Chinpal Kim

ECTO-NOX target for the anticancer isoflavene phenoxodiol.

Phenoxodiol, a synthetic isoflavene with clinical efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated tNOX. Purified recombinant tNOX bound phenoxodiol with high affinity (Kd of 50 nM). The tNOX protein appeared to be both necessary and sufficient for the cancer-specific cytotoxicity of phenoxodiol. Growth inhibition of fibroblasts from embryos of mice expressing a tNOX transgene, but not from wild-type mice, was inhibited by phenoxodiol followed by apoptosis. Both the oxidative and protein disulfide-thiol interchange activities that alternate to generate the complex set of oscillations with a period length of 22 min (24 min for the constitutive counterpart CNOX) that characterize ECTO-NOX proteins respond to phenoxodiol. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked by phenoxodiol. In contrast, the protein disulfidethiol interchange activity measured either by the restoration of activity to scrambled and inactive RNase or from the cleavage of dithiodipyridine (EC50 of 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ECTO-NOX (CNOX) forms of either cancer or noncancer cells were unaffected by phenoxodiol to help explain how the cytotoxic effects of phenoxodiol may be restricted to cancer cells.

IS THE DRUG-RESPONSIVE NADH OXIDASE OF THE CANCER CELL PLASMA MEMBRANE A MOLECULAR TARGET FOR ADRIAMYCIN?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC50 for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.

Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients

Abstract. Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide–thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl-N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide–thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.

Purification and Characterization of a Doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) Reductase from Rat Liver Plasma Membranes

Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochromeb 5 reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochromeb 5 reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q0 reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5..) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q0 reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.

Is the cancer protective effect correlated with growth inhibitions by green tea (−)-epigallocatechin gallate mediated through an antioxidant mechanism?

The preferential inhibition by (-)-epigallocatechin gallate (EGCg) of growth of cancer cells (e.g. HeLa) in culture correlates with the ability of EGCg to inhibit a growth-related, cell surface hydroquinone oxidase with protein disulfide-thiol interchange activity (tNOX) measured as an NADH oxidase and specifically associated with tumorigenically-transformed cells and tissues. tNOX is reduced or absent from the surface of non-cancer cells. Various oxidizing conditions known to render other antioxidants such as thiols, ascorbate and vitamin E ineffective did not reduce the effectiveness of EGCg in inhibiting either the tNOX activity or the growth of HeLa cells. Only after Fenton reaction with iron catalysis in the presence of hydrogen peroxide was the effectiveness of the EGCg reduced. We conclude that it is unlikely that the anticancer action of green tea EGCg on the tNOX protein is mediated through antioxidant properties of EGCg.

Cancer Site-Specific Isoforms of ENOX2 (tNOX), A Cancer-Specific Cell Surface Oxidase

IntroductionAll neoplastic cells express one or more members of a unique family of tumor-associated cell surface ubiquinone (NADH) oxidase proteins with protein disulfide-thiol interchange activity (ENOX2 or tNOX proteins) that are characteristically blocked by quinone site inhibitors with anti-cancer activity.MethodsAnalyses using two-dimensional gel electrophoresis with detection on western blots using a pan ENOX2 recombinant antibody revealed unique ENOX2 isoforms or unique combinations of isoforms of differing molecular weights and/or isoelectric points in sera of patients with cancers of different cellular or tissue origins.Results and DiscussionIsoform presence provides for broad-range cancer detection. The specific patterns and molecular weights of the isoforms present allows for identification of the cell type and/or tissue of origin of the neoplasm. ENOX2 isoform presence and relative amounts are largely independent of stage but may be proportional to tumor burden to provide indications of response to therapy and disease progression.

STRUCTURAL CHANGES REVEALED BY FOURIER TRANSFORM INFRARED AND CIRCULAR DICHROISM SPECTROSCOPIC ANALYSES UNDERLIE tNOX PERIODIC OSCILLATIONS

A recurring pattern of spectral changes indicative of periodic changes in the proportion of β-structure and a-helix of a recombinant ECTO-NOX fusion protein of tNOX, with a cellulose binding domain peptide, was demonstrated by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic analyses. The pattern of structural changes correlated with oscillatory patterns of enzymatic activities exhibited by the protein previously interpreted as indicative of a clock function. The pattern consisted of a repeating pattern of oscillations with a period length of 21 min with five maxima (two separated by 5 min and 3 separated by 4 to 4.5 min) within each 21 min repeat. Oscillatory patterns were not obvious in comparable FTIR or CD spectra of albumin, ribonuclease or concanavalin A. The period length was constant at 5, 15, 25, 35 and 45° C (temperature compensated) and oscillations occurred independently of substrate presence. Spectra obtained in deuterium oxide yielded a longer period length of 26 min both for oscillations in enzymatic activity and absorbance ratios determined by FTIR. Taken together the findings suggest that the regular patterns of oscillations exhibited by the ECTO-NOX proteins are accompanied by recurrent global changes in the conformation of the protein backbone that directly modulate enzymatic activity.