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Biological Description

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

Targets&IC50: mTOR:0.1 nM (HEK293 cells)

In vitro: 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. 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 μg/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 μg/ml). HUVECs were very sensitive to rapamycin, with a significant effect at 0.01 μg/ml. VEGF-induced HUVEC tubular formation was completely abrogated by concentrations of rapamycin as low as 0.01 μg/ml. 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.

In vivo: 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. 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. 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.

Kinase Assay: HEK293 cells were plated at 2-2.5 × 10^5 cells per well of a 12-well plate and serum-starved for 24 h in DMEM only. Cells were mock-treated or treated with rapamycin (0.05-50 nM), iRap (0.5-500 nM), or AP21967 (0.5-500 nM) for 15 minutes at 37 °C. Serum was added to a final concentration of 20% for 30 minutes at 37 °C. Cells were lysed as described and cell lysates were separated by SDS-PAGE. Resolved proteins were transferred to a PVDF membrane and immunoblotted with a phosphospecific primary antibody against Thr389 of p70 S6 kinase. Data were analyzed using ImageQuant and KaleidaGraph.

Cell Research: To determine the effects of rapamycin and rapamycin plus LY294002 or UCN-01 on tumor cells, we determined cell viability after the treatments. We used a trypan blue dye exclusion assay as described previously. Tumor cells in exponential growth were harvested and seeded at 5 × 10^3 cells per well (0.1 mL) in 96-well flat-bottomed plates and incubated overnight at 37°C. The cells were then incubated for 72 hours with or without rapamycin or with rapamycin plus LY294002 or UCN-01. After the cells were collected by trypsinization, they were stained with trypan blue, and the viable cells in each well were counted. The viability of the untreated cells (the control) was considered 100%. Survival fractions were calculated from the mean cell viability of the treated cells.

Animal Research: Animals were randomized to treatment or vehicle groups so that the mean starting body weights of each group were equal. Drug treatment began on the day of surgery or on the first day of reloading after the 14-day suspension. Rapamycin was delivered once daily by intraperitoneal injection at a dose of 1.5 mg/kg, dissolved in 2% carboxymethylcellulose. CsA was delivered once daily by subcutaneous injection at a dose of 15 mg/kg, dissolved in 10% methanol and olive oil. FK506 was delivered once daily via subcutaneous injection at a dose of 3 mg kg?1, dissolved in 10% ethanol, 10% cremophor and saline.

Chemical Properties

Molecular Weight: 914.18

Formula: C51H79NO13

CAS No.: 53123-88-9

Storage & Solubility Information

Storage

Powder: -20°C for 3 years

In solvent: -80°C for 2 years

Solubility Information

Ethanol: 18.3 mg/mL (20 mM)

H2O: Insoluble

DMSO: 45.7 mg/mL (50 mM)

( < 1 mg/ml refers to the product slightly soluble or insoluble )

Catalog #: T1537

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References and Literature

1. Edwards SR, et al. The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain. J Biol Chem. 2007 May 4;282(18):13395-401.

2. Guba M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med. 2002 Feb;8(2):128-35.

3. Takeuchi H, et al. Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res. 2005 Apr 15;65(8):3336-46.

4. Bodine SC, et al. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol. 2001 Nov;3(11):1014-9.

5. Marin TM, et al. Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation. J Clin Invest. 2011 Mar;121(3):1026-43.

6. Zhang JW, et al. Metformin synergizes with rapamycin to inhibit the growth of pancreatic cancer in vitro and in vivo. Oncol Lett. 2018 Feb;15(2):1811-1816.

7. Gao C, Wang H, Wang T, et al. Platelet CLEC‐2 regulates neuroinflammation and restores blood brain barrier integrity in a mouse model of traumatic brain injury[J]. Journal of Neurochemistry. 2020: e14983.

8. Zhang T, Tian C, Wu J, et al. . MicroRNA‐182 exacerbates blood‐brain barrier (BBB) disruption by downregulating the mTOR/FOXO1 pathway in cerebral ischemia[J].  The FASEB Journal. 2020, 34(10): 13762-13775.

9. Shang Z, Zhang T, Jiang M, et al. High-carbohydrate, High-fat Diet-induced Hyperlipidemia Hampers the Differentiation Balance of Bone Marrow Mesenchymal Stem Cells by Suppressing Autophagy via the AMPK/mTOR Pathway in Rat Models[J]. 2020.

10. Zhao, Ming, et al. GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein. Nature Communications . 12.1 (2021): 1-14.