雷帕霉素

产品描述

Rapamycin, a macrolide compound obtained from Streptomyces hygroscopicus, is a potent and specific mTOR inhibitor (IC50: 0.1 nM in HEK293 cells).

靶点活性

mTOR,~0.1nM

实验溶液

0.5% CMC+0.25% Tween 80: 30 mg/mL

体外活性

HEK293 cells were treated with rapamycin (0.05-50 nM), iRap (0.5-500 nM), or AP21967 (0.5-500 nM) under conditions that stimulate mTOR kinase activity. All three compounds were found to inhibit endogenous mTOR, with IC50 values of ?0.1 nM for rapamycin, ?5 nM for iRap and ?10 nM for AP21967 [1]. Rapamycin inhibited the secretion of VEGF in CT-26 adenocarcinoma cell. Amounts of VEGF mRNA were slightly lower (4-fold) in rapamycin-treated (0.1 ?g/ml) B16 tumor cells than in controls. CT-26 and B16 tumor cell proliferation decreased moderately in the presence of rapamycin, but only at the highest concentration tested (1 ?g/ml). HUVECs were very sensitive to rapamycin, with a significant effect at 0.01 ?g/ml. VEGF-induced HUVEC tubular formation was completely abrogated by concentrations of rapamycin as low as 0.01 ?g/ml [2]. Rapamycin-induced autophagy but not apoptosis in rapamycin-sensitive malignant glioma U87-MG and T98G cells by inhibiting the function of mTOR. In contrast, in rapamycin-resistant U373-MG cells, the inhibitory effect of rapamycin was minor, although the phosphorylation of p70S6 kinase, a molecule downstream of mTOR, was remarkably inhibited [3].

体内活性

Rapamycin in vivo did not alter the phosphorylation or activity of Akt itself or of its mTOR-independent target GSK-3β, but instead specifically blocked targets known to be downstream of mTOR12–17. Rapamycin in vivo almost completely prevented the hypertrophic increases in plantaris muscle weight and fiber size at 7 and 14 days [4]. Cardiomyocytes isolated from rapamycin-treated (2 mg/kg body weight, i.p.) LS/+ mice had significantly decreased cell size as compared with that of vehicle-treated LS/+ cells [5]. Mice receiving relatively low doses of RAPA (1.5 mg/kg/d) showed a slightly delayed increase in tumor size during the first 20 days. The lowest dose of RAPA (0.15 mg/kg/d) delayed tumor growth slightly, but the mice died by day 23. A high dose of RAPA (15 mg/kg/d) caused a more pronounced delay in tumor development during the first 3 weeks, but after this, the tumors began to grow again rapidly and the mice died shortly thereafter [2].

激酶实验

Immunoblotting for the mTOR kinase assay: HEK293 cells are plated at 2-2.5×105 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% 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 KaleidaGr

细胞实验

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 μg/mL) for 15 minutes, and examined under a fluorescence microscope. To quantify the development of AVOs, cells are stained with acridine orange (1 μg/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. (Only for Reference)

细胞系： U87-MG, T98G, and U373-MG

动物实验

动物模型：Athymic Nu/Nu mice inoculated subcutaneously with VEGF-A-expressing C6 rat glioma cells