ascorbic-acid and prolinedithiocarbamate

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, gamma-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.

Topics: Animals; Antineoplastic Agents; Antioxidants; Ascorbic Acid; ATP Binding Cassette Transporter, Subfamily B, Member 1; Catalase; Docosahexaenoic Acids; Doxorubicin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; gamma-Linolenic Acid; Glutathione Peroxidase; Glutathione Transferase; Leukemia P388; Mice; Proline; Superoxide Dismutase; Thiocarbamates; Tumor Cells, Cultured

Proinflammatory cytokines, altered connective tissue metabolism, and overexpression of matrix metalloproteinases (MMPs) such as stromelysin compared to tissue inhibitors of metalloproteinases (TIMPs) result in synovial inflammation and erosion of arthritic cartilage. Tumor necrosis factor alpha (TNF-alpha) is a major synovial inflammatory mediator responsible for inhibiting extracellular matrix (ECM) synthesis and stimulating degradation of cartilage ECM by activated MMPs in arthritic joints. To suppress these effects and to gain insight into the mechanism of TNF-alpha action, we identified the inhibitors of TNF-alpha stimulation of stromelysin gene expression. In bovine synovial fibroblasts, TNF-alpha did not affect a recently identified inhibitor, TIMP-3, but induced stromelysin mRNA expression in a dose- and time-dependent fashion (3- to 5-fold) which required de novo protein synthesis. Stimulation by TNF-alpha was potently inhibited (99-100%) by the synthetic glucocorticoid, dexamethasone. Sodium salicylate dose-dependently inhibited (100%) the TNF-alpha action. Indomethacin and ibuprofen were partially inhibitory. Free radical scavenger antioxidant, N-acetylcysteine (but not other antioxidants) also suppressed the TNF-alpha induction (36-100%) of stromelysin suggesting involvement of reactive oxygen species in the induction process. TNF-alpha induction of stromelysin gene expression can therefore be inhibited at the gene expression level by several pharmacological agents which are likely to function via arachidonic acid metabolites, free radical scavenging or interference with the activator protein 1, polyoma virus enhancer A-binding protein 3, and nuclear factor kappaB classes of transcription factors. Our results may help to elucidate the mechanism of TNF-alpha action and explain the beneficial role of these agents in the treatment of inflammatory diseases.

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Cattle; Cells, Cultured; Dexamethasone; Fibroblasts; Free Radical Scavengers; Gene Expression Regulation, Enzymologic; Ibuprofen; Indomethacin; Matrix Metalloproteinase 3; Proline; Sodium Salicylate; Synovial Membrane; Thiocarbamates; Tissue Inhibitor of Metalloproteinase-3; Transcription Factors; Transcription, Genetic; Tumor Necrosis Factor-alpha; Vitamin E