TABLE 3.

GX15-070 restores the synergy of sorafenib + valproate toxicity in cells with blocked extrinsic pathway signaling

Pancreatic cancer (PANC1) cells were infected 12 h after plating at an approximate multiplicity of infection of 50 with either a control empty vector recombinant adenovirus (CMV) or a recombinant virus to express the caspase 8 inhibitor c-FLIP-s. Twenty-four hours after infection, infected cells were plated as single cells (250-1500 cells/well) in sextuplicate; 12 h after this plating, the infected cells were treated with vehicle (DMSO), sorafenib (2.25-3.75 μM), sodium valproate (0.75-1.25 mM), or both drugs combined, as indicated, at a fixed concentration ratio to perform median dose-effect analyses for the determination of synergy. Cells were in parallel treated with either vehicle (DMSO) or GX15-070 (50 nM). After drug exposure (24 h), the medium was changed and cells were cultured in drug-free medium for an additional 10 to 14 days. Cells were fixed and stained with crystal violet, and colonies of >50 cells/colony were counted. Colony formation data were entered into the Calcusyn program, and combination index (CI) values were determined. A CI value of less than 1.00 indicates synergy. Treatment of CMV-infected cells with GX15-070 reduced colony formation as a single agent by 0.63 ± 0.02. Treatment of c-FLIP-s infected cells with GX15-070 reduced colony formation as a single agent by 0.72 ± 0.01.

Treatments	Sorafenib	Valproate	Fa	CI
	μM	mM
CMV + DMSO	2.25	0.75	0.43	0.72
	3.00	1.00	0.51	0.70
	3.75	1.25	0.66	0.66
CMV + 50 nM GX15-070	2.25	0.75	0.43	0.31
	3.00	1.00	0.51	0.32
	3.75	1.25	0.58	0.26
c-FLIP-s + DMSO	2.25	0.75	0.10	4.55
	3.00	1.00	0.13	3.54
	3.75	1.25	0.19	2.08
c-FLIP-s + 50 nM GX15-070	2.25	0.75	0.43	0.58
	3.00	1.00	0.47	0.5
	3.75	1.25	0.56	0.49