Role of autophagy in differential sensitivity of hepatocarcinoma cells to sorafenib

Figure 1 Cell death with sorafenib. The effect of varying doses (1, 5 and 10 µmol/L) of SFN on hepatocellular carcinoma cell viability was determined by the MTT assay at 24, 48 and 72 h in (A) Hep3B and (B) Huh7 cells. PARP expression was analyzed by immunoblotting analysis (C). SFN: Sorafenib; RAPA: Rapamycin.

Figure 2 Autophagic response to sorafenib. A: LC3 expression in hepatocellular carcinoma cells was analyzed in the presence of SFN for up to 6 h. Representative blot (left panel) and quantitative analysis of LC3-II by densitometry (middle and right panel). ^aP < 0.05; B: LC3 expression was analyzed in the presence of SFN with and without CQ to determine autophagy flux after 4 and 24 h; C: Autophagic response to nutrient-rich (control) or nutrient-depleted medium was analyzed by immunoblotting of LC3 in Hep3B and Huh7 cells for up to 4 h; D: LC3 expression was analyzed in the presence of 5 nmol/L RAPA after 4 h treatment with and without CQ to examine autophagy flux. NS: Non-significant; SFN: Sorafenib; CQ: Chloroquine; RAPA: Rapamycin.

Figure 3 Altered autophagy signaling in hepatocellular carcinoma cells. A: Hepatocellular carcinoma cells were treated with 5 and 10 μmol/L SFN for 4 and 24 h and the expression of autophagy initiating proteins was analyzed with immunoblotting; B: Quantitative analysis of p-AMPK. ^aP < 0.05; C: Quantitative analysis of LKB1. ^aP < 0.05; D: Quantitative analysis of ULK1. ^bP < 0.001. SFN: Sorafenib.

Figure 4 Gene silencing for AMPK. A: Huh7 cells were treated with siRNA for AMPK for 24 h. After 4 h treatment with SFN, LC3 was assessed with immunoblotting assay; B, C: The effect of either Met or SFN on hepatocellular carcinoma cell viability was determined by the MTT assay at 24 h in (B) Hep3B and (C) Huh7 cells. Some cells were treated with both agents. ^aP < 0.05, ^bP < 0.001. Met: Metformin; SFN: Sorafenib; CQ: Chloroquine.