A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid

Auerbach, Bruce J.; Kiely, John S.; Cornicelli, Joseph A.

doi:10.1016/0003-2697(92)90354-a

Cited by 98 publications

(74 citation statements)

References 7 publications

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“…Similar results with regard to caspase activation, mitochondrial cytochrome C release, and induction of intracellular oxidative stress were obtained, when G361 melanoma cells were exposed to 25 μM MB (data not shown). Moreover, no induction of cellular oxidative stress and apoptosis were observed, when G361 melanoma cells were exposed to N-benzoyl-leucomethylene blue, a redox inactive leuco-derivative of MB [41]. Taken together, these data provide evidence for a mechanism of PRC-induced melanoma cell apoptosis that depends on the initial generation of cellular oxidative stress by formation of ROS that triggers the mitochondrial pathway of apoptosis with cytochrome C release and activation of caspase-3.…”

Section: Prc-induced Intracellular Oxidative Stress Loss Of Mitochonmentioning

confidence: 75%

NQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apoptosis

Wondrak

2007

Free Radical Biology and Medicine

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Altered redox signaling and regulation in cancer cells represent a chemical vulnerability that can be targeted by selective chemotherapeutic intervention. Here, we demonstrate that 3,7-diaminophenothiazinium-based redoxcyclers (PRC) induce selective cancer cell apoptosis by NAD (P)H:quinone oxidoreductase (NQO1)-dependent bioreductive generation of cellular oxidative stress. Using PRC lead compounds including toluidine blue against human metastatic G361 melanoma cells, apoptosis occurred with phosphatidylserine-externalization, loss of mitochondrial transmembrane potential, cytochrome C release, caspase-3 activation, and massive ROS production. Consistent with reductive activation and subsequent redoxcycling as the mechanism of PRC cytotoxicity, co-incubation with catalase achieved cell protection, whereas reductive antioxidants enhanced PRC-cytotoxicity. Unexpectedly, human A375 melanoma cells were resistant to PRCinduced apoptosis, and PRC-sensitive G361 cells were protected by preincubation with the NQO1-inhibitor dicoumarol. Indeed, NQO1 specific enzymatic activity was nine fold higher in G361 than in A375 cells. The critical role of NQO1 in PRC-bioactivation and cytotoxicity was confirmed, when NQO1-transfected breast cancer cells (MCF7-DT15) stably overexpressing active NQO1 displayed strongly enhanced PRC-sensitivity as compared to vector-control transfected cells with base line NQO1 activity. Based on the known overexpression of NQO1 in various tumors these findings suggest the feasibility of developing PRC lead compounds into tumor-selective bioreductive chemotherapeutics.

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Section: Prc-induced Intracellular Oxidative Stress Loss Of Mitochonmentioning

confidence: 75%

NQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apoptosis

Wondrak

2007

Free Radical Biology and Medicine

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“…Lipid hydroperoxides were determined using a spectrophotometric assay (21). Paraoxonase activity was measured as described earlier (22).…”

Section: Methodsmentioning

confidence: 99%

ApoA-I Mimetic Peptides with Differing Ability to Inhibit Atherosclerosis Also Exhibit Differences in Their Interactions with Membrane Bilayers

Handattu

Garber

Horn

et al. 2007

Journal of Biological Chemistry

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Two homologous apoA-I mimetic peptides, 3F-2 and 3F 14 , differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404 -51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 g/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 ؎ 1000 m 2 , n ‫؍‬ 29), whereas 3F 14 does not (lesion area 20,400 ؎ 1000 m 2 , n ‫؍‬ 26) compared with control saline administered (19,900 ؎ 1400 m 2 , n ‫؍‬ 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F 14 preferentially associates with apoB-containing particles. After intraperitoneal injection of 14 C-labeled peptides, 3F 14 reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F 14 . In contrast, only 3F 14 affects the terminal methyl group of the acyl chain, decreasing the 2 H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F 14 can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.

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“…The conversion of benzoyl-leuco-methylene blue by 13-HPOD according to Reaction 1 requires hemoglobin as a catalyst [11]. The direct reduction of hemoglobin at electrode surfaces is reported to occur at À 0.05 to À 0.3 V depending on the pH and the electrode used [17,18].…”

Section: Resultsmentioning

confidence: 99%

“…We discovered that hemoglobin is electroactive at the platinum electrodes utilized by us near the potential for reduction of MB (À 0.22 V), masking the peak of interest. Given that the catalytic activity of myoglobin and hemoglobin is similar [11], we have used myoglobin to replace hemoglobin as a catalyst in this reaction. As myoglobin did not demonstrate any electroactivity in the potential window of interest (0.1 to À 0.4 V), yet acts as a catalyst for Reaction 1.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, an indirect UV-visible spectrophotometric assay has been reported for the assay of lipoxygenase inhibitors [11]. This method is a variation of previous methods [12,13] and relies upon the detection at 660 nM of methylene blue, formed by the catalytic action of hydroperoxide, the product of the lipoxygenase reaction, on benzoyl-leuco methylene blue according to Reaction 1:…”

Section: Introductionmentioning

confidence: 99%

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A Voltammetric Assay of Antioxidants and Inhibitors of Soybean Lipoxygenase

Pérusse

Leech

2003

Electroanalysis

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Antioxidant activity and/or the inhibition of lipoxygenases may contribute to the cell and tissue protective properties of flavonoids. Soybean lipoxygenase-1 (LOX-1), is a useful paradigm for lipoxygenases isolated from different sources, and can be used to devise assays of oxygenase inhibitors and antioxidants. Here we describe the voltammetric detection of methylene blue (MB) to assay for antioxidant and lipoxygenase inhibition activity. MB is formed by the catalytic action of hydroperoxide, produced by soybean lipoxygenase reaction on linoleic acid, on benzoyl-leuco methylene blue (BLMB). Every compound which blocks either the enzymatic reaction, lipoxygenase inhibitors, or the oxidation of BLMB, antioxidants, can thus be detected using this method. The assay involves reaction for a period of one hour of linoleic acid, lipoxygenase, BLMB and inhibitor, followed by injection into a flow-injection system and detection by square-wave voltammetry of the methylene blue produced. The problem of irreproducibility induced by adsorption at the solid platinum working electrode is alleviated using the method of alternate injections of control and assay solutions. Assays of several known lipoxygenase inhibitors were undertaken. All of these inhibitors were also, however, shown to be antioxidants. Extension of the assay to the rapid evaluation of novel therapeutic antioxidants and lipoxygenase inhibitors is proposed.

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A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid

Cited by 98 publications

References 7 publications

NQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apoptosis

NQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apoptosis

ApoA-I Mimetic Peptides with Differing Ability to Inhibit Atherosclerosis Also Exhibit Differences in Their Interactions with Membrane Bilayers

A Voltammetric Assay of Antioxidants and Inhibitors of Soybean Lipoxygenase

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