TRAIL-R3 surface expression was detectable in both cell lines after Doxo treatment. TRAIL-R4 was upregulated in Hep-G2 and unaffected in Huh7 cells (Figure (Figure1D1D). Sensitivity of HCC cells towards different TRAIL compounds Next, we determined sensitivity of HCC cells towards TRAIL-induced apoptosis. Firstly, we analyzed the effect of recombinant thenthereby TRAIL in concentrations from 20 up to 200 ng/mL (combined with Enhancer, 1 ��g/mL). Hep-G2 cells were sensitive towards recombinant TRAIL in a dose-dependent manner, whereas Huh7 were resistant (Figure (Figure2A).2A). Next, we tested LBY135, a chimeric monoclonal antibody targeting TRAIL-R2, in concentrations from 50 to 2000 ng/mL, together with the cross linker F(ab)��2 (2 ��g/mL).

To optimize the interaction between LBY135 and F(ab)��2, we coated the plates with the F(ab)��2-fragment before seeding the cells. Hep-G2 showed moderate sensitivity towards LBY135-induced apoptosis in a dose-dependent manner, whereas Huh7 cells were resistant (Figure (Figure2B).2B). To discover whether resistance was due to impaired interactions between enhancer or F(ab)��2, the ligand and the receptor, we included SkTRAIL in our study. SkTRAIL interacts effectively with TRAIL receptors without additional supplements. Again, Hep-G2 cells revealed a dose-dependent sensitivity to SkTRAIL in concentrations from 10 to 200 ng/mL. In contrast, Huh7 cells were resistant to SkTRAIL (Figure (Figure2C2C). Figure 2 TRAIL-induced apoptosis in hepatocellular carcinoma (HCC) cells. Huh7 and Hep-G2 cells were seeded onto 96-well plates and treated on day one after seeding with different TRAIL compounds.

A: Cells were treated for 48 h with rec. TRAIL + 1 ��g/mL … Treatment of HCC cells with TRAIL in combination with chemotherapy Next, we analyzed whether HCC cells were sensitized to TRAIL-induced apoptosis by co-treatment with the chemotherapeutic drugs 5-FU and Doxo. As a first step, we analyzed whether the chemotherapeutics induced loss of viability if applied alone: after 48 h treatment of Huh7 and Hep-G2 cells with 5-FU (50, 100 and 200 ��g/mL) and Doxo (0.1, 0.5, 1 and 2 ��mol/L), we observed a dose-dependent decrease of cell viability (Figure (Figure3A).3A). Next, we applied these agents in concentrations which exhibited less significant cytotoxic effects, in combination with recombinant TRAIL (+ Enhancer 1 ��g/mL).

5-FU (50 ��g/mL) or TRAIL (100 ng/mL) did not induce apoptosis in Huh7 cells when administered alone. However, combination of 5-FU and TRAIL induced apoptosis in 62% of Huh7 cells. In Hep-G2 cells, TRAIL (100 ng/mL) induced apoptosis in 15% of cells. 5-FU treatment alone triggered apoptosis of 12% of Hep-G2 cells. 5-FU and TRAIL Dacomitinib co-treatment of Hep-G2 resulted in 93% apoptotic cells (Figure (Figure3B,3B, upper panel). Next, we tested the combination of Doxo (0.5 ��mol/L) and TRAIL (100 ng/mL).