Manufacturer |
Selleckchem
|
Category |
|
Type |
Inhibitors |
Specific against |
other |
Amount |
25 mg |
Item no. |
S1039-25 |
Targets |
MTOR |
CASRN |
53123-88-9 |
eClass 6.1 |
30220300 |
eClass 9.0 |
32160605 |
Available |
|
Administration |
Injection i.p. |
Animal Models |
Athymic Nu/Nu mice inoculated subcutaneously with VEGF-A-expressing C6 rat glioma cells |
Cell lines |
U87-MG, T98G, and U373-MG |
Chemical Name |
(3S, 6R, 7E, 9R, 10R, 12R, 14S, 15E, 17E, 19E, 21S, 23S, 26R, 27R, 34aS)-9, 10, 12, 13, 14, 21, 22, 23, 24, 25, 26, 27, 32, 33, 34, 34a-Hexadecahydro-9, 27-dihydroxy-3-[(1R)-2-[(1S, 3R, 4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10, 21-dimethoxy-6, 8, 12, 14, 20, 26-hexamethyl-23, 27-epoxy-3H-pyrido[2, 1-c][1, 4]oxaazacyclohentriacontine-1, 5, 11, 28, 29(4H, 6H, 31H)-pentone |
Clinical Trials |
A Phase II study of Rapamycin to erase angiofibromas in patients with Tuberous Sclerosis Complex (TSC) is currently ongoing. |
Concentrations |
Dissolved in DMSO, final concentrations ca.25 uM |
Description |
Rapamycin (Sirolimus, AY-22989, WY-090217) is a specific mTOR inhibitor with IC50 of ca.0.1 nM. |
Dosages |
ca.4 mg/kg/day |
Formulation |
Dissolved in solvent solution (0.2% carboxymethylcellulose and 0.25% Tween-80 in sterile H2O) |
IC50 |
ca.0.1 nM [1], ca.0.1 nM [1], ca.0.1 nM [1], ca.0.1 nM [1], ca.0.1 nM [1], ca.0.1 nM [1] |
In vitro |
Rapamycin inhibits endogenous mTOR activity in HEK293 cells with IC50 of ca.0.1 nM, more potently than iRap and AP21967 with IC50 of ca.5 nM and ca.10 nM, respectively. [1] In Saccharomyces cerevisiae, Rapamycin treatment induces a severe G1/S cell cycle arrest and inhibition of translation initiation to levels below 20% of control. [2] Rapamycin significantly inhibits the cell viability of T98G and U87-MG in a dose-dependent manner with IC50 of 2 nM and 1 uM, respectively, while displaying little activity against U373-MG cells with IC50 of >25 uM despite the similar extent of the 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 the function of mTOR. [3] |
In vivo |
Treatment with Rapamycin in vivo specifically blocks targets known to be downstream of mTOR such as the phosphorylation and activation of p70S6K and the release of inhibition of eIF4E by PHAS-1/4E-BP1, leading to complete blockage of the hypertrophic increases in plantaris muscle weight and fibre size. [4] Short-term Rapamycin treatment, even at the lowest dose of 0.16 mg/kg, produces profound inhibition of p70S6K activity, which correlates with increased tumor cell death and necrosis of the Eker renal tumors. [5] Rapamycin inhibits metastatic tumor growth and angiogenesis in CT-26 xenograft models by reducing the production of VEGF and blockage of VEGF-induced endothelial cell signaling. [6] Rapamycin treatment at 4 mg/kg/day significantly reduces tumor growth of C6 xenografts, and tumor vascular permeability. [7] |
Incubation Time |
72 hours |
Kinase Assay |
Immunoblotting for the mTOR kinase assay, HEK293 cells are plated at 2-2.5105 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% for 30 minutes at 37 C. Cells are lysed, and cell lysates are separated by SDS-PAGE. Resolved proteins are transferred to a polyvinylidene difluoride membrane and immunoblotted with a phosphospecific primary antibody against Thr-389 of p70 S6 kinase. Data are analyzed using ImageQuant and KaleidaGraph. |
Method |
Cells are exposed to various concentrations of Rapamycin for 72 hours. For the assessment of cell viability, cells are collected by trypsinization, stained with trypan blue, and the viable cells in each well are counted. For the determination of cell cycle, 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 apoptosis detection, 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 acidic vesicular organelles (AVO), cells are stained with acridine orange (1 g/mL) for 15 minutes, and examined under a fluorescence microscope. To quantify the development of AVOs, cells are stained with acridine orange (1 g/mL) for 15 minutes, removed from the plate with trypsin-EDTA, and analyzed using 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 are then observed under a fluorescence microscope. |
Molecular Weight (MW) |
914, 18 |
Picture ChemicalStructure Description |
Rapamycin (Sirolimus) Chemical Structure |
Picture Description 1 |
Data from [Biochem Pharmacol , 2011, 82, 216-226], Rapamycin (Sirolimus)purchased from Selleck, Inhibition of mTOR activity may be responsible for sorafenib-induced down-regulation of survivin. H1299 cells were treated with the indicated concentration of RAD001 or Rapamycin for 48 h. Then H1299 cells were incubated with or without 5 M sorafenib, with or without 5 M RAD001, and with or without 2 M rapamycin for 48 h. The indicated protein levels were determined by Western blot analysis. -Actin protein levels were measured as loading controls. |
Picture Description 2 |
Data from [J Lipid Res , 2011, 52, 1617-1625], Rapamycin (Sirolimus)purchased from Selleck, Rapamycin (RPM) inhibits OA-induced lipogenesis. Male SD rats were divided into three groups, and then treated as follows for seven days: group I (control, Ctrl), normal diet + vehicle, group II (OA), 1% OA diet + vehicle, group III (OA + RPM), 1% OA diet + RPM. (A) Hepatic TG concentrations were determined using the serum triglyceride determination kit. (B) Fixed liver sections were subjected to HE and Oil Red O staining as well as immunohistochemistry assays with SREBP-1 antibody. Representative microphotographs of liver specimens from all groups of rats are shown. (C) The levels of mRNA of the indicated genes involved in lipogenesis were determined by real-time PCR. Each bar represents the mean SE of the results obtained from six rats. * P, 0.05, ** P, 0.01, *** P, 0.001. |
Solubility (25C) |
DMSO 20 mg/mL, Water <1 mg/mL, Ethanol 4 mg/mL |
Storage |
2 years -20CPowder, 2 weeks4Cin DMSO, 2 months-80Cin DMSO |