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Research Articles: Therapeutics, Targets, and Development

Extrinsic pathway- and cathepsin-dependent induction of mitochondrial dysfunction are essential for synergistic flavopiridol and vorinostat lethality in breast cancer cells

Departments of ¹ Biochemistry and ² Medicine, Virginia Commonwealth University, Richmond, Virginia; ³ Departments of Pathology, Neurosurgery and Urology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York; and ⁴ Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama

Requests for reprints: Paul Dent, Department of Biochemistry, Box 980035, Virginia Commonwealth University, Richmond, VA 23298-0035. Phone: 804-628-0861; Fax: 804-827-1309. E-mail: pdent{at}vcu.edu

Abstract

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. [Mol Cancer Ther 2007;6(12):3101–12]

Grant support: The Goodwin Foundation, PHS grants (R01-CA88906, R01-DK52825, P01-CA104177, Core B for virus production), Department of Defense Awards (DAMD17-03-1-0262; to P. Dent); PHS grants (R01-CA63753; R01-CA77141) and a Leukemia Society of America grant 6405-97 (S. Grant). These studies were also funded in part by a grant from The Jim Valvano "V" Foundation. P. Dent is the holder of the Universal Inc. Professorship in Signal Transduction Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/14/07; revised 10/ 1/07; accepted 10/24/07.

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