diamide and dihydrolipoic-acid

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or alpha-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or alpha-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce alpha-lipoate, while Q7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce alpha-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of alpha-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit alpha-lipoate reduction in Q7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.

Topics: Animals; Arsenicals; Arsenites; Carmustine; CHO Cells; Cricetinae; Cystine; Diamide; Disulfides; Dithionitrobenzoic Acid; Glutathione; Humans; Oxidation-Reduction; Rats; Selenium Compounds; Selenium Oxides; Sulfhydryl Compounds; Thioctic Acid; Tumor Cells, Cultured

NF-kappa B transcription factor regulates a wide variety of cellular and viral genes including the human immunodeficiency virus type 1. Here, we demonstrate that dihydrolipoate/alpha-lipoate redox couple which is a cofactor for mitochondrial dehydrogenases reactions, influences the DNA binding activity of NF-kappa B. The elimination of dithiothreitol in the electrophoretic mobility shift assay protocol resulted in the inability to detect DNA binding activity of activated NF-kappa B. The DNA binding activity was restored by the addition of dihydrolipoate in the binding reaction mixture. Inhibition of NF-kappa B DNA binding activity by in vitro exposure to a sulfhydryl oxidizing agent, diamide was also blocked by dihydrolipoate. In contrast, the addition of the oxidized form, alpha-lipoate inhibited the NF-kappa B DNA binding activity. Coincidentally, preincubation of Jurkat cells with dihydrolipoate potentiated and alpha-lipoate inhibited the okadaic acid-induced NF-kappa B activation as detected by assessing its DNA binding activity. These results suggest the redox exchange between lipoate and NF-kappa B molecules. Furthermore, since the inhibition of AP-1 DNA binding activity by diamide was also blocked by dihydrolipoate, this natural reductant may participate in the redox regulation of transcription factors by enhancing the DNA-protein interactions.

Topics: Base Sequence; Cell Nucleus; Diamide; Dithiothreitol; DNA; DNA Probes; Ethers, Cyclic; Humans; Molecular Sequence Data; NF-kappa B; Okadaic Acid; Oxidation-Reduction; Thioctic Acid; Transcription Factor AP-1; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha