Vorinostat and sorafenib synergistically kill tumor cells via FLIP suppression and CD95 activation

Abstract

Purpose and design: Mechanism(s) by which the multikinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal, and pancreatic adenocarcinoma cells has been defined.

Results: Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal, and pancreatic adenocarcinoma cells in multiple short-term viability (24-96 h) and in long-term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase-8 and, to a lesser extent, by inhibition of caspase-9. Twenty-four hours after exposure, the activities of extracellular signal-regulated kinase 1/2, AKT, and nuclear factor-kappaB were only modestly modulated by sorafenib and vorinostat treatment. However, 24 h after exposure, sorafenib- and vorinostat-treated cells exhibited markedly diminished expression of c-FLIP-s, full-length BID, BCL-2, BCL-XL, MCL-1, XIAP, increased expression of BIM, and increased activation of BAX, BAK, and BAD. Expression of eIF2alpha S51A blocked sorafenib- and vorinostat-induced suppression of c-FLIP-s levels and overexpression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 association with caspase-8. Knockdown of CD95 or FADD expression significantly reduced sorafenib/vorinostat-mediated lethality.

Conclusions: These data show that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple antiapoptotic proteins and promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the proapoptotic signals from CD95 to promote mitochondrial dysfunction and death.

Figure 1. Sorafenib and Vorinostat interact in a synergistic fashion to kill pancreatic, liver and kidney tumor cells in colony formation assays

Panel A. Pancreatic (PANC-1, MiaPaca2) and hepatoma (HEP3B, HEPG2) cells were plated as single cells (250–1500 cells/well) in sextuplicate and 12h after plating treated with vehicle (VEH, DMSO), sorafenib (Sor., 3.0–6.0 µM) or vorinostat (Vor. 250–500 nM), or with both drugs combined, as indicated at a fixed concentration ratio to perform median dose effect analyses for the determination of synergy. After drug exposure (48h), the media was changed and cells cultured in drug free media for an additional 10–14 days. Cells were fixed, stained with crystal violet and colonies of > 50 cells / colony counted. The true percentage inhibition of colony formation is plotted from the means of sextuplicate plates from two separate experiments ± SEM (n = 3 total studies). * p < 0.05 greater cell killing than compared to any other treatment condition. Colony formation data were also entered into the Calcusyn program and combination index (CI) values determined. A CI value of less than 1.00 indicates synergy: CI values for PANC-1, MiaPaca2, HEP3B, HEPG2 were all below 0.70. Panel B. HEPG2 cells were plated in triplicate, treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. After drug exposure (48h) cells were isolated, and either: (a) spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods. The percentage of TUNEL positive cells ± SEM was determined. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition; or (b) treated with annexin and propidium iodide according to manufacturer’s instructions, and stained using these established methods. Cells were subjected to flow cytometery to determine the numbers of annexin and propidium iodide positive cells ± SEM. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition. Panel C. UOK121LN, Mia Paca2 and HEPG2 cells were plated in triplicate, treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. After drug exposure (48h, 96h) cells were isolated and stained using trypan blue indicative of later stages of cell death / plasma membrane disruption as described in the Methods. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition.

Figure 1. Sorafenib and Vorinostat interact in a synergistic fashion to kill pancreatic, liver and kidney tumor cells in colony formation assays

Panel A. Pancreatic (PANC-1, MiaPaca2) and hepatoma (HEP3B, HEPG2) cells were plated as single cells (250–1500 cells/well) in sextuplicate and 12h after plating treated with vehicle (VEH, DMSO), sorafenib (Sor., 3.0–6.0 µM) or vorinostat (Vor. 250–500 nM), or with both drugs combined, as indicated at a fixed concentration ratio to perform median dose effect analyses for the determination of synergy. After drug exposure (48h), the media was changed and cells cultured in drug free media for an additional 10–14 days. Cells were fixed, stained with crystal violet and colonies of > 50 cells / colony counted. The true percentage inhibition of colony formation is plotted from the means of sextuplicate plates from two separate experiments ± SEM (n = 3 total studies). * p < 0.05 greater cell killing than compared to any other treatment condition. Colony formation data were also entered into the Calcusyn program and combination index (CI) values determined. A CI value of less than 1.00 indicates synergy: CI values for PANC-1, MiaPaca2, HEP3B, HEPG2 were all below 0.70. Panel B. HEPG2 cells were plated in triplicate, treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. After drug exposure (48h) cells were isolated, and either: (a) spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods. The percentage of TUNEL positive cells ± SEM was determined. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition; or (b) treated with annexin and propidium iodide according to manufacturer’s instructions, and stained using these established methods. Cells were subjected to flow cytometery to determine the numbers of annexin and propidium iodide positive cells ± SEM. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition. Panel C. UOK121LN, Mia Paca2 and HEPG2 cells were plated in triplicate, treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. After drug exposure (48h, 96h) cells were isolated and stained using trypan blue indicative of later stages of cell death / plasma membrane disruption as described in the Methods. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition.

