• Effect of Methotrexate and Tea Polyphenols on the Viability and Oxidative Stress in MDA-MB-231 Breast Cancer Cells

      Owusu-Apenten, Richard K.; Kelly, Theresa; University of Ulster (SCIENCEDOMAIN International, 2015-03-24)
      Aim: To determine the effect of tea polyphenols and methotrexate on viability and reactive oxygen species (ROS) in a naturally resistant breast cancer cell line MDA-MB-231. Methodology: MDA-MB-231 cells were selected as a model for methotrexate resistant breast cancer. Drug tests were performed over 72 hours at concentrations 0-100 µM. Pre-treatments were with quercetin (QE) or epigallocatechin gallate (EGCG) for 5 hours followed by methotrexate. Cytotoxicity was measured using the MTT assay or resazurin fluorescence assay. ROS was determined using the 2’, 7’-dichlorofluorescein diacetate assay. Intracellular GSH was measured using the monochlorobimane assay. Results: Methotrexate was cytotoxic to MDA-MB-231 cells with IC50 of 35±4 µM. The IC50 value was 68±9.4 µM with QE and 83±16 µM for EGCG. The pre-treatment with QE and EGCG lowered the IC50 for methotrexate by 28% (P =0.009) and 16% (P=0.2027). Intracellular ROS concentrations increased after treatment with methotrexate, QE or EGCG singly and ROS decreased with combination treatment compared with the response for methotrexate only. There were no significant changes in intracellular GSH. Conclusion: Pretreatment with tea polyphenols partially sensitized breast cancer cells towards methotrexate and decreases intracellular ROS. More research is needed to optimize the sensitizing effect of tea phenols on the breast cancer cell response to methotrexate.
    • Effect of pH on the Radical Quenching Capacity of Tea Infusions Using the ABTS•+ Assay

      Chan, Yuk Man; Cheng, Nga Kwan; Nigam, Poonam S.; Owusu-Apenten, Richard K.; University of Chester, University of Ulster (Sciencedomain International, 2016-06-22)
      Aims: The aims of this study were to assess the impact of pH on the free radical quenching activity of tea infusions using a modified 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay and three antioxidant compounds as reference. Study Design: In-vitro method. Place and Duration of Study: Faculty of Life and Health Science, School of Biomedical Sciences, Ulster University, UK. From Sept 2014 and May 2016. Methodology: Free radical quenching capacity of tea (Earl grey, black tea, Ceylon tea, & green tea) infusions were investigated using persulfate activated ABTS with acetate buffer (pH 4.5) or phosphate buffer saline (pH 7.0) as solvent. Tests were performed using 96-well microplates, 20 µl of sample and 280 µl of ABTS reagent, and calibrated using ascorbic acid, trolox or gallic acid as reference antioxidants. Results: Gallic acid free radical quenching was pH dependent and unsuitable as reference. The free radical quenching capacity of trolox and ascorbic acid was not significantly different at pH 4.5 and pH 7.0. The radical quenching capacity of tea infusions expressed as Trolox Equivalent Antioxidant Capacity (TEAC) or Ascorbic Acid Equivalent Antioxidant Capacity (AAEAC) was greater by 50-300% at pH 7 compared to pH 4.5. Conclusion: The modified ABTS assay is suitable for examining the influence of pH on free radical quenching ability of tea samples. Gallic acid was not a suitable reference compound. The radical quenching capacity of tea infusions increases with rising pH.
    • Effects of ascorbic acid, dehydroascorbic acid and methotrexate on breast cancer cell viability.

      Dosunmu, Yewande; Owusu-Apenten, Richard K.; University of Chester, University of Ulster (Sciencedomain international, 2017-10-28)
      Aims: To examine the effects of ascorbic acid (AA), dehydroascorbic acid (DHA) and methotrexate (MTX) combined treatments on (MDA-MB-231) breast cancer cell viability and intracellular reactive oxygen species (ROS). Study Design: In-vitro method. Place and Duration of Study: Biomedical Sciences Research Institute, University of Ulster, Coleraine, BT52 1SA, United Kingdom. September 2016-2017 Methodology: Cytotoxicity tests were performed with MTX (0.01- 1000 µmol/l) alone or in combination with AA or DHA, for 72 h. Cell viability was measured by 3-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) or Sulforhodamine B (SRB) assays. Intracellular ROS was measured by 2’,7’-dichlorofluroscein diacetate assay. Results: Treatments of MDA-MB231 cells with single agents, showed dose dependent response with 50% inhibition of cell viability (IC50) of 110.5-201.4 µmol/l (MTX), 2237-5703 µmol/l (AA) or 2474 µmol/l (DHA). Combination studies showed clear synergisms for MTX (~10 µmol/l) and DHA or AA (1100 µmol/l) but weak or no interactions at other concentrations. Three days combination treatment of DHA showed decrease of ROS, which was reversed by MTX (>10 µmol/l). Conclusions: Co-treatment of methotrexate with AA or DHA showed synergism (C1<1.0) and enhanced cytotoxicity of the anti-folate towards MDA-MB-231 breast cancer cells. Intracellular ROS decreased with AA and DHA treatment, which might be useful for reducing MTX-related oxidative stress.