Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123

Hempel, S. L.; Buettner, Garry R.; O’Malley, Yunxia; Wessels, D. A.; Flaherty, Dawn M.

doi:10.1016/s0891-5849(99)00061-1

Cited by 685 publications

(442 citation statements)

References 31 publications

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“…Increased cellular oxidative stress could be detected after 3h PRC-exposure of G361 melanoma cells and was even more pronounced after 24h treatment as obvious from an approximately fourfold increase in green fluorescence intensity over untreated control cells exposed to DCFH-DA only. The exact molecular nature of the oxidizing species involved in PRC-induced cytotoxicity was not examined at this point, and various ROS and other reactive species including PRC metabolites could participate in the intracellular oxidation of the nonfluorescent precursor dye [40]. However, H 2 O 2 is a likely candidate mediator of PRCinduced intracellular oxidative stress, a hypothesis consistent with earlier reports that document H 2 O 2 formation by PRC redox cycling [26].…”

Section: Prc-induced Intracellular Oxidative Stress Loss Of Mitochonsupporting

confidence: 56%

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 Mitochonsupporting

confidence: 56%

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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“…40 It is of interest that DCFH is readily oxidized by superoxide formation whereas DHR shows little reactivity to this species. 39 EGCG had an antiproliferative effect on unirradiated HaCaT cells as expressed by a fall in cell survival upon subsequent plating. EGCG has been reported to exert a growth inhibitory effect on a number of tumour cell lines 7,8,41 and on immortalized but non-tumorigenic bronchial epithelial cells.…”

Section: Discussionmentioning

confidence: 94%

“…There is an increasing trend to use DCFH or DHR oxidation as evidence of H 2 O 2 production 7,37 and although the probes do react with peroxide, they are sensitive to a variety of other free radical species. 39 The fact that there is an increase in DCFH oxidation in UVA-irradiated, EGCGtreated cells suggests that DCFH may react with reactive species generated as a result of the oxidation of EGCG through scavenging UVA ROS or with an EGCG radical itself. Although the scavenging mechanisms for EGCG are still poorly understood, there is recent evidence pointing to the production of superoxide (O 2Ϫ ) by EGCG in the process of scavenging peroxyl radicals.…”

Section: Discussionmentioning

confidence: 99%

The green tea polyphenol, epigallocatechin‐3‐gallate, protects against the oxidative cellular and genotoxic damage of UVA radiation

Tobi

Gilbert

Paul

et al. 2002

Intl Journal of Cancer

101

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A number of biological activities have been ascribed to the major green tea polyphenol epigallocatechin-3-gallate (EGCG) to explain its chemopreventive properties. Its antioxidant properties emerge as a potentially important mode of action. We have examined the effect of EGCG treatment on the damaging oxidative effects of UVA radiation in a human keratinocyte line (HaCaT). Using the ROS-sensitive probes dihydrorhodamine 123 (DHR) and 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA), we detected a reduction in fluorescence in UVA-irradiated (100 kJ/m 2 ) cells in the case of the former but not the latter probe after a 24-hr treatment with EGCG (e.g., 14%, [p < 0.05] after 10 M EGCG). In the absence of UVA, however, both DHR and DCFH detected a pro-oxidant effect of EGCG at the highest concentration used of 50 M. Measurements of DNA damage in UVA-exposed cells using the single cell gel electrophoresis assay (comet assay) also showed the protective effects of EGCG. A concentration of 10 M EGCG decreased the level of DNA single strand breaks and alkali-labile sites to 62% of the level observed in non-EGCG, irradiated cells (p < 0.001) with a 5-fold higher concentration producing little further effect. Correspondingly, EGCG ablated the mutagenic effects of UVA (500 kJ/m 2 ) reducing an induced hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant frequency of (3.39 ؎ 0.73) ؋ 10 ؊6 to spontaneous levels (1.09 ؎ 0.19) ؋ 10 ؊6 . Despite having an antiproliferative effect in the absence of UVA, EGCG also served to protect against the cytotoxic effects of UVA radiation. Our data demonstrate the ability of EGCG to modify endpoints directly relevant to the carcinogenic process in skin. © 2002 Wiley-Liss, Inc. Key words: epigallocatechin-3-gallate; antioxidant; ultraviolet AThere has been considerable recent interest in the chemopreventive properties of the polyphenols or catechins of green tea that have been shown to inhibit tumorigenesis in a variety of organs in rodent models. 1-3 The major polyphenol component by mass, (Ϫ)-epigallocatechin-3-gallate (EGCG) is an effective protectant against the mutagenicity of a number of chemical carcinogens including benzo[a]pyrene (B[a]P), aflatoxin B1 and 3-hydroxyamino-1-methyl-5H-pyrido [4,3-b]indole. 4 -6 EGCG, together with other green tea polyphenols also exhibits growth inhibitory properties in a variety of tumour cell lines. 7-9 Several mechanisms have been proposed by which these compounds exert their anti-tumorigenic action: these include the blockade of growth factors binding to their receptors, 10 phosphorylation (activation) of mitogen-activated protein kinases (MAPKs) 11,12 possibly resulting in the observed induction of Phase II drug-metabolizing enzymes, 13,14 and the generation of oxidative stress leading to apoptosis. 7,9 In the face of an oxidative challenge, however, it is now well documented that green tea catechins act as antioxidants. For example, EGCG abrogates oxidation by hydrogen peroxide both in a cell free system 15 and in terms of DNA single-s...

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“…The fluorogenic substrate DCFH-DA was used to monitor intracellular generation of ROS (Hempel et al, 1999). As positive control, cells were treated with 100 mM t-BOOH (15 min, 371C).…”

Section: Intracellular Rosmentioning

confidence: 99%

Regulation of normal cell cycle progression by flavin-containing oxidases

Prakash¹,

Toledo²,

Pandey³

et al. 2007

Oncogene

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Mechanisms underlying the role of reactive oxygen species (ROS) generated by flavin-containing oxidases in regulating cell cycle progression were examined in human and rodent fibroblasts. Incubation of confluent cell cultures with nontoxic/nonclastogenic concentrations of the flavoprotein inhibitor, diphenyleneiodonium (DPI), reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity and basal ROS levels, but increased proteolysis of cyclin D1, p21 Waf1 and phospho-p38 MAPK . When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G 1 delay was observed with concomitant activation of p53/p21 Waf1 signaling and reduced phosphorylation of mitogen-activated kinases. Compensation for decreased oxidant generation by simultaneous exposure to DPI and nontoxic doses of the ROS generators, c-radiation or t-butyl-hydroperoxide, attenuated the G 1 delay. Whereas the DPI-induced G 1 checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G 1 to S progression was not affected when another flavin-containing enzyme, nitric oxide synthase, was inhibited nor was it associated with changes in mitochondrial membrane potential. Proliferating cells treated with DPI also experienced a significant but attenuated delay in G 2 . We propose that ATM performs a critical function in mediating normal cellular proliferation that is regulated by nonphagocytic NAD(P)H oxidase enzymes activity, which may serve as a novel target for arresting cancer cells in G 1 .

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Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′,7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123

Cited by 685 publications

References 31 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

The green tea polyphenol, epigallocatechin‐3‐gallate, protects against the oxidative cellular and genotoxic damage of UVA radiation

Regulation of normal cell cycle progression by flavin-containing oxidases

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