期刊
JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY
卷 95, 期 1, 页码 46-57出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.jphotobiol.2008.12.010
关键词
NADH; Hs578T; Hs578Bst; Two-photon FLIM; Associated anisotropy; Dehydrogenase
资金
- National Institute of Health [AG030949]
- Johnson Johnson
- Penn State Materials Research Institute
- Center for Optical Technologies
- Direct For Biological Sciences [1048936] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience [1048936] Funding Source: National Science Foundation
Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result. there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular Processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed non-invasive assay, we estimate the average NADH concentration in cancer cells (168 +/- 49 mu M) to be similar to 1.8-fold higher than in breast normal cells (99 +/- 37 mu M). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 +/- 0.08) and enzyme-bound (0.82 +/- 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different front those in breast cancer cells (free: 0.25 +/- 0.08: bound: 0.75 +/- 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in Solution mimic Our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as it conventional biochemical assays. (C) 2009 Elsevier B.V. All rights reserved.
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