Description
Rapamycin (AY-22989, Rapamune, Sirolimus, NSC-2260804) is a specific mTOR inhibitor with an IC50 of ~ 0.1 nM for HEK293 cells.
Goals
- mTOR
- (HEK293 cells)
- ~ 0.1 nM
In vitro
Rapamycin inhibits endogenous mTOR activity in HEK293 cells with IC50 of ~ 0.1 nM, more potent than iRap and AP21967 with IC50 of ~ 5 nM and ~ 10 nM, respectively. In Saccharomyces cerevisiae, treatment with rapamycin induces severe G1 / S cell cycle arrest and inhibition of translation initiation at levels below 20% of control.
Rapamycin significantly inhibits the cell viability of T98G and U87-MG in a dose-dependent manner with IC50s of 2 nM and 1 μM, respectively, while showing little activity against U373-MG cells with IC50s of> 25 μM despite the similar degree of inhibition of mTOR signaling. Rapamycin (100 nM) induces G1 arrest and autophagy, but not apoptosis in rapamycin-sensitive U87-MG and T98G cells by inhibiting mTOR function.
In vivo
Treatment with rapamycin in vivo specifically blocks targets known to be downstream of mTOR, such as phosphorylation and activation of p70S6K and the release of eIF4E inhibition by PHAS-1 / 4E-BP1, leading to a complete blockade of hypertrophic augmentations in plantaris. muscle weight and fiber size. Short-term rapamycin treatment, even at the lowest dose of 0.16 mg/kg, produces profound inhibition of p70S6K activity, which is correlated with increased tumor cell death and necrosis of Eker’s renal tumors.
Rapamycin inhibits metastatic tumor growth and angiogenesis in CT-26 xenograft models by reducing VEGF production and blocking VEGF-induced endothelial cell signaling. Treatment with rapamycin at 4 mg/kg/day significantly reduces tumor growth of C6 xenografts and tumor vascular permeability.
Kinase Assay:
Immunoblot for mTOR kinase assay:
HEK293 cells are plated at 2-2.5 x 105 cells / well of a 12-well plate and serum-starved for 24 hours in DMEM. Cells are treated with increasing concentrations of rapamycin (0.05-50 nM) for 15 minutes at 37 ° C. Serum is added to a final concentration of 20% over 30 minutes at 37 ° C. Cells are lysed and cell lysates are separated by SDS-PAGE. The resolved proteins are transferred to a polyvinylidene difluoride membrane and immunoblotted with a phosphospecific primary antibody against Thr-389 of the p70 S6 kinase.
Cell research:
Cell lines: U87-MG, T98G, and U373-MG
Concentrations: dissolved in DMSO, final concentrations ~ 25 μM
Incubation time: 72 hours.
Method:
Cells are exposed to various concentrations of rapamycin for 72 hours. For evaluation of cell viability, cells are harvested by trypsinization, stained with trypan blue, and viable cells are counted in each well. For cell cycle determination, cells are trypsinized, fixed with 70% ethanol, and stained with propidium iodide using a flow cytometry reagent set. Samples are analyzed for DNA content using a FACScan flow cytometer and CellQuest software.
For the detection of apoptosis, cells are stained with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique using an ApopTag Apoptosis Detection Kit. To detect the development of acid vesicular organelles (AVO), cells are stained with acridine orange (1 µg / ml) for 15 minutes and examined under a fluorescence microscope.
To quantify AVO development, cells are stained with acridine orange (1 µg / ml) for 15 minutes, removed from the plate with trypsin-EDTA, and analyzed with the FACScan flow cytometer and CellQuest software. To analyze the autophagic process, cells are incubated for 10 minutes with 0.05 mM monodansylcadaverine at 37 ° C and then observed under a fluorescence microscope.