The Effect Of Phyllanthus Debilis Extract On Dna Methylation And Mirna Regulation In Ht-29 Colorectal Cancer Cell Line
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Date
2021-03
Authors
Zain, Siti Nur Dalila Mohd
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Phyllanthus is one of the herbal plants that have been found to have anticancer property. Unfortunately, there is lack of study on the effect of the plant natural products on the epigenetics of cancer. Hence, this study was carried out to determine the effect of Phyllanthus debilis extract on DNA methylation and miRNA regulation in HT-29 colorectal cancer cell line. The study was started by determining the most suitable Phyllanthus species to be used in the epigenetic regulation of HT-29 cell line. Three species of Phyllanthus named Phyllanthus urinaria, Phyllanthus niruri and Phyllanthus debilis were used. The phytochemicals of each Phyllanthus species were then extracted using 100% methanol and 100% water. The extracts were then tested for Total Phenolic Content (TPC) and Total Flavonoid content (TFC) and the antioxidant activities of the plant extracts using DPPH and ABTS assays. Then, the extracts were screened with HPLC to detect the presence of targeted compounds. The therapeutic index of each Phyllanthus extract was then measured on cancer cell lines (breast and colorectal cell lines) versus normal cell line (normal breast cell line). P. debilis methanolic extract showed highest therapeutic index when compared to other extracts. P. debilis methanolic extract was then used for further study to investigate its mechanisms in regulating DNA methylation, gene and miRNA expressions in HT-29 cell line that led to the cancer cell death. The TPC of the extracts ranged from 107.09 to 308.71 mg GAE/g DW while their TFC ranged from 7.07 to 35.86 mg QE/g DW. P. urinaria methanolic extract had the highest TPC and TFC and it was the most potent scavenger of DPPH and ABTS radicals. HPLC analysis revealed that P. urinaria methanolic extracts had caffeic acid, p-coumaric acid and myricetin while the water extract of this species only had myricetin. P. niruri methanolic extract had p-coumaric acid and naringenin, and water extract of this species had caffeic acid, p-coumaric acid, myricetin and naringenin. Similarly, the compounds found in P. niruri water extract were also found in P. debilis methanolic extract. In P. debilis water extract, caffeic acid and p-coumaric acid were found. The therapeutic index decreased in the following order P. debilis methanol extract (4.6581) > P. debilis water extract (2.8308)>P. niruri water extract (2.3572)> P. niruri methanol extract (0.9819)> P. urinaria water extract (0.8971)>P. urinaria methanol extract (0.8356). In Alu and LINE-1 global methylation, P. debilis methanolic extract caused a significant increase of DNA methylation at Alu repeat sequence (P < 0.05) and significant increase in LINE-1 methylation status (p< 0.01). In TAC1 gene specific methylation, P. debilis methanolic extract caused a significant reduction of DNA methylation at site 1, site 2, and site 3 (p < 0.05). The decrease of TAC1 methylation status did not have any significant correlation with the expression of TAC1 gene (p>0.05). However, P. debilis methanolic extract did significantly increase the expression of miR-125a-5p by 2.255-folds and miR-320a by 1.629-folds. In conclusion, Phyllanthus sp. possesses high antioxidants which include polyphenolic compounds. These compounds exert cytotoxicity activity of Phyllanthus sp. and regulate DNA methylation, gene expression and miRNA expression of HT-29 cell.