Figure 1. Sorafenib and Vorinostat interact in a synergistic fashion to kill pancreatic, liver and kidney tumor cells in colony formation assays

Panel A. Pancreatic (PANC-1, MiaPaca2) and hepatoma (HEP3B, HEPG2) cells were plated as single cells (250–1500 cells/well) in sextuplicate and 12h after plating treated with vehicle (VEH, DMSO), sorafenib (Sor., 3.0–6.0 µM) or vorinostat (Vor. 250–500 nM), or with both drugs combined, as indicated at a fixed concentration ratio to perform median dose effect analyses for the determination of synergy. After drug exposure (48h), the media was changed and cells cultured in drug free media for an additional 10–14 days. Cells were fixed, stained with crystal violet and colonies of > 50 cells / colony counted. The true percentage inhibition of colony formation is plotted from the means of sextuplicate plates from two separate experiments ± SEM (n = 3 total studies). * p < 0.05 greater cell killing than compared to any other treatment condition. Colony formation data were also entered into the Calcusyn program and combination index (CI) values determined. A CI value of less than 1.00 indicates synergy: CI values for PANC-1, MiaPaca2, HEP3B, HEPG2 were all below 0.70. Panel B. HEPG2 cells were plated in triplicate, treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. After drug exposure (48h) cells were isolated, and either: (a) spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods. The percentage of TUNEL positive cells ± SEM was determined. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition; or (b) treated with annexin and propidium iodide according to manufacturer’s instructions, and stained using these established methods. Cells were subjected to flow cytometery to determine the numbers of annexin and propidium iodide positive cells ± SEM. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition. Panel C. UOK121LN, Mia Paca2 and HEPG2 cells were plated in triplicate, treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. After drug exposure (48h, 96h) cells were isolated and stained using trypan blue indicative of later stages of cell death / plasma membrane disruption as described in the Methods. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from a representative of 3 independent studies. * p < 0.05 greater cell killing than compared to any other treatment condition.

Figure 2. Inhibition of caspase 8 and cathepsin function suppresses sorafenib and vorinostat lethality in GI tumor cells

Panels A and B. HEPG2 and MiaPaca2 cells plated in triplicate, as indicated, were either treated with: left graphical panels, vehicle (DMSO), zVAD (25 µM), cathepsin inhibitor (1 µM) or zVAD and cathepsin inhibitor, or: right graphical panels, infected to express empty vector (CMV), dominant negative caspase 9, caspase 8 inhibitor CRM A or dominant negative caspase 9 and CRM A. Cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel C. SV40 Large T antigen transformed mouse embryonic fibroblasts lacking expression of various pro-apoptotic genes were plated in triplicate and treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells.

Figure 2. Inhibition of caspase 8 and cathepsin function suppresses sorafenib and vorinostat lethality in GI tumor cells

Panels A and B. HEPG2 and MiaPaca2 cells plated in triplicate, as indicated, were either treated with: left graphical panels, vehicle (DMSO), zVAD (25 µM), cathepsin inhibitor (1 µM) or zVAD and cathepsin inhibitor, or: right graphical panels, infected to express empty vector (CMV), dominant negative caspase 9, caspase 8 inhibitor CRM A or dominant negative caspase 9 and CRM A. Cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel C. SV40 Large T antigen transformed mouse embryonic fibroblasts lacking expression of various pro-apoptotic genes were plated in triplicate and treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells.

Figure 2. Inhibition of caspase 8 and cathepsin function suppresses sorafenib and vorinostat lethality in GI tumor cells

Panels A and B. HEPG2 and MiaPaca2 cells plated in triplicate, as indicated, were either treated with: left graphical panels, vehicle (DMSO), zVAD (25 µM), cathepsin inhibitor (1 µM) or zVAD and cathepsin inhibitor, or: right graphical panels, infected to express empty vector (CMV), dominant negative caspase 9, caspase 8 inhibitor CRM A or dominant negative caspase 9 and CRM A. Cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel C. SV40 Large T antigen transformed mouse embryonic fibroblasts lacking expression of various pro-apoptotic genes were plated in triplicate and treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells.

