Curcumin restores glutathione-S-transferase activity for LNCaP prostate cancer cells

Prostate cancer is a leading cause of death in males aged fifty and over. Glutathione transferase (GST) activity is depressed in prostate cancer cells. The aim of this study was to assess GST reactivation in LNCaP prostate cancer cells treated with curcumin or 5-azacitidine (5-Aza) which is a known hypomethylation agent. GST activity was determined using monochlorobimane (MCB). Cell viability was assessed with resazurin (Vision blue TM) or 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium-bromide (MTT). From the results, treatment with >5 μM of curcumin or 5-Aza for 3 or 6 days depressed LNCaP cell viability. The concentrations of curcumin leading to 50% reduction of LNCaP cell viability (IC50) was 10-25 μM or 2-3 μM for 3 days or 6 days of treatment, respectively. The IC50 with 5-Aza was 17-23 μM (3 days) or 50-52 μM (6 days). Combination treatment using curcumin and 5-Aza showed complimentary interactions affecting cell viability. Low levels of curcumin or 5-Aza had no effect on GST activity. By contrast, cytotoxic doses of curcumin or 5-Aza increased GST activity by 450-750 % (3days) or 161-2800 % (6days). In conclusion, GST reactivation was feasible but only when LNCaP prostate cancer cells were treated with cytotoxic doses of curcumin or 5-azacytidne

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

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

Effects of ascorbic acid, dehydroascorbic acid and methotrexate on breast cancer cell viability.

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

Total Phenols, Antioxidant Capacity and Antibacterial Activity of Manuka Honey Extract.

Aims: To evaluate total phenols content (TPC), antioxidant capacity (TAC) and antibacterial activity of Manuka honey extract (MHE) and to compare such properties with those for unfractionated Manuka honey. Study Design: In vitro study. Place and Duration of Study: School of Biomedical Sciences, Ulster University, Coleraine, UK. Between September 2016 and September 2017. Methodology: MHE was prepared by solvent extraction using ethyl acetate. TPC was determined by Folin-Ciocalteu assay. The iron (III) reducing antioxidant capacity (IRAC) method was used to determine TAC. Antibacterial activity was evaluated using disc diffusion assay and 96-well microtiter plate methods with absorbance measured at 600 nm. Results: The TPC for MHE was 30-fold higher than the value for Manuka honey (33420±1685 mg vs. 1018±78 mg GAE/kg) while TAC values were~ 100-times greater (83,198±7064 vs. 793±104 TEAC, respectively). Antibacterial activity assessed by disc diffusion for Manuka honey (18.5 mm on S. aureus and 20 mm on E. coli) was two times greater than for MHE (9mm for both S. aureus and E. coli). The 96-well microtiter plate assay confirmed the greater antibacterial activity for Manuk

Antioxidant and genoprotective activity of selected cucurbitaceae seed extracts and LC–ESIMS/MS identification of phenolic components

Cucurbitaceae are one of most widely used plant species for human food but lesser known members have not been examined for bioactive components. The purpose of this study was to evaluate the antioxidant and genoprotective activities from three cucurbitaceae seeds extracts and to identify phenolic components by LC–ESIMS/MS analysis. From the results, the yield of seeds extract was 20–41% (w/w) and samples had 16–40% total phenols as gallic acid equivalents (GAE). Compared with methanol solvent, using acidified methanol led to increased extraction yield by 1.4 to 10-fold, higher phenolic content (149.5 ± 1.2 to 396.4 ± 1.9 mg GAE/g), higher DPPH radical quenching and enhanced enoprotective activity using the pBR322 plasmid assay. LC–ESI-MS/MS analysis led to identification of 14–17 components, based on authentic standards and comparison with literature reports, as mainly phenolic acids and esters, flavonol glycosides. This may be the first mass spectrometric profiling of polyphenol components from cucurbitaceae seeds

Determination of Iron (III) Reducing Antioxidant Capacity for Manuka Honey and Comparison with ABTS and Other Methods

Aims: Applying multiple assays with trolox as the sole reference compound is a recent AOAC proposal to improve the reliability of total antioxidant capacity determinations. The aim of this study was to evaluate, iron (III) reducing antioxidant capacity (iRAC) for Manuka honey samples and comparisons with ABTS and other well-known assays. Study Design: In-vitro, laboratory-based study. Place and Duration of Study: School of Biomedical Sciences, Faculty of Life and Health Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK; September 2015-May 2016. Methodology: Manuka honey rated Unique Manuka Factor (UMF) 5+, 10+, 15+, 18+ and a nonrated (NR) sample were analysed using five assays for total antioxidant capacity namely, iRAC, ABTS, DPPH, FRAP, and Folin assays. Values for total antioxidant capacity were normalized as Trolox Equivalent Antioxidant capacity (TEAC) for comparison within and between assays. Results: The TAC were correlated for all methods (R2 = 0.83-0.99) and also correlated with the total phenols content. Actual TEAC value for a given honey ranged by 21-70-fold depending on the assay method with the following general order of increase; DPPH < FRAP (pH 3.6) < iRAC (pH 7.0) <ABTS (pH7) < Folin (pH ~11). The trends in TAC values are discussed alongside of TEAC values for 50 food items and some challenges for comparing different antioxidant methods are highlighted. Conclusion: Total antioxidant capacity of Manuka honey changes in a regular manner probably affected by assay pH. The findings are important for attempts to standardize antioxidant methods as currently applied to foods, beverages and dietary supplements. Further research is recommended to examine the effect of normalizing antioxidant methods for solvent composition and pH

Comparison of Iron (III) Reducing Antioxidant Capacity (iRAC) and ABTS Radical Quenching Assays for Estimating Antioxidant Activity of Pomegranate

Pomegranate juice (PJ) has total antioxidant capacity which is reportedly higher compared to other common beverages. This short study aimed to assess the total antioxidant capacity of commercial PJ and pomegranate fruit using a newly described method for iron (III) reducing antioxidant capacity (iRAC) and to compare with the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and Folin–Ciocalteu assays. Commercial PJ, freeze-dried pomegranate, and oven-dried pomegranate were analyzed. The calibration results for iRAC were comparable to ABTS and Folin–Ciocalteu methods in terms of linearity (R2 > 0.99), sensitivity and precision. The total antioxidant capacity for PJ expressed as trolox equivalent antioxidant capacity (TEAC) was 33.4 ± 0.5 mM with the iRAC method and 36.3 ± 2.1 mM using the ABTS method. For dried pomegranates, total antioxidant capacity on a dry weight basis (DB) was 89–110 mmol/100 g DB or 76.0 ± 4.3 mmol/100 g DB using iRAC and ABTS methods, respectively. Freeze-dried pomegranate had 15% higher total antioxidant capacity compared with oven-dried pomegranate. In conclusion, pomegranate has high total antioxidant capacity as evaluated by the iRAC and ABTS methods, though variations occur due to the type of cultivar, geographic origin, processing and other factors. The study is relevant for attempts to refine food composition data for pomegranate and other functional food