Supplementary figure 1.
(A) The list of apoptosis-related proteins measured by the Human Apoptosis Array kit
(Proteome ProfilerTM, R&D Systems, Minneapolis, MN).
(B) Evaluation of effects of different drug combinations on expression of
apoptosis-related proteins. Hep3B cells were treated with different drug
combinations (NVP-AEW541 (1M) + MK2206 (5M), NVP-AEW541 (1M)
+ BEZ235 (0.1M), and NVP-AEW541 (1M) + RAD001 (0.1M)) for 48
hours. Whole-cell lysates were incubated with the protein array membranes
according to the manufacturer’s instructions.
1
Supplementary figure 2.
Survivin is a possible mediator of antitumor synergy between NVP-AEW541 and
MK2206, BEZ235, and RAD001.
Hep3B cells were transfected with V-Survivin (pCMV6-Myc-DKK-survivin) or
empty vectors for 8 hours, and then treated with different drug combination as
indicated. The percentages of apoptotic cells were measured by flow cytometry after
48-hour drug treatment. **, p < 0.01.
2
Supplementary figure 3.
In vivo tumor angiogenesis between NVP-AEW541, and MK2206 and BEZ235
inhibitors. Hep3B and Huh7 cells were subcutaneously injected into male BALB/c
athymic nude mice. Mice were treated as indicated (NVP, NVP-AEW-541 30
mg/kg/day by gavage; MK2206, MK2206 100 mg/kg/week by intraperitoneal (i.p.)
injection; BEZ, BEZ235 25 mg/kg/day by gavage). Tumor angiogenesis (microvessel
density (MVD)).*, p < 0.05; **, p < 0.01, compared with the control (vehicle-treated)
group. Values presented are the means ± SD (n = 4 in each group).
3
Supplementary figure 4.
Assessment of the in vivo toxicities. Male BALB/c athymic nude mice were treated as
indicated (NVP, NVP-AEW-541 30 mg/kg/day by gavage; MK2206, MK2206 100
mg/kg/week by intraperitoneal (i.p.) injection; BEZ, BEZ235 25 mg/kg/day by
gavage) after subcutaneous tumor implantation with Huh-7 cells. Values presented are
the means ± SD (n = 5 in each group).
(A) Body weight was recorded every five days.
(B) Blood samples were obtained via cardiac puncture after 28 days of drug treatment.
White blood cell counts (WBC), hemoglobin (Hb), and platelet (PLT) counts were
measured by a quantitative automated hematology analyzer (the Sysmex XT2000i,
Sysmex America). Creatinine (Cre), alanine aminotransferase (ALT), and glucose
(Glu) were measured by a quantitative automated Chemistry Analyzer (the Advia
1800, Siemens America).
4
B
5
Supplementary table 1.
Median dose effect analysis of synergistic growth inhibitory effects of
NVP-AEW541/ MK2206, NVP-AEW541/ BEZ235 and NVP-AEW541/ RAD001.
The dose levels of different treatments combining NVP-AEW541, MK2206, BEZ235,
and RAD001 used and the corresponding CI (combination index) were listed in the
tables.
NVP-AEW541 (μM)
MK2206 (μM)
Hep3B
CI Value
Huh7
CI Value
PLC5
CI Value
HUVEC
CI Value
0.28
2.79
0.74
0.54
2.14
0.98
0.34
3.35
0.88
0.54
1.08
0.99
0.40
4.02
0.79
0.98
1.08
0.94
0.48
4.82
0.92
0.82
1.31
0.82
0.58
5.79
0.95
0.82
1.50
0.82
0.69
6.94
0.79
0.76
1.40
0.91
0.83
8.33
0.28
0.76
0.69
0.82
1.00
10.00
0.07
2.79
0.54
0.81
NVP-AEW541 (μM)
BEZ235 (μM)
Hep3B
CI Value
Huh7
CI Value
PLC5
CI Value
HUVEC
CI Value
0.16
0.02
1.74
0.79
2.77
7.33
0.21
0.02
0.91
0.53
1.98
3.37
0.27
0.03
0.80
0.56
1.27
1.76
0.35
0.04
0.74
0.43
0.81
0.80
0.46
0.05
0.72
0.40
0.77
0.84
0.59
0.06
0.63
0.43
0.52
0.54
0.77
0.08
0.44
0.62
0.31
0.55
1.00
0.10
0.40
0.58
0.29
0.49
NVP-AEW541 (μM)
RADOO1 (μM)
Hep3B
CI Value
Huh7
CI Value
PLC5
CI Value
HUVEC
CI Value
0.16
0.19
0.62
0.08
0.56
0.13
0.21
0.25
0.52
0.11
0.69
0.14
0.27
0.32
0.47
0.11
0.43
0.18
0.35
0.42
0.39
0.11
0.53
0.25
0.46
0.55
0.25
0.20
0.58
0.20
0.59
0.71
0.43
0.13
0.63
0.13
0.77
0.92
0.40
0.13
0.64
0.40
1.00
1.20
0.35
0.13
0.68
0.48
6