title: Irofulven induces apoptosis in breast cancer cells regardless of caspase-3 status.
abstract: Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacyl-fulvene, HMAF. MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48 h incubation at 1 microM irofulven (approximately 3 x GI50), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24 h but decreased markedly after 48 h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48 h. Normal HMEC cells were refractory to 1 microM drug with only approximately 3-9% fragmented DNA after 12-48 h, although apoptosis was observed at drug levels >3 microM. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20 microM with nearly complete abrogation of apoptosis at 100 microM. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).
authors.affiliation: Cancer Therapy and Research Center, The University of Texas, San Antonio, USA.
authors.forename: Maryanne C S
authors.initials: MC
authors.lastname: Herzig
authors.forename: Huiyun
authors.initials: H
authors.lastname: Liang
authors.forename: Anne E
authors.initials: AE
authors.lastname: Johnson
authors.forename: Barbara
authors.initials: B
authors.lastname: Woynarowska
authors.forename: Jan M
authors.initials: JM
authors.lastname: Woynarowski
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000473:pathology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007700:Kinetics; D012717:Sesquiterpenes / Q000633:toxicity*; D014407:Tumor Cells, Cultured
chemical_list: D018906:Antineoplastic Agents, Alkylating; D012717:Sesquiterpenes; C102714:irofulven; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases