Cezar R. Pestana

Antioxidant activity of flavonoids in isolated mitochondria

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 µmol/L) on Fe2+/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 (IC50 = 1.23 ± 0.27 and 2.39 ± 0.79 µmol/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. Copyright

(+)α-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo

The vitamin E derivative (+)α-tocopheryl succinate (α-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-RARα transgenic mice, we demonstrated that α-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that α-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, α-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 α-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler.

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: ensuing energetic and oxidative stress implications.

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 μM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca²⁺ efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds.

SET protein accumulates in HNSCC and contributes to cell survival: Antioxidant defense, Akt phosphorylation and AVOs acidification

OBJECTIVES Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages. MATERIALS AND METHODS SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250μM) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively. RESULTS SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death. CONCLUSION SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy.

Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHMs inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 microM Ca(2+), DHM (50-250 microM) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 microM) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline.

Effects on mitochondria of mitochondria-induced nitric oxide release from a ruthenium nitrosyl complex.

The ruthenium nitrosyl complex trans-[Ru(NO)(NH(3))(4)(py)](PF(6))(3) (pyNO), a nitric oxide (NO) donor, was studied in regard to the release of NO and its impact both on isolated mitochondria and HepG2 cells. In isolated mitochondria, NO release from pyNO was concomitant with NAD(P)H oxidation and, in the 25-100 microM range, it resulted in dissipation of mitochondrial membrane potential, inhibition of state 3 respiration, ATP depletion and reactive oxygen species (ROS) generation. In the presence of Ca(2+), mitochondrial permeability transition (MPT), an unspecific membrane permeabilization involved in cell necrosis and some types of apoptosis, was elicited. As demonstrated by externalization of phosphatidylserine and activation of caspase-9 and caspase-3, pyNO (50-100 microM) induced HepG2 cell death, mainly by apoptosis. The combined action of the NO itself, the peroxynitrite yielded by NO in the presence of reactive oxygen species (ROS) and the oxidative stress generated by the NAD(P)H oxidation is proposed to be involved in cell death by pyNO, both via respiratory chain inhibition and ROS levels increase, or even via MPT, if Ca(2+) is present.

Enantioselective analysis of oxybutynin and N-desethyloxybutynin with application to an in vitro biotransformation study.

An enantioselective method using liquid-phase microextraction (LPME) followed by HPLC analysis was developed for the determination of oxybutynin (OXY) and its major metabolite N-desethyloxybutynin (DEO) in rat liver microsomal fraction. The LPME procedure was optimized using multifactorial experiments. Under the optimal extraction conditions, the mean recoveries were 61 and 55% for (R)-OXY and (S)-OXY, respectively, and 70 and 76% for (R)-DEO and (S)-DEO, respectively. The validated method was employed to an in vitro biotransformation study using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of OXY.

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca2+-induced cysteine relative mobility/mitochondrial permeability transition pore

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca2+-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys56 relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca2+-induced ANT conformational change and mitochondrial swelling. ANT-Pro61 isomerization increased ANT-Cys56 relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca2+ induced ANT “c” conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca2+-induced ANT “c” conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca2+ induces the ANT “c” conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.