Abstract
We examined whether the multikinase inhibitor sorafenib and histone deacetylase inhibitors (HDACI) interact to kill pancreatic carcinoma cells and determined the impact of inhibiting BCL-2 family function on sorafenib and HDACI lethality. The lethality of sorafenib was enhanced in pancreatic tumor cells in a synergistic fashion by pharmacologically achievable concentrations of the HDACIs vorinostat or sodium valproate. Overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s) or knockdown of CD95 suppressed the lethality of the sorafenib/HDACI combination (sorafenib + HDACI). In immunohistochemical analyses or using expression of fluorescence-tagged proteins, treatment with sorafenib and vorinostat together (sorafenib + vorinostat) promoted colocalization of CD95 with caspase 8 and CD95 association with the endoplasmic reticulum markers calnexin, ATG5, and Grp78/BiP. In cells lacking CD95 expression or in cells expressing c-FLIP-s, the lethality of sorafenib + HDACI exposure was abolished and was restored when cells were coexposed to BCL-2 family inhibitors [ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA14-1), obatoclax (GX15-070)]. Knockdown of BCL-2, BCL-XL, and MCL-1 recapitulated the effects of GX15-070 treatment. Knockdown of BAX and BAK modestly reduced sorafenib + HDACI lethality but abolished the effects of GX15-070 treatment. Sorafenib + HDACI exposure generated a CD95- and Beclin1-dependent protective form of autophagy, whereas GX15-070 treatment generated a Beclin1-dependent toxic form of autophagy. The potentiation of sorafenib + HDACI killing by GX15-070 was suppressed by knockdown of Beclin1 or of BAX + BAK. Our data demonstrate that pancreatic tumor cells are susceptible to sorafenib + HDACI lethality and that in tumor cells unable to signal death from CD95, use of a BCL-2 family antagonist facilitates sorafenib + HDACI killing via autophagy and the intrinsic pathway.
Footnotes
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This work was funded by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK52825]; the National Institutes of Health National Cancer Institute [Grants P01-CA104177, R01-CA108520, R01-CA63753, R01-CA77141; R01-CA93738]; by The Jimmy V Foundation; and by The Goodwin Foundation for Cancer Research (to Massey Cancer Center). P.D. is the holder of the Universal Inc. Professorship in Signal Transduction Research.
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A.P.M., M.A.P., and C.M. contributed equally to this work.
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ABBREVIATIONS: MEK, mitogen-activated extracellular-regulated kinase; ERK, extracellular regulated kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; c-FLIP-s, cellular FLICE-like inhibitory protein; ER, endoplasmic reticulum; dn, dominant negative; ca, constitutively active; PERK, PKR-like endoplasmic reticulum kinase; PKR, protein kinase regulated by RNA; HDACI, histone deacetylase inhibitor; GX15-070; HA14-1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; GFP, green fluorescent protein; YFP, yellow fluorescent protein; DMSO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; PE, phycoerythrin; CMV, cytomegalovirus; siRNA, small interfering RNA; siSCR, scrambled siRNA; DISC, death-inducing signal complex; FADD, FAS-associated death domain; IHC, immunohistochemistry; MEF, mouse embryonic fibroblast.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. - Accepted May 29, 2009.
- Received March 18, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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