These results are in concordance with previous reports where ERK inhibition

The response of the HCC cell lines tested to individual MTAs did not differ significantly from one another. BEZ235 appeared to be the most potent inhibitor of PI3KAktmTOR signaling activity. BEZ235 inhibited Akt, GSK3B, and P70S6K phosphorylation at submicromolar range, consist ent with its growth inhibitory effects. On the other hand, although RAD001 inhibited ABT-888 分子量 the downstream P70S6K phosphorylation at submicromolar levels, the Akt and GSK3Bphosphorylation appeared increased after RAD001 treatment, suggesting compensatory activation of up stream signaling activities. This finding may explain the relatively poor growth inhibitory effects of RAD001 in the HCC cells tested.<br><br> To investigate the potential synergistic antitumor effects of vertical blockade Afatinib 構造 of the IGFRPI3KAkt mTOR signaling pathway, median effect analysis was performed to measure the combination index of different treatments combining NVP AEW541, MK2206, BEZ235, and RAD001, with CI values 1 indicating syn ergy. Synergistic growth inhibitory effects were seen for most of the combinations tested in all three HCC cell lines and in HUVECs. Synergistic apoptosis inducing effects, measured by flow cytometry and Western blotting, were most consistent when NVP AEW541 was combined with the Akt inhibi tor MK2206. BEZ235 and RAD001 could enhance apoptosis in Hep3B and HUVECs only when combined with NVP AEW541. Survivin is an important downstream mediator of anti tumor synergy To explain the differential effects on apoptosis induction by different drug combination, we first compared the effects of these combinations on activity of PI3KAkt mTOR pathway in Hep3B and Huh7 cells.<br><br> As shown in Figure 3A, all the combinations, including NVP AEW541 MK2206, NVP AEW541 BEZ235, and NVP AEW541 RAD001, inhibited the phosphorylation of Akt, P70S6K, and 4EBP 1 to a similar extent in Hep3B and Huh7 cells. Therefore, the difference in apoptosis in duction by different AG-1478 溶解度 drug combination in the 2 cell lines cannot be explained by their inhibitory effects on PI3K AktmTOR signaling activity alone. Similarly, the effects of these drug combinations on expression of apoptosis related proteins, including mcl 1, bcl 2, bim, bad, and bax, were also similar in the 2 cell lines. The apoptosis protein array was then used to explore potential mediators of anti tumor activity of different drug combinations targeting the IGFRAKTmTOR pathway.<br><br> Survivin was identified as the candidate mol ecule because it showed the most consistent inhibition when NVP AEW541 was combined with MK2206, BEZ235, or RAD001 and correlated with the anti tumor synergy. This finding was confirmed by Western blotting. Furthermore, in Huh7 cells inhibition of survivin expression was more prominent in NVP AEW541MK2206 combination than in NVP AEW541BEZ235 and NVP AEW541RAD001 combinations. This finding suggested that the differential effects of different drug combinations in HCC cells can be explained at least in part by their inhibitory effects on survivin expression. The role of survivin as an important downstream mediator was further explored by siRNA knockdown and transient over expression experiments.