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Selective induction of apoptosis by capsaicin in transformed cells: the role of reactive oxygen species and calcium

Capsaicin is a vanilloid quinone analog that inhibits the plasma membrane electron transport (PMOR) system and induces apoptosis in transformed cells. Using a cytofluorimetric approach we have determined that capsaicin induces a rapid increase of reactive oxygen species (ROS) followed by a subsequent disruption of the transmembrane mitochondrial potential (ΔΨm) and DNA nuclear loss in transformed cell lines and in mitogen activated human T cells. This apoptotic pathway is biochemically different from the typical one induced by either ceramide or edelfosine where, in our system, the ΔΨm dissipation precedes the generation of reactive oxygen species. Neither production of ROS nor apoptosis was found in capsaicin-treated resting T cells where the activity of the PMOR system is minimal when compared with mitogen activated or transformed T cells. Capsaicin also induces Ca2+ mobilization in activated but not in resting T cells. However, preincubation of cells with BAPTA-AM, which chelate cytosolic free calcium, did not prevent ROS generation or apoptosis induced by capsaicin, suggesting that ROS generation in capsaicin treated cells is not a consequence of calcium signaling and that the apoptotic pathway may be separated from the one that mobilizes calcium. Moreover, we present data for the implication of a possible vanilloid receptor in calcium mobilization, but not in ROS generation. These results provide evidence that the PMOR system may be an interesting target to design antitumoral and anti-inflammatory drugs.

Coenzyme Q deficiency triggers mitochondria degradation by mitophagy

Coenzyme Q10 (CoQ) is a small lipophilic molecule critical for the transport of electrons from complexes I and II to complex III in the mitochondrial respiratory chain. CoQ deficiency is a rare human genetic condition that has been associated with a variety of clinical phenotypes. With the aim of elucidating how CoQ deficiency affects an organism, we have investigated the pathophysiologic processes present within fibroblasts derived from 4 patients with CoQ deficiency. Assays of cultured fibroblasts revealed decreased activities of complex II+III, complex III, and complex IV, reduced expression of mitochondrial proteins involved in oxidative phosphorylation, decreased mitochondrial membrane potential, increased production of reactive oxygen species (ROS), activation of mitochondrial permeability transition (MPT), and reduced growth rates. These abnormalities were partially restored by CoQ supplementation. Moreover, we demonstrate that CoQ deficient fibroblasts exhibited increased levels of lysosomal markers (β-galactosidase, cathepsin, LC3, and Lyso Tracker), and enhanced expression of autophagic genes at both transcriptional and translational levels, indicating the presence of autophagy. Electron microscopy studies confirmed a massive degradation of the altered mitochondria by mitophagy. Autophagy in CoQ deficient fibroblasts was abolished by antioxidants or cyclosporin treatments suggesting that both ROS and MPT participate in this process. Furthermore, prevention of autophagy in CoQ deficient fibroblasts by 3-methyl adenine or wortmannin, as well as the induction of CoQ deficiency in cells lacking autophagy (by means of genetic knockout of the Atg5 gene in mouse embryonic fibroblasts) resulted in apoptotic cell death, suggesting a protective role of autophagy in CoQ deficiency.

Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants.

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery inSaccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q6 by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-lesscoq7 mutants derived from four distinct laboratory yeast strains. Q6 supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the fourcoq7 mutant strains. Following culture in medium containing dextrose, the added Q6 was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q6 in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q6. These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q6 into the plasma membrane. The two coq7 mutant strains rescued by Q6 supplementation for growth on ethanol contained mitochondrial Q6 levels similar to wild type. However, the content of Q6 in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q6, succinate-cytochrome creductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q6 content, succinate-cytochromec reductase activity, and steady state levels of the cytochrome c 1 polypeptide. These studies show that transport of extracellular Q6 to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc 1complex is unstable. These results indicate that delivery of exogenous Q6 to mitochondria is required fore activity and stability of the bc 1 complex in yeast coqmutants.