Figure 3. Sorafenib and vorinostat treatment modulates the expression of c-FLIP-s, BCL-XL and MCL-1 in tumor cells

Panel A. HEPG2 and UOK121LN cells were plated and treated 24h after plating with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to SDS PAGE followed by immunoblotting to determine the expression of BID, pro-caspase 8, pro-caspase 3, XIAP, BCL-2, BCL-XL, MCL-1, c-FLIP-s, P-eIF2α S51, GAPDH and ERK2. Data are from a representative study (n = 3). Panel B. HEPG2 cells infected to express empty vector (CMV), BCL-XL, XIAP or c-FLIP-s. Cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Inset panel upper right: HEPG2 cells were plated and treated 24h after plating with vehicle (DMSO), sorafenib (3.0 µM), vorinostat (500 nM) or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to immunoprecipitation and/or SDS PAGE followed by immunoblotting to determine the expression of BIM, pro-caspase 8, BAD S112 phosphorylation, BAX activation, BAK activation and GAPDH. Data are from a representative study (n = 3). Panel C. UOK121LN cells were infected to express empty vector (CMV), BCL-XL, XIAP or c-FLIP-s. Cells were treated with vehicle (DMSO), sorafenib (3.0 µM), vorinostat (500 nM) or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Inset panel upper right: UOK121LN cells were plated and treated 24h after plating with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to immunoprecipitation and/or SDS PAGE followed by immunoblotting to determine the expression of BIM, pro-caspase 8, BAD S112 phosphorylation, BAX activation, BAK activation and GAPDH. Data are from a representative study (n = 3).

Figure 3. Sorafenib and vorinostat treatment modulates the expression of c-FLIP-s, BCL-XL and MCL-1 in tumor cells

Panel A. HEPG2 and UOK121LN cells were plated and treated 24h after plating with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to SDS PAGE followed by immunoblotting to determine the expression of BID, pro-caspase 8, pro-caspase 3, XIAP, BCL-2, BCL-XL, MCL-1, c-FLIP-s, P-eIF2α S51, GAPDH and ERK2. Data are from a representative study (n = 3). Panel B. HEPG2 cells infected to express empty vector (CMV), BCL-XL, XIAP or c-FLIP-s. Cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Inset panel upper right: HEPG2 cells were plated and treated 24h after plating with vehicle (DMSO), sorafenib (3.0 µM), vorinostat (500 nM) or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to immunoprecipitation and/or SDS PAGE followed by immunoblotting to determine the expression of BIM, pro-caspase 8, BAD S112 phosphorylation, BAX activation, BAK activation and GAPDH. Data are from a representative study (n = 3). Panel C. UOK121LN cells were infected to express empty vector (CMV), BCL-XL, XIAP or c-FLIP-s. Cells were treated with vehicle (DMSO), sorafenib (3.0 µM), vorinostat (500 nM) or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Inset panel upper right: UOK121LN cells were plated and treated 24h after plating with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to immunoprecipitation and/or SDS PAGE followed by immunoblotting to determine the expression of BIM, pro-caspase 8, BAD S112 phosphorylation, BAX activation, BAK activation and GAPDH. Data are from a representative study (n = 3).

Figure 3. Sorafenib and vorinostat treatment modulates the expression of c-FLIP-s, BCL-XL and MCL-1 in tumor cells

Panel A. HEPG2 and UOK121LN cells were plated and treated 24h after plating with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to SDS PAGE followed by immunoblotting to determine the expression of BID, pro-caspase 8, pro-caspase 3, XIAP, BCL-2, BCL-XL, MCL-1, c-FLIP-s, P-eIF2α S51, GAPDH and ERK2. Data are from a representative study (n = 3). Panel B. HEPG2 cells infected to express empty vector (CMV), BCL-XL, XIAP or c-FLIP-s. Cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Inset panel upper right: HEPG2 cells were plated and treated 24h after plating with vehicle (DMSO), sorafenib (3.0 µM), vorinostat (500 nM) or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to immunoprecipitation and/or SDS PAGE followed by immunoblotting to determine the expression of BIM, pro-caspase 8, BAD S112 phosphorylation, BAX activation, BAK activation and GAPDH. Data are from a representative study (n = 3). Panel C. UOK121LN cells were infected to express empty vector (CMV), BCL-XL, XIAP or c-FLIP-s. Cells were treated with vehicle (DMSO), sorafenib (3.0 µM), vorinostat (500 nM) or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Inset panel upper right: UOK121LN cells were plated and treated 24h after plating with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Six and 24 hours after drug exposure, cells were isolated and subjected to immunoprecipitation and/or SDS PAGE followed by immunoblotting to determine the expression of BIM, pro-caspase 8, BAD S112 phosphorylation, BAX activation, BAK activation and GAPDH. Data are from a representative study (n = 3).

