Extrinsic pathway- and cathepsin-dependent induction of mitochondrial dysfunction are essential for synergistic flavopiridol and vorinostat lethality in breast cancer cells.
The present studies have determined whether interactions between the cyclin-dependent kinase inhibitor flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat; Zolinza) occur in breast cancer cells. MDA-MB-231 and MCF7 cells were treated with flavopiridol (25-100 nmol/L) and vorinostat (125-500 nmol/L) in vitro, and mechanisms of cell killing were determined. Concurrent treatment of cells with flavopiridol and vorinostat or treatment of cells with flavopiridol followed by vorinostat promoted cell killing in a greater than additive fashion. Similar data were obtained with the CDK inhibitor roscovitine. Flavopiridol suppressed c-FLIP-l/s and BCL-xL expression, whereas vorinostat reduced expression of BCL-xL, and combined exposure to flavopiridol and vorinostat reduced MCL-1 and X-chromosome-linked inhibitor of apoptosis protein (XIAP) levels. Pharmacologic or genetic inhibition of caspase-8 reduced flavopiridol toxicity, but abolished killing by vorinostat and cell death caused by the vorinostat/flavopiridol regimen. Loss of BAX/BAK function or loss of BID function modestly reduced flavopiridol toxicity, but abolished vorinostat-mediated potentiation of flavopiridol toxicity, as did inhibition of caspase-9. Inhibition and/or deletion of cathepsin B function significantly attenuated vorinostat/flavopiridol lethality. Flavopiridol suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT activity and expression of activated forms of AKT and mitogen-activated protein/ERK kinase 1 maintained c-FLIP-l/s, BCL-xL, and XIAP expression and protected cells against flavopiridol/vorinostat lethality. Overexpression of c-FLIP-s and BCL-xL abolished the lethality of flavopiridol/vorinostat. Collectively, these data argue that flavopiridol enhances the lethality of vorinostat in breast cancer cells in part through the inhibition of AKT and ERK1/2 function, leading to reduced expression of multiple inhibitors of the extrinsic and intrinsic apoptosis pathways, as well as activation of cathepsin protease-dependent pathways.
Mitchell
Clint
C
Department of Biochemistry, Box 980035, Virginia Commonwealth University, Richmond, VA 23298-0035, USA.
Park
Maragret A
MA
Zhang
Guo
G
Yacoub
Adly
A
Curiel
David T
DT
Fisher
Paul B
PB
Roberts
John D
JD
Grant
Steven
S
Dent
Paul
P
D000970:Antineoplastic Agents / Q000494:pharmacology*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D002401:Cathepsin B / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D004357:Drug Synergism; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D010880:Piperidines / Q000494:pharmacology*; D000077337:Vorinostat; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000970:Antineoplastic Agents; D051023:BH3 Interacting Domain Death Agonist Protein; D005419:Flavonoids; D006877:Hydroxamic Acids; D010880:Piperidines; D051028:bcl-2-Associated X Protein; C077990:alvocidib; D000077337:Vorinostat; D002401:Cathepsin B