Mitochondria are important intracellular sources and targets of reactive oxygen species (ROS), while flavonoids, a large group of secondary plant metabolites, are important antioxidants. Following our previous study on the energetics of mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, the present work addressed the antioxidant activity of these compounds (1-50 micromol/L) on Fe(2+)/citrate-mediated membrane lipid peroxidation (LPO) in isolated rat liver mitochondria, running in parallel studies of their antioxidant activity in non-organelle systems. Only quercetin inhibited the respiratory chain of mitochondria and only galangin caused uncoupling. Quercetin and galangin were far more potent than taxifolin and catechin in affording protection against LPO (IC(50) = 1.23 +/- 0.27 and 2.39 +/- 0.79 micromol/L, respectively), although only quercetin was an effective scavenger of both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals. These results, together with the previous study, suggest that the 2,3-double bond in conjugation with the 4-oxo function in the flavonoid structure are major determinants of the antioxidant activity of flavonoids in mitochondria, the presence of an o-di-OH structure on the B-ring, as occurs in quercetin, favours this activity via superoxide scavenging, while the absence of this structural feature in galangin, favours it via a decrease in membrane fluidity and/or mitochondrial uncoupling.
The vitamin E derivative (þ)a-tocopheryl succinate (a-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RARa transgenic mice, we demonstrated that a-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that a-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, a-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for a-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.
SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 μM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.
The hSuv3 (human Suv3) helicase has been shown to be a major player in mitochondrial RNA surveillance and decay, but its physiological role might go beyond this functional niche. hSuv3 has been found to interact with BLM (Bloom’s syndrome protein) and WRN (Werner’s syndrome protein), members of the RecQ helicase family involved in multiple DNA metabolic processes, and in protection and stabilization of the genome. In the present study, we have addressed the possible role of hSuv3 in genome maintenance by examining its potential association with key interaction partners of the RecQ helicases. By analysis of hSuv3 co-IP (co-immunoprecipitation) complexes, we identify two new interaction partners of hSuv3: the RPA (replication protein A) and FEN1 (flap endonuclease 1). Utilizing an in vitro biochemical assay we find that low amounts of RPA inhibit helicase activity of hSuv3 on a forked substrate. Another single-strand-binding protein, mtSSB (mitochondrial single-strand-binding protein), fails to affect hSuv3 activity, indicating that the functional interaction is specific for hSuv3 and RPA. Further in vitro studies demonstrate that the flap endonuclease activity of FEN1 is stimulated by hSuv3 independently of flap length. hSuv3 is generally thought to be a mitochondrial helicase, but the physical and functional interactions between hSuv3 and known RecQ helicase-associated proteins strengthen the hypothesis that hSuv3 may play a significant role in nuclear DNA metabolism as well.
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