Huh7 cells were being grown on sterile coverslips in Fingolimod six-nicely plates at the density of 3. Cells have been then Fingolimod addressed by various doses of sorafenib in the presence or absence of FTY720 for 24 h. In order to examine the mechanisms behind the enhanced cytotoxicity following combined treatment, further studies were performed in the Huh7 cell line, which, similarly to a majority of primary HCC, and in contrast to HepG2, harbor mutated p53 (Bressac et al. 1990). To investigate whether the cell cycle distribution was affected by the combined treatment, Huh7 cells were treated with sorafenib in the presence or absence of FTY720 for 24 or 48 h. After both 24 and 48 h, combined treatment with 2 μmol/L of sorafenib and 8 μmol/L of FTY720 caused a significant accumulation of cells in the G1 phase and a concomitant decrease of the number of cells in the S phase compared to treatment with sorafenib alone, FigureFigure2.2. In contrast, at higher doses of sorafenib, cotreatment with 8 μmol/L of FTY720 caused a significant decrease or increase of cells in the G2 phase after 24 or 48 h, respectively. At the latter time point, FTY720 also significantly increased the sub G1 fraction, representing dead cells, observed after treatment with 6 μmol/L or 10 μmol/L doses of sorafenib alone, FigureFigure22.
To further investigate the mechanisms behind the cytotoxic effect of combined treatment with sorafenib and FTY720, early signs of apoptosis were assessed by flow cytometry analysis of cells labeled with Annexin-V-Fluos and PI. A significant increase in presumably apoptotic, secondary necrotic and necrotic cells in the lower right, upper right and upper left quadrants, respectively, was observed after combined treatment with 10 μmol/L of sorafenib and 8 μmol/L of FTY720 as compared to treatment with sorafenib alone for 6 hours, FigureFigure3.3. Condensation of nuclei was observed after treatment with 10 and 20 μmol/L of sorafenib for 24 h, and was further enhanced after cotreatment with 8 μmol/L of FTY, FigureFigure4A.4A. In order to investigate different apoptotic pathways, cleaved PARP and cytosolic AIF, markers for caspase-dependent and -independent apoptosis, respectively, were analyzed by western blotting. Treatment with a high dose of sorafenib (20 μmol/L) for 24 h caused cleavage of PARP in the absence of FTY720, whereas lower doses did not, FigureFigure4B.4B. The ratio of cleaved PARP increased when the cells were treated with 20 μmol/L of sorafenib together with 8 μmol/L of FTY720. Furthermore, a weak band representing cleaved PARP was observed when 10 μmol/L of sorafenib was combined with 8 μmol/L of FTY720. Treatment with either 2 or 10 μmol/L of sorafenib or 8 μmol/L of FTY720 alone for 24 h induced a slight release of AIF into the cytosol, which was further increased after combination treatment, FigureFigure4C.4C. The purity of the mitochondrial and cytosolic fractions was confirmed using the mitochondrial and cytoskeletal markers COX IV and β-actin, respectively. The expression of COX IV, declined after treatment with 10 μmol/L sorafenib ±8 μmol/L FTY720. The role of mitochondria-mediated apoptosis was supported by release of Cyt c into the cytosol.