Rapamycin is an antibiotic, anti-cancer, and immunosuppressant compound that can induce cancer cell death by inhibiting the activity of the mammalian target of rapamycin (mTOR). Cancer cells often use the mTOR pathway as a mechanism to enhance their proliferation and growth.
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How does rapamycin affect T cells?
That is to say, by slowing down the speed at which cells replicate, we extend how long a cell survives. Slowing biological age also involves reducing the rate of functional decline. Rapamycin does precisely this. It slows down cell proliferation. At the same time, it maintains the cell’s potential to do so.
What is the concentration of rapamycin used in cell culture?
Sirolimus. Sirolimus ( rapamycin) is a novel immunosuppressive agent that impairs cytokine-induced lymphocyte proliferation. The prolonged half-life of sirolimus means that once-daily dosing is sufficient. It is metabolized via the cytochrome P-450 system; thus, the potential for multiple drug interactions exists.
What is rapamycin used to treat?
· Dr. Peter Attia: 5 mg taken as an oral tablet once every 7-14 days to reduce side effects, and because Rapamycin’s effects can last for a very long time post-dose .
Does rapamycin cause erythrocyte microcytosis?
In the experiments that cell confluence was required to induce cilia formation, the cultured cells were incubated with media containing 2% FBS and 0, 1 or 10 μM of rapamycin for 1, 3, and 8 …
What happens with treatment of cells with rapamycin?
Cells treated with rapamycin demonstrated lower EdU incorporation, hence lower proliferation rate, compared to control cells (58.90% vs. vehicle 88.50%; P < 0.001 (Fig. 2A). The change in proliferation with rapamycin paralleled the results of the cell cycle analysis.
How does rapamycin enter the cell?
Upon entering the cells, rapamycin binds a small protein receptor called FKBP12 (FK506-binding protein 12 kDa). The rapamycin/FKBP12 complex specifically binds to TOR and potently interferes with its function, causing cell growth arrest.
What is rapamycin cell signaling?
Rapamycin is a bacterial macrolide with antifungal and immunosuppressant activities (1). Rapamycin forms a complex with the immunophilin FKBP12 which then inhibits the activity of FRAP/ mTOR (TOR in yeast) (2,3). Rapamycin treatment of cells leads to the dephosphorylation and inactivation of p70 S6 kinase.
Does mTOR increase cell growth?
Once activated, mTORC1 promotes cell growth by regulating several anabolic processes including protein, lipid and nucleotide synthesis, which contribute to the increase in cell biomass.
How do I activate mTOR?
mTOR is known to be activated by insulin, and the mechanisms involved are well documented. The ways by which exercise and AA lead to mTOR activation remain partially unclear. Exercise and AA use different signalling pathways upstream of mTOR.
What is rapamycin used for?
Rapamycin (Rapamune, Sirolimus) is a macrolide exhibiting potent antitumor and immunosuppressive activity [261,262]. Rapamycin is thus used in clinical settings to prevent rejection in organ transplantation and to treat certain types of cancer.
Is Rapamycin a protein?
Rapamycin and TOR The intracellular rapamycin receptor in all eukaryotes is a small, ubiquitous protein termed FKBP12 (FK506-binding protein, molecular mass of 12 kDa; refs. 13, 14, 15).
What does mTOR do in autophagy?
The mTOR–autophagy axis. MTORC1 blocks the early steps in autophagy by phosphorylation-dependent inhibition of Atg13 and ULK1 and also restrains the degradative capacity of the cell by inhibiting the activity of TFEB family members.
How does mTOR regulate autophagy?
mTORC1 tightly regulates autophagy by suppressing autophagy induction via phosphorylation-dependent inhibition of ULK1/2 and the VPS34 complex and by preventing global expression of lysosomal and autophagy genes through TFEB phosphorylation.
Does rapamycin inhibit mTOR?
Rapamycin is an acute inhibitor of mTOR complex 1 (mTORC1), which phosphorylates substrates including S6 kinase 1 (S6K1), eIF4E-binding protein 1 (4E-BP1), transcription factor EB (TFEB), unc-51-like autophagy-activating kinase 1 (Ulk1), and growth factor receptor-bound protein 10 (GRB-10).
Does fasting inhibit mTOR?
Because mTOR is a nutrient-sensing pathway, it can be deactivated by fasting and severe calorie restriction (CR), which exert metabolic effect that are somewhat similar, but not identical, to those of rapamycin42.
What is mTOR treatment?
Mechanistic target of rapamycin (mTOR) is a protein kinase regulating cell growth, survival, metabolism, and immunity. mTOR is usually assembled into several complexes such as mTOR complex 1/2 (mTORC1/2).
What is rapamycin used for?
Rapamycin is thus used in clinical settings to prevent rejection in organ transplantation and to treat certain types of cancer. Rapamycin inhibits the response to IL-2, and thereby blocks activation and proliferation of T and B cells [263]. Treatment of human T lymphoid cells with rapamycin resulted in a marked diminution of HIV-1 transcription [264]. In addition, rapamycin synergistically enhances the anti-HIV activity of entry inhibitors such as vicriviroc, aplaviroc and enfuvirtide in vitro [265]. It was also shown that rapamycin inhibits HIV-1 replication in vitro through other different mechanisms, including downregulation of CCR5 on lymphocytes and monocytes [265,266]. All these effects support the use of rapamycin in addition to ART. Its efficacy remains, however, to be established in clinical trials.
What is the role of rapamycin in immunosuppression?
The basis for the immunosuppressive activity of these agents is their action in blocking interleukin-2 stimulation of lymphocyte proliferation.
What is Sirolimus rapamycin?
Sirolimus, or rapamycin, is a macrolide antibiotic with immunosuppressive properties. Sirolimus inhibits a protein, target of rapamycin (TOR), a key regulatory kinase controlling cytokine-mediated cellular proliferation.
Is sirolimus a cytokine?
Sirolimus ( rapamycin) is a novel immunosuppressive agent that impairs cytokine-induced lymphocyte proliferation. The prolonged half-life of sirolimus means that once-daily dosing is sufficient. It is metabolized via the cytochrome P-450 system; thus, the potential for multiple drug interactions exists. Adverse effects of sirolimus include hyperlipidemia, anemia, thrombocytopenia, diarrhea, and interstitial pneumonitis. Short- and medium-term data suggest sirolimus may have a useful role as a CNI-sparing agent. There is growing evidence that sirolimus has important antineoplastic effects in vivo.
Is rapamycin cytotoxic?
Rapamycin was tested by the Developmental Therapeutic Branch, National Cancer Institute (NCI) and identified as a noncytotoxic agent that delays tumor proliferation, finding evidence of cytostatic activity against several human cancers in vitro and in vivo (http://129.43.7.110/index.html ).
Does rapamycin inhibit cancer growth?
Rapamycin was shown to inhibit the growth of several murine and human cancer cell lines in a concentration-dependent manner, both in tissu e culture and xenograft models: B16 melanoma, P388 leukemia, MiaPaCa-2, and Panc-1 human pancreatic carcinomas, and others Busca et al (1996), Eng et al (1984), Grewe et al (1999).
Does rapamycin affect renal blood flow?
Animal studies in Wistar rats and pigs have demonstrated that unlike treatment with calcineurin inhibitors, rapamycin treatment alters neither renal blood flow nor glomerular filtration rate Ryffel et al (1994), Golbaekdal et al (1994). For a review of preclinical results of rapamycin treatment in transplant models see Stepkowski (2003).
How much rapamycin should I use for whole worms?
I have worked with rapamycin in whole worms. My optimizations showed that in whole worm planaria it works at a 100nM (5 injections of 20nM rap amycin) concentration, while for C. elegans we use 100uM concentrations.
How many ml of rapamycin is in DMSO?
I prepared the rapamycin in DMSO (5mg in 5 mL, that is roughly 1 mM), then aliquot to 100 ul, I diluted it in serum free DMEM before use (10-fold serial dilution), then treated the cells 1h/6h. But neither my target protein nor mTOR phosphorylation is affected. The concentration I used are 20nM, 50nM, 100nM, 200nM.
Is rapamycin selective?
Rapamycin is highly selective and potent. In human primary cells and co-cultures, rapamycin has the same phenotypic impact (changes in biomarkers) from 0.5 nM to 1 microM. 200 nM should be fine (PMID:24088371). Cite.
Is rapamycin dose dependent?
The attached chapter discusses the genotoxicity of a wide range of drugs among them rapamycin. The experiment with rapamycin was conducted in different concentrations and it was found that the effect is dose dependent. Details on the concentrations range with illustration by figures are shown.
Does rapamycin increase LC3-II?
Rapamycin does not increase LC3-II level - can you help?
Does rapamycin help with IPEX?
Treatment with rapamycin can restore regulatory T-cell function in IPEX patients. Rapamycin is able to affect Treg suppressive function via a FOXP3-independent mechanism, thus sustaining the clinical improvement observed in patients with IPEX syndrome under rapamycin treatment.
Does rapamycin suppress T cells?
Rapamycin has been shown to suppress peripheral T cells while sparing Treg cells expressing wild-type FOXP3, thereby proving beneficial in the clinical setting of immune dysregulation. However, the mechanisms of immunosuppression selective to Treg cells in patients with IPEX syndrome are unclear.
Does rapamycin affect iPSC?
High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adher ens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differ …
Does bafilomycin block autophagy?
Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner.
Does rapamycin cause EB formation?
Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products.
How to prepare rapamycin in DMSO?
There is trick to prepare rapamycin (probably other substance dissolve in DMSO will do the same way). First dilute rapamycin in DMSO (mine is 10 mM to what ever you want, I did 2 folds series dilution). Second, make sure you pour DMEM to rapamycin not the opposite.
How much DMSO to use in rapa?
For this time (I re-made the rapa DMSO stock solution), 5 mg in 550 ul DMSO (10 mM), shaker >3h RT and vortex intermittently, cover in foil paper the whole, then aliquot 100 ul per tube, store at -20C.
Can you pour DMEM to rapamycin?
Second, make sure you pour DMEM to rapamycin not the opposite. I did a a little test, if you add rapamycin to DMEM, unless your concentration is low enough (uM level), you will see precipitate, if you pour DMEM to rapamycin, there will be MUCH LESS precipitate forms, if you use diluted rapamycin, no precipitate seen.
Is rapa a negative control?
Only insulin group can serve as positive control, and the negative control should be the group that add nothing .
Is rapamycin dependent on phosphatidic acid?
The efficacy of rapamycin is dependent on the level of phosphatidic acid. Maybe your cells have too much?
Is rapa dissolving fine?
Seems that the rapa is dissolving fine. My protocol was indeed to first make a DMSO stock and then dilute it further with preheated cell culture medium.
Does rapamycin dissolve in DMSO?
There is trick to prepare rapamycin (probably other substance dissolve in DMSO will do the same way).
