu and LINE 1. Similar changes in methylation were also seen in the 5-aza treatment. These epigenetic changes by P. debilis methanolic extract may contribute to its anti-cancer properties.