Enhancement of depsipeptide-mediated apoptosis of lung or esophageal cancer cells by flavopiridol: activation of the mitochondria-dependent death-signaling pathway.
OBJECTIVE
Treating cancer cells with depsipeptide, a novel antitumor agent currently in a phase II clinical trial, causes potent upregulation of p21/WAF1 expression and cell arrest at G1 and G2 checkpoints. p21/WAF1 upregulation, however, impedes the ability of depsipeptide to induce significant apoptosis. This study was designed to determine whether flavopiridol, a synthetic cyclin-dependent kinase inhibitor known to inhibit p21 expression in tumor cells, could enhance depsipeptide-mediated apoptosis in cultured lung and esophageal cancer cells.


METHODS
Lung or esophageal cancer cells were exposed to depsipeptide, flavopiridol, or a combination of depsipeptide and flavopiridol. Cytotoxicity and apoptosis were quantitated by means of (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-based assays, respectively. Cytosolic cytochrome c levels, caspase 9 activity, mitochondrial membrane depolarization, and dependence of apoptosis on caspase 9 in treated cells were studied to determine the role of the mitochondria in mediating apoptosis induced by this drug combination.


RESULTS
Flavopiridol completely abolished depsipeptide-mediated dose-dependent upregulation of p21/WAF1 expression. Combining flavopiridol with depsipeptide resulted in a 3- to 8-fold reduction of depsipeptide inhibitory concentration of 50% values that was closely paralleled by synergistic enhancement of apoptosis (4- to 10-fold higher than levels of cell death induced by either drug alone) in all cancer cell lines. The essential role of mitochondria in mediating cell death was indicated by robust translocation of cytochrome c from the mitochondria into the cytosol, 2.5- to 5-fold activation of caspase 9, severe disruption of mitochondrial inner membrane potential, and complete inhibition of apoptosis by the selective caspase 9 inhibitor. More important, this drug combination was not toxic to primary normal epithelial cells derived from the airway or skin.


CONCLUSIONS
The depsipeptide plus flavopiridol combination exhibits powerful and selective cytocidal activity against cancer but not normal cells. Apoptosis induced by this combination is mediated by the mitochondria-dependent death pathway.
Nguyen
Dao M
DM
Section of Thoracic Oncology, Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Dao_Nguyen@nih.gov
Schrump
William D
WD
Tsai
Wilson S
WS
Chen
Aaron
A
Stewart
John H
JH
Steiner
Federico
F
Schrump
David S
DS
D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000187:drug effects*; D047630:Depsipeptides; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D004938:Esophageal Neoplasms / Q000188:drug therapy* / Q000473:pathology; D005419:Flavonoids / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology; D008928:Mitochondria / Q000187:drug effects; D010456:Peptides, Cyclic / Q000494:pharmacology* / Q000627:therapeutic use; D010880:Piperidines / Q000494:pharmacology* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D005419:Flavonoids; D010456:Peptides, Cyclic; D010880:Piperidines; C077990:alvocidib; C087123:romidepsin; D018844:Cyclin-Dependent Kinases