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Cancer Cell Sensitivity to Redox-Cycling Quinones is Influenced by NAD(P)H: Quinone Oxidoreductase 1 Polymorphism.

ABSTRACT: BACKGROUND:Cancer cell sensitivity to drugs may be associated with disturbed antioxidant enzymes expression. We investigated mechanisms of resistance by using oxidative stress-resistant MCF-7 breast cancer cells (Resox cells). Since nicotinamide adenine dinucleotide phosphate (NAD(P)H): quinone oxidoreductase-1 (NQO1) is modified in tumors and oxidative stress-resistant cells, we studied its role in cells exposed to ?-lapachone, menadione, and doxorubicin. METHODS:Normal mammary epithelial 250MK, MCF-7, and Resox cells were employed. NQO1 expression and enzyme activity were determined by quantitative polymerase chain reaction (RT-PCR), immunoblotting, and biochemical assays. Dicoumarol and gene silencing (siRNA) were used to modulate NQO1 expression and to assess its potential drug-detoxifying role. MTT (3-(4,5-dimethylthia-zolyl-2)-2,5-diphenyltetrazolium bromide) or clonogenic assays were used to investigate cytotoxicity. NQO1 variants, NQO1*1 (wt), and NQO1*2 (C609T), were obtained by transfecting NQO1-null MDA-MB-231 cell line. RESULTS:Resox cells have higher NQO1 expression than MCF-7 cells. In 250MK cells its expression was low but enzyme activity was higher suggesting a variant NQO1 form in MCF-7 cells. MCF-7 and Resox cells are heterozygous NQO1*1 (wt)/ NQO1*2 (C609T). Both NQO1 polymorphism and NQO1 overexpression are main determinants for cell resistance during oxidative stress. NQO1 overexpression increases cell sensitivity to ?-lapachone whereas NQO1*2 polymorphism triggers quinone-based chemotherapies-sensitivity. CONCLUSIONS:NQO1 influences cancer cells redox metabolism and their sensitivity to drugs. We suggest that determining NQO1 polymorphism may be important when considering the use of quinone-based chemotherapeutic drugs.

SUBMITTER: Glorieux C

PROVIDER: S-EPMC6770057 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Cancer Cell Sensitivity to Redox-Cycling Quinones is Influenced by NAD(P)H: Quinone Oxidoreductase 1 Polymorphism.

Glorieux Christophe C Buc Calderon Pedro P

Antioxidants (Basel, Switzerland) 20190902 9

<h4>Background</h4>Cancer cell sensitivity to drugs may be associated with disturbed antioxidant enzymes expression. We investigated mechanisms of resistance by using oxidative stress-resistant MCF-7 breast cancer cells (Resox cells). Since nicotinamide adenine dinucleotide phosphate (NAD(P)H): quinone oxidoreductase-1 (NQO1) is modified in tumors and oxidative stress-resistant cells, we studied its role in cells exposed to β-lapachone, menadione, and doxorubicin.<h4>Methods</h4>Normal mammary e ...[more]

PMID: 31480790

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Project description:NAD(P)H:quinone oxidoreductase type I (NQO1) is a target enzyme for triggered delivery of drugs at inflamed tissue and tumor sites, particularly those that challenge traditional therapies. Prodrugs, macromolecules, and molecular assemblies possessing trigger groups that can be cleaved by environmental stimuli are vehicles with the potential to yield active drug only at prescribed sites. Furthermore, quinone propionic acids (QPAs) covalently attached to prodrugs or liposome surfaces can be removed by application of a reductive trigger stimulus, such as that from NQO1; their rates of reductive activation should be tunable via QPA structure. We explored in detail the recombinant human NAD(P)H:quinone oxidoreductase type I (rhNQO1)-catalyzed NADH reduction of a family of substituted QPAs and obtained high precision kinetic parameters. It is found that small changes in QPA structure-in particular, single atom and function group substitutions on the quinone ring at R(1)-lead to significant impacts on the Michaelis constant (K(m)), maximum velocity (V(max)), catalytic constant (k(cat)), and catalytic efficiency (k(cat)/K(m)). Molecular docking simulations demonstrate that alterations in QPA structure result in large changes in QPA alignment and placement with respect to the flavin isoalloxazine ring in the active site of rhNQO1; a qualitative relationship exists between the kinetic parameters and the depth of QPA penetration into the rhNQO1 active site. From a quantitative perspective, a very good correlation is observed between log(k(cat)/K(m)) and the molecular-docking-derived distance between the flavin hydride donor site and quinone hydride acceptor site in the QPAs, an observation that is in agreement with developing theories. The comprehensive kinetic and molecular modeling knowledge obtained for the interaction of recombinant human NQO1 with the quinone propionic acid analogues provides insight into the design and implementation of the QPA trigger groups for drug delivery applications.

Project description:The effects of quinone-generated active oxygen species on rat hepatocyte protein kinase C were investigated. The specific activity of cytosolic protein kinase C was increased 2-3-fold in hepatocytes incubated with the redox-cycling quinones, menadione, duroquinone or 2,3-dimethoxy-1,4-naphthoquinone, without alterations in particulate protein kinase C specific activity or Ca2+- and lipid-independent kinase activities. Redox-cycling quinones did not stimulate translocation of protein kinase C; however, activated protein kinase C was redistributed from cytosol to the particulate fraction when quinone-treated hepatocytes were exposed to 12-O-tetradecanoylphorbol 13-acetate (TPA). Quinone treatment did not alter cytosolic phorbol 12,13-dibutyrate (PDBu) binding capacity, and the cytosol of both control and quinone-treated hepatocytes exhibited a Kd for PDBu binding of 2 nM. Quinone-mediated activation of cytosolic protein kinase C was reversed by incubation with 10 mM-beta-mercaptoethanol, dithiothreitol or GSH, at 4 degrees C for 24 h. Furthermore, protein kinase C specific activity in control cytosol incubated in air increased by over 100% within 3 h; this increase was reversed by thiol-reducing agents. Similarly, incubation of partially-purified rat brain protein kinase C in air, or with low concentrations of GSSG in the presence of GSH, resulted in a 2-2.5-fold increase in Ca2+- and lipid-dependent kinase activity. In contrast with the effects of the redox-cycling quinones, when hepatocytes were treated with the thiol agents N-ethylmaleimide (NEM), p-benzoquinone (pBQ) or p-chloromercuribenzoic acid (pCMB), the cytosolic Ca2+- and lipid-dependent kinase activity was significantly inhibited, but the particulate-associated protein kinase C activity was unaffected. The Ca2+- and lipid-independent kinase activity of both the cytosolic and particulate fractions was significantly stimulated by NEM, but was unaffected by pBQ and pCMB. These results show that hepatocyte cytosolic protein kinase C is activated to a high-Vmax form by quinone-generated active oxygen species, and this effect is due to a reduction-sensitive modification of the thiol/disulphide status of protein kinase C.

Dataset Information

Cancer Cell Sensitivity to Redox-Cycling Quinones is Influenced by NAD(P)H: Quinone Oxidoreductase 1 Polymorphism.

Publications

Cancer Cell Sensitivity to Redox-Cycling Quinones is Influenced by NAD(P)H: Quinone Oxidoreductase 1 Polymorphism.

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