Haploinsufficiency of COQ4 causes coenzyme Q10 deficiency

Background COQ4 encodes a protein that organises the multienzyme complex for the synthesis of coenzyme Q10 (CoQ10). A 3.9 Mb deletion of chromosome 9q34.13 was identified in a 3-year-old boy with mental retardation, encephalomyopathy and dysmorphic features. Because the deletion encompassed COQ4, the patient was screened for CoQ10 deficiency. Methods A complete molecular and biochemical characterisation of the patients fibroblasts and of a yeast model were performed. Results The study found reduced COQ4 expression (48% of controls), CoQ10 content and biosynthetic rate (44% and 43% of controls), and activities of respiratory chain complex II+III. Cells displayed a growth defect that was corrected by the addition of CoQ10 to the culture medium. Knockdown of COQ4 in HeLa cells also resulted in a reduction of CoQ10. Diploid yeast haploinsufficient for COQ4 displayed similar CoQ deficiency. Haploinsufficency of other genes involved in CoQ10 biosynthesis does not cause CoQ deficiency, underscoring the critical role of COQ4. Oral CoQ10 supplementation resulted in a significant improvement of neuromuscular symptoms, which reappeared after supplementation was temporarily discontinued. Conclusion Mutations of COQ4 should be searched for in patients with CoQ10 deficiency and encephalomyopathy; patients with genomic rearrangements involving COQ4 should be screened for CoQ10 deficiency, as they could benefit from supplementation.

Silencing of ubiquinone biosynthesis genes extends life span in Caenorhabditis elegans

Ubiquinone (coenzyme Q; Q) is a key factor in the mitochondria electron transport chain, but it also functions as an antioxidant and as a cofactor of mitochondrial uncoupling proteins. Furthermore, Q isoforms balance in Caenorhabiditis elegans is determined by both dietary intake and endogenous biosynthesis. In the absence of synthesis, withdrawal of dietary Q8 in adulthood extends life span. Thus, Q plays an important role in the aging process and understanding its synthesis acquires a new impetus. We have identified by RNA interference (RNAi) eight genes, including clk‐1, involved in ubiquinone biosynthesis in C. elegans feeding animals with dsRNA‐containing Escherichia coli HT115 strains. Silenced C. elegans showed lower levels of both endogenous Q9 and Q8 provided by diet, produced less superoxide without a significant modification of mitochondrial electron chain, and extended life span compared with non‐interfered animals. E. coli strains harboring dsRNA also interfered with their own Q8 biosynthesis. These findings suggest that more efficient electron transport between a lower amount of Q and electron transport capacity of the mitochondrial complexes leads to less production of reactive oxygen species that contributes to extension of life span in the nematode C. elegans.

Coenzyme Q deficiency in muscle

PURPOSE OF REVIEW Coenzyme Q (CoQ) is a vital component of the mitochondrial respiratory chain. A number of patients with CoQ deficiency presented with different clinical phenotypes, often affecting skeletal muscle, and responded well to CoQ supplementation. We discuss recent advances in this field with special attention to muscle involvement. RECENT FINDINGS The identification of genetic defects causing CoQ deficiency has allowed to distinguish primary forms, due to mutations in biosynthetic genes, from secondary defects caused either by mutations in genes unrelated to CoQ biosynthesis or by nongenetic factors. To date, none of the patients with genetically proven primary deficiency presented with an exclusively (or prominently) myopathic phenotype. Most patients with myopathy were found to harbor other genetic defects (mutations in electron-transferring-flavoprotein dehydrogenase or mitochondrial DNA). The majority of patients with CoQ deficiency still lack a genetic diagnosis. The pathogenesis of CoQ deficiency cannot be attributed solely to the bioenergetic defect, suggesting that other roles of CoQ, including its antioxidant properties or its role in pyrimidine metabolism, may also play crucial roles. SUMMARY Early recognition of CoQ deficiency is essential to institute appropriate and timely treatment, thus avoiding irreversible tissue damage.

