Paclitaxel triggers cell death primarily via caspase-independent routes in the non-small cell lung cancer cell line NCI-H460.
OBJECTIVE
Here we report on the role of mitochondria, death receptors (DRs), and caspases in exerting the cytotoxic effect of clinically relevant concentrations of paclitaxel in the non-small cell lung cancer cell line NCI-H460.


METHODS
We have characterized paclitaxel-induced cell death with annexin V, propidium iodide staining, and poly(ADP-ribose) polymerase cleavage assays. The involvement of the mitochondria pathway was studied by monitoring cytochrome c release and using H460 cells stable in overexpressing Bcl-2 or Bcl-x(L). DR dependency was analyzed in FADD dominant-negative or cytokine response modifier A-overexpressing cells, and a possible role for DR4 and DR5 was investigated by antagonistic antibodies. Caspase activity and cleavage assays and treatment with the synthetic inhibitor zVAD-fmk were used to determine the involvement of caspases.


RESULTS
Paclitaxel-treated cells displayed several features of apoptosis, including annexin V staining and poly(ADP-ribose) polymerase cleavage. The sequence of events suggested the involvement of a DR, as indicated by an early role for Fas-associated death domain and caspase-8, followed by cleavage of Bid and the disruption of mitochondria; nonetheless, we failed to demonstrate the involvement of DR4 and DR5. Interestingly, inhibition of either one of these routes only resulted in a 30% reduction of cell death that was in line with the observed small effect of caspase inhibition by zVAD-fmk on H460 cell survival.


CONCLUSIONS
Paclitaxel triggers cell death in H460 cells mainly via a currently unidentified caspase-independent mechanism in which the basic apoptotic machinery is merely coactivated. This finding is in sharp contrast with the largely caspase-dependent response elicited by DNA-damaging agents in these cells. We speculate on therapeutic implications.
Huisman
Cynthia
C
Department of Medical Oncology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands.
Ferreira
Carlos G
CG
Bröker
Linda E
LE
Rodriguez
Jose A
JA
Smit
Egbert F
EF
Postmus
Pieter E
PE
Kruyt
Frank A E
FA
Giaccone
Giuseppe
G
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D029681:Arabidopsis Proteins; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism / Q000494:pharmacology*; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004249:DNA Damage; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044943:Fatty Acid Desaturases / Q000235:genetics; D005799:Genes, Dominant; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy*; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D017239:Paclitaxel / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011419:Propidium / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D000972:Antineoplastic Agents, Phytogenic; D029681:Arabidopsis Proteins; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011419:Propidium; D044943:Fatty Acid Desaturases; C084083:Fad7 protein, Arabidopsis; D011065:Poly(ADP-ribose) Polymerases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D017239:Paclitaxel