Various Concentrations Of Rapamycin Research Articles

Inflammation Is More Sensitive than Cell Proliferation in Response to Rapamycin Treatment in Polycystic Kidney Disease

Introduction: It has been reported that rapamycin inhibited inflammation in renal interstitial diseases. We therefore hypothesized that rapamycin could attenuate inflammation in polycystic kidney disease (PKD). Methods: Han:SPRD rats were treated with rapamycin by daily gavage from 4 weeks to 12 weeks of age at the dosage of 0.5 mg/kg/day (low dose) or 1 mg/kg/day (high dose). WT9-12 human PKD cells were treated with various concentrations of rapamycin. Results: Two-kidney/total body weight ratio and cystic index in Cy/+ kidneys were significantly reduced with the treatment of low-dose rapamycin and further reduced by the treatment with high-dose rapamycin. However, the renal function of Cy/+ rats was equally improved by the treatment with either low-dose or high-dose rapamycin. The renal cell proliferation was significantly decreased in Cy/+ kidneys with the treatment of low-dose rapamycin and was further decreased with the treatment of high-dose rapamycin as examined by Ki67 staining. The phosphorylation of S6K in cystic kidneys was decreased by low-dose rapamycin and further decreased by high-dose rapamycin. Both low-dose and high-dose rapamycin treatment decreased macrophage infiltration and the expression of complement factor B (CFB), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-α) to a similar level. The expression of CFB, MCP-1, and TNF-α and phosphorylation of S6K were inhibited in WT9-12 cells treated with 10 nm rapamycin at 24 h and 48 h, respectively. Moreover, the phosphorylation of Akt was not increased by 1 nm and 10 nm of rapamycin and enhanced by 1 μm rapamycin treatment. Interestingly, WT9-12 cell proliferation could be inhibited by 1 μm rapamycin. Conclusion: Low dose of rapamycin could inhibit inflammation and protect renal function in PKD. Inflammation is more sensitive than cell proliferation in response to rapamycin treatment in PKD.

Rapamycin inhibits lung squamous cell carcinoma growth by downregulating glypican-3/Wnt/β-catenin signaling and autophagy.

There is not much progress in the treatment for lung squamous cell carcinoma LSCC in the past few years. Rapamycin Rapa, an inhibitor of mammalian target of rapamycin mTOR, has exhibited antitumor efficacy in a variety of malignant tumors. It has recently been reported that Rapamycin can induce autophagy signaling pathway in lung cancer and Glypican-3GPC3 can promote the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling pathway. The aim of this study is to investigate the mechanisms of rapamycin's antitumor efficacy in relation to GPC3/Wnt/β-catenin pathway and autophagy in LSCC. SK-MES-1 cells, a LSCC cell line, were treated with various concentrations of rapamycin with or without Glypican-3 GPC3-targeting siRNA. SK-MES-1 cell proliferation was determined by MTT assay. Protein expression levels of GPC3, β-catenin, Beclin-1 were checked via western blotting. We established the xenograft mice model to investigate the suppression effect of rapamycin on LSCC. In addition, we further testified the metabolism protein of autophagy process using the xenograft tumor tissue. Rapamycin could inhibit the SK-MES-1 cell proliferation in a concentration-dependent manner both in vitro and in vivo by decreasing the GPC3 expression and downregulating the glypican-3/Wnt/β-catenin signaling pathway. In addition, we found that GPC3 silencing can activate the glypican-3/Wnt/β-catenin pathway and autophagy, which contribute to the suppression of tumor growth both in vitro and in vivo. Rapamycin suppresses the growth of lung cancer through down-regulating glypican-3/Wnt/β-catenin signaling, which mediates with activation of autophagy. This study suggests GPC3 is a new promising target for rapamycin in the treatment of lung cancer.

Rapamycin and abundant TCR stimulation are required for the generation of stable human induced regulatory T cells.

ObjectivesRegulatory T cells (Tregs) are a vital sub‐population of CD4+ T cells with major roles in immune tolerance and homeostasis. Given such properties, the use of regulatory T cells for immunotherapies has been extensively investigated, with a focus on adoptive transfer of ex vivo expanded natural Tregs (nTregs). For immunotherapies, induced Tregs (iTregs), generated in vitro from naïve CD4+ T cells, provide an attractive alternative, given the ease of generating cell numbers required for clinical dosage. While the combination of TGF‐β, ATRA and rapamycin has been shown to generate highly suppressive iTregs, the challenge for therapeutic iTreg generation has been their instability. Here, we investigate the impact of rapamycin concentrations and α‐CD3/CD28 bead ratios on human iTreg stability.MethodsWe assess iTregs generated with various concentrations of rapamycin and differing ratios of α‐CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg‐specific demethylation region (TSDR) status.ResultsiTregs generated in the presence of TGF‐β, ATRA, rapamycin and a higher ratio of α‐CD3/CD28 beads were highly suppressive and stable upon in vitro re‐stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation.ConclusionThis work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re‐stimulation. Further validation in pre‐clinical models will be needed to ensure its suitability for applications in adoptive transfer.

Effect of rapamycin treatment during post-activation and/or in vitro culture on embryonic development after parthenogenesis and in vitro fertilization in pigs.

This study investigated the effects of early induction of autophagy on embryonic development in pigs. For this, oocytes or embryos were treated with an autophagy inducer, rapamycin (RP), during post-activation (Pa), in vitro fertilization (IVF) and/or in vitro culture (IVC). When parthenogenesis (PA) embryos were untreated (control) or treated with various concentrations of RP for 4hr during Pa, 100nm RP showed a higher blastocyst formation (48.8±2.7%) than the control (34.6±3.0%). When PA embryos were treated during the first 24hr of IVC, blastocyst formation was increased (p<.05) by 1 and 10nm RP (61.9±3.0 and 59.6±3.0%, respectively) compared to the control (43.2±1.8%) and 100nm RP (47.8±3.2%), with a higher embryo cleavage in response to 10nm RP (87.3±2.4%) than the control (74.1±3.2%). RP treatment during IVC and Pa + IVC showed increased blastocyst formation (44.7±2.5 and 44.1±2.0%, respectively) compared to the control (33.2±2.0%). In addition, RP treatment during Pa and/or IVC increased glutathione content and inversely reduced reactive oxygen species. In IVF, RP treatment for 6hr during IVF significantly increased embryonic development (34.0±2.6%) compared to the control (24.8±1.6%), but treatment during IVC for 24hr with RP did not (23.0±3.8%). Autophagy was significantly increased in PA oocytes by the RP treatment during Pa but not altered by the treatment during the first 24hr of IVC. Overall, RP treatment positively regulated the pre-implantation development of pig embryos, probably by regulating cellular redox state and stimulating autophagy.

Down-regulation of mitogen-activated protein kinases and nuclear factor-κB signaling is involved in rapamycin suppression of TLR2-induced inflammatory response in monocytic THP-1 cells.

Tripalmitoyl-S-glycero-Cys-(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signal pathway. Rapamycin can suppress TLR-induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2-induced inflammatory responses was investigated. It was found that Pam3CSK4-induced pro-inflammatory cytokines were significantly down-regulated at both the mRNA and protein levels in THP-1 cells pre-treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3-kinase/protein kinase-B (PI3K/AKT) signaling did not suppress the expression of pro-inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT-PCR showed that Erk and NF-κB signal pathways are related to the production of pro-inflammatory cytokines. Inhibition of Erk or NF-κB signaling significantly down-regulated production of pro-inflammatory cytokines. Additionally, western blot showed that pre-treatment of THP-1 cells with rapamycin down-regulates MAPKs and NF-κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4-induced pro-inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2-induced inflammatory responses by down-regulation of Erk and NF-κB signaling.

Rapamycin inhibiting Jurkat T cells viability through changing mRNA expression of serine/threonine protein phosphatase 2A

Aims In this study, we analyzed the mRNA expression of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) in the human leukemic T-cell line Jurkat cells treated with rapamycin, to determine whether rapamycin inhibiting cell viability is accompanied with the change of mRNA expression of PP2A. Methods and results Jurkat cells were incubated with various concentrations of rapamycin and cultured for different hours. Cell viability was assessed by MTT assay. The mRNA expressions of PP2A subunits were measured by quantitative real-time polymerase chain reaction (PCR). We found that rapamycin had an inhibitory effect on cell viability. IC50 was 343.3 nM at 48 h.We also found rapamycin had a dose and time-dependent effect on the gene expression of PP2A. When setting the concentration of rapamycin 500 nM, the mRNA expressions of PP2A subunits (Aa, Aβ, PR55a, PR55δ, PR61γ, PR70, Ca and Cβ) were declined significantly at 48 h. When treated with various concentrations of rapamycin for 48 h, the mRNA expressions of PP2A subunits were down-regulated in the range from 10 nM to500 nM. Conclusions Rapamycin inhibiting Jurkat T cells viability may be related to the reduction of PP2A mRNA expressions.

Rapamycin-mediated mTOR inhibition attenuates survivin and sensitizes glioblastoma cells to radiation therapy

Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.

Evaluation of the mammalian target of rapamycin pathway and the effect of rapamycin on target expression and cellular proliferation in osteosarcoma cells from dogs

To investigate activation of the mammalian target of rapamycin (mTOR) pathway and the antitumor effect of rapamycin in canine osteosarcoma cells. 3 established primary canine osteosarcoma cell lines generated from naturally developing tumors. Expression of total and phosphorylated mTOR and p70S6 kinase was assessed by use of western blot analysis in canine osteosarcoma cells with and without the addition of rapamycin. A clonogenic assay was performed to determine the surviving fraction of osteosarcoma cells at various concentrations of rapamycin. Total and phosphorylated mTOR and p70S6 kinase expression was evident in all 3 cell lines evaluated, which was indicative of activation of this pathway. Treatment with rapamycin resulted in a time-dependent decrease in phosphorylated mTOR expression and a lack of detectable phosphorylated p70S6 kinase. No detectable change in expression of total mTOR and total p70S6 kinase was identified after rapamycin treatment. The clonogenic assay revealed a significant dose-dependent decrease in the surviving fraction for all 3 cell lines when treated with rapamycin. These data indicated that mTOR and its downstream product are present and active in canine osteosarcoma cells. The pathway can be inhibited by rapamycin, and treatment of cells with rapamycin decreased the surviving tumor cell fraction. These data support the molecular basis for further investigation into the use of mTOR inhibitors as an antineoplastic approach for dogs with osteosarcoma.

Large Scale Expansion of Human Dendritic Cells with Regulatory Function from Peripheral Blood Monocytes Employing Leukapheresis and Rapamycin for Clinical Trials.

Regulatory T cells (Treg) exert pivotal roles on the induction and maintenance of peripheral tolerance. Adoptive transfer of Treg is a promising strategy to control acute GVHD. However, in vitro expansion of Treg is costly, laborious and time-consuming (more than 3 weeks). As an alternative source, dendritic cells (DC) with regulatory function (DCreg) seem to be attractive, since DC are potent antigen presenting cells capable of regulating the initiation of various immune responses and can be generated in a relatively short term culture from peripheral blood (PB) monocytes. In the present study, we examined the feasibility of generating DCreg on a large scale from PB monocytes obtained by leukapheresis. PB mononuclear cells (range 1.8×109 to 3.1×109) were obtained from 5 healthy volunteers with informed consent by leukapheresis for 1–2 hours using a COBE Spectra apparatus. Immature DC were generated from isolated CD14+ monocytes by culturing with GM-CSF and IL4 with or without various concentrations of rapamycin for 5–7days. For maturation, immature DC were cultured with the addition of TNFα for 2 days. The yield of immature DC cultured with rapamycin (10ng/ml) was 8.4±3.6×107, constituting 4.2±1.8% of the initial PB mononuclear cells. FACS analysis showed that DC cultured with rapamycin (DC-rapa) had immature DC phenotype with CD14−, CD1a+, CD83−. But the surface expression of CD86, CD40, HLA-DR was much lower compared with control DC (DC-ctr) cultured without rapamycin. The reduction of the expression of CD86, CD40, HLA-DR was dependent of rapamycin dose and resistant to maturational stimulation of TNFα. DC-rapa showed very weak allostimulatory activity in a mixed lymphocyte culture (MLC) compared with DC-ctr, corresponding to the reduced expression of HLA-DR and costimulatory molecules. To further determine the types of cells involved, CSFE-labelled non-isolated CD4+ T cells were cultured with DC-rapa or DC-ctr for 7 days and the proliferating cells were identified by FACS analysis. DC-rapa inhibited the proliferation of CD4+CD25− alloreactive T cells by approximately 60% while the inhibitory effect of DC-rapa on CD4+ CD25+ cells was minimal. Neither the irradiation of DC nor the addition of IL2 altered the inhibition. Stimulation with both anti-CD3 and ani-CD28 moonoclonal antibodies coated on microbeads reversed the inhibition completely, suggesting the importance of the signals via T cell receptors and costimulatory molecules in the inhibition by DC-rapa. Double staining with anti-CD25 and anti-Foxp3 monoclonal antibodies revealed that DC-rapa increased the percentage and number of CD25+Foxp3+ Treg by approximately 2.6 times compared with DC-ctr during 7-day culture. In conclusion, these results demonstrated that immature DC induced with rapamycin have a potent immunoregulatory function including preferential expansion of Treg. To the best of our knowledge, this is the first demonstration that immunoregulatory DC can be generated on a large scale in relatively short term culture with rapamycin following leukapheresis. These findings have an important clinical implication to facilitate DC-based immunotherapy for acute GVHD.

Rapamycin Enhances the Cytotoxicity of Bortezomib and Rituximab on Mantle Cell Lymphoma (MCL) Cell Lines.

[Background]: MCL is an aggressive non-Hodgkin lymphoma (NHL) with a median survival of 3-4 years. New agents with mechanisms of action different than standard chemotherapy are needed to improve the outcome of MCL. Recent studies have documented single agent activity of bortezomib and rituximab in MCL. Analogs of rapamycin (temsirolimus) have also shown single agent activity in MCL (J Clin Oncol, June 27, 2005 epub). In order to provide rationale for a clinical trial combining rapamycin analogs with bortezomib or rituximab, we performed in vitro studies of the effect of combinations of these drugs on human MCL cell lines.[Methods]: Two human lymphoma cell lines - Granta 519 and M0258 - were incubated with various concentrations of rapamycin, bortezomib, and rituximab to determine the LD50 of each drug alone. For the experiments with rituximab a secondary goat anti-human (GAH) IgG Fc antibody was used to crosslink cell bound rituximab and induce cell killing. Percent viability was assessed after 48 hours using annexin/propidium iodide staining and flow cytometry. Tests for drug synergy used combinations of rapamycin and bortezomib and the Granta 519 cell line. Fixed ratios of the two drugs were tested and the results were evaluated for synergism (combination index <1 in median effect analysis) in inducing cell death. The drug concentrations used spanned the LD50 of each drug determined from the single-agent studies. In the studies combining rituximab with bortezomib or rapamycin, a single fixed dose of rituximab and GAH Fc was tested with various concentrations of the other drugs to detect additive anti-tumor effects. Studies were performed at least in triplicate.[Results]: All three drugs exhibited single-agent activity in vitro against the two cell lines. The LD50 for rapamycin ranged from 17.5 – 25 micromolar for the Granta 519 cell line and 7.5–10 micromolar for M0258. The LD50 for bortezomib ranged from 2–5 ng/ml in both cell lines. Incubation of the cells with various concentrations of rituximab and GAH Fc had mild cytotoxic activity (reduced viability to 50–70% compared to GAH Fc control). This activity was not concentration dependent indicating saturation of CD20 by rituximab; therefore, a fixed concentration of rituximab was used for the combination experiments. When rituximab was added to bortezemib the % viability decreased by up to 37% (compared to bortezomib alone), and the bortezomib LD50 typically decreased to <1–2 ng/ml (LD50 was 2–5 ng/ml with bortezomib alone). The addition of rapamycin to rituximab increased the cytotoxicity by 23% compared to either drug alone and decreased the rapamycin LD50. When rapamycin and bortezomib were combined as treatment of Granta 519 cells, rapamycin was synergistic with bortezomib with a combination index <1. Sequencing the rapamycin either before or after the bortezomib treatment did not effect synergy in either direction.[Conclusion]: In vitro, rapamycin synergizes with bortezomib to enhance its cytotoxicity in MCL lines. Rituximab enhances the cytotoxicity of both of rapamycin and bortezomib. These in vitro studies provide the rationale to test combinations of these active agents in patients with MCL.

Rapamycin inhibits human renal epithelial cell proliferation: Effect on cyclin D3 mRNA expression and stability

Recent data have suggested that rapamycin use during the initial period after transplantation is associated with prolonged delayed graft function (DGF). Because of the known effects of rapamycin in other cell types, we speculated that this action may be secondary to human renal epithelial cells (HRECs) inhibition of proliferation. Primary cultures of HRECs were incubated with various concentrations of rapamycin. Cell proliferation was evaluated by cytotoxicity assays. The cell cycle was analyzed by flow cytometry. Protein expression levels were assessed by Western blot. Cyclin D3 mRNA levels were measured by quantitative real-time polymerase chain reaction (PCR). The transcriptional activity of the cyclin D3 gene was evaluated using transient transfection. Rapamycin exerted a significant concentration-dependent antiproliferative effect on growing HRECs by inhibiting the G(1) to S transition. The p70(S6) kinase pathway leading to cell cycle progression was found to be active, and low concentrations of rapamycin dramatically reduced p70(S6) kinase phosphorylation. Rapamycin completely inhibited the increase in cyclin D3 protein expression and mRNA accumulation induced by fetal calf serum, but did not affect cyclin E or cdk-inhibitor expression levels. This regulation of cyclin D3 protein expression is mainly due to a destabilization of its mRNA. Rapamycin reduced the mRNA half-life by 26% (4.8 +/- 1.3 hours vs. 6.5 +/- 1.0 hours, P < 0.001). Rapamycin inhibits the proliferative response of HRECs to mitogenic stimuli, and causes cell cycle arrest in the early G(1) phase, not only by a nonspecific process due to inhibition of the p70(S6k) pathway, but also by a direct effect on cyclin D3 mRNA stability.

Enhancement of human platelet aggregation and secretion induced by rapamycin.

Rapamycin is a new immunosuppressive drug of the macrolide type. Despite binding to one of the FK-binding proteins as the initial step in intracellular action, further effects differ from those of the other fungally derived macrolides, cyclosporine and tacrolimus. We have previously demonstrated an enhancement of agonist-mediated platelet activation by cyclosporine and tacrolimus which was associated with increased phosphorylation of two intracellular platelet proteins, p20 and p40. Because rapamycin utilizes the same class of binding proteins as tacrolimus, but its action is not associated with the inhibition of calcineurin, we postulated that if the stimulatory effect of cyclosporine or tacrolimus was due to calcineurin inhibition, rapamycin should not affect platelets in a similar fashion. Normal, washed human platelets were treated with various concentrations of rapamycin (from ng to microg/ml), and pre-incubated at 37 degrees C with rapamycin for various periods (1-30 min). Several platelet functional parameters were measured in samples treated with rapamycin and these parameters were compared with control platelet samples treated with the vehicle for the same period. Platelet aggregations following exposure to ADP or to the thrombin equivalent, TRAP-6, were measured as changes in optical transmission in a Chronolog lumi-aggregometer. Each experiment was repeated at three or more times and the mean results were used for statistical comparison. Rapamycin-treated platelets demonstrated an increase in their dose- and time-dependent sensitivity to ADP, resulting in a significantly enhanced primary wave of ADP-induced platelet aggregation followed by a secondary wave of aggregation, indicative of granule secretion. Furthermore, rapamycin-treated platelets showed significantly enhanced sensitivity to TRAP-6 as demonstrated by an increase in the initial velocity of aggregation, an increase in their maximal extent of aggregation and an enhancement of granular ATP secretion. Concentrations of rapamycin in the ng range, as well as short pre-incubation times (within min), were sufficient to cause significant enhancement of agonist-induced platelet aggregation and secretion (P < 0.001) as compared with their vehicle controls. Rapamycin significantly potentiates agonist-induced platelet aggregation in a time- and dose-dependent manner. As these findings are similar to those observed with the other fungal macrolides, we hypothesize that inhibition of calcineurin may not be necessary for the increase in intracellular protein phosphorylation observed following exposure of platelets to cyclosporine or tacrolimus. Whether the rapamycin-induced enhancement of sensitivity to agonists and platelet hyperaggregability explains the thrombocytopenia observed in patients when high doses of rapamycin are administered in the clinical setting, and whether these effects are synergistic with cyclosporine, are questions which remain to be investigated.