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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: J Cell Physiol. 2015 Jan;230(1):131–139. doi: 10.1002/jcp.24689

Figure 6. Sorafenib and lapatinib treatment kills through the extrinsic and intrinsic apoptosis pathways.

Figure 6

(A) and (B) GBM5 and GBM12 cells were infected with a control empty vector adenovirus (CMV, 50 m.o.i.) or viruses to express dominant negative caspase 9, BCL-XL or c-FLIP-s. Twenty four h after infection cells were treated with vehicle (DMSO), sorafenib (6 μM), lapatinib (2 μM) or the drugs combined. Cells were isolated 24h after exposure and viability determined by trypan blue exclusion (n = 3, +/− SEM) #p < 0.05 less than corresponding value in CMV control; ## p < 0.05 less than corresponding value in dominant negative caspase 9 expressing cells. (C) GBM12 cells were transfected with a control scrambled siRNA (siSCR) or siRNA molecules to knock down the expression of BAX, BAK, NOXA, PUMA. Thirty six h after transfection cells were treated with vehicle (DMSO), sorafenib (6 μM), lapatinib (2 μM) or the drugs combined. Cells were isolated 24h after exposure and viability determined by trypan blue exclusion (n = 3, +/− SEM) #p < 0.05 less than corresponding value in siSCR control. (D) GBM12 cells were transfected with a scrambled siRNA (siSCR) or siRNA molecules to knock down expression of CD95 (siCD95) or FADD (siFADD). Thirty six h after transfection cells were treated with vehicle (DMSO), sorafenib (6 μM) and lapatinib (2 μM) combined. Cells were isolated 24h after exposure and viability determined by trypan blue exclusion (n = 3, +/− SEM) #p < 0.05 less than corresponding value in siSCR control. (E) GBM12 cells in 96 well plates were pre-treated with vehicle, the ceramide synthase inhibitor Fumonisin B1 (25 μM) or the ROS quenching agent N-acetyl cysteine (10 mM). Cells were then treated with vehicle (DMSO), sorafenib (6 μM) and lapatinib (2 μM) combined. Six h after treatment cells were fixed in situ without permeabilization. The levels of cell surface CD95 were assessed after immunohistochemical detection of CD95 using a Hermes Wiscan instrument and associated WiSoft densitometry software (n = 3, +/− SEM) #p < 0.05 less than corresponding value in vehicle control. (F) GBM12 cells were infected with a control empty vector adenovirus (CMV, 50 m.o.i.) or viruses to express activated AKT (caAKT) and/or activated MEK1 (caMEK1). Twenty four h after infection cells were treated with vehicle (DMSO), sorafenib (6 μM), lapatinib (2 μM) or the drugs combined. Cells were isolated 24h after exposure and viability determined by trypan blue exclusion (n = 3, +/− SEM) #p < 0.05 less than corresponding value in CMV control; ## p < 0.05 less than corresponding value in caAKT or caMEK1 expressing cells. (G) GBM12 cells were transfected with a control empty vector plasmid (CMV) or plasmids to express activated p70 S6K (ca-p70) and/or activated mTOR (ca-mTOR). Twenty four h after infection cells were treated with vehicle (DMSO), sorafenib (6 μM), lapatinib (2 μM) or the drugs combined. Cells were isolated 24h after exposure and viability determined by trypan blue exclusion (n = 3, +/− SEM) #p < 0.05 less than corresponding value in CMV control; ## p < 0.05 less than corresponding value in ca-p70 expressing cells. (H) GBM6 and GBM12 cells were pre-treated with vehicle (DMSO) or with the JNK inhibitory peptide (10 μM). Cells were then treated with vehicle (DMSO), sorafenib (6 μM), lapatinib (2 μM) or the drugs combined. Cells were isolated 24h after exposure and viability determined by trypan blue exclusion (n = 3, +/− SEM) #p < 0.05 less than corresponding value in vehicle control. (I) Upper: GBM12 cells were pre-treated with vehicle (DMSO) or with the JNK inhibitory peptide (10 μM). Cells were then treated with vehicle (DMSO), sorafenib (6 μM), lapatinib (2 μM) or the drugs combined. Cells were isolated 12h after exposure and lysates generated in CHAPS buffer followed by immunoprecipitation and SDS PAGE to determine activated BAX levels. Lower: GBM12 cells were transfected with a scrambled siRNA (siSCR) or an siRNA molecule to knock down expression of CD95 (siCD95). Thirty six h after transfection cells were treated with vehicle (DMSO), sorafenib (6 μM), lapatinib (2 μM) or the drugs combined. Cells were isolated 12h after treatment and the levels of P-JNK determined.