Figure 4. Effect of FTY720 on the activities of major signaling pathways, and effect of FTY720/mitotane combination on the proliferation of ACC cells.

(A) The antibodies against PI3K, phospho-PI3K^Y607 (p-PI3K), AKT, phospho-AKT^S473 (p-AKT), ERK, phospho-ERK ^{pT202/pY204 + pT185/pY187} (p-ERK) were used to determine the effect of FTY720 on the activities of PI3K/Akt and MAPK signaling. Western blotting analysis revealed that FTY720 significantly reduced the levels of p-PI3K, p-AKT and p-ERK in a dose dependently manner in H295R cells; (B) Western blotting analysis revealed that FTY720 significantly reduced the levels of p-PI3K, p-AKT and p-ERK in a dose dependently manner in SW13 cells; (C) Cell viability after single or combined mitotane and FTY720 treatment. Cytotoxic response to mitotane and the combination of mitotane and FTY720 in H295R and SW13 cell lines.; (D) The combination of mitotane and FTY720 showed a significant synergistic anti-proliferative effect (CI = 0.90 ± 0.08) in SW13 cells that potentiated the cytotoxic effect observed by using FTY720 alone.