Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells.

INTRODUCTION PrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity. Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts. METHODS The anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1-200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol--a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB). RESULTS At higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC50 values compared to other modes of measurement, however, the statistically significant differences in EC50 values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract>resveratrol>walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay. DISCUSSION In conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.

[1]  W. Klaffke,et al.  Comparison of In Vitro Assays of Cellular Toxicity in the Human Hepatic Cell Line HepG2 , 2006, Journal of biomolecular screening.

[2]  Yu-cai Fu,et al.  Cellular and molecular effects of resveratrol in health and disease , 2012, Journal of cellular biochemistry.

[3]  B. Szende,et al.  Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures , 2000, Experimental & Molecular Medicine.

[4]  K. Azadmanesh,et al.  Cytotoxicity effects of various Juglans regia (walnut) leaf extracts in human cancer cell lines , 2012, Pharmaceutical biology.

[5]  Satoru Takahashi,et al.  Differential effect of resveratrol on nitric oxide production in endothelial f-2 cells. , 2009, Biological & pharmaceutical bulletin.

[6]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[7]  S. Ahmed,et al.  A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. , 1994, Journal of immunological methods.

[8]  N. Lall,et al.  Viability Reagent, PrestoBlue, in Comparison with Other Available Reagents, Utilized in Cytotoxicity and Antimicrobial Assays , 2013, International journal of microbiology.

[9]  R. Pijnenborg,et al.  The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. , 2007, Human reproduction.

[10]  Fields Rd,et al.  Dual-attribute continuous monitoring of cell proliferation/cytotoxicity. , 1993, American biotechnology laboratory.

[11]  Irena Cosic,et al.  Biological Effects of a De Novo Designed Myxoma Virus Peptide Analogue: Evaluation of Cytotoxicity on Tumor Cells , 2011, PloS one.

[12]  H. Wysokińska,et al.  An unusual taxodione derivative from hairy roots of Salvia austriaca. , 2012, Fitoterapia.

[13]  A. Alshatwi,et al.  Anti-proliferative effects of organic extracts from root bark of Juglans Regia L. (RBJR) on MDA-MB-231 human breast cancer cells: role of Bcl-2/Bax, caspases and Tp53. , 2011, Asian Pacific journal of cancer prevention : APJCP.

[14]  M. Hansen,et al.  Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. , 1989, Journal of immunological methods.

[15]  A. Szuławska,et al.  Biological evaluation of novel Pt(II) and Pd(II) complexes with pyrazole-containing ligands. , 2004, European journal of pharmacology.

[16]  I. Wilson,et al.  Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. , 2000, European journal of biochemistry.

[17]  J. Pereira,et al.  Human cancer cell antiproliferative and antioxidant activities of Juglans regia L. , 2010, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[18]  Michael R Hamblin,et al.  Low‐level laser therapy (LLLT) reduces oxidative stress in primary cortical neurons in vitro , 2012, Journal of biophotonics.

[19]  Henrik Stapelfeldt,et al.  Light and heat sensitivity of red cabbage extract in soft drink model systems , 2001 .

[20]  W. E. Hillis,et al.  The phenolic constituents of Prunus domestica. I.—The quantitative analysis of phenolic constituents , 1959 .

[21]  D. Doughman,et al.  A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability. , 1997, Investigative ophthalmology & visual science.

[22]  J. F. Stevens,et al.  Xanthohumol and related prenylflavonoids from hops and beer: to your good health! , 2004, Phytochemistry.

[23]  M. Zavatti,et al.  Pharmacognostic and pharmacological profile of Humulus lupulus L. , 2008, Journal of ethnopharmacology.

[24]  N. A. Taha,et al.  Utility and importance of walnut, Juglans regia Linn: A review , 2011 .

[25]  Michael K Pugsley,et al.  An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. , 2011, Proceedings of the Western Pharmacology Society.

[26]  Lihui Wei,et al.  Inhibition of tumor angiogenesis by Scutellaria barbata D. Don via suppressing proliferation, migration and tube formation of endothelial cells and downregulation of the expression of VEGF-A in cancer cells , 2011 .

[27]  E. Heiss,et al.  Effect of resveratrol on endothelial cell function: Molecular mechanisms , 2010, BioFactors.

[28]  D. Knorr,et al.  Structure-antioxidant efficiency relationships of phenolic compounds and their contribution to the antioxidant activity of sea buckthorn juice. , 2003, Journal of agricultural and food chemistry.

[29]  D. Noonan,et al.  Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF‐κB and Akt as targets , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  M. Ehlers,et al.  Resazurin Reducing Time as an Indicator of Bovine Semen Fertilizing Capacity , 1950 .

[31]  H. Ashino,et al.  Inhibition of angiogenesis by humulone, a bitter acid from beer hop. , 2001, Biochemical and biophysical research communications.

[32]  A. Akim,et al.  Protective effects of Tinospora crispa extracts on H 2 O 2 - induced oxidative stress and TNF-α-induced inflammation on human umbilical vein endothelial cells (HUVECs) , 2012 .

[33]  Ratmir Derda,et al.  Portable self-contained cultures for phage and bacteria made of paper and tape. , 2012, Lab on a chip.

[34]  T. Hsieh,et al.  Differential regulation of proliferation, cell cycle control and gene expression in cultured human aortic and pulmonary artery endothelial cells by resveratrol. , 2010, International journal of molecular medicine.

[35]  L. Terry,et al.  Biochemical profiling and chemometric analysis of seventeen UK-grown black currant cultivars. , 2008, Journal of agricultural and food chemistry.