Abstract
Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment. We have used Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify mitochondrial metabolic response in PCa cells by tracking auto-fluorescent NAD(P)H, FAD and tryptophan (Trp) lifetimes and their enzyme-bound fractions as markers, before and after treatment with anti-cancer drug doxorubicin. A 3-channel FLIM assay and quantitative analysis of these markers for cellular metabolism show in response to doxorubicin, NAD(P)H mean fluorescence lifetime (τm) and enzyme-bound (a2%) fraction increased, FAD enzyme-bound (a1%) fraction was decreased, NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio and ROS increased, followed by induction of apoptosis. For the first time, a FRET assay in PCa cells shows Trp-quenching due to Trp-NAD(P)H interactions, correlating energy transfer efficiencies (E%) vs NAD(P)H-a2%/FAD-a1% as sensitive parameters in predicting drug response. Applying this FLIM assay as early predictor of drug response would meet one of the important goals in cancer treatment.
Highlights
The fluorescence lifetimes of NAD(P)H and FAD are sensitive to changes in pH, temperature, their conformational state and proximity to quenchers[6]
The direct link between defective oxidative phosphorylation (OXPHOS) activity in cancer cell metabolism and apoptosis having been demonstrated in published work, we have focused in this manuscript on the metabolic responses to doxorubicin drug treatment of aggressive (African-American, E006AA) and more responsive (Caucasian-American, LNCaP) Prostate cancer (PCa) cells using our Fluorescence Lifetime Imaging Microscopy (FLIM)-FRET assay, hypothesizing that FLIM-based markers should show the correction of impaired OXPHOS
Metabolic changes in the mitochondria were investigated in LNCaP and E006AA PCa cells before and after doxorubicin treatment using our 3-channel FLIM-FRET assay
Summary
The fluorescence lifetimes of NAD(P)H and FAD are sensitive to changes in pH, temperature, their conformational state and proximity to quenchers[6]. The direct link between defective OXPHOS activity in cancer cell metabolism and apoptosis having been demonstrated in published work, we have focused in this manuscript on the metabolic responses to doxorubicin drug treatment of aggressive (African-American, E006AA) and more responsive (Caucasian-American, LNCaP) PCa cells using our FLIM-FRET assay, hypothesizing that FLIM-based markers should show the correction of impaired OXPHOS. The goals were to identify (i) the molecular events associated with the changes in mitochondrial energy metabolism/OXPHOS activity upon doxorubicin treatment, and (ii) the early predictors of drug response by following the lifetimes (τ1, τ2, τm) and their relative fractions (a1%, a2%) of NAD(P)H, FAD, Trp; NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio, NAD(P)H-Trp FRET interactions (E%), E% vs NAD(P) H-a2%/FAD-a1% median correlation along-with biochemically quantifying the generation of ROS and caspase-3 activity. We demonstrate that along with NAD(P)H and FAD lifetime changes, Trp-quenching due to Trp-NAD(P)H FRET interactions, and E% vs NAD(P)H-a2%/FAD-a1% median correlation are sensitive parameters in predicting the drug response of PCa cells
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