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

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

^* To whom correspondence should be addressed. 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):OF1–12]

Key Words: Breast cancer, Signal transduction, CDKs and CDK inhibitors, bcl-2 pathways: anti- and proapoptotic, Preclinical toxicology

This article has been cited by other articles:

				G. Zhang, M. A. Park, C. Mitchell, H. Hamed, M. Rahmani, A. P. Martin, D. T. Curiel, A. Yacoub, M. Graf, R. Lee, et al. Vorinostat and Sorafenib Synergistically Kill Tumor Cells via FLIP Suppression and CD95 Activation Clin. Cancer Res., September 1, 2008; 14(17): 5385 - 5399. [Abstract] [Full Text] [PDF]