Prolonged Rapamycin Treatment Research Articles

Rapamycin suppresses inflammation and increases the interaction between p65 and IκBα in rapamycin-induced fatty livers.

Rapamycin treatment significantly increases lifespan and ameliorates several aging-related diseases in mice, making it a potential anti-aging drug. However, there are several obvious side effects of rapamycin, which may limit the broad applications of this drug. Lipid metabolism disorders such as fatty liver and hyperlipidemia are some of those unwanted side effects. Fatty liver is characterized as ectopic lipid accumulation in livers, which is usually accompanied by increased inflammation levels. Rapamycin is also a well-known anti-inflammation chemical. How rapamycin affects the inflammation level in rapamycin-induced fatty liver remains poorly understood. Here, we show that eight-day rapamycin treatment induced fatty liver and increased liver free fatty acid levels in mice, while the expression levels of inflammatory markers are even lower than those in the control mice. Mechanistically, the upstream of the pro-inflammatory pathway was activated in rapamycin-induced fatty livers, however, there is no increased NFκB nuclear translocation probably because the interaction between p65 and IκBα was enhanced by rapamycin treatment. The lipolysis pathway in the liver is also suppressed by rapamycin. Liver cirrhosis is an adverse consequence of fatty liver, while prolonged rapamycin treatment did not increase liver cirrhosis markers. Our results indicate that although fatty livers are induced by rapamycin, the fatty livers are not accompanied by increased inflammation levels, implying that rapamycin-induced fatty livers might not be as harmful as other types of fatty livers, such as high-fat diet and alcohol-induced fatty livers.

Rapamycin Cannot Reduce Seizure Susceptibility in Infantile Rats with Malformations of Cortical Development Lacking mTORC1 Activation.

The mechanistic target of the rapamycin (mTOR) pathway is involved in cortical development. However, the efficacy of mTOR inhibitors in malformations of cortical dysplasia (MCD) outside of the tuberous sclerosis complex is unknown. We selected the MCD rat model with prenatal MAM exposure to test the efficacy of mTOR inhibitors in MCDs. We explored the early cortical changes of mTOR pathway protein expression in rats aged P15. We also monitored the early treatment effect of the mTOR inhibitor, rapamycin, on N-methyl-D-aspartate (NMDA)-induced spasms at P15 and their behavior in the juvenile stage. In vivo MR spectroscopy was performed after rapamycin treatment and compared with vehicle controls. There was no difference in mTORC1 pathway protein expression between MAM-exposed MCD rats and controls at P15, and prolonged treatment of rapamycin had no impact on NMDA-induced spasms despite poor weight gain. Prenatal MAM-exposed juvenile rats treated with rapamycin showed increased social approaching and freezing behavior during habituation. MR spectroscopy showed altered neurometabolites, including Gln, Glu+Gln, Tau, and Cr. Despite behavioral changes and in vivo neurometabolic alteration with early prolonged rapamycin treatment, rapamycin had no effect on spasms susceptibility in prenatal MAM-exposed infantile rats with MCD without mTORC1 activation. For MAM-exposed MCD rats without mTORC1 activation, treatment options outside of mTOR pathway inhibitors should be explored.

Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension.

Rationale: Enhanced proliferation and distal migration of human pulmonary arterial smooth muscle cells (hPASMCs) both contribute to the progressive increases in pulmonary vascular remodeling and resistance in pulmonary arterial hypertension (PAH). Our previous studies revealed that Rictor deletion, to disrupt mTOR Complex 2 (mTORC2), over longer periods result in a paradoxical rise in platelet-derived growth factor receptor (PDGFR) expression in PASMCs. Thus, the purpose of this study was to evaluate the role of combination therapy targeting both mTOR signaling with PDGFR inhibition to attenuate the development and progression of PAH. Methods and Results: Immunoblotting analyses revealed that short-term exposure to rapamycin (6h) significantly reduced phosphorylation of p70S6K (mTORC1-specific) in hPASMCs but had no effect on the phosphorylation of AKT (p-AKT S473, considered mTORC2-specific). In contrast, longer rapamycin exposure (>24 h), resulted in differential AKT (T308) and AKT (S473) phosphorylation with increases in phosphorylation of AKT at T308 and decreased phosphorylation at S473. Phosphorylation of both PDGFRα and PDGFRβ was increased in hPASMCs after treatment with rapamycin for 48 and 72 h. Based on co-immunoprecipitation studies, longer exposure to rapamycin (24–72 h) significantly inhibited the binding of mTOR to Rictor, mechanistically suggesting mTORC2 inhibition by rapamycin. Combined exposure of rapamycin with the PDGFR inhibitor, imatinib significantly reduced the proliferation and migration of hPASMCs compared to either agent alone. Pre-clinical studies validated increased therapeutic efficacy of rapamycin combined with imatinib in attenuating PAH over either drug alone. Specifically, combination therapy further attenuated the development of monocrotaline (MCT)- or Hypoxia/Sugen-induced pulmonary hypertension (PH) in rats as demonstrated by further reductions in the Fulton index, right ventricular systolic pressure (RVSP), pulmonary vascular wall thickness and vessel muscularization, and decreased proliferating cell nuclear antigen (PCNA) staining in PASMCs. Conclusion: Prolonged rapamycin treatment activates PDGFR signaling, in part, via mTORC2 inhibition. Combination therapy with rapamycin and imatinib may be a more effective strategy for the treatment of PAH.

