Irofulven (6-hydroxymethylacylfulvene, MGI 114) induces caspase 8 and 9-mediated apoptosis in human pancreatic adenocarcinoma cells.
BACKGROUND
Irofulven (MGI 114) is a novel, clinically active sesquiterpene whose mechanism of action is not fully understood. We sought to identify apoptotic effectors induced by this agent in human pancreatic cancer cells.


METHODS
MTT assay was used to assess IC50-Apoptosis was quantitated by flow cytometry and DAPI staining. Caspase activation was identified by western blot analysis.


RESULTS
Irofulven was cytotoxic against all pancreatic cancer cell lines tested (IC50 1-18 microM), and induced 10-fold (4%+/- 2, vs. 41% +/- 5) induction of apoptosis. Irofulven-treated cells also demonstrated PARP3 cleavage and DAPI staining. Apoptosis was reduced to baseline levels by Z-VAD-FMK, a broad-spectrum caspase inhibitor. Western blot analysis revealed that caspases-3, -7, -8, and -9 were activated by irofulven. Time course evaluation demonstrated that caspases-8 and -9 were the initial species activated.


CONCLUSIONS
Our data demonstrate that the cytotoxicity of irofulven in human pancreatic carcinoma cell lines is mediated by caspase-induced apoptosis.
Wang
W
W
Department of Surgery, University of Texas Health Science Center at San Antonio, 78229, USA.
Waters
S J
SJ
MacDonald
J R
JR
Von Hoff
D D
DD
Strodel
W E
WE
Miller
A R
AR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D004396:Coloring Agents / Q000494:pharmacology; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D007211:Indoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000473:pathology*; D011419:Propidium / Q000494:pharmacology; D012717:Sesquiterpenes / Q000494:pharmacology*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D018906:Antineoplastic Agents, Alkylating; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D007211:Indoles; D012717:Sesquiterpenes; D013778:Tetrazolium Salts; D013844:Thiazoles; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011419:Propidium; C007293:DAPI; C102714:irofulven; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C022616:thiazolyl blue