With NADPH as the electron donor, rat liver NADPH cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) catalyzes the single-electron reduction of several quinone antibiotics to a semiquinone or free radical state. The benzanthraquinones adriamycin, daunorubicin, carminomycin, 7-O-methylnogalarol, and aclacinomycin A and the N-heterocyclic quinones streptonigrin and mitomycin C are activated to free radical intermediates which can transfer their single electron to molecular oxygen to form superoxide. The overall Km range for this electron transfer is 0.4 to 42.1 X 10-4 M. We postulate that the formation of the "site-specific free radical" intermediate is central to the cytotoxic action of these antibiotics.An unusually high proportion of anticancer agents contain quinone groups. Such quinone-containing anticancer agents include adriamycin, daunorubicin, mitomycin C, streptonigrin, lapachol, and analogs of these agents. Although investigators have identified several potential biochemical sites of action for these drugs, preponderant evidence indicates that the agents act primarily by interfering with DNA and RNA replication(1-4).We have established that these quinone agents are catalytically activated to a free radical state by a microsomal system requiring NADPH as an electron donor (5, 6). Normal microsomes as well as microsomes from murine leukemia cells catalyze augmented oxygen consumption with the quinone antibiotics, indicative of free radical formation. By using effectors of this microsomal system we obtained indirect evidence that a flavoprotein catalyst of the microsomes was involved and that cytochrome P-450 was not. Iyanagi and Yamazaki (7) have shown that NADPH cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) reduces quinone substrates to semiquinones; and Goodman and Hochstein (8) reported that NADPH cytochrome P-450 reductase catalyzes production of superoxide from adriamycin. In the present paper, we describe and characterize the catalysis of drug free-radical formation bhey -tochrome P-450 reductase from rat-iver microsomes and propose a mechanism for the cytotoxic action of these agents.METHODS AND MATERIALS Adriamycin, N-dimethyladriamycin, daunorubicin, N-acetyldaunorubicin, 7-iminodaunorubicin, aclacinomycin A, mitomycin C, streptonigrin, and lapachol were kindly provided by the Drug Synthesis and Chemistry Branch and the Natural Products Branch (Division of Cancer Treatment, National Cancer Institute, National Institutes of Health). Daunorubicinol (9) and adriamycin (Fe3+)4 (10) were prepared as described. Carminomycin was provided by G. F. Gause (Institute of New Antibiotics, Moscow). Rubidazone was supplied by Rene Maral (Rhone-Poulenc, Vitry-Sur-Seine, France). Nogalamycin, 7-O-methylnogarol, and steffimycin were provided by G. Neil (Upjohn Co.); and N-trifluoroacetyladriamycin-14-valerate (AD-32) and N-trifluoroacetyladriamycin (AD-41) were supplied by M. Israel, (Farber Cancer Center, Boston, MA).NADPH cytochrome P-450 reduct...

Key Points• IL1RAP is overexpressed on candidate AML stem cells and is a promising target for antibody-based therapy.IL1RAP, a co-receptor for interleukin (IL)-1 and IL-33 receptors, was previously found to be highly upregulated on candidate chronic myeloid leukemia stem cells, allowing for leukemia-selective killing using IL1RAP-targeting antibodies. We analyzed IL1RAP expression in a consecutive series of 29 patients with acute myeloid leukemia (AML) and, based on the level of expression in mononuclear cells (MNCs), we divided the samples into 3 groups: IL1RAP low (n 5 6), IL1RAP intermediate (n 5 11), and IL1RAP high (n 5 12). Within the CD341CD382 population, the intermediate and high groups expressed higher levels of IL1RAP than did corresponding normal cells. With the aim to target AML stem cells, an anti-IL1RAP monoclonal antibody was generated followed by isotype switching for improved antibody-dependent, cell-mediated cytotoxicity activity. Using this antibody, we achieved selective killing of AML MNC, CD341CD381, and CD341CD382 cells. Our findings demonstrate that IL1RAP is a promising new therapeutic target in AML. (Blood. 2013;121(18):3709-3713)

Blackcurrant (Ribes nigrum L.) is a widely grown commercial crop valued for its high vitamin C (l-ascorbic acid, AsA) content. In the present study, a systematic analysis of the mechanism of fruit AsA accumulation was undertaken. AsA accumulation occurred during fruit expansion and was associated with high in situ biosynthetic capacity via the l-galactose pathway and low rates of turnover. Cessation of AsA accumulation was associated with reduced biosynthesis and increased turnover. Translocation of AsA from photosynthetic or vegetative tissues contributed little to fruit AsA accumulation. Manipulation of substrate availability by defoliation had no effect on fruit AsA concentration but significantly reduced fruit yields. Supply of the AsA precursor l-galactono-1,4-lactone to intact, attached fruit transiently increased fruit AsA concentration which rapidly returned to control levels after removal of the compound. These data suggest strong developmental, metabolic and genetic control of AsA accumulation in blackcurrant fruit and indicate the potential for breeding high AsA cultivars.

Background: The presently available pharmaceutical aids in smoking cessation possess a rather limited effectiveness. Therefore, we have synthesized a series of immunoconjugates that stimulate the induction of antibodies which may bind nicotine in the blood, thereby preventing it from passing the blood-brain barrier. Thus, the reinforcing action of nicotine in the brain, which is the driving force in tobacco smoking, should be abolished. Objective: The present study was undertaken to test this notion in a long-term relapse model in rats, measuring the reinstatement of nicotine-seeking behavior, following active immunization with IP18-KLH, one of our immunoconjugates. Methods: Male Wistar rats were immunized with a nicotine-KLH conjugate (nicotine immunogen) and Freund’s adjuvant after having been trained to meet the criteria of stable nicotine self-administration on a fixed ratio (FR3) schedule. The rats were subsequently extinguished from nicotine self-administration behavior and finally, as extinction was completed, they were exposed to small, priming doses of nicotine, which previously have been shown to reinstate the nicotine-seeking behavior. The antibody titers were measured by ELISA. Results: It was found that rats with high titers (>1:10,000) of antibodies against nicotine, in contrast to those with low/no nicotine selective antibodies, do not reinstate nicotine self-administration behavior when they are exposed to nicotine. Conclusions: Our findings indicate that active immunization against nicotine may effectively abolish the reinforcing action of nicotine in brain, an effect which is critical for relapse in nicotine dependence. These data suggest the potential utility of active immunization in smoking cessation programs.

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