Figure 4. Sorafenib and vorinostat interact to kill tumor cells via activation of CD95 and suppression of c-FLIP-s expression

Panel A. HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel B. UOK121LN cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel C. Upper blotting panels: HEPG2 cells were plated and treated 24h after plating with vehicle or with sorafenib and vorinostat. Cells were isolated at the indicated time points after sorafenib and vorinostat exposure and CD95 immunoprecipitated from the cell lysate. SDS PAGE followed by immunoblotting of CD95 immunoprecipitates was performed to determine the association of pro-caspase 8 and caspase 8 with CD95. Data are from a representative study (n = 3). Lower immunohistochemistry panels: HEPG2 and UOK121LN cells were plated on glass slides and treated 24h after plating with vehicle (DMSO) or with sorafenib and vorinostat. Six hours after drug exposure, cells were fixed in situ. Fixed cells were blocked, incubated overnight with anti-CD95 antibody, then incubated with a 488nm-tagged fluorescent secondary antibody. Cells were analyzed on a fluorescent microscope (X100 mag.). The intensity of CD95 staining was determined at 50 random points per cell for a total of 5 cells ± SEM (n = 3 separate studies). Panel D. Blotting section: HEP3B cells were transfected with either an empty vector plasmid (CMV) or to express dominant negative eIF2α S51A. Twenty four hours after plating, cells were treated with vehicle (DMSO), or with sorafenib (S, 3.0 µM) and vorinostat (V, 500 nM). Cells were isolated 6h or 24h, as indicated, after drug exposure and the expression of c-FLIP-s, MCL-1 and GAPDH determined at each time point. A representative study (n = 2) is shown. Graphical Section: transformed mouse embryonic fibroblasts (wild type; expressing eIF2α S51A) were plated in triplicate and treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells.

Figure 4. Sorafenib and vorinostat interact to kill tumor cells via activation of CD95 and suppression of c-FLIP-s expression

Panel A. HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel B. UOK121LN cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel C. Upper blotting panels: HEPG2 cells were plated and treated 24h after plating with vehicle or with sorafenib and vorinostat. Cells were isolated at the indicated time points after sorafenib and vorinostat exposure and CD95 immunoprecipitated from the cell lysate. SDS PAGE followed by immunoblotting of CD95 immunoprecipitates was performed to determine the association of pro-caspase 8 and caspase 8 with CD95. Data are from a representative study (n = 3). Lower immunohistochemistry panels: HEPG2 and UOK121LN cells were plated on glass slides and treated 24h after plating with vehicle (DMSO) or with sorafenib and vorinostat. Six hours after drug exposure, cells were fixed in situ. Fixed cells were blocked, incubated overnight with anti-CD95 antibody, then incubated with a 488nm-tagged fluorescent secondary antibody. Cells were analyzed on a fluorescent microscope (X100 mag.). The intensity of CD95 staining was determined at 50 random points per cell for a total of 5 cells ± SEM (n = 3 separate studies). Panel D. Blotting section: HEP3B cells were transfected with either an empty vector plasmid (CMV) or to express dominant negative eIF2α S51A. Twenty four hours after plating, cells were treated with vehicle (DMSO), or with sorafenib (S, 3.0 µM) and vorinostat (V, 500 nM). Cells were isolated 6h or 24h, as indicated, after drug exposure and the expression of c-FLIP-s, MCL-1 and GAPDH determined at each time point. A representative study (n = 2) is shown. Graphical Section: transformed mouse embryonic fibroblasts (wild type; expressing eIF2α S51A) were plated in triplicate and treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells.

Figure 4. Sorafenib and vorinostat interact to kill tumor cells via activation of CD95 and suppression of c-FLIP-s expression

Panel A. HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel B. UOK121LN cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel C. Upper blotting panels: HEPG2 cells were plated and treated 24h after plating with vehicle or with sorafenib and vorinostat. Cells were isolated at the indicated time points after sorafenib and vorinostat exposure and CD95 immunoprecipitated from the cell lysate. SDS PAGE followed by immunoblotting of CD95 immunoprecipitates was performed to determine the association of pro-caspase 8 and caspase 8 with CD95. Data are from a representative study (n = 3). Lower immunohistochemistry panels: HEPG2 and UOK121LN cells were plated on glass slides and treated 24h after plating with vehicle (DMSO) or with sorafenib and vorinostat. Six hours after drug exposure, cells were fixed in situ. Fixed cells were blocked, incubated overnight with anti-CD95 antibody, then incubated with a 488nm-tagged fluorescent secondary antibody. Cells were analyzed on a fluorescent microscope (X100 mag.). The intensity of CD95 staining was determined at 50 random points per cell for a total of 5 cells ± SEM (n = 3 separate studies). Panel D. Blotting section: HEP3B cells were transfected with either an empty vector plasmid (CMV) or to express dominant negative eIF2α S51A. Twenty four hours after plating, cells were treated with vehicle (DMSO), or with sorafenib (S, 3.0 µM) and vorinostat (V, 500 nM). Cells were isolated 6h or 24h, as indicated, after drug exposure and the expression of c-FLIP-s, MCL-1 and GAPDH determined at each time point. A representative study (n = 2) is shown. Graphical Section: transformed mouse embryonic fibroblasts (wild type; expressing eIF2α S51A) were plated in triplicate and treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells.

Figure 4. Sorafenib and vorinostat interact to kill tumor cells via activation of CD95 and suppression of c-FLIP-s expression

Panel A. HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel B. UOK121LN cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells. Panel C. Upper blotting panels: HEPG2 cells were plated and treated 24h after plating with vehicle or with sorafenib and vorinostat. Cells were isolated at the indicated time points after sorafenib and vorinostat exposure and CD95 immunoprecipitated from the cell lysate. SDS PAGE followed by immunoblotting of CD95 immunoprecipitates was performed to determine the association of pro-caspase 8 and caspase 8 with CD95. Data are from a representative study (n = 3). Lower immunohistochemistry panels: HEPG2 and UOK121LN cells were plated on glass slides and treated 24h after plating with vehicle (DMSO) or with sorafenib and vorinostat. Six hours after drug exposure, cells were fixed in situ. Fixed cells were blocked, incubated overnight with anti-CD95 antibody, then incubated with a 488nm-tagged fluorescent secondary antibody. Cells were analyzed on a fluorescent microscope (X100 mag.). The intensity of CD95 staining was determined at 50 random points per cell for a total of 5 cells ± SEM (n = 3 separate studies). Panel D. Blotting section: HEP3B cells were transfected with either an empty vector plasmid (CMV) or to express dominant negative eIF2α S51A. Twenty four hours after plating, cells were treated with vehicle (DMSO), or with sorafenib (S, 3.0 µM) and vorinostat (V, 500 nM). Cells were isolated 6h or 24h, as indicated, after drug exposure and the expression of c-FLIP-s, MCL-1 and GAPDH determined at each time point. A representative study (n = 2) is shown. Graphical Section: transformed mouse embryonic fibroblasts (wild type; expressing eIF2α S51A) were plated in triplicate and treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells.

Figure 5. Delayed inactivation of ERK1/2 and AKT correlates with profound and long-term loss of c-FLIP-s expression: delayed activation of NFκB is a toxic signal following sorafenib and vorinostat exposure

Panel A. section (i): HEPG2 cells, 24h after plating, were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Cells were isolated 24–96h after drug exposure, subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2, JNK1/2, p38 MAPK and AKT (S473) as well as total ERK2. A representative of 3 separate studies is shown; section (ii): HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 as well as total ERK2 levels. A representative of 3 separate studies is shown. Panel B. Upper blotting panel to left: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. After infection (24h) cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 and AKT (S473) as well as total ERK2 levels. Upper blotting panel to right: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), or viruses to express constitutively active AKT and constitutively active MEK1 EE. Twenty four hours after infection cells were treated with vehicle or with sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the expression of c- FLIP-s as well as total ERK2 levels. Lower Graphical Panel: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. Twenty four hours after infection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells; ## p < 0.05 less cell killing than compared to parallel condition in caAKT or caMEK1 EE treatment cells. Panel C. graphical panel to the left; HEPG2 and UOK121LN cells, 24h after plating in triplicate, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs. Parallel control plates of cells, not shown, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs and co-transfected with a plasmid to express dominant negative IκB or with a plasmid to express a constitutively activated form of NFκB as internal negative and positive controls. Thirty six hours after transfection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. At the indicated times cells were isolated and the amount of luciferase per cell and amount of β-galactosidase per cell determined as described in the Methods (± SEM, a representative from 2 separate studies). Control studies demonstrated that over-expression of dominant negative IκB blocked vorinostat-induced activation of NFκB-luciferase activity (not shown). graphical panel to the right; HEPG2 cells, 24h after plating in triplicate, were infected with control empty vector virus (CMV) or a recombinant virus to express dominant negative IκB S32A S36A (dn IκB). Twenty four hours after infection, cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated, spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods. The percentage of TUNEL positive cells ± SEM was determined. Data shown are from a representative of 2 independent studies. # p < 0.05 less cell killing than compared to the same condition in CMV infected cells. Panel D. HEP3B cells were injected (10⁷) into the flanks of athymic mice and tumors permitted to form. The tumor take-rate was approximately 20%. Tumors were permitted to grow to ~150 mm³ after which time tumors were segregated based on volume into relatively normalized groups. Animals / tumors in triplicate were subjected to vehicle / drug administration by oral gavage once daily for three consecutive days. BAY 54-9085 was administered at a dose of 45 mg/kg. Vorinostat was administered at a dose of 25 mg/kg. Twelve hours after the final gavage dosing, the animals were humanely sacrificed, the tumors were removed and sectioned. One portion of the tumor was subjected to immunohistochemical analyses (panels to the left) to determine eIF2α phosphorylation, ERK1/2 phosphorylation, AKT (S473) phosphorylation, c-FLIP-s expression, MCL-1 expression, caspase 3 cleavage status, TUNEL positivity, and tumor morphology by H&E staining. Another portion of the tumor was macerated and digested to obtain individual tumor cells. Individual viable isolated tumor cells were plated 2,000–10,000 cells per 10 cm radius dish and colonies permitted to form over the following 10 days. Cells were fixed, stained with crystal violet and colonies of > 50 cells / colony counted. The colony formation for Vorinostat treated cells was 0.78 ± 0.04 and the colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib was 0.58 ± 0.06; # p < 0.05 colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib less than colony formation for Vorinostat treated cells.

Figure 5. Delayed inactivation of ERK1/2 and AKT correlates with profound and long-term loss of c-FLIP-s expression: delayed activation of NFκB is a toxic signal following sorafenib and vorinostat exposure

Panel A. section (i): HEPG2 cells, 24h after plating, were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Cells were isolated 24–96h after drug exposure, subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2, JNK1/2, p38 MAPK and AKT (S473) as well as total ERK2. A representative of 3 separate studies is shown; section (ii): HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 as well as total ERK2 levels. A representative of 3 separate studies is shown. Panel B. Upper blotting panel to left: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. After infection (24h) cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 and AKT (S473) as well as total ERK2 levels. Upper blotting panel to right: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), or viruses to express constitutively active AKT and constitutively active MEK1 EE. Twenty four hours after infection cells were treated with vehicle or with sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the expression of c- FLIP-s as well as total ERK2 levels. Lower Graphical Panel: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. Twenty four hours after infection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells; ## p < 0.05 less cell killing than compared to parallel condition in caAKT or caMEK1 EE treatment cells. Panel C. graphical panel to the left; HEPG2 and UOK121LN cells, 24h after plating in triplicate, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs. Parallel control plates of cells, not shown, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs and co-transfected with a plasmid to express dominant negative IκB or with a plasmid to express a constitutively activated form of NFκB as internal negative and positive controls. Thirty six hours after transfection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. At the indicated times cells were isolated and the amount of luciferase per cell and amount of β-galactosidase per cell determined as described in the Methods (± SEM, a representative from 2 separate studies). Control studies demonstrated that over-expression of dominant negative IκB blocked vorinostat-induced activation of NFκB-luciferase activity (not shown). graphical panel to the right; HEPG2 cells, 24h after plating in triplicate, were infected with control empty vector virus (CMV) or a recombinant virus to express dominant negative IκB S32A S36A (dn IκB). Twenty four hours after infection, cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated, spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods. The percentage of TUNEL positive cells ± SEM was determined. Data shown are from a representative of 2 independent studies. # p < 0.05 less cell killing than compared to the same condition in CMV infected cells. Panel D. HEP3B cells were injected (10⁷) into the flanks of athymic mice and tumors permitted to form. The tumor take-rate was approximately 20%. Tumors were permitted to grow to ~150 mm³ after which time tumors were segregated based on volume into relatively normalized groups. Animals / tumors in triplicate were subjected to vehicle / drug administration by oral gavage once daily for three consecutive days. BAY 54-9085 was administered at a dose of 45 mg/kg. Vorinostat was administered at a dose of 25 mg/kg. Twelve hours after the final gavage dosing, the animals were humanely sacrificed, the tumors were removed and sectioned. One portion of the tumor was subjected to immunohistochemical analyses (panels to the left) to determine eIF2α phosphorylation, ERK1/2 phosphorylation, AKT (S473) phosphorylation, c-FLIP-s expression, MCL-1 expression, caspase 3 cleavage status, TUNEL positivity, and tumor morphology by H&E staining. Another portion of the tumor was macerated and digested to obtain individual tumor cells. Individual viable isolated tumor cells were plated 2,000–10,000 cells per 10 cm radius dish and colonies permitted to form over the following 10 days. Cells were fixed, stained with crystal violet and colonies of > 50 cells / colony counted. The colony formation for Vorinostat treated cells was 0.78 ± 0.04 and the colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib was 0.58 ± 0.06; # p < 0.05 colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib less than colony formation for Vorinostat treated cells.

Figure 5. Delayed inactivation of ERK1/2 and AKT correlates with profound and long-term loss of c-FLIP-s expression: delayed activation of NFκB is a toxic signal following sorafenib and vorinostat exposure

Panel A. section (i): HEPG2 cells, 24h after plating, were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Cells were isolated 24–96h after drug exposure, subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2, JNK1/2, p38 MAPK and AKT (S473) as well as total ERK2. A representative of 3 separate studies is shown; section (ii): HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 as well as total ERK2 levels. A representative of 3 separate studies is shown. Panel B. Upper blotting panel to left: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. After infection (24h) cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 and AKT (S473) as well as total ERK2 levels. Upper blotting panel to right: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), or viruses to express constitutively active AKT and constitutively active MEK1 EE. Twenty four hours after infection cells were treated with vehicle or with sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the expression of c- FLIP-s as well as total ERK2 levels. Lower Graphical Panel: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. Twenty four hours after infection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells; ## p < 0.05 less cell killing than compared to parallel condition in caAKT or caMEK1 EE treatment cells. Panel C. graphical panel to the left; HEPG2 and UOK121LN cells, 24h after plating in triplicate, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs. Parallel control plates of cells, not shown, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs and co-transfected with a plasmid to express dominant negative IκB or with a plasmid to express a constitutively activated form of NFκB as internal negative and positive controls. Thirty six hours after transfection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. At the indicated times cells were isolated and the amount of luciferase per cell and amount of β-galactosidase per cell determined as described in the Methods (± SEM, a representative from 2 separate studies). Control studies demonstrated that over-expression of dominant negative IκB blocked vorinostat-induced activation of NFκB-luciferase activity (not shown). graphical panel to the right; HEPG2 cells, 24h after plating in triplicate, were infected with control empty vector virus (CMV) or a recombinant virus to express dominant negative IκB S32A S36A (dn IκB). Twenty four hours after infection, cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated, spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods. The percentage of TUNEL positive cells ± SEM was determined. Data shown are from a representative of 2 independent studies. # p < 0.05 less cell killing than compared to the same condition in CMV infected cells. Panel D. HEP3B cells were injected (10⁷) into the flanks of athymic mice and tumors permitted to form. The tumor take-rate was approximately 20%. Tumors were permitted to grow to ~150 mm³ after which time tumors were segregated based on volume into relatively normalized groups. Animals / tumors in triplicate were subjected to vehicle / drug administration by oral gavage once daily for three consecutive days. BAY 54-9085 was administered at a dose of 45 mg/kg. Vorinostat was administered at a dose of 25 mg/kg. Twelve hours after the final gavage dosing, the animals were humanely sacrificed, the tumors were removed and sectioned. One portion of the tumor was subjected to immunohistochemical analyses (panels to the left) to determine eIF2α phosphorylation, ERK1/2 phosphorylation, AKT (S473) phosphorylation, c-FLIP-s expression, MCL-1 expression, caspase 3 cleavage status, TUNEL positivity, and tumor morphology by H&E staining. Another portion of the tumor was macerated and digested to obtain individual tumor cells. Individual viable isolated tumor cells were plated 2,000–10,000 cells per 10 cm radius dish and colonies permitted to form over the following 10 days. Cells were fixed, stained with crystal violet and colonies of > 50 cells / colony counted. The colony formation for Vorinostat treated cells was 0.78 ± 0.04 and the colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib was 0.58 ± 0.06; # p < 0.05 colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib less than colony formation for Vorinostat treated cells.

Figure 5. Delayed inactivation of ERK1/2 and AKT correlates with profound and long-term loss of c-FLIP-s expression: delayed activation of NFκB is a toxic signal following sorafenib and vorinostat exposure

Panel A. section (i): HEPG2 cells, 24h after plating, were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Cells were isolated 24–96h after drug exposure, subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2, JNK1/2, p38 MAPK and AKT (S473) as well as total ERK2. A representative of 3 separate studies is shown; section (ii): HEPG2 cells were transfected with a non-specific scrambled control (siSCR) siRNA molecule, or molecules to knock down the expression of CD95 or FADD, according to the manufacturer’s instructions. Cells were treated 24h after transfection with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 as well as total ERK2 levels. A representative of 3 separate studies is shown. Panel B. Upper blotting panel to left: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. After infection (24h) cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the phosphorylation of ERK1/2 and AKT (S473) as well as total ERK2 levels. Upper blotting panel to right: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), or viruses to express constitutively active AKT and constitutively active MEK1 EE. Twenty four hours after infection cells were treated with vehicle or with sorafenib and vorinostat. Forty eight hours after drug exposure cells were isolated, and subjected to SDS PAGE and immunoblotting to determine the expression of c- FLIP-s as well as total ERK2 levels. Lower Graphical Panel: HEPG2 cells, 24h after plating, were infected with empty vector control virus (CMV), a virus to express constitutively active AKT, a virus to express constitutively active MEK1 EE, or both the active AKT and active MEK1 EE viruses. Twenty four hours after infection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Ninety six hours after drug exposure cells were isolated and viability determined by trypan blue assay. The percentage of trypan blue positive cells ± SEM was determined. Data shown are from the mean of 3 independent studies. # p < 0.05 less cell killing than compared to parallel condition in vehicle treatment cells; ## p < 0.05 less cell killing than compared to parallel condition in caAKT or caMEK1 EE treatment cells. Panel C. graphical panel to the left; HEPG2 and UOK121LN cells, 24h after plating in triplicate, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs. Parallel control plates of cells, not shown, were transfected with NFκB-luciferase and β-galactosidase constitutive reporter constructs and co-transfected with a plasmid to express dominant negative IκB or with a plasmid to express a constitutively activated form of NFκB as internal negative and positive controls. Thirty six hours after transfection cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. At the indicated times cells were isolated and the amount of luciferase per cell and amount of β-galactosidase per cell determined as described in the Methods (± SEM, a representative from 2 separate studies). Control studies demonstrated that over-expression of dominant negative IκB blocked vorinostat-induced activation of NFκB-luciferase activity (not shown). graphical panel to the right; HEPG2 cells, 24h after plating in triplicate, were infected with control empty vector virus (CMV) or a recombinant virus to express dominant negative IκB S32A S36A (dn IκB). Twenty four hours after infection, cells were treated with vehicle, sorafenib, vorinostat or both sorafenib and vorinostat. Forty eight hours after drug exposure, cells were isolated, spun onto glass slides and stained using established methods for double stranded DNA breaks indicative of apoptosis (TUNEL) as described in the Methods. The percentage of TUNEL positive cells ± SEM was determined. Data shown are from a representative of 2 independent studies. # p < 0.05 less cell killing than compared to the same condition in CMV infected cells. Panel D. HEP3B cells were injected (10⁷) into the flanks of athymic mice and tumors permitted to form. The tumor take-rate was approximately 20%. Tumors were permitted to grow to ~150 mm³ after which time tumors were segregated based on volume into relatively normalized groups. Animals / tumors in triplicate were subjected to vehicle / drug administration by oral gavage once daily for three consecutive days. BAY 54-9085 was administered at a dose of 45 mg/kg. Vorinostat was administered at a dose of 25 mg/kg. Twelve hours after the final gavage dosing, the animals were humanely sacrificed, the tumors were removed and sectioned. One portion of the tumor was subjected to immunohistochemical analyses (panels to the left) to determine eIF2α phosphorylation, ERK1/2 phosphorylation, AKT (S473) phosphorylation, c-FLIP-s expression, MCL-1 expression, caspase 3 cleavage status, TUNEL positivity, and tumor morphology by H&E staining. Another portion of the tumor was macerated and digested to obtain individual tumor cells. Individual viable isolated tumor cells were plated 2,000–10,000 cells per 10 cm radius dish and colonies permitted to form over the following 10 days. Cells were fixed, stained with crystal violet and colonies of > 50 cells / colony counted. The colony formation for Vorinostat treated cells was 0.78 ± 0.04 and the colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib was 0.58 ± 0.06; # p < 0.05 colony formation for Vorinostat and Sorafenib treated cells corrected for the toxicity of sorafenib less than colony formation for Vorinostat treated cells.