Effect of Phyllanthus amarus Extract on 5-Fluorouracil-Induced Perturbations in Ribonucleotide and Deoxyribonucleotide Pools in HepG2 Cell Line

Abstract

Abstract:The aim of this study was to investigate the antitumor activities of Phyllanthus amarus (PHA) and its potential of herb-drug interactions with 5-Fluorouracil (5-FU). Cell viability, ribonucleotides (RNs) and deoxyribonucleotides (dRNs) levels, cell cycle distribution, and expression of thymidylate synthase (TS) and ribonucleotide reductase (RR) proteins were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) method, flow cytometry and Western blot analysis, respectively. Our standardized PHA extract showed toxicity to HepG2 cells at high concentrations after 72 h exposure and induced G2/M cell cycle arrest. Combined use of 5-FU with PHA resulted in significant decreases in ATP, CTP, GTP, UTP and dTTP levels, while AMP, CMP, GMP and dUMP levels increased significantly compared with use of 5-FU alone. Further, PHA could increase the role of cell cycle arrest at S phase induced by 5-FU. Although PHA alone had no direct impact on TS and RR, PHA could change the levels of RNs and dRNs when combined with 5-FU. This may be due to cell cycle arrest or regulation of key enzyme steps in intracellular RNs and dRNs metabolism.

Fast and sensitive HPLC–MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells

“…We have demonstrated the samples can be successfully diluted, which can be useful in measuring samples with higher concertation than the upper limit of our calibration curve. Additionally, this method was able to chromatographically separate ATP and dGTP, whereas previous methods were unable to achieve separation [10,19,28,35], had to use run times of up to 1 hour and switch ion modes [20], or utilize ion-pairing reagents [36] for successful separation. In summary, the assay is a fast, reproducible, and robust method that efficiently extracts nucleotide from small tissue samples and quantifies four intracellular deoxynucleosides triphosphates simultaneously.…”

Chemical Components and Biological Activities of the Genus Phyllanthus: A Review of the Recent Literature

“…It was further explained that P. amarus extracts have potential to be utilized at a commercial scale for the isolation of natural antioxidants and some novel anticancer agents [ 52 ]. P. amarus extracts have good anti-tumor activity and good drug-herb interactions with 5-fluorouracil (5-FU), and at higher concentrations show greater toxicity to HepG2 cells by inducing G2/M cell cycle arrest or modulation of major enzymes involved in intracellular ribonucleotide and deoxyribonucleotide metabolism [ 64 ]. Apoptotic cells are one of the main biomarkers used to predict and define what type of treatment should be given to patients suffering colorectal cancer and monitor the end results.…”