Cell-cycle specific cytotoxicity mediated by stizophyllin (2 alpha,3 beta,12 beta-trihydroxypregna-4,7,16-trien-20-one), a novel electrophilic pregnane isolated from Stizophyllum riparium.
A structurally-novel pregnane derivative, 2 alpha,3 beta,12 beta-trihydroxypregna-4,7,16-trien-20-one (stizophyllin), was isolated from an extract of Stizophyllum riparium (H.B.K.) Sandw. on the basis of bioactivity-guided fractionation and confirmed to mediate a potent cytotoxic response with cultured P-388 cells. We presently report a detailed isolation procedure and the results of studies designed to examine its mechanism of action. By means of a Michael-type addition, stizophyllin formed adducts with nucleophilic substances such as L-cysteine and beta-mercaptoethanol. The adduct with beta-mercaptoethanol was isolated, structurally characterized, and found to be 20-fold less cytotoxic than stizophyllin. Stizophyllin interacted with DNA, but no mutagenicity was observed with Salmonella typhimurium strain TM677 or cultured Chinese hamster ovary cells, and no in vitro reaction occurred with guanosine. Relative to control cell cultures, the total biosynthesis of DNA, RNA or protein was reduced when P-388 cells were treated with stizophyllin. However, the cell number did not increase in the presence of inhibitory stizophyllin concentrations (e.g., 4 micrograms/ml), and the DNA content (per cell) actually doubled after approximately 48 h. Consistent with this, stizophyllin blocked the cells in the G2 + M compartment of the cycle, and the block appeared to be specific for the G2 phase. Accordingly, stizophyllin did not modulate in vitro tubulin polymerization reactions nor did it affect the morphology of dibutyryl cAMP-treated astrocytoma cells in culture. These data suggest that the cytotoxic activity of stizophyllin is mediated by covalent reaction with a cellular component (such as a sulfhydryl-containing protein) by means of a Michael-type addition. Based on the cell-cycle specificity of the response, it appears that this interaction prevents the cells from progressing through mitosis.
Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois, Chicago 60612.
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D000818:Animals; D001254:Astrocytoma; D016466:CHO Cells; D002453:Cell Cycle / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects*; D007700:Kinetics; D007941:Leukemia P388; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D009152:Mutagenicity Tests; D009153:Mutagens / Q000494:pharmacology*; D010946:Plants, Medicinal; D011281:Pregnatrienes / Q000302:isolation & purification / Q000494:pharmacology*; D012486:Salmonella typhimurium / Q000187:drug effects; D013936:Thymidine / Q000378:metabolism; D014404:Tubulin / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009153:Mutagens; D011281:Pregnatrienes; D014404:Tubulin; C071134:stizophyllin; D004247:DNA; D013936:Thymidine