These results clearly demon strated that Mcl 1 reduction is

TUNEL assay was performed to measure the extent of tumor cell apoptosis, as previously described. Statistical analysis All data used were representative of at least three independ ent experiments. INK 128 臨床試験 Quantitative data are expressed as mean SD. Comparisons were analyzed using the Students t test and ANOVA. Significance was defined as p 0. 05. Results In vitro anti tumor efficacy of IGFR PI3K Akt mTOR inhibition The growth inhibitory effects of NVP AEW541, MK2206, BEZ235, and RAD001 on HCC cells and HUVEC were shown in Figure 1A. 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 PI3K Akt mTOR signaling activity. BEZ235 inhibited Akt, GSK3B, and P70S6K phosphorylation at submicromolar range, consist ent with its growth inhibitory effects.<br><br> On the other hand, although RAD001 inhibited 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. To KU-57788 臨床試験 investigate the potential synergistic antitumor effects of vertical blockade of the IGFR PI3K Akt 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.<br><br> Synergistic apoptosis inducing effects, measured Linsitinib 分子量 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 PI3K Akt mTOR pathway in Hep3B and Huh7 cells. 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.<br><br> Therefore, the difference in apoptosis in duction by different drug combination in the 2 cell lines cannot be explained by their inhibitory effects on PI3K Akt mTOR 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 IGFR AKT mTOR pathway. 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.