Next Article in Journal
Multiple Export Mechanisms for mRNAs
Next Article in Special Issue
NAD+-Metabolizing Ectoenzymes in Remodeling Tumor–Host Interactions: The Human Myeloma Model
Previous Article in Journal
Spatiotemporal Regulation of Nuclear Transport Machinery and Microtubule Organization
Article Menu

Export Article

Open AccessArticle
Cells 2015, 4(3), 427-451; doi:10.3390/cells4030427

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

1
Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia
2
Neurosurgical clinic and polyclinic, Leipzig University Clinic, Leipzig 04103, Germany
3
Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119234, Russia
4
Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia
*
Author to whom correspondence should be addressed.
Academic Editor: Ross Grant
Received: 14 July 2015 / Revised: 11 August 2015 / Accepted: 12 August 2015 / Published: 21 August 2015
(This article belongs to the Special Issue NAD+ Metabolism and Signaling)
View Full-Text   |   Download PDF [2613 KB, uploaded 21 August 2015]   |  

Abstract

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. View Full-Text
Keywords: glioblastoma viability; cellular NAD(P)H-dependent oxidoreductase; metabolon; thiamin; oxythiamin; 2-oxo acid dehydrogenase; phosphonate analog of 2-oxo acid; resazurin; T98G; U87 glioblastoma viability; cellular NAD(P)H-dependent oxidoreductase; metabolon; thiamin; oxythiamin; 2-oxo acid dehydrogenase; phosphonate analog of 2-oxo acid; resazurin; T98G; U87
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

Aleshin, V.A.; Artiukhov, A.V.; Oppermann, H.; Kazantsev, A.V.; Lukashev, N.V.; Bunik, V.I. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays. Cells 2015, 4, 427-451.

Show more citation formats Show less citations formats

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Cells EISSN 2073-4409 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top