Plasma Membrane NADH Oxidase Research Articles

The plasma membrane NADH oxidase of soybean has vitamin K 1 hydroquinone oxidase activity

Isolated plasma membrane vesicles and the plasma membrane NADH oxidase partially purified from soybean plasma membrane vesicles exhibited a cyanide-insensitive vitamin K 1 hydroquinone oxidase activity with isolated plasma membrane vesicles. Reduced vitamin K 1 (phylloquinol) was oxidized at a rate of about 10 nmol/min/mg protein as determined by reduced vitamin K 1 reduction or oxygen consumption. The K m for reduced K 1 was 350 μM. With the partially purified enzyme, reduced vitamin K 1 was oxidized at a rate of about 600 nmol/min/mg protein and the K m was 400 μM. When assayed in the presence of 1 mM KCN, activities of both plasma membrane vesicles and of the purified protein were stimulated (0.1 μM) or inhibited (0.1 mM) by the synthetic auxin growth factor 2,4-dichlorophenoxyacetic acid. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via reduced vitamin K 1 to acceptors (molecular oxygen or protein disulfides) at the cell surface.

Plasma membrane NADH oxidase is gravi-responsive

NADH oxidase activities measured with intact tissue sections and with isolated plasma membrane vesicles from etiolated hypocotyls of soybean (Glycine max) respond to gravity and imposed centrifugal forces. The response is one of inhibition of activity with tissue sections lying flat for 20 min or less at 1 × g and one of stimulation with times of lying flat of 30 min or longer at 1 × g. Turning the tissue sections upside down resulted in stimulation of the activity with a lag of about 30 min. Returning the sections to the normal upright position resulted in a return to initial rates with a lag of less than 20 min. Both the stimulated and non-stimulated activities oscillate with a period of 24 min, precluding a more precise analyses of lag times. The activity was stimulated reversibly to a maximum of about 2-fold both in tissue sections and in isolated plasma membrane vesicles when subjected to centrifugal forces of 10 to 400 × g for 0.5 to 4 min duration. The findings are the first description of a gravi-responsive enzymatic activity related to the growth response in plants.

Plasma membrane NADH oxidase is gravi-responsive

Plasma membrane NADH oxidase is gravi-responsive

The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity

The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q 10 (Q 10H 2=ubiquinol) was oxidized at a rate of 15±6 nmol min −1 mg protein −1 depending on degree of purification. The apparent K m for Q 10H 2 oxidation was 33 μM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY181984). With coenzyme Q 0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q 0 and Q 0H 2 forms. With the mixture, a rate of Q 0H 2 oxidation of 8–17 nmol min −1 mg protein −1 was observed with an apparent K m of 0.22 mM. The rate of Q 10H 2 oxidation was not stimulated by addition of equal amounts of Q 10 and Q 10H 2. However, addition of Q 0 to the Q 10H 2 did stimulate. The oxidation of Q 10H 2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q 0H 2 may involve Q 0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.

Glaucarubolone and Simalikalactone D, Respectively, Preferentially Inhibit Auxin‐Induced and Constitutive Components of Plant Cell Enlargement and the Plasma Membrane NADH Oxidase

Glaucarubolone, a naturally occurring quassinoid from the root bark of Castela polyandra, Simaroubaceae, with a C(8), C(11) hemiacetal bridge, is demonstrated to be a potent inhibitor of both the auxin‐induced component of the plasma membrane NADH oxidase and of plant cell enlargement in soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), tomato (Lycopersicum esculentum), and Sorghum (Sorghum vulgare). Auxin‐stimulated NADH oxidase activity of isolated vesicles of soybean, A. thaliana, and tomato plasma membranes were inhibited half maximally by ca. 0.1 nM glaucarubolone. Auxin‐induced enlargement of stem segments of soybean and elongation growth of A. thaliana seedlings were inhibited half maximally by ca. 10 nM glaucarubolone with significant inhibition of auxin‐stimulated growth even at nannomolar glaucarubolone concentrations. Seedlings of A. thaliana and tomato treated with sublethal concentrations of 1–10 μM glaucarubolone remained alive but failed to elongate for periods of two to several months. Once seedlings recovered from the effects of the inhibitor, they resumed growth, flowered, and produced fruits. Treated A. thaliana produced viable seeds that germinated and developed into normal‐appearing progeny. In contrast to glaucarubolone, which inhibited preferentially auxin‐induced growth and plasma membrane NADH oxidase, simalikalactone D, a quassinoid with a C(8), C(13) epoxymethano bridge, inhibited the constitutive NADH oxidase activity and growth and had little or no effect on the 2,4‐D‐responsive NADH oxidase and growth. The findings provide correlative evidence for a functional role of both the constitutive and the auxin‐stimulated plasma membrane NADH oxidase activities in plant cell enlargement.

Annonaceous acetogenins: recent progress.

The Annonaceous acetogenins are promising new antitumor and pesticidal agents that are found only in the plant family Annonaceae. Chemically, they are derivatives of long-chain fatty acids. Biologically, they exhibit their potent bioactivities through depletion of ATP levels via inhibiting complex I of mitochondria and inhibiting the NADH oxidase of plasma membranes of tumor cells. Thus, they thwart ATP-driven resistance mechanisms. This review presents the progress made in the chemistry, biology, and development of these compounds since December 1995.

Mode of action of the anticancer quassinoids — Inhibition of the plasma membrane NADH oxidase

A plasma membrane-associated NADH oxidase of transformed cells was shown to be inhibited by nanomolar and subnanomolar concentrations of the antitumor quassinoid, glaucarubolone. The inhibition was seen with plasma membrane vesicles of HeLa cells at two log orders less glaucarubolone than with plasma membrane vesicles of rat liver. Assignment of a drug-binding site to the external surface of the HeLa cell plasma membrane was supported by findings where full activity of the glaucarubolone in the inhibition of NADH oxidase activity of isolated plasma membrane vesicles and of growth of HeLa cells was given on a molar glaucarubolone basis by an impermeant conjugate of glaucarubolone in which the glaucarubolone moiety was linked via the C-15 hydroxyl to amino polyethyleneglycol (ave Mr 5,000). The activity of the conjugate, and to a lesser extent, of free glaucarubolone was modulated by the redox environment of the cells and of the plasma membrane vesicles. Activity, both in the inhibition of NADH oxidase activity and in the Inhibition of growth, was enhanced by oxidizing conditions in the presence of oxidized glutathione compared to reducing conditions in the presence of reduced glutathione.

Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1

Plasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase, the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADH-oxidase also inhibited WST-1 reduction, but the impermeative SH-blocking reagentpara-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils.

Transferrin and transferrin receptor gene expression and iron uptake in hepatocellular carcinoma in the rat.

Iron plays an important role in cell growth and metabolism. In preneoplastic liver nodules, a rise in the number of transferrin receptors (Tf-R) is associated with decreased endocytosis of the Fe2-Tf/Tf-R complex. Because nodules are precursors of hepatocellular carcinoma (HCC), the question arises whether changes in iron uptake by nodules persist in HCC. Current work showed up-regulation of Tf messenger RNA (mRNA) production in preneoplastic nodules, 12 to 37 weeks after initiation, and down-regulation in atypical nodules (at 45 and 50 weeks) and HCCs, induced in rats by the "resistant hepatocyte" model. Tf-R gene expression increased in nodules and HCCs. Tf-R numbers increased, without changes in affinity constant, in HCC. Iron uptake was higher in HCC than in normal liver, 5 to 40 minutes after injection of 59Fe2-Tf, with preferential accumulation in cytosol of tumor cells and in microsomes of normal liver. Purification through Percoll gradient of mitochondria plus lysosomes allowed the identification in liver and HCC of an endosomal compartment sequestering injected 125I-Tf. This subfraction was not seen when 59Fe2-Tf was injected into rats, and 59Fe was found in particulate material of both tissues. Liver and HCC exhibited comparable basal activities of plasma membrane NADH oxidase, an enzyme involved in iron uptake and cell growth. Stimulation of this activity by Fe2-Tf was higher in HCC than in normal liver. These results indicate that Tf expression may be a marker of preneoplastic liver progression to malignancy. Differently from nodules, HCC may sequester relatively high iron amounts, necessary for fast growth, both through the endocytic pathway and the reduced form of nicotinamide adenine dinucleotide (NADH) oxidase system.

NADH oxidase activity of soybean plasma membranes inhibited by submicromolar concentrations of ATP.

The activity of an auxin-stimulated NADH oxidase activity from soybean hypocotyls was inhibited by submicromolar concentrations of ATP. Auxins are plant growth regulators that increase the rate of cell enlargement in plant stems. A synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was used. The inhibition was half maximal at 1 nM ATP and was not observed with other nucleotides and nucleosides. The inhibition was the result of an increase in the Km for NADH from about 60 microM to > 100 microM and was noncompetitive. The decrease in Km due to ATP was enhanced by the addition of 1 microM 2,4-D. The Vmax of the plasma membrane NADH oxidase was approximately doubled (1.5-2.8-fold) by ATP and by 1 microM 2,4-D. No further increase in the Vmax was observed by the combination of 1 nM to 0.1 mM ATP in the presence of 1 microM 2,4-D. The results demonstrate a response of the NADH oxidase activity of isolated vesicles of soybean plasma membranes to ATP distinct from that observed previously with other nucleotide di- and triphosphates. The results are suggestive either of control of the cell surface NADH oxidase by phosphorylation or a direct response to ATP binding at nanomolar concentrations of ATP.

Relationship between IAA-induced elongation growth and plasma membrane NADH oxidase in soybean (Glycine max Merr.) hypocotyls

A relationship between the activity of NADH oxidase of the plasma membrane and the IAA-induced elongation growth of hypocotyl segments in etiolated soybean (Glycine max Merr.) seedlings was investigated. The plasma membrane NADH oxidase activity increased in parallel to IAA effect on elongation growth in hypocotyl segments. Actually, NADH oxidase activity was stimulated 3-fold by 1 u,M IAA, and the elongation rate of segments was stimulated 10-fold by 10 iM IAA. The short-term elongation growth kinetics, however, showed that the IAA-induced elongation of hypocotyl segments was completely inhibited by plasma membrane redox inhibitors such as actinomycin D and adriamycin, at 80 μM and 50 μM respectively. In addition, 1 mM actinomycin D inhibited the IAA-stimulated NADH oxidase activity by about 80%. However, adriamycin had no effect on NADH oxidase activity of plasma membrane vesicles. Based on these results, the plasma membrane redox reactions seemed to be involved in IAA-induced elongation growth of hypocotyls, and the redox component responding to IAA was suggested to be NADH oxidase.

Impermeant antitumor sulfonylurea conjugates that inhibit plasma membrane NADH oxidase and growth of HeLa cells in culture. Identification of binding proteins from sera of cancer patients

The antitumor sulfonylurea LY237868 ( N-(4-aminophenyl-sulfonyl)- N′-(4-chlorophenyl)urea) was conjugated through the A ring to α-cyclodextrin or agarose bead material (Affigel 10) to prepare impermeant conjugates for activity measurements and affinity isolation of binding proteins from serum. When conjugated to α-cyclodextrin, the resulting LY237868 conjugate inhibited both NADH oxidase activity and growth of HeLa cells in culture. The conjugate was at least one order of magnitude more potent as an inhibitor than the parent compound. These findings confirm previous results that demonstrate an antitumor sulfonylurea-binding protein with NADH oxidase activity at the external plasma membrane surface of HeLa cells that is shed into culture media conditioned by growth of HeLa cells. A comparable activity, responsive to sulfonylurea, was present in sera of cancer patients. LY237868 conjugated to agarose beads as the affinity support bound a large number of serum proteins. However, compared to serum from normal patients, the affinity support bound two proteins of M r approx. 33.5 and 29.5 not found in sera of normal patients. The 33.5 kDa protein from human sera reacted with antisera to a 33.5 kDa protein from culture media conditioned by growth of HeLa cells that blocked and immunoprecipitated the sulfonylurea-responsive activity from HeLa cell plasma membranes. The results point to the 33.5 kDa protein from cancer patient sera that bound to the sulfonylurea affinity support as representing the circulating equivalent of the previously identified 34 kDa sulfonylurea-binding protein, with NADH oxidase activity at the external cell surface of cultured HeLa cells and a corresponding 33.5 kDa protein shed into culture media conditioned by growth of HeLa 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.

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids.

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10(-8) to 10(-5) M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 microM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 microM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture.

Isolation and Identification of a Protein with Capsaicin-Inhibited NADH Oxidase Activity from Culture Media Conditioned by Growth of HeLa Cells

A ca. 33.5-kDa protein has been identified as a soluble NADH oxidase activity of culture media conditioned by growth of HeLa cells. The protein appears to be derived from a 34-kDa protein of the HeLa plasma membrane. Both proteins are characterized by an ability to oxidize NADH in the absence of exogenous electron acceptors. The activity is inhibited by 8-methyl-N-vanillyl-6-noneamide (capsaicin). The soluble and the plasma membrane forms of the activity exhibit a similar EC50of about 5 nMfor inhibition of the activity by capsaicin. The activity was purified from culture media conditioned by growth of HeLa cells using DEAE ion exchange chromatography, G-200 size exclusion chromatography, and preparative SDS–PAGE. Purification was monitored on the basis of the capsaicin-inhibited oxidation of NADH, including the final electrophoretic purification. Activity was restored following SDS–PAGE by reduction with dithiothreitol or reduced glutathione in the presence of NADH followed by the addition of 0.03% hydrogen peroxide and preincubation in the presence of NADH for 5–15 min. For affinity purification, the vanillylamine portion of capsaicin was linked to agarose. The agarose-linked vanillylamine bound a ca. 33.5-kDa protein band with capsaicin-inhibited NADH activity from total defined culture media conditioned by growth of HeLa cells. The NADH oxidase activity of both the soluble and the plasma membrane-associated form of the activity was inhibited by antisera corresponding to the 33.5-kDa protein. The antisera also immunoprecipitated and reacted on Western blots with both the soluble (33.5 kDa) and plasma membrane (34 kDa)-associated forms of the capsaicin-inhibited activity. The results identify the capsaicin-inhibited NADH oxidase of the conditioned media of HeLa cells as being a ca. 33.5-kDa shed form of the previously reported capsaicin-inhibited NADH oxidase of the HeLa cell plasma membrane.

Capsaicin inhibits plasma membrane NADH oxidase and growth of human and mouse melanoma lines

Hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane is thought to be involved in the control of normal cell proliferation. The aim of this study was to determine the effect of the naturally occurring quinone analogue capsaicin (8-methyl-N-vanillyl-6-noneamide) on the NADH oxidase activity of plasma membranes and cell growth of human primary melanocytes, the A-375 and SK-MEL-28 human melanoma cell cultures. NADH oxidase activity was inhibited preferentially in the A-375 melanoma cells but not in the primary melanocytes, by capsaicin. Inhibition of growth and the NADH oxidase by capsaicin could be induced in resistant SK-MEL-28 melanoma cells by co-administration of capsaicin with t-butyl hydroperoxide, a mild oxidising agent. Death of the inhibited cells was accompanied by nuclear changes suggestive of apoptosis. With B16 mouse melanoma, capsaicin inhibited both the NADH oxidase activity and growth in culture. Growth of B16 melanoma, transplanted in C57BL/6 mice, was significantly inhibited by capsaicin injected directly into the tumour site when co-administered with t-butyl hydroperoxide. The findings correlate the inhibition of cell surface NADH oxidase activity with inhibition of growth and capsaicin-induced apoptosis, and also suggest that the extent of inhibition may relate to the oxidation state of the plasma membrane.

NADH oxidase activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY 181984) at an external site

NADH oxidase activity from HeLa plasma membranes was inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′(4-chlorophenyl)urea (LY181984). With sealed right side-out vesicles, the drug inhibited half maximally at about 30 nM and the inhibition was nearly complete. A closely related but growth-inactive sulfonylurea, N-(4-methylphenylsulfonyl)- N′-(phenyl)urea (LY181985), did not inhibit the activity. With plasma membranes first solubilized with 2% Triton X-100, activity also was inhibited by LY181984 and not by LY181985 but the maximum inhibition at 10 μM LY181984 was only 50%. When sealed right side-out plasma membrane vesicles were frozen and thawed repeatedly to evert some of the vesicles into an inside-out configuration, the NADH oxidase activity again was only about 50% inhibited by 1 μM LY181984. In such preparations, the right side-out vesicles exhibited an electrophoretic mobility greater than that of the inside-out vesicles. Sidedness was confirmed by measurements of ATPase latency and binding of immunogold-labeled concanavalin A. When the two vesicle populations were resolved by preparative free-flow electrophoresis, the active antitumor sulfonylurea LY181984 inhibited only the NADH oxidase activity of the right side-out vesicles. These findings suggested two NADH sites or activity isoforms for the plasma membrane NADH oxidase. One activity, inhibited by LY181984, appeared to be accessible to external NADH only with sealed right side-out vesicles. The other, not inhibited by LY181984, was accessible to NADH only with inside-out vesicles or after membrane disruption by Triton X-100. The findings demonstrate that the NADH oxidation site inhibited as a result of binding the active antitumor sulfonylurea LY181984 is at the external cell surface. Plasma membrane vesicles from HeLa cells are able to oxidize NADH supplied to either membrane surface but only with inside-out vesicles is NADH oxidation sensitive to inhibition by the antitumor sulfonylurea.

Chlorsulfuron blocks 2,4-D-induced cell enlargement and NADH oxidase in excised sections of soybean hypocotyls

Elongation of soybean hypocotyl sections induced by the auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was blocked by micromolar concentrations of the sulfonylurea herbicide chlorsulfuron (2-chloro- N-[[(4-methoxy-6-methyl-1,3,5-trizin-2yl)amino]carbonyl]benzenesulfonamide). The inhibition at equimolar concentrations of 2,4-D and chlorsulfuron was overcome by a 10- or 100-fold excess of 2,4-D. Stimulation by 2,4-D of an 2,4-D-responsive NADH-oxidase activity of the soybean plasma membrane also was blocked by the presence of concentrations of chlorsulfuron equimolar to the 2,4-D. Chlorsulfuron alone was largely without effect on either cell elongation or the auxin-stimulated NADH-oxidase activity over a range of chlorsulfuron concentrations. The results show a clear correlation between inhibition of auxin-stimulated cell elongation and the inhibition of an auxin-stimulated component of a plasma membrane NADH-oxidase activity.

Determination of structure-activity relationships of Annonaceous acetogenins by inhibition of oxygen uptake in rat liver mitochondria

A new group of natural compounds, the Annonaceous acetogenins, have recently been determined to inhibit ATP production at a similar site of action and higher levels of potency as rotenone, i.e., at NADH-ubiquinone oxido-reductase, complex I of the mitochondrial electron-transport chain. The acetogenins had earlier been determined to be pesticidal, antimalarial, antimicrobial, anti-parasitic, cytotoxic, and in vivo active as potentially new antitumor agents. In order to determine structural activity relationships (SARs) among these compounds, at the subcellular level, several available acetogenins have been tested. Data obtained , from the inhibition of oxygen consumption by rat liver mitochondria, demonstrated that all of the twenty acetogenins tested are active with IC 50 values in the range of 15–800 nM/mg protein. The IC 50 value of rotenone was 17 nM/mg protein. The bis-adjacent THF ring acetogenins and the bis-nonadjacent THF ring compounds are about ten times more active than the mono-THF ring acetogenins. Overall, 30-OH and 31-OH-bullatacinone were the most active and were slightly more active than rotenone. The least active were the 4-deoxy bis-adjacent THF ring compounds followed by the mono-THF ring group. There was some variation between the groups, e.g., within the bis-adjacent and mono-THF ring groups, the α,β-unsaturated-γ-lactones were less active than the keto-lactones, but this observation was reversed for one of the pairs of bis-nonadjacent THF ring acetogenins. Additional hydroxylations, to a maximum of three, seemed to increase activity within all of the groups. Before final decisions on SARs can be made, additional comparisons of the results of this subcellular assay (as an in vitro assay) with the results of in vivo assays should be made. Also, future investigations into the exact site of action within complex I and other possible sites of action (such as the NADH oxidase of plasma membranes) need to be conducted for a more complete understanding of the utility and potential of this new group of very potent compounds.

Capsaicin inhibits preferentially the NADH oxidase and growth of transformed cells in culture.

A hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells, all of human origin, by the naturally occurring quinone analog capsaicin (8-methyl-N-vanillyl-6-noneamide), compared with plasma membranes from human mammary epithelial, rat liver, normal rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. With cells in culture, capsaicin preferentially inhibited growth of HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells but was largely without effect on the mammary epithelial cells, rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. Inhibited cells became smaller and cell death was accompanied by a condensed and fragmented appearance of the nuclear DNA, as revealed by fluorescence microscopy with 4',6-diamidino-2-phenylindole, suggestive of apoptosis. The findings correlate capsaicin inhibition of cell surface NADH oxidase activity and inhibition of growth that correlate with capsaicin-induced apoptosis.