Cytotoxicity of mitochondria-targeted resveratrol derivatives: Interactions with respiratory chain complexes and ATP synthase

https://doi.org/10.1016/j.bbabio.2014.06.010Get rights and content
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Highlights

  • Mitochondriotropic resveratrol derivatives inhibit respiratory chain (RC) complexes.

  • Effectiveness as RC inhibitors and as promoters of ROS generation correlate

  • “Capping” free hydroxyls increases the effectiveness of the derivative.

  • They also inhibit the ATPase and may turn it into a conduit for uncoupled proton flux.

Abstract

We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low μM range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H2O2 produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. “Capping” free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca2 +] “spikes” in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F0F1 ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore.

Abbreviations

B-CLL
B-cell chronic lymphocytic leukemia
BSA
bovine serum albumin
BTPI
4-triphenylphosphoniumbutyl
CoQ
coenzyme Q
CsA
cyclosporin A
DCPI
2,6-dichlorophenolindophenol
Δψm
mitochondrial membrane potential
DMEM
Dulbecco's Modified Eagle Medium
Δμ̃H
proton electrochemical gradient
ECAR
extracellular acidification rate
FACS
fluorescence-activated cell scanner
FBS
fetal bovine serum
FCCP
carbonyl cyanide p-trifluoromethoxy-phenylhydrazone
HBSS
Hank's Balanced Salt Solution
H2DCF-DA
2′,7′-dichlorodihydrofluorescein-diacetate
IMM
inner mitochondrial membrane
LDH
lactate dehydrogenase
MPT
mitochondrial permeability transition
OCR
oxygen consumption rate
OL
oligomycin
PEG-SOD
polyethyleneglycol-superoxide dismutase
PEG-CAT
polyethyleneglycol-catalase
PEP
phosphoenolpyruvate
PK
pyruvate kinase
R-4′BTPI
4′-(4-triphenylphosphoniumbutyl) resveratrol iodide
R-3BTPI
3-(4-triphenylphosphoniumbutyl) resveratrol iodide
RDA-4′BTPI
3,5-diacetyl-4′-(4-triphenylphosphoniumbutyl) resveratrol iodide
RDA-3BTPI
4′,5-diacetyl-3-(4-triphenylphosphoniumbutyl) resveratrol iodide
RDM-4′BTPI
3,5-dimethyl-4′-(4-triphenylphosphoniumbutyl) resveratrol iodide
RDM-3BTPI
4′,5-dimethyl-3-(4-triphenylphosphoniumbutyl) resveratrol iodide
RLM
rat liver mitochondria
ROI
regions of interest
ROS
reactive oxygen species
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Keywords

Resveratrol
Triphenylphosphonium
ROS
Mitochondria
Respiratory chain
ATP synthase

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