BCL-2 Family Inhibitors Enhance Histone Deacetylase Inhibitor and Sorafenib Lethality via Autophagy and Overcome Blockade of the Extrinsic Pathway to Facilitate Killing

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 ϩ HDACI). In immunohistochemical analyses or using expression of fluorescence-tagged proteins, treatment with sorafenib and vorinostat together (sorafenib ϩ 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 ϩ 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 ϩ HDACI lethality but abolished the effects of GX15-070 treatment. Sorafenib ϩ 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 ϩ HDACI killing by GX15-070 was suppressed by knockdown of Beclin1 or of BAX ϩ BAK. Our data demonstrate that pancreatic tumor cells are susceptible to sorafenib ϩ HDACI lethality and that in tumor cells unable to signal death from CD95, use of a BCL-2 family antagonist facilitates sorafenib ϩ HDACI killing via autophagy and the intrinsic pathway.In the United States, pancreatic carcinomas have 5-year survival rates of less than 5% (Bardeesy and DePinho, 2002;Parkin et al., 2005). This statistic emphasizes the need to develop original therapies using novel targeted agents against this lethal malignancy.Tumor cells use a wide variety of mechanisms to maintain cell viability, including loss of death receptor expression (e.g., CD95) by reducing expression of proapoptotic BH3 domain proteins (e.g., BAX) or by increasing expression of antiapoptotic BCL-2 family members (e.g., MCL-1) (Lessene et al.,

Enhancing CHK1 inhibitor lethality in glioblastoma

“…4D). 29,30 In addition to MEK1/2 inhibitors, we have also observed in other tumor types that inhibitors of SRC family kinases interact with CHK1 inhibitors to kill tumor cells.…”

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

“…13,14 Recent findings have also revealed that sorafenib generated a protective form of autophagy in gastrointestinal and pancreatic tumor cells. 15,16 However, both how these therapies induce autophagy and whether such autophagy contributes to tumor cell death or is a mechanism of resistance remains uncertain and varies depending on the cell type and stimulus. 17 In the present study, we found that direct stimulation of ER stress by sorafenib in HCC induced autophagy via upregulation of the IRE1 pathway and that inhibition of autophagy promoted ER stress-related apoptosis of HCC cells in vivo and in vitro.…”

Vorinostat-Induced Apoptosis in Mantle Cell Lymphoma Is Mediated by Acetylation of Proapoptotic BH3-Only Gene Promoters

“…A total of 10,000 to 25,000 stained cells per sample were acquired and analyzed in a FACScan or FACSCalibur flow cytometer by using CellQuest and Paint-A-Gate softwares (Becton Dickinson). Lethal dose 50 (LD 50 ) was defined as the concentration of drug required to reduce cell viability by 50%. For drug combinations, combination indexes (CI) were calculated with the CalcuSyn software version 2.0 (Biosoft) by using the Chou and Talalay algorithm.…”