mTOR-mediated Signaling Pathway Research Articles

Decreased Treg cells induced by bisphenol A is associated with up-regulation of PI3K/Akt/mTOR signaling pathway and Foxp3 DNA methylation in spleen of adolescent mice

The current study aimed to explore whether bisphenol A (BPA) exposure aggravated the decrease in Tregs induced by ovalbumin (OVA) in adolescent female mouse models of asthma, and whether the process was associated with mTOR-mediated signaling pathways and DNA methylation levels. A total of 40 female C57BL/6 mice at the age of four weeks were used and divided into five groups after 1 week of domestication. Each group consisted of eight mice: the control group, OVA group, OVA + BPA (0.1 μg mL−1) group, OVA + BPA (0.2 μg mL−1) group, and OVA + BPA (0.4 μg mL−1) group. Results revealed that Foxp3 protein levels decreased in the spleens of mice exposed to BPA compared to those in the OVA group. After an elevation in BPA dose, the mRNAs of methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) were gradually upregulated. The mechanism was related to the activity of TLR4/NF-κB and PI3K/Akt/mTOR signaling pathways and the enhancement of Foxp3 DNA methylation. Our results, collectively, provided a new view for studying the mechanisms underlying BPA exposure-induced immune dysfunction. Investigation of the regulatory mechanisms of DNA methylation in the abnormal Th immune response caused by BPA exposure could help reveal the causes and molecular mechanisms underlying the high incidence of allergic diseases in children in recent years.

Canagliflozin interrupts mTOR-mediated inflammatory signaling and attenuates DMBA-induced mammary cell carcinoma in rats

BackgroundBreast cancer prevalence has been globally increasing, therefore, introducing novel interventions in cancer treatment is of a significant importance. The present study was designed to investigate the anti-cancer effect of Canagliflozin (CNG) in an experimental model of DMBA-induced mammary carcinoma in female rats. Methods18 female rats were divided into three experimental groups: Normal control, DMBA control, DMBA+ CNG treated group. DMBA (7.5 mg/kg) was injected subcutaneously in the mammary cells twice weekly for 4 weeks and CNG (10 mg/kg) was orally administered daily for an additional 3 weeks while DMBA control rats only received the vehicle for 3 weeks. Tumors’ weight and volume were measured, BRCA-1 and TAC were quantified in serum samples, mTOR, caspase-1, NFκB, IL-1β, NLRP3, GSDMD and MDA were quantified in tumors’ homogenates. Results: CNG treatment increased the BRCA-1 expression, suppressed mTOR inflammatory pathway, attenuated tumor inflammatory mediators; NLRP3, GSDMD, NFκB, IL-1β, suppressed the oxidative stress and inhibited tumor expression of the proliferation biomarker; Ki67. ConclusionCNG modulated mTOR-mediated signaling pathway and attenuated pyroptotic, inflammatory pathways, suppressed oxidative stress and eventually inhibited DMBA-induced mammary carcinoma proliferation.

A novel targeted co-delivery nanosystem for enhanced ovarian cancer treatment via multidrug resistance reversion and mTOR-mediated signaling pathway

BackgroundMultidrug resistance (MDR) is the main challenge of successful chemotherapy for ovarian cancer patients, with 50% to 75% of ovarian cancer patients eventually relapsed due to it. One of the effective strategies for treating MDR and improving therapeutic efficiency of ovarian cancer is to use nanotechnology-based targeted drug delivery systems. In this study, a novel nano targeted co-delivery system modified by hyaluronic acid (HA) was developed by using gold nanorods coated with functionalized mesoporous silica nanoparticles (HA-PTX/let-7a-GNR@MSN) for combined delivery of hydrophobic chemotherapy drug Paclitaxel (PTX) and lethal-7a (let-7a), a microRNA (miR), to overcome MDR in ovarian cancer. Furthermore, we also analyzed the molecular mechanism of this nanotherapeutic system in the treatment of ovarian cancer.ResultsHA-modified nanocomplexes can specifically bind to the CD44 receptor, which is highly expressed in SKOV3/SKOV3TR cells, achieving effective cell uptake and 150% enhancement of tumor site permeability. The nanosystem realized the stable combination and protective transportation of PTX and miRs. Analysis of drug-resistant SKOV3TR cells and an SKOV3TR xenograft model in BALB/c-nude mice showed significant downregulation of P-glycoprotein in heterogeneous tumor sites, PTX release, and subsequent induction of apoptosis. More importantly, this nanosystem could synergistically inhibit the growth of ovarian tumors. Further studies suggest that mTOR-mediated signaling pathways play an important role in reversing drug resistance and inducing apoptosis.ConclusionsTo sum up, these data provide a model for overcoming PTX resistance in ovarian cancer.Graphical

Autophagy protects against retinal cell death in mouse model of cytomegalovirus retinitis

BackgroundExtensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis (CMV). Our previous results have shown that there is a functional relationship between autophagy and apoptosis during MCMV infection of retinal pigment epithelium (RPE). The purpose of this study was to determine whether autophagy plays a significant role in the death of retinal cells during MCMV retinitis.MethodsThe retinas of adult BALB/c mice were infected with MCMV via supraciliary injection. Rapamycin, a mTOR inhibitor, was injected to MCMV-infected BALB/c mice intraperitoneally. Immunohistochemistry and western blot were performed to observe the spread pattern of virus in retinas and the levels of targeted proteins. Plaque assay was performed to determine the virus titer in different groups. Since Atg5 is a key gene regulating autophagy, we bred Atg5flox/flox; Nestin-Cre mice to deeply elucidate the role of autophagy during MCMV retinitis. Atg5flox/flox; Nestin-Cre mice were genotyped and infected with MCMV. Immunohistochemistry was performed to observe the type of virus-infected cells and apoptosis in retinas during MCMV retinitis.ResultsIn MCMV mouse model, MCMV infection in outer nuclear layer (ONL) and inner nuclear layer (INL) in the retinas caused cleaved caspase 3 positive apoptosis, which is not co-localized with early antigen (EA) positive virus infected cells in rapamycin treated group. Rapamycin treatment increased the levels of LC3B-II by inhibiting mTOR and decreased the levels of cleaved caspase-3 during MCMV retinitis. However, virus propagation was not affected by rapamycin. In Atg5flox/flox; Nestin-Cre mice, RPE and glial cells were the main targets of viral infection, and number of EA positive retinal cells and TUNEL positive retinal cells was significantly increased compared to Atg5flox/+; Nestin-Cre mice though there was no difference of virus propagation between Atg5flox/flox; Nestin-Cre mice and Atg5flox/+; Nestin-Cre mice.ConclusionsAutophagy protects retinal cells from MCMV infection induced apoptosis through mTOR-mediated signaling pathway.

Overexpression of Kinase-Dead mTOR Impairs Glucose Homeostasis by Regulating Insulin Secretion and Not β-Cell Mass.

Regulation of glucose homeostasis by insulin depends on β-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in β-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in β-cells. We examined glucose homeostasis and β-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in β-cell function without alterations in β-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their β-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and β-cell insulin secretion defect but showed no changes in β-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to β-cell growth but is involved in regulating β-cell function in normal and diabetogenic conditions.

SH003 suppresses breast cancer growth by accumulating p62 in autolysosomes.

Drug markets revisits herbal medicines, as historical usages address their therapeutic efficacies with less adverse effects. Moreover, herbal medicines save both cost and time in development. SH003, a modified version of traditional herbal medicine extracted from Astragalus membranaceus (Am), Angelica gigas (Ag), and Trichosanthes Kirilowii Maximowicz (Tk) with 1:1:1 ratio (w/w) has been revealed to inhibit tumor growth and metastasis on highly metastatic breast cancer cells, both in vivo and in vitro with no toxicity. Meanwhile, autophagy is imperative for maintenance cellular homeostasis, thereby playing critical roles in cancer progression. Inhibition of autophagy by pharmacological agents induces apoptotic cell death in cancer cells, resulting in cancer treatment. In this study, we demonstrate that SH003-induced autophagy via inhibiting STAT3 and mTOR results in an induction of lysosomal p62/SQSTM1 accumulation-mediated reactive oxygen species (ROS) generation and attenuates tumor growth. SH003 induced autophagosome and autolysosome formation by inhibiting activation of STAT3- and mTOR-mediated signaling pathways. However, SH003 blocked autophagy-mediated p62/SQSTM1 degradation through reducing of lysosomal proteases, Cathepsins, resulting in accumulation of p62/SQSTM1 in the lysosome. The accumulation of p62/SQSTM1 caused the increase of ROS, which resulted in the induction of apoptotic cell death. Therefore, we conclude that SH003 suppresses breast cancer growth by inducing autophagy. In addition, SH003-induced p62/SQSTM1 could function as an important mediator for ROS generation-dependent cell death suggesting that SH003 may be useful for treating breast cancer.

MTOR, a Potential Target to Treat Autism Spectrum Disorder.

Mammalian target of rapamycin (mTOR) is a key regulator in various cellular processes, including cell growth, gene expression, and synaptic functions. Autism spectrum disorder (ASD) is frequently accompanied by monogenic disorders, such as tuberous sclerosis complex, phosphatase and tensin homolog tumor hamartoma syndrome, neurofibromatosis 1, and fragile X syndrome, in which mTOR is hyperactive. Mutations in the genes involved in the mTOR-mediated signaling pathway have been identified in some cases of syndromic ASD. Evidences indicate a pathogenic role for hyperactive mTOR-mediated signaling in ASD associated with these monogenic disorders, and mTOR inhibitors are a potential pharmacotherapy for ASD. Abnormal synaptic transmission through metabotropic glutamate receptor 5 may underlie in a part of ASD associated with hyperactive mTOR-mediated signaling. In this review, the relationship between mTOR and ASD is discussed.

Study on the Mechanism of mTOR-Mediated Autophagy during Electroacupuncture Pretreatment against Cerebral Ischemic Injury.

This study is aimed at investigating the association between the electroacupuncture (EA) pretreatment-induced protective effect against early cerebral ischemic injury and autophagy. EA pretreatment can protect cerebral ischemic and reperfusion injuries, but whether the attenuation of early cerebral ischemic injury by EA pretreatment was associated with autophagy is not yet clear. This study used the middle cerebral artery occlusion model to monitor the process of ischemic injury. For rats in the EA pretreatment group, EA pretreatment was conducted at Baihui acupoint before ischemia for 30 min for 5 consecutive days. The results suggested that EA pretreatment significantly increased the expression of autophagy in the cerebral cortical area on the ischemic side of rats. But the EA pretreatment-induced protective effects on the brain could be reversed by the specific inhibitor 3-methyladenine of autophagy. Additionally, the Pearson correlation analysis indicated that the impact of EA pretreatment on p-mTOR (2481) was negatively correlated with its impact on autophagy. In conclusion, the mechanism of EA pretreatment at Baihui acupoint against cerebral ischemic injury is mainly associated with the upregulation of autophagy expression, and its regulation of autophagy may depend on mTOR-mediated signaling pathways.

Roles of mTOR Signaling in Brain Development.

mTOR is a serine/threonine kinase composed of multiple protein components. Intracellular signaling of mTOR complexes is involved in many of physiological functions including cell survival, proliferation and differentiation through the regulation of protein synthesis in multiple cell types. During brain development, mTOR-mediated signaling pathway plays a crucial role in the process of neuronal and glial differentiation and the maintenance of the stemness of neural stem cells. The abnormalities in the activity of mTOR and its downstream signaling molecules in neural stem cells result in severe defects of brain developmental processes causing a significant number of brain disorders, such as pediatric brain tumors, autism, seizure, learning disability and mental retardation. Understanding the implication of mTOR activity in neural stem cells would be able to provide an important clue in the development of future brain developmental disorder therapies.

Abstract A168: Hepatitis B virus pre-S2 mutant stimulates aerobic glycolysis through mammalian target of rapamycin signaling in tumorigenesis.

Abstract Hepatitis B virus (HBV) pre-S2 mutant identified in ground glass hepatocytes (GGHs) in patients with chronic HBV infection has been proposed as viral oncoprotein and can induce hepatocellular carcinoma (HCC). Previously, we observed the activation of mammalian target of rapamycin (mTOR) by HBV pre-S2 mutant in GGHs and HCCs. The frequent association of metabolic syndrome with HCC raises the possibility that pre-S2 mutant-induced mTOR activation plays a driving role in the development of metabolic disorders in HBV-related HCC patients. To address this issue, we developed transgenic mice harboring pre-S2 mutant and analyzed gene expression profiles of the transgenic livers. In support of our hypothesis, the pre-S2 mutant transgenic mice showed glycogen depletion and glycolysis elevation in HCCs, a phenotype that has been implicated in highly proliferative malignancy. We identified that pre-S2 mutant could initiate an mTOR-dependent glycolytic pathway, involving Yin Yang 1, c-Myc, and glucose transporter 1 to upregulate aerobic glycolysis. This mTOR-mediated signaling pathway was significantly activated in the pre-S2 mutant transgenic livers and HCCs and contributed to aberrant glucose uptake and lactate production in vitro. Furthermore, we demonstrated that pre-S2 mutant could promote proliferation and invasion of HCC cells by sustaining the aerobic glycolysis. Activation of this glycolysis-associated mTOR signaling cascade was also observed in human HBV-related HCCs. Collectively, our results support the role of mTOR activation in the metabolic reprogramming of hepatocytes in chronic HBV infection and tumorigenesis, which should be valuable for chemoprevention design for HCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A168. Citation Format: Chiao-Fang Teng, Han-Chieh Wu, Wen-Chuan Hsieh, Hung-Wen Tsai, Ih-Jen Su. Hepatitis B virus pre-S2 mutant stimulates aerobic glycolysis through mammalian target of rapamycin signaling in tumorigenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A168.

Abstract 4097: Targeting of mTOR mediated signaling pathways in papillary thyroid cancer cell.

Abstract The mammalian target rapamycin (mTOR) signaling cascade is a key regulatory pathway controlling initiation of mRNA in mammalian cells. Torin2 is a potent, selective and orally available mTOR inhibitor for treatment of cancer. Dysregulation of mTOR signaling has been found in many cancers, however its role Papillary Thyroid Cancer (PTC) has not been explained. Therefore in this study, we investigated the role of mTOR and its associated molecules in PTC cell lines using MTT, flow cytometry and DNA fragmentation assays and western blotting. Our data showed that Torin2 caused a dose dependent growth inhibition in all cell lines studied. Cell cycle assay showed that the sub-G1 population in TPC-1 cell increased from 12.44% in untreated control sample to 31.50% after 200nM of Torin2 treatment and 44.08% after 400nM of Torin2 treatment for 48 hours. Similarly BCPAP cells increased from 8.47% in untreated control sample to 33.54% after 200nM of Torin2 treatment and 41.16% after 400nM of Torin2 treatment for 48 hours. Further confirmation using FITC-conjugated Annexin V and PI assay showed that apoptotic cell increased from 8.89% in untreated cells to 58.54% and 73.54% after 200nM and 400nM treatment in TPC-1 cells, and from 13.24% in control to 46.74% and 70.64% in BCPAP cells, indicating a dose dependent apoptosis induced in both cell lines. Treatment of PTC cell lines with Torin2 inactivated downstream targets of mTOR; p70S6 and 4E-BP1. Torin2 treatment of PTC cell lines also induced mitochondrial dependent apoptosis via activation and cleavage of caspase-9, and 3, and cleavage of PARP. Furthermore Torin2 positively regulated autophagy by disrupting the Beclin-1 complex, releasing Beclin-1 to induce autophagy. Our results suggest mTOR and its associated signaling pathway play a critical role in PTC tumorigenesis, therefore targeting of mTOR expression may be a viable therapeutic intervention for treatment of these cancers. Citation Format: Maqbool Ahmed, Azhar R. Hussain, Fouad Al Dayel, Shahab Uddin, Khawla S. Al-Kuraya. Targeting of mTOR mediated signaling pathways in papillary thyroid cancer cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4097. doi:10.1158/1538-7445.AM2013-4097

Abstract 4354: Dihydroartemisinin inhibits mTORC1 signaling in tumor cells.

Abstract Dihydroartemisinin (DHA) is a derivative of artemisinin, a natural product isolated from the plant, Artemisia annua. DHA is also the active metabolite of first-generation artemisinin derivatives (artemisinin, artesunate, artemether, etc.). Artemisinins have been clinically used to treat malaria for decades. Recent studies have demonstrated that artemisinin and its derivatives possess anticancer activity. This study was set to investigate the anticancer mechanism of DHA. We found that DHA inhibited proliferation in a spectrum of tumor cells, by arresting cells in G1/G0 and G2/M phases, which was linked to downregulation of cyclin D1, CDK4 and CDC25A, as well as CDK1 and CDC25C expression, respectively. We also observed that DHA induced caspase-dependent apoptosis, which was related to downregulation of survivin, Mcl-1 and Bcl2. As the mammalian target of rapamycin (mTOR) is a master kinase that regulates cell proliferation and survival, we further tested whether DHA inhibits mTOR signaling. Interestingly, we observed that DHA inhibited phosphoryaltion of mTOR and its downstream effector molecules, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), in a concentration- and time-dependent manner, but did not obviously affect mTORC2-mediated phosphorylation of Akt, which is in contrast to the conventional mTORC1 inhibitor, rapamycin. The results suggest that DHA may represent a novel class of mTORC1 inhibitor, and may execute its anticancer activity primarily by blocking mTOR-mediated signaling pathways in the tumor cells. Citation Format: Yoshinobu Odaka, Baoshan Xu, Shile Huang. Dihydroartemisinin inhibits mTORC1 signaling in tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4354. doi:10.1158/1538-7445.AM2013-4354

Calcium Signaling Is Involved in Cadmium-Induced Neuronal Apoptosis via Induction of Reactive Oxygen Species and Activation of MAPK/mTOR Network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+]i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+]i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+]i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+]i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+]i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Cardiac Restricted Overexpression of Kinase-dead Mammalian Target of Rapamycin (mTOR) Mutant Impairs the mTOR-mediated Signaling and Cardiac Function

Mammalian target of rapamycin (mTOR) is a key regulator for cell growth through modulating components of the translation machinery. Previously, numerous pharmacological studies using rapamycin suggested that mTOR has an important role in regulating cardiac hypertrophic growth. To further investigate this assumption, we have generated two lines of cardiac specific mTOR transgenic mice, kinase-dead (kd) mTOR and constitutively active (ca) mTOR, using alpha-myosin heavy chain promoter. alpha-Myosin heavy chain (alphaMHC)-mTORkd mice had a near complete inhibition of p70 S6k and 4E-BP1 phosphorylation, whereas alphaMHC-mTORca had a significant increase in p70 S6k and 4E-BP1 phosphorylation. Although the cardiac function of alphaMHC-mTORkd mice was significantly altered, the cardiac morphology of these transgenic mice was normal. The cardiac hypertrophic growth in response to physiological and pathological stimuli was not different in alphaMHC-mTORkd and alphaMHC-mTORca transgenic mice when compared with that of nontransgenic littermates. These findings suggest that the mTOR-mediated signaling pathway is not essential to cardiac hypertrophic growth but is involved in regulating cardiac function. Additional analysis of cardiac responses to fasting-refeeding or acute insulin administration indicated that alphaMHC-mTORkd mice had a largely impaired physiological response to nutrient energy supply and insulin stimulation.

Curcumin inhibits the mammalian target of rapamycin‐mediated signaling pathways in cancer cells

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Here we show that curcumin inhibited growth of rhabdomyosarcoma cells (Rh1 and Rh30) (IC50 = 2-5 microM) and arrested cells