title: Selective induction of apoptosis in Hep 3B cells by topoisomerase I inhibitors: evidence for a protease-dependent pathway that does not activate cysteine protease P32.
abstract: Progress in the treatment of hepatocellular carcinoma (HCC), a common tumor worldwide, has been disappointing. Inhibitors of topoisomerases are being widely studied as potential inducers of tumor cell apoptosis. Our aims were to determine whether topoisomerase-directed drugs would induce apoptosis in a human HCC cell line (Hep 3B) and, if so, to investigate the mechanism. The topoisomerase I poison camptothecin (CPT) induced apoptosis of Hep 3B cells in a time- and concentration-dependent manner. In contrast, the topoisomerase II poison etoposide failed to induce apoptosis despite the apparent stabilization of topoisomerase II-DNA complexes. Unexpectedly, CPT-induced apoptosis in this cell type occurred without any detectable cleavage of poly(ADP-ribose) polymerase or lamin B, polypeptides that are commonly cleaved in other cell types undergoing apoptosis. Likewise, Hep 3B cell apoptosis occurred without a detectable increase in interleukin-1beta-converting enzyme (ICE)-like or cysteine protease P32 (CPP32)-like protease activity. In contrast, trypsin-like protease activity (cleavage of Boc-Val-Leu-Lys-chloromethylaminocoumarin in situ) increased threefold in cells treated with CPT but not etoposide. Tosyl-lysyl chloromethyl ketone inhibited the trypsin-like protease activity and diminished CPT-induced apoptosis. These data demonstrate that (a) apoptosis is induced in Hep 3B cells after stabilization of topoisomerase I-DNA complexes but not after stabilization of topoisomerase II-DNA complexes as measured by alkaline filter elution; (b) Hep 3B cell apoptosis occurs without activation of ICE-like and CPP32-like protease activity; and (c) a trypsin-like protease activity appears to contribute to apoptosis in this cell type.
authors.affiliation: Division of Gastroenterology and Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
authors.forename: P N
authors.initials: PN
authors.lastname: Adjei
authors.forename: S H
authors.initials: SH
authors.lastname: Kaufmann
authors.forename: W Y
authors.initials: WY
authors.lastname: Leung
authors.forename: F
authors.initials: F
authors.lastname: Mao
authors.forename: G J
authors.initials: GJ
authors.lastname: Gores
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D011480:Protease Inhibitors / Q000494:pharmacology*; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D010450:Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1; D002166:Camptothecin