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| Three-dimensional responses of MCF7/IGF-1R cells to TAM (1 μM), E2 and IGF-1. Compared to parental MCF7 cells (a), MCF7/IGF-1R cells (b) in three-dimensional (3D) culture formed bigger acini in response to IGF-1 stimulation and displayed significant TAM resistance when treated with TAM (1 μM) + E2 + IGF-1, which was removable by kinase inhibitors BMS-536924, U0126 and BEZ235 (c). Cells (10,000/well) were seeded in 96-well plates. Acini were formed on 100% Matrigel and cultured for 14 days in starving medium containing 2% Matrigel and 5% charcoal/dextran-stripped fetal bovine serum with the treatments as indicated. Concentrations used: TAM (1 μM), E2 (1 nM) and IGF-1 (100 ng/mL). Confocal image original magnification, × 20. Red, rhodamine phalloidin (actin). Blue, Hoechst blue stain. Results are representative of two individual experiments. |
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Rating |
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| Source |
Proc Natl Acad Sci USA , 2011, 108, 6067-6072. PLX-4720 purchased from Selleck |
| Method |
Western blotting |
| Cell Lines |
Cells overexpressing myc-CRAF and BRAF |
| Concentrations |
10/50 µM |
| Incubation Time |
1/2 h |
| Results |
GDC0879 but not PLX4720 induced BRAF/CRAF dimer formation (Fig. A). However, both drugs induced complexes between KSR1 and CRAF and enhanced interactions between KSR1 and BRAF (Fig. B and C), which suggested that KSR1/RAF complexes induced by the drug might explain the effects ofthe type I BRAF specific inhibitors. As reported previously, treatment of wild-type cells with either drug strongly induced ERK activation at low to intermediate doses but inhibited ERK activation at higher doses (Fig. D and E). Similar results were obtained with cells expressing constitutively active RAS (Fig. D and E) or after serum treatment. Strikingly, in KSR deficient cells, ERK activation was significantly attenuated after PLX4720 or GDC0879 treatment (Fig. D and E), which demonstrates that the ability of PLX4720 and GDC0879 to activate ERK requires the presence of KSR1. We found that, when KSR1 was overexpressed withCRAF, MEK activation was induced by PLX4720 or GDC0879 treatment (Fig. F). this result suggested that induction of the CRAF/KSR1 complex might be important in the activation of MEK. In vitro kinase activity toward MEK was detected but only after drug treatment (Fig. G), which suggests KSR1/CRAF complex formation kinase activity toward MEK. |
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(A) MCF7 cells pre-treated with 100 nM siRNA for 72 h were re-seeded with normal growth media and grown overnight, then further transfected by 100nM of fresh siRNA. Twenty-(A) four hours after transfection, the cells were further treated with PI-103 for 24 h and the cell lysates were immunoblotted with the indicated antibodies.
(B) Breast cancer cells carrying BRCA1 mutations were treated with 1 mM of PI-103 for 24h (left) or increasing amounts of PI-103 for 24 h (right). Cell lysates were analyzed by Western blotting with the indicated antibodies. |
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Rating |
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| Source |
Mol Carcinog, 2012.April, ahead of print. PI-103 purchased from Selleck |
| Method |
Western blot |
| Cell Lines |
MCF7 cells |
| Concentrations |
0-1 μM |
| Incubation Time |
24 h |
| Results |
In BRCA1-KD MCF7 cells, treatment of PI-103, a PI3K/mTOR inhibitor, abolished phosphorylation of AKT and its substrate GSK3b, in a dose dependent manner (Figure A). Treatment of PI-103 reduced the phosphorylation of AKT in all BRCA1 mutant breast cancer cells tested (Figure B). Phosphorylations of downstream targets of AKT, such as phospho-GSK3b (S9) and phospho-BAD(S112) were also reduced by PI-103 treatment. Phosphorylation of mTOR at S2448, which is also known to be phosphorylated by AKT, was also reduced by PI-103 resulting in reduced phosphorylation of S6 ribosomal protein at S235/236 (Figure B). The effect of PI-103 was much more potent than LY294002 in MDA-MB-436 cells (Figure B) |
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| After starved in serum-free medium for 24h,A549 cells incubated with the indicated concentrations of A66 for 3h,followed by 20-minute stimolation of 100ng/ml EGF. |
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| Source |
A66 purchased from Selleck |
| Method |
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| Cell Lines |
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| Concentrations |
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| Incubation Time |
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| Results |
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| We treated all of drugs in T47D which has a PI3KCA H1044R mutation with the concentration shown below for 1 hour and performed western blot analysis using antibodies to phospho-AKT(SERINE 472), and total AKT. |
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Rating |
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| Source |
Saraswati Sukumar of Johns Hopkins University School of Medicine. AS-605240 purchased from Selleck |
| Method |
Western blot |
| Cell Lines |
T47D cells |
| Concentrations |
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| Incubation Time |
1 h |
| Results |
AS-605240 treatment resulted in a reduction of AKT phosphorylation in T47D cells. |
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| After starved in serum-free medium for 24h, Breast cancer cells incubated with the indicated concentrations of AS-605240 for 3h,followed by 15-minute stimolation of 100ng/ml EGF |
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Rating |
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| Source |
Dr. Zhang of Tianjin Medical University. AS-605240 purchased from Selleck |
| Method |
Western blot |
| Cell Lines |
Breast cancer cells |
| Concentrations |
0-20 μM |
| Incubation Time |
3 h |
| Results |
AS-605240 treatment resulted in a reduction of AKT phosphorylation in a concentration-dependent manner. |
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| After starved in serum-free medium for 24h,A549 cells incubated with the indicated concentrations of CAL-101 for 3h,followed by 20-minute stimolation of 100ng/ml EGF. |
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Rating |
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| Source |
Dr. Zhang of Tianjin Medical University. CAL-101 (GS-1101) purchased from Selleck |
| Method |
Western blot |
| Cell Lines |
A549 cells |
| Concentrations |
0-1 μM |
| Incubation Time |
3 h |
| Results |
Reduction of AKT phosphorylation in A549 cells treated with CAL-101 was observed. |
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| Knockdown of BRCA1 sensitizes cells to PI3K/AKT pathway inhibitors. MCF7 cells transfected with either BRCA1-siRNA or control-siRNA were treated with increasing amounts of inhibitors targeting the PI3K/AKT pathway for 48 h in triplicate. Viable cells were measured by MTT assay. |
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Rating |
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| Source |
Mol Carcinog, 2012.April, ahead of print. PIK-75 purchased from Selleck |
| Method |
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| Cell Lines |
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| Concentrations |
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| Incubation Time |
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| Results |
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