title: Multiphoton fluorescence lifetime imaging microscopy reveals free-to-bound NADH ratio changes associated with metabolic inhibition.
abstract: Measurement of endogenous free and bound NAD(P)H relative concentrations in living cells isa useful method for monitoring aspects of cellular metabolism, because the NADH∕NAD⁺ reduction-oxidation pair is crucial for electron transfer through the mitochondrial electron transport chain. Variations of free and bound NAD(P)H ratio are also implicated in cellular bioenergetic and biosynthetic metabolic changes accompanying cancer. This study uses two-photon fluorescence lifetime imaging microscopy (FLIM) to investigate metabolic changes in MCF10A premalignant breast cancer cells treated with a range of glycolysis inhibitors: namely, 2 deoxy-D-glucose, oxythiamine, lonidamine, and 4-(chloromethyl) benzoyl chloride, as well as the mitochondrial membrane uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Through systematic analysis of FLIM data from control and treated cancer cells, we observed that all glycolytic inhibitors apart from lonidamine had a slightly decreased metabolic rate and that the presence of serum in the culture medium generally marginally protected cells from the effect of inhibitors. Direct production of glycolytic L-lactate was also measured in both treated and control cells. The combination of these two techniques gave valuable insights into cell metabolism and indicated that FLIM was more sensitive than traditional biochemical methods, as it directly measured metabolic changes within cells as compared to quantification of lactate secreted by metabolically active cells.
authors.affiliation: Macquarie University, MQ BioFocus Research Centre/Department of Physics and Astronomy, North Ryde, New South Wales, 2109 Australia.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Drozdowicz-Tomsia
authors.affiliation: Macquarie University, MQ BioFocus Research Centre/Department of Physics and Astronomy, North Ryde, New South Wales, 2109 Australia.
authors.forename: Ayad G
authors.initials: AG
authors.lastname: Anwer
authors.affiliation: Charles Sturt University, School of Biomedical Sciences, Wagga Wagga, New South Wales, 2678 Australia.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cahill
authors.affiliation: University of Baghdad, Institute of Laser for Postgraduate Studies, Baghdad 10071, Iraq.
authors.forename: Kaiser N
authors.initials: KN
authors.lastname: Madlum
authors.affiliation: University of Baghdad, Institute of Laser for Postgraduate Studies, Baghdad 10071, Iraq.
authors.forename: Amel M
authors.initials: AM
authors.lastname: Maki
authors.affiliation: Macquarie University, Department of Chemistry & Biomolecular Sciences, North Ryde, New South Wales, 2109 Australia.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Baker
authors.affiliation: Macquarie University, MQ BioFocus Research Centre/Department of Physics and Astronomy, North Ryde, New South Wales, 2109 AustraliaeMacquarie University, ARC Centre of Excellence in Nanoscale Biophotonics, North Ryde, New South Wales, 2109 Australia.
authors.forename: Ewa M
authors.initials: EM
authors.lastname: Goldys
mesh_terms: D001943:Breast Neoplasms / Q000201:enzymology*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009243:NAD / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D009243:NAD