Fig. 1. The chemical structure of 2-amino-3-{[2-(2-benzyloxy-phenyl)-1H- benzoimidazole-4-carbonyl]-amino}-benzoic acid methyl ester (ACP-93).
Fig. 1. The chemical structure of 2-amino-3-{[2-(2-benzyloxy-phenyl)-1H-
benzoimidazole-4-carbonyl]-amino}-benzoic acid methyl ester (ACP-93).
N
N H
BnO N
H
NH 2 O
OMe
O
Fig. 2. Comparison of apoptosis between the securin-wild type and securin-null HCT116 cells after treatment with ACP-93.
3.2%±0.4
HCT116 cells after treatment with ACP-93. (A) The cells were treated with 0-32 μM
ACP-93 for 24 h. After treatment, the cells were re-cultured in fresh medium for 48 h.
Apoptosis was determined by Annexin V-PI staining using flow cytometry analysis. The cell population of Annexin V+/PI- was indicated early apoptosis (lower right). The fraction of Annexin V+/PI+ was indicated late apoptosis (upper right). (B) The percentage of total apoptosis populations (combine early and late apoptosis) were quantified by CellQuest software. The results were obtained from four to seven experiments and the bar represents the mean ± S.E.M. p < 0.05 (*), indicates significant difference between control and ACP-93 treated samples.
HCT116 securin (+/+)
49
Fig. 3. Comparison of apoptosis-related protein expression between the securin-wild type and securin-null HCT116 cells after treatment with ACP-93.
0 4 8 16 32
HCT116 securin (+/+) HCT116 securin (-/-) (M, 24 h) ACP-93
Fig. 3. Comparison of apoptosis-related protein expression between the securin-wild
type and securin-null HCT116 cells after treatment with ACP-93. (A) The cells were
treated with 0-32 μM ACP-93 for 24 h. After treatment, the total protein extracts were subjected to Western blot analysis using anti-bax, anti-caspase 3, anti-PARP and anti-actin antibodies. Representative Western blot data are shown from one of three separate experiments with similar findings. (B) The relative protein intensity of bax, active caspase 3 and cleaved-PARP were from Western blot by semi-quantification. The results were obtained from three experiments and the bar represents the mean ± S.E.M. p < 0.05 (*), indicates significant difference between the control and ACP-93 treated samples.
0 4 8 16 32
Relative intensity of active caspase 3 0
1 Relative intensity of cleaved-PARP
0
Fig. 4. Comparison of the caspases 8 activation between the securin-wild type and securin-null HCT116 cells after treatment with ACP-93. HCT116 securin (+/+) HCT116 securin (-/-)
0 4 8 16 32 0 4 8 16 32
Fig. 4. Comparison of the caspases 8 activation between the securin-wild type and
securin-null HCT116 cells after treatment with ACP-93. (A) The cells were treated
with 0-32 μM ACP-93 for 24 h. After treatment, the total protein extracts were subjected to Western blot analysis using anti-caspase 8 and anti-actin antibides. Representative Western blot data are shown from one of three separate experiments with similar findings. (B) The relative protein intensity of active caspase 8 was from Western blot by semi-quantification. The results were obtained from three experiments and the bar represents the mean ± S.E.M. p < 0.05 (*), indicates significants difference between the control and ACP-93 treated samples.
(50/55 KDa)
(36/40 KDa)
1 1.3 1.5 2.4 2.7 1 1.9 1.9 2.4 2.2 fold
ACP-93 (M) Relative intensity of active caspase 8
0
Fig. 5. Comparison of γ-H2AX protein expression between the securin-wild type and securin-null HCT116 cells after treatment with ACP-93. HCT116 securin (+/+) HCT116 securin (-/-)
0 4 8 16 32 0 4 8 16 32
treated with 0-32 μM ACP-93 for 24 h. After treatment, the total protein extracts were subjected to Western blot analysis using anti-phosph-histone H2AX (ser139) and anti-actin antibodies. Representative Western blot data are shown from one of three
separate experiments with similar findings. (B) The relative protein intensity of γ-H2AX was from Western blot by semi-quantification. The results were obtained from
three experiments and the bar represents the mean ± S.E.M. p < 0.05 (*), indicates significant difference between the control and ACP-93 treated samples.
ACP-93 (M)
Fig. 6. Comparison of ATF-3 protein expression between the securin-wild type and securin-null HCT116 cells after treatment with ACP-93.
0 4 8 16 32 0 4 8 16 32 HCT116 securin (+/+) HCT116 securin (-/-) ACP-93
Fig. 6. Comparison of ATF-3 protein expression between the securin-wild type and
securin-null HCT116 cells after treatment with ACP-93. (A) The cells were treated
with 0-32 μM ACP-93 for 24 h. After treatment, the total protein extracts were subjected to Western blot analysis using anti-ATF-3 and anti-actin antibodies.
Representative Western blot data are shown from one of three separate experiments with similar findings. (B) The relative protein intensity of ATF-3 was from Western blot by semi-quantification. The results were obtained from three experiments and the bar represents the mean ± S.E.M. p < 0.05 (*), indicates significant difference between the control and ACP-93 treated samples.
1 1.1 1.3 5.0 5.7 1 2.1 2.0 5.6 4.2 fold
Fig. 7. Effect of ACP-93 on the ROS generation in the securin-wild type and securin-null HCT116 cells.
HCT116 securin (+/+) HCT116 securin (-/-)
Fig. 7. Effect of ACP-93 on the ROS generation in the securin-wild type and
securin-null HCT116 cells. The cells were treated with 0-32 μM ACP-93 for 24 h.
After treatment, the cells were incubated with 50 nM H2DCF-DA and then analyzed by flow cytometer. Representative flow data are shown from one of four separate experiments with similar findings.
－ Control
－ ACP-93 32μM
－ ACP-93 16μM
－ ACP-93 4μM
－ ACP-93 8μM
－ Control
－ ACP-93 16μM
－ ACP-93 32μM
－ ACP-93 4μM
－ ACP-93 8μM
Fig. 8. Effects of ACP-93 on cell growth in the securin-wild type and securin-null HCT116 cells.
Fig. 8. Effects of ACP-93 on cell growth in the securin-wild type and securin-null
HCT116 cells. The cells were plated at a density of 106 cells/p60 Petri dish for 20 h.
Then the cells were treated with or without 32 μM ACP-93 for 24 h. After treatment, the cells were incubated for various times before they were counted by hemocytometer.
The results were obtained from three experiments and the bar represents the mean ± S.E.M. p < 0.05 (*), indicates significant difference between control and ACP-93 treated securin-wild type samples. p < 0.05 (#), indicates significant difference between control and ACP-93 treated securin-null samples.
HCT116 cells
Fig. 9. Effect of ACP-93 on cell cycle progression in the securin-wild type and
securin-null HCT116 cells. (A) The cells were treated with 0-32 μM ACP-93 for 24 h.
After treatment, the cells were trypsinized and then subjected to flow cytometry analysis. Representative flow data are shown from one of three to four separate experiments with similar findings. (B) The percentages of G1, S, G2/M and sub-G1
fractions were quantified by ModFit LT software. The results were obtained from three to four experiments and the bar represents the mean ± S.E.M. p < 0.05 (*), indicates significant difference between control and ACP-93 treated samples.
HCT116 securin (-/-)
Fig. 10. Effect of ACP-93 on anti-apoptotic protein expression in the securin-wild type HCT116 cells.
0 4 8 16 32 (μM, 24 h) p-AKT
AKT
Actin Bcl-2
survivin
ACP-93
Fig. 10. Effect of ACP-93 on anti-apoptotic protein expression in the
securin-wild type HCT116 cells. The cells were treated with 0-32 μM ACP-93 for
24 h. After treatment, the total protein extracts were subjected to Western blot analysis using anti-phospho-AKT (ser473), anti-AKT, anti-bcl-2, anti-survivin and anti-actin antibodies. Representative Western blot data are shown from one of three separate experiments with similar findings.
Fig. 11. Effect of ACP-93 on anti-apoptotic protein expression with various time treatment in the securin-wild type HCT116 cells.
0 4 8 12 24 36 (h, 32 μM)
AKT p-AKT
1 1.1 1.9 2.8 3.3 2.7 fold
Bcl-2
survivin
1 2.4 2.7 3.1 5.8 4.6 fold
1 3.9 4.0 3.8 2.3 0.5 fold
Actin
Fig. 11. Effect of ACP-93 on anti-apoptotic protein expression with various time
treatment in the securin-wild type HCT116 cells. The cells were treated with 32 μM
ACP-93 for 0-36 h. After treatment, the total protein extracts were subjected to Western blot analysis using anti-phospho-AKT (ser473), anti-AKT, anti-bcl-2, anti-survivin and anti-actin antibodies. Representative Western blot data are shown from one of three separate experiments with similar findings. The relative protein level of phospho-AKT, bcl-2 and survivin were from Western blot by semi-quantification.
ACP-93
Fig. 12. Effect of a PI3K/AKT inhibitor (wortmannin) on the cell viability in the securin-wild type HCT116 cells by ACP-93 treatment.
Fig. 12. Effect of a PI3K/AKT inhibitor (wortmannin) on the cell viability in the
securin-wild type HCT116 cells by ACP-93 treatment. The cells were co-treated
with 16 μM ACP-93 and 1 μM wortmannin for 24 h. At the end of treatment, the cells were washed with PBS and then re-cultured in fresh medium for 48 h. The cell viability was determined by MTT assay. The results were from six independent experiments and the bars represented mean ± S.E.M. p < 0.001 (***), indicates significant difference between control and ACP-93 treated samples. p < 0.01 (##), indicates significant difference between the ACP-93 treated samples and co-treatment with wortmannin samples.
Fig. 13. Comparison of tumor formation between the securin-wild type and mice in each group and the bar represents the mean ± S.E.M. p < 0.01 (**), indicates significant difference between the control and ACP-93 treated samples. (B) The visible tumors were separated from mice.
Fig. 14. Inhibition of tumorigenesis by ACP-93 in xenograft tumor of SCID mouse
Fig. 14. Inhibition of tumorigenesis by ACP-93 in xenograft tumor of SCID mouse model. (A) HCT116 securin-wild type cells were pre-treated with or without 32 μM ACP-93 for 24 h. After treatment, the cells were subcutaneously injected with 1 × 106 cells to the four-week-old SCID mice. Each group contained five mice. The tumor volume was measured every four days during total 40 days. (B) The four-week-old SCID mice were subcutaneously injected with HCT116 1 × 106 cells. The mice bearing HCT116 xenografts were tumor injected with vehicle control (corn oil) or 30 mg/kg of ACP-93 once per four days from day 14 to day 22. Each group contained nine to ten mice. The tumor volume was measured every four days. The results were obtained from five to ten mice and the bar represents the mean ± S.E.M. p < 0.001 (***), indicates significant difference between the control and ACP-93 treated samples.
Fig. 15. Inhibition of tumorigenesis by ACP-93 in xenograft tumor of nude mouse model.
Fig. 15. Inhibition of tumorigenesis by ACP-93 in xenograft tumor of nude mouse model. The five-week-old nude mice were subcutaneously injected with HCT116 1 × 106 cells. The mice bearing HCT116 xenografts were tumor injected with vehicle control (corn oil) or 30 mg/kg of ACP-93 once per four days from day 24 to day 32.
Each group contained three mice. The tumor volume was measured every four days.
The results were obtained from three mice and the bar represents the mean ± S.E.M. p
< 0.001 (***), indicates significant difference between the control and ACP-93 treated samples.
Days after inoculation
4 8 12 16 20 24 28 32
Tum or si ze (mm 3 )
0 20 40 60 80 100 120 140
160 control
30 mg/kg ACP-93
***
Fig. 16. Proposed model of apoptosis induction and anti-tumorigenesis by ACP-93.
Securin
ACP-93
apoptosis tumorigenesis
Fig. 16. Proposed model of apoptosis induction and anti-tumorigenesis by ACP-93.
Apoptotic signal proteins Anti-apoptotic signal proteins
-H2AX Caspase 8 Caspase 3 PARP cleavage
AKT Bcl-2 survivin