N-acetylcysteine, coenzyme Q10 and superoxide dismutase mimetic prevent mitochondrial cell dysfunction and cell death induced by D-galactosamine in primary culture of human hepatocytes

D-Galactosamine (D-GalN) induces reactive oxygen species (ROS) generation and cell death in cultured hepatocytes. The aim of the study was to evaluate the cytoprotective properties of N-acetylcysteine (NAC), coenzyme Q(10) (Q(10)) and the superoxide dismutase (SOD) mimetic against the mitochondrial dysfunction and cell death in D-GalN-treated hepatocytes. Hepatocytes were isolated from liver resections. NAC (0.5 mM), Q(10) (30 microM) or MnTBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (1mg/mL) were co-administered with D-GalN (40 mM) in hepatocytes. Cell death, oxidative stress, mitochondrial transmembrane potential (MTP), ATP, mitochondrial oxidized/reduced glutathione (GSH) and Q(10) ratios, electronic transport chain (ETC) activity, and nuclear- and mitochondria-encoded expression of complex I subunits were determined in hepatocytes. d-GalN induced a transient increase of mitochondrial hyperpolarization and oxidative stress, followed by an increase of oxidized/reduced GSH and Q(10) ratios, mitochondrial dysfunction and cell death in hepatocytes. The cytoprotective properties of NAC supplementation were related to a reduction of ROS generation and oxidized/reduced GSH and Q(10) ratios, and a recovery of mitochondrial complexes I+III and II+III activities and cellular ATP content. The co-administration of Q(10) or MnTBAP recovered oxidized/reduced GSH ratio, and reduced ROS generation, ETC dysfunction and cell death induced by D-GalN. The cytoprotective properties of studied antioxidants were related to an increase of the protein expression of nuclear- and mitochondrial-encoded subunits of complex I. In conclusion, the co-administration of NAC, Q(10) and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death induced by D-GalN in cultured hepatocytes.

Genetic Evidence for Coenzyme Q Requirement in Plasma Membrane Electron Transport

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3Δ, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q6 and contained greatly diminished levels of NADH–ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q6 in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase. Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q6 and NADH–ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3Δ strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3Δ strain grown in the presence of coenzyme Q6. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3–14. The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3Δ strain. Ascorbate free radical reductase was not altered significantly in mutants atp2Δ and cor1Δ which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3Δ mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH–ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.

Neutral magnesium-dependent sphingomyelinase from liver plasma membrane: purification and inhibition by ubiquinol.

Plasma membranes isolated from pig liver contained almost no acid sphingomyelinase but significant neutral magnesium-dependent sphingomyelinase that was activated by phosphatidylserine. We report here the purification to apparent homogeneity of neutral sphingomyelinase of about 87 kDa from liver plasma membranes. The purified enzyme strictly required magnesium and had a neutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma membrane was not inhibited by GSH and, strikingly, it was not activated by phosphatidylserine. Liver sphingomyelinase was inhibited by several lipophilic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective than α-tocopherol, α-tocopherylquinone, α-tocopherylquinone, and ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with different levels of protection against lipid peroxidation. The purified sphingomyelinase was not inhibited significantly by ubiquinone-10 and ubiquinol-10, but ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our results demonstrate a direct inhibitory effect of ubiquinol on the plasma membrane n-SMase and support the participation of this molecule in the regulation of ceramide-mediated signaling.

Acute oxidant damage promoted on cancer cells by amitriptyline in comparison with some common chemotherapeutic drugs

Oxidative therapy is a relatively new anticancer strategy based on the induction of high levels of oxidative stress, achieved by increasing intracellular reactive oxygen species (ROS) and/or by depleting the protective antioxidant machinery of tumor cells. We focused our investigations on the antitumoral potential of amitriptyline in three human tumor cell lines: H460 (lung cancer), HeLa (cervical cancer), and HepG2 (hepatoma); comparing the cytotoxic effect of amitriptyline with three commonly used chemotherapeutic drugs: camptothecin, doxorubicin, and methotrexate. We evaluated apoptosis, ROS production, mitochondrial mass and activity, and antioxidant defenses of tumor cells. Our results show that amitriptyline produces the highest cellular damage, inducing high levels of ROS followed by irreversible serious mitochondrial damage. Interestingly, an unexpected decrease in antioxidant machinery was observed only for amitriptyline. In conclusion, based on the capacity of generating ROS and inhibiting antioxidants in tumor cells, amitriptyline emerges as a promising new drug to be tested for anticancer therapy.