A Novel Combination Treatment with Honokiol and Rapamycin Effectively Restricts c-Met-Induced Growth of Renal Cancer Cells, and also Inhibits the Expression of Tumor Cell PD-L1 Involved in Immune Escape.

The mTOR inhibitor Rapamycin has tumor inhibitory properties; and it is also used as an immunosuppressive agent after organ transplantation. However, prolonged Rapamycin treatment re-activates Akt and can promote cancer growth. Honokiol is a natural compound with both anti-tumorigenic and anti-inflammatory properties. Here, we assessed the anti-tumor effects of Rapamycin and Honokiol combination in renal cell carcinoma (RCC). Receptor tyrosine kinase c-Met-mediated signaling plays a major role in RCC growth. We observed that compared with Rapamycin alone, Rapamycin + Honokiol combination can effectively down-regulate c-Met-induced Akt phosphorylation in renal cancer cells; and it markedly inhibited Ras activation and cell proliferation and promoted G1 phase cell cycle arrest. The combination treatment significantly induced ROS generation and cancer cell apoptosis even when c-Met is activated. Importantly, Honokiol, but not Rapamycin, decreased c-Met-induced expression of the co-inhibitory molecule PD-L1, implied in the immune escape of renal cancer cells. In mouse renal cancer cells and Balb/c splenocytes co-culture assay, Rapamycin + Honokiol markedly potentiated immune-cell-mediated killing of cancer cells, possibly through the down-regulation of PD-L1. Together, Honokiol can effectively overcome the limitation of Rapamycin treatment alone; and the combination treatment can markedly restrict the growth of RCC, with particular importance to post-transplantation renal cancer.

Rapamycin rescues mitochondrial myopathy via coordinated activation of autophagy and lysosomal biogenesis.

The mTOR inhibitor rapamycin ameliorates the clinical and biochemical phenotype of mouse, worm, and cellular models of mitochondrial disease, via an unclear mechanism. Here, we show that prolonged rapamycin treatment improved motor endurance, corrected morphological abnormalities of muscle, and increased cytochrome c oxidase (COX) activity of a muscle‐specific Cox15 knockout mouse (Cox15 sm/sm). Rapamycin treatment restored autophagic flux, which was impaired in naïve Cox15 sm/sm muscle, and reduced the number of damaged mitochondria, which accumulated in untreated Cox15 sm/sm mice. Conversely, rilmenidine, an mTORC1‐independent autophagy inducer, was ineffective on the myopathic features of Cox15 sm/sm animals. This stark difference supports the idea that inhibition of mTORC1 by rapamycin has a key role in the improvement of the mitochondrial function in Cox15 sm/sm muscle. In contrast to rilmenidine, rapamycin treatment also activated lysosomal biogenesis in muscle. This effect was associated with increased nuclear localization of TFEB, a master regulator of lysosomal biogenesis, which is inhibited by mTORC1‐dependent phosphorylation. We propose that the coordinated activation of autophagic flux and lysosomal biogenesis contribute to the effective clearance of dysfunctional mitochondria by rapamycin.

Yeast m6A Methylated mRNAs Are Enriched on Translating Ribosomes during Meiosis, and under Rapamycin Treatment.

Interest in mRNA methylation has exploded in recent years. The sudden interest in a 40 year old discovery was due in part to the finding of FTO’s (Fat Mass Obesity) N6-methyl-adenosine (m6A) deaminase activity, thus suggesting a link between obesity-associated diseases and the presence of m6A in mRNA. Another catalyst of the sudden rise in mRNA methylation research was the release of mRNA methylomes for human, mouse and Saccharomyces cerevisiae. However, the molecular function, or functions of this mRNA ‘epimark’ remain to be discovered. There is supportive evidence that m6A could be a mark for mRNA degradation due to its binding to YTH domain proteins, and consequently being chaperoned to P bodies. Nonetheless, only a subpopulation of the methylome was found binding to YTHDF2 in HeLa cells.The model organism Saccharomyces cerevisiae, has only one YTH domain protein (Pho92, Mrb1), which targets PHO4 transcripts for degradation under phosphate starvation. However, mRNA methylation is only found under meiosis inducing conditions, and PHO4 transcripts are apparently non-methylated. In this paper we set out to investigate if m6A could function alternatively to being a degradation mark in S. cerevisiae; we also sought to test whether it can be induced under non-standard sporulation conditions. We find a positive association between the presence of m6A and message translatability. We also find m6A induction following prolonged rapamycin treatment.

Catalytic mTOR inhibitors can overcome intrinsic and acquired resistance to allosteric mTOR inhibitors.

We tested the antitumor efficacy of mTOR catalytic site inhibitor MLN0128 in models with intrinsic or acquired rapamycin-resistance. Cell lines that were intrinsically rapamycin-resistant as well as those that were intrinsically rapamycin-sensitive were sensitive to MLN0128 in vitro. MLN0128 inhibited both mTORC1 and mTORC2 signaling, with more robust inhibition of downstream 4E-BP1 phosphorylation and cap-dependent translation compared to rapamycin in vitro. Rapamycin-sensitive BT474 cell line acquired rapamycin resistance (BT474 RR) with prolonged rapamycin treatment in vitro. This cell line acquired an mTOR mutation (S2035F) in the FKBP12-rapamycin binding domain; mTORC1 signaling was not inhibited by rapalogs but was inhibited by MLN0128. In BT474 RR cells, MLN0128 had significantly higher growth inhibition compared to rapamycin in vitro and in vivo. Our results demonstrate that MLN0128 may be effective in tumors with intrinsic as well as acquired rapalog resistance. mTOR mutations are a mechanism of acquired resistance in vitro; the clinical relevance of this observation needs to be further evaluated.

Prolonged Rapamycin treatment led to beneficial metabolic switch

Prolonged Rapamycin treatment led to beneficial metabolic switch

Duration of Rapamycin Treatment Has Differential Effects on Metabolism in Mice

The evolutionarily conserved target of rapamycin (TOR) signaling controls growth, metabolism, and aging. In the first robust demonstration of pharmacologically-induced life extension in mammals, longevity was extended in mice treated with rapamycin, an inhibitor of mechanistic TOR (mTOR). However, detrimental metabolic effects of rapamycin treatment were also reported, presenting a paradox of improved survival despite metabolic impairment. How rapamycin extended lifespan in mice with such paradoxical effects was unclear. Here we show that detrimental effects of rapamycin treatment were only observed during the early stages of treatment. These effects were reversed or diminished in mice treated for 20 weeks, with better metabolic profiles, increased oxygen consumption and ketogenesis, and markedly enhanced insulin sensitivity. Thus, prolonged rapamycin treatment lead to beneficial metabolic alterations, consistent with life extension previously observed. Our findings provide a likely explanation of the "rapamycin paradox" and support the potential causal importance of these metabolic alterations in longevity.

Platelet-derived growth factor-induced Akt phosphorylation requires mTOR/Rictor and phospholipase C-γ1, whereas S6 phosphorylation depends on mTOR/Raptor and phospholipase D

Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.

Regulation of Protein Kinase C Inactivation by Fas-associated Protein with Death Domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.

Phospholipase D Regulates Myogenic Differentiation through the Activation of Both mTORC1 and mTORC2 Complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.

GSK3β inactivation in podocytes results in decreased phosphorylation of p70S6Kaccompanied by cytoskeletal rearrangements and inhibited motility

The inhibition of mTOR kinase after renal transplantation has been associated with podocyte injury and proteinuria; however, the signaling pathways regulating these effects are not well understood. We found that prolonged rapamycin treatment in podocytes leads to an increase in glycogen synthase kinase 3β (GSK3β) phosphorylation, resulting in inactivation of total GSK3β kinase activity. To investigate the cellular consequences of the inactivation of GSK3β, we used two inhibitors reducing kinase activity and studied the cross talk between GSK3 function and the Akt/mammalian target of rapamycin (mTOR) pathway. Both GSK3 inhibitors reduced the phosphorylation of the mTOR downstream target, p70(S6K), indicating that GSK3 inhibition in podocytes is able to cause similar effects as treatment with rapamycin. Moreover, GSK3 inhibition was accompanied by the reduced expression of slit diaphragm-associated proteins and resulted in an altered cytoskeletal structure and reduced motility of podocytes, suggesting that GSK3 kinase can modulate Akt/mTOR-dependent signaling in podocytes.

