Article Figures & Data
Figures
- Figure 1.
Structure of AT7867. A, chemical structure of AT7867. B, X-ray structure of AT7867 bound to the ATP binding site of AKT2.
- Figure 2.
AT7867 inhibits proliferation of human cancer cell lines and reduces phosphorylation of GSK3β. A, PTEN-negative U87MG human glioblastoma cells were treated with a range of concentrations of AT7867 for 72 hours. Alamar blue cell viability assays were done to measure cell growth inhibition. B, cells were incubated with a range of concentrations of AT7867 for 1 hour, and the in-cell phosphorylated GSK3β ELISA was done in parallel with Western blot of cell lysates in U87MG cells. No addition (NA) and DMSO (D) vehicle were used as negative controls, and LY294002 (20 μmol/L; LY) was used as a positive control.
- Figure 3.
AT7867 suppresses AKT signaling in U87MG human glioblastoma cells in a concentration-dependent and time-dependent manner and induces apoptosis. A, PTEN-negative U87MG human glioblastoma cells were incubated with AT7867 for 1 hour, and Western blot analyses were done to assess the phosphorylation and expression of AKT pathway proteins. GAPDH was used as a loading control. No addition (NA) and DMSO (D) vehicle were used as negative controls, and LY294002 (20 μmol/L; LY) was used as a positive control. B, U87MG cells were treated with 16 μmol/L AT7867 for, 1, 2, 4, 8, and 24 hours, and Western blot analyses were done for AKT pathway proteins as indicated and the appearance of cleaved PARP. GAPDH was used as a loading control. No addition (NA) and DMSO (D) vehicle were used as negative controls, and LY294002 (20 μmol/L; LY) was used as a positive control. Okadaic acid (100 nmol/L) for 24 hour (OA) was used as a positive control for apoptosis. C, Annexin V staining of U87MG cells following treatment with 8, 16, or 24 μmol/L AT7867 or DMSO vehicle (Control) for 24 or 48 hours.
- Figure 4.
The plasma pharmacokinetic profile of AT7867 in vivo and comparison of expression of AKT pathway proteins in the MES-SA human uterine versus U87MG glioblastoma and MCF-7 and MDA-MB-468 breast cell lines. A, plasma pharmacokinetic in mice following administration of AT7867 at 20 mg/kg p.o. and 5 mg/kg i.v. B, Western blot analysis of AKT pathway proteins in lysates prepared from MES-SA, U87MG, MCF7, and the MDA-MB-468 cell lines. GAPDH was used as a loading control.
- Figure 5.
The pharmacodynamic and pharmacokinetic profiles of AT7867. Athymic BALB/c mice bearing MES-SA human uterine xenograft tumors were treated with a single dose of AT7867 at 20 mg/kg i.p. and 90 mg/kg p.o. Plasma and tumors were harvested at 2, 6, or 24 hours after dosing. A and B, phosphorylated and total GSK3β, S6RP, cleaved PARP, and GAPDH were assessed from tumor protein lysates by Western blotting at each dose as indicated. C1, control 1; C2, control 2. C and D, plasma and tumor concentrations of AT7867 were assessed by liquid chromatography tandem mass spectrometry at each dose as indicated.
- Figure 6.
AT7867 suppresses MES-SA human uterine and U87MG human glioblastoma xenograft tumor growth. A, athymic BALB/c mice bearing MES-SA xenograft tumors were given AT7867 20 mg/kg i.p. once every 3 days for 16 days. B, athymic BALB/c mice bearing MES-SA xenograft tumors were given AT7867 at 90 mg/kg p.o. once every 3 days for 12 days. C, athymic BALB/c mice bearing U87MG xenograft tumors were given AT7867 at 20 mg/kg i.p. once every 3 days for 13 days. Tumor size was monitored three times a week, and relative tumor volumes were calculated.
Tables
- Table 1.
Activity of AT7867 against selected kinases as determined by in vitro kinase assays
Kinase IC50 (nmol/L) AKT1 32 AKT2 17 AKT3 47 PKA 20 p70S6K 85 RSK1 >100 CDK2 >1,000 GSK3β >1,000 c-SRC >1,000 CHK1 >1,000 EGFR >1,000 FGFR3 >1,000 MEK1 >1,000 PDK1 >1,000 PI3K-β >1,000 PLK3 >1,000 RET >1,000 SGK >1,000 TIE2 >1,000 - Table 2.
Inhibition of cell growth across a panel of human cell lines from various tumor types; IC50 values are shown as the mean (n = 3) with SEM
Cell line Cancer type Proliferation IC50 ± SEM (μmol/L) MES-SA Uterine 0.94 ± 0.14 MDA-MB-468 Breast 2.26 ± 0.54 MCF-7 Breast 1.86 ± 0.09 U87MG Glioma 8.22 ± 1.15 PC-3 Prostate 10.37 ± 0.79 DU145 Prostate 11.86 ± 0.67 HCT116 Colon 1.76 ± 0.36 HT29 Colon 3.04 ± 0.45 - Table 3.
A panel of human tumor cell lines were exposed to AT7867, the in-cell phosphorylated GSK3β ELISA was done, and the IC50 for phosphorylated Ser9 GSK3β signal was determined
Cell line Cancer type pSer9 GSK3β ELISA IC50 ± SEM (μmol/L) MES-SA Uterine 2.31 ± 0.61 MDA-MB-468 Breast 4.45 ± 1.47 MCF-7 Breast 2.78 ± 1.34 U87MG Glioma 2.08 ± 0.28 PC-3 Prostate 2.65 ± 0.79 DU145 Prostate 3.21 ± 0.28 HCT116 Colon 3.3 ± 0.46 HT29 Colon 3.09 ± 1.46 - Table 4.
U87MG cells were treated with 0.1 to 20 μmol/L AT7867 for 1 h; lysates were prepared, and analysis of AKT pathway proteins was done by electrochemiluminescent immunoassay (MSD) as indicated
IC50 (μmol/L) GSK3β pSer9 P70S6K pThr421/Ser424 S6RP pSer235/236 S6RP pSer240/244 AT7867 7.1 >20 10.7 12.3
Volume 9, Issue 5
