Treatment of Hep-G2 with Doxo alone induced apoptosis in 24% of cells, whereas Doxo and TRAIL in combination led to apoptosis rates of 43% exactly (Figure (Figure3B,3B, lower panel). Figure 3 Treatment of HCC cells with TRAIL in combination with 5-FU and doxorubicin. Values are expressed as mean �� SD. A: Huh7 and Hep-G2 cells were analyzed for cell viability after treatment with the chemotherapeutic agents 5-FU and doxorubicin alone. … Treatment of HCC cells with TRAIL in combination with specific kinase inhibitors Antiapoptotic pathways such as PI3K/Akt, epidermal growth factor receptor (EGFR), [mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) kinase] (MEK)/ERK are well known to be activated in malignant cells, thus contributing to cell cycle progression and tumor growth.

Therefore, we analyzed whether inhibition of kinases involved in these pathways could overcome resistance towards TRAIL-mediated apoptosis. Firstly, we applied the multi-kinase inhibitor Sorafenib to inhibit RAF/MEK/ERK signaling, in escalating concentrations (2.5, 5 and 10 ��mol/L). A dose-dependent decrease of cell viability in Huh7 and Hep-G2 was observed (Figure (Figure4A,4A, upper panel). In a second step, we analyzed the impact of Sorafenib on TRAIL treatment. In Huh7 cells, Sorafenib (10 ��mol/L) induced apoptosis rates of 50%. Strikingly, the combination of SkTRAIL (50 ng/mL) and Sorafenib (10 ��mol/L) induced apoptosis in 80% of the cells. In Hep-G2 cells Sorafenib caused only minor apoptosis rates (33%). However, combination of TRAIL and Sorafenib led to 98% apoptotic cells (Figure (Figure4A,4A, lower panel).

Figure 4 Treatment of HCC cells with TRAIL in combination with specific kinase inhibitors. Viability of HCC cells treated with kinase inhibitors alone (upper panels). On day one after seeding of Huh7 and Hep-G2 cells onto 96-well plates, cells were treated with … Next, we inhibited the PI3K/Akt pathway by application of the PI3K inhibitor LY294002. A slight decrease of cell viability was observed in Huh7 and Hep-G2 after 48 h treatment in concentrations lower than 50 ��mol/L (Figure (Figure4B,4B, upper panel). However, combination of LY294002 (10 ��mol/L) and SkTRAIL (50 ng/mL) doubled apoptosis rates in Hep-G2 cells to 59% compared to SkTRAIL treatment alone.

In Huh7, we observed an increased rate of apoptosis after treatment with the combination of LY294002 and SkTRAIL compared to SkTRAIL alone (23% vs 11%, respectively. Figure Figure4B,4B, lower panel). Furthermore, we used AG1478 to inhibit EGFR kinase. Interestingly, inhibition of EGFR Dacomitinib kinase increased cell viability in Hep-G2 cells in concentrations up to 5 ��mol/L. In Huh7 cells, AG1478 caused no significant changes in cell viability when applied in low concentrations (Figure (Figure4C,4C, upper panel).