Figure 5

Mcl-1 interference sensitizes HCC cells towards combined treatment modalities. (A) Huh7 cells were transfected with siRNA specific for Mcl-1 (grey bars) or transfected with siRNA specific for GFP as control (light bars). After 24 h, cells were treated with chemotherapeutic drugs for another 24 h: cisplatin, 1 μg/ml, epirubicin, 200 ng/ml, 5-FU, 150 μg/ml (+ valproic acid (VA), 100 μg/ml), and mitomycin C, 5 μM. Some cultures were pre-incubated for 1 h with the kinase inhibitors LY294002 (25 μM, blocking PI3K), PD98059 (50 μM, blocking MEK1), AG1478 (5 μM, blocking EGF receptor tyrosine kinase) and a Raf I-kinase inhibitor (100 nM). Apoptosis was assessed by FACS analysis. *p < 0.05, **p < 0.001. n.s., not significant. (B) and (C) Huh7 cells were transfected with siRNA (40 nM) as described above and treated 24 h thereafter. In (B), cells were treated with 5-FU (150 μg/ml) and VA (100 μg/ml) for 8 or 24 h, as indicated. Cells were harvested and caspase-3 (upper panel) or caspase-9 activities (lower panel) were measured by applying caspase-specific fluorogenic substrates as described in the materials and methods section. *p < 0.01, **p < 0.001. (C) Cells were treated with 5-FU (150 μg/ml) for 48 h with or without simultaneous treatment with TRAIL as indicated. Apoptosis was measured using flow cytometrical analysis. Values are means + SD. *p < 0.05, **p < 0.001, n.s., not significant.