Abstract 427: Targeted inhibition of the mTOR complex components, RAPTOR and RICTOR, attenuates progression of colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E-BP1, while the mTOR-RICTOR complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. The purpose of the present study was to determine the effect of targeting mTORC1 and mTORC2 upon progression of CRCs. METHODS. HCT116 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with shRNA directed against RAPTOR or RICTOR. Effects on migration (measured by wound healing assay) and invasion (measured by transwell matrigel assay) were analyzed. Changes in actin cytoskeleton and cell-cell contacts were determined by immunofluorescence microscopy. RESULTS. (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR, are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iii) Stable shRNA-mediated knockdown of RAPTOR and RICTOR significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Stable shRNA-mediated knockdown of RAPTOR and RICTOR leads to mesenchymal-to-epithelial transition, as evidenced by increased E-cadherin and decreased vimentin levels. (v) Stable shRNA-mediated knockdown of RAPTOR and RICTOR leads to characteristic rearrangements in actin cytoskeleton as well as increased cell-cell contact. CONCLUSIONS. The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR, are overexpressed in both primary CRCs as well as matched liver metastases. Although the mTOR signaling axis is primarily associated with growth of normal and cancer cells, we demonstrate that stable inhibition of both mTORC1 and mTORC2 decreases migration and invasion capabilities of CRC cells as well as causing a mesenchymal-to-epithelial change in phenotype. The mechanism for this decrease in migration and invasion may be via modulation of actin cytoskeletal rearrangement and cell-cell contact. Thus, targeted inhibition of mTOR signaling represents a novel therapeutic strategy to attenuate the progression and metastasis of CRCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 427.

Abstract B15: Targeted inhibition of mTOR signaling attenuates the growth and progression of colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E-BP1, while the mTOR-RICTOR-mSin1 complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. Furthermore, rapamycin mediated mTORC1 inhibition leads to loss of mTOR-S6K-IRS1 dependent feedback inhibition, thereby resulting in increased Akt activation. The purpose of the present study was to determine: (i) sensitivity of CRC cells to rapamycin treatment, and (ii) effect of targeting mTORC1 and mTORC2 upon CRC proliferation, apoptosis, cell cycle progression, migration, invasion and xenograft growth. Methods: HCT116, KM20, Caco-2 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with siRNA/shRNA directed against mTOR, RAPTOR or RICTOR. Effects on cell proliferation (measured by Coulter counter cell counts), apoptosis (measured by detecting the level of histone-associated DNA fragments), cell cycle progression (measured by fluorescence-activated cell sorting), migration (measured by wound healing assay), invasion (measured by transwell matrigel assay) and subcutaneous growth were analyzed. Results: (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the proliferation of HCT116 and KM20 cells (rapamycin sensitive); however, rapamycin did not alter SW480 or Caco-2 proliferation (rapamycin resistant). (iii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Transient siRNA-mediated knockdown of RAPTOR decreased proliferation of KM20 cells only, while knockdown of RICTOR decreased proliferation of all four cell lines. (v) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 components leads to decreased proliferation, increased apoptosis and inhibition of G1-S phase cell cycle progression in HCT116 cells. (vi) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 leads to decreased proliferation, but only mTORC2 knockdown leads to increased apoptosis in SW480 cells. (vii) Targeted inhibition of mTORC1 and mTORC2 attenuates growth of rapamycin-sensitive and — resistant CRCs in xenografted nude mice. Conclusions: The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in CRCs. Transient inhibition of mTORC2, but not mTORC1, decreased the proliferation of rapamycin-sensitive and —resistant CRC cells. Stable inhibition of mTORC2 decreased proliferation and induced apoptosis in rapamycin-sensitive and —resistant CRC cells. Finally, targeted inhibition of both mTORC1 and mTORC2 attenuates subcutaneous growth of CRCs in vivo. Thus, inhibition of mTORC2 activity represents a novel therapeutic strategy for treatment of rapamycin-sensitive and, more importantly, rapamycin-resistant CRCs. Citation Information: Cancer Res 2009;69(23 Suppl):B15.

Effects of rapamycin on prostate cancer PC-3 cells

The mammalian target of rapamycin (mTOR) signaling network regulates cell growth, proliferation, survival and apoptosis. This study was to investigate the effect and the underlying mechanism of rapamycin on prostate cancer PC-3 cells. PC-3 cells were treated with 1 nmol/L rapamycin. The proliferation of PC-3 was examined by MTT. The cell cycle distribution of PC-3 was measured by FCM. The protein levels of raptor, rictor, Akt, pS6k1-T389, pAkt-s473 in PC-3 were examined by western blot. Rapamycin increased the proliferation of PC-3 at 24 h, however, it remarkably inhibited cell proliferation after 36 h (P<0.01), which became more obviously at 72 h. Although incubation with rapamycin slightly induced cell arrest at the S phase at 24 h, this gradually increased PC-3 cells at the G1 phase at 36 h and 48 h. Compared with the control group, the protein levels of raptor and pS6k1-T389 were significantly decreased (P<0.01), and those of rictor and Akt remained unchanged after the treatment with rapamycin for 24 h; the protein level of pAkt-s473 was significantly increased at 24 h (P<0.01), but was obviously inhibited at 36 h and almost completely inhibited at 72 h (P<0.01). Prolonged rapamycin treatment inhibits the proliferation of PC-3 cells. This may be caused by rapamycin-induced cell cycle arrest at the G(1) phase and inhibition of Akt phosphorylation.

MTOR regulates expression of slit diaphragm proteins and cytoskeleton structure in podocytes

The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors can cause proteinuria, especially in kidney and heart transplanted patients. Podocytes play a major role in establishing the selective permeability of the blood-urine filtration barrier. Damage of these cells leads to proteinuria, a hallmark of most glomerular diseases. Interestingly, podocyte damage and focal segmental glomerulosclerosis can occur after treatment with an mTOR inhibitor in some transplant patients. To investigate the mechanisms of mTOR inhibitor-induced podocyte damage, we analyzed the effect of rapamycin on mTOR signaling and cellular function in human podocytes. We found that prolonged rapamycin treatment reduced the expression of total mTOR, which correlates with diminished levels of mTOR phosphorylation at Ser(2448) and Ser(2481). In addition, treatment with rapamycin reduced rictor expression and mTORC2 formation, resulting in a reduced phosphorylation of protein kinase B at Ser(473). The expression level of the slit-diaphragm proteins nephrin and transient receptor potential cation channel 6 as well as the cytoskeletal adaptor protein Nck significantly decreased. Moreover, rapamycin reduced cell adhesion and cell motility, which was accompanied by an enhanced formation of dot-like actin-rich structures. Our data provide new molecular insights explaining which pathways and molecules are affected in podocytes by an imbalanced mTOR function because of rapamycin treatment.

SREBP Activity Is Regulated by mTORC1 and Contributes to Akt-Dependent Cell Growth

SummaryCell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). Here we show that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. We also show that silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of dSREBP in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. Our results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth.

Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4+CD25+FoxP3+ T cells

Graft-versus-host-disease (GVHD) is the most common cause of poor outcome after allogeneic stem cell transplantation (SCT). Of late, exploitation of FOXP3(+) regulatory T-cell (T(REG)) function is emerging as a promising strategy in suppression of GVHD, while preserving graft-versus-leukemia (GVL). Cyclosporine and rapamycin reduce the expansion of effector T cells by blocking interleukin (IL)-2, but signaling by IL-2 is pivotal for T(REG) homeostasis. The resolution of GVHD is critically dependent on thymus-dependent reconstitution of the immunoregulatory system. Thus, there has been concern about the impact of blocking IL-2 signaling by immunosuppressive agents on T(REG) homeostasis. Here we demonstrate in a mouse model that in contrast to rapamycin, cyclosporine compromises not only the thymic generation of CD4(+)CD25(+)FoxP3(+) T cells but also their homeostatic behavior in peripheral immune compartments. Treatment with cyclosporine resulted in a sharp reduction of peripheral CD25(+)FoxP3(+) T cells in all immune compartments studied. Prolonged rapamycin treatment allowed for thymic generation of CD4(+)FoxP3(+) T cells, whereas treatment with cyclosporine led to a reduced generation of these cells. In conclusion, cyclosporine and rapamycin differentially affect homeostasis of CD4(+)FoxP3(+) T(REG) in vivo. As peripheral tolerance induction is a prerequisite for successful treatment outcome after allogeneic SCT, these findings are of potential clinical relevance.

Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB

The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. Here we show that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. Our work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types.