Biochemical pharmacology
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Biochemical pharmacology · Dec 2000
Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines.
One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1-1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH-adriamycin conjugates. ⋯ These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1-1 at pH 7.2. Indeed, their synthetic C(7)-aglycon-GSH conjugates exerted a strong inhibitory effect on GST P1-1, with K(i) at 25 degrees in the 1-2 microM range, scarcely dependent on their stereochemistry at C(7).
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Biochemical pharmacology · Sep 2000
Inhibition of C5a-induced neutrophil chemotaxis and macrophage cytokine production in vitro by a new C5a receptor antagonist.
A cyclic peptide, Phe-[Orn-Pro-D-Cyclohexylalanine-Trp-Arg] (F-[OPdChaWR]), was recently shown in vitro to antagonise the binding of C5a to its receptor (CD88) on human polymorphonuclear leukocytes (PMNs) and in vivo to inhibit the neutropenia associated with septic shock induced by lipopolysaccharide (LPS) in rats. The aim of this study was to investigate whether F-[OPdChaWR] inhibits C5a-mediated chemotaxis of human PMNs using a modified Boyden chamber and C5a-stimulated release of cytokines from human monocytes in vitro. Approximately 50% of the chemotactic activity induced by 10 nM C5a was inhibited by 76 nM F-[OPdChaWR]. ⋯ This co-stimulation was inhibited by F-[OPdChaWR] with IC(50)s of 0.8 and 6.9 nM for release of TNF-alpha and IL-1beta, respectively. No agonist activity was detected for F-[OPdChaWR] in either the chemotaxis or cytokine release assays at concentrations up to 50 microM. These results show that F-[OPdChaWR] inhibits several important inflammatory activities of C5a and suggest that C5a receptor antagonists may be effective in the treatment of inflammatory diseases mediated by C5a.
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Biochemical pharmacology · Sep 2000
Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome c-mediated activation of caspase-3 protease.
Ginseng saponins exert various important pharmacological effects with regard to the control of many diseases including cancer. The novel intestinal bacterial metabolites of ginseng protopanaxadiol saponins have recently been found and isolated after the oral administration of ginseng extract in human and rats. 20-O-(beta-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH-901) formed from ginsenosides Rb1, Rb2, and Rc is of particular interest in cancer chemoprevention and treatment. We investigated the effects of IH-901 on the human myeloid leukemia cell line HL-60 in terms of inhibition of proliferation and induction of apoptosis. ⋯ IH-901 did not affect the expression of antiapoptotic protein Bcl-2 but did cause a release of mitochondrial cytochrome c into cytosol. In conclusion, our results demonstrate that IH-901 dramatically suppresses HL-60 cell growth by inducing programed cell death through activation of caspase-3 protease, which occurs via mitochondrial cytochrome c release independently of Bcl-2 modulation. These results may provide a pivotal mechanism for the use of IH-901 in the prevention and treatment of leukemia.
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Biochemical pharmacology · May 2000
Differential cytotoxicity and induction of apoptosis in tumor and normal cells by hydroxymethylacylfulvene (HMAF).
This investigation compared the effects of hydroxymethylacylfulvene (HMAF), a novel antitumor drug with alkylating properties, in eight human tumor (prostate, colon, and leukemia) cell lines, and five human normal (prostate and renal proximal tubule epithelial, colon mucosa, fibroblasts, and endothelial) cell lines. Drug-induced growth inhibition paralleled the uptake of HMAF into both tumor and normal cells, although normal cells were 3- to 4-fold more tolerant to the accumulated drug. In both tumor and normal cells, approximately two-thirds of internalized [(14)C]HMAF-derived radioactivity was bound covalently to macromolecules. ⋯ This resistance of normal cells to apoptosis could not be accounted for by differences in drug accumulation or drug covalent binding to macromolecules. The qualitatively different responses of the tumor and normal cells studied suggest a greater tolerance of normal cells to HMAF-macromolecular adducts. The demonstrated differential cytotoxic/cytostatic and apoptotic effects of HMAF can be of significance for the clinical use of this promising new agent.
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Biochemical pharmacology · May 2000
Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant.
In the present study, we demonstrate a model cell system for evaluating the specificity of inhibitors of serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step of sphingolipid biosynthesis. The LY-B strain is a Chinese hamster ovary (CHO) cell mutant defective in SPT, and the LY-B/cLCB1 strain is a genetically corrected revertant of the mutant. Although LY-B cells grew only slightly in sphingolipid-deficient medium, their growth was restored to the level of LY-B/cLCB1 cells under sphingosine-supplied conditions, indicating that, in CHO cells, the growth inhibition caused by SPT inactivation was rescued almost fully by the metabolic complementation of sphingolipids. ⋯ Similar cytotoxic patterns of these inhibitors also were observed on the growth of SPT-defective LY-B cells cultured under sphingosine-supplied conditions. The SPT inhibitors did not affect metabolic conversion of exogenous [(3)H]sphingosine to complex sphingolipids. Thus, the cytotoxicity of sphingofungin B and ISP-1, but not L-cycloserine or beta-chloro-L-alanine, is due largely to inhibition of sphingolipid synthesis by inhibiting the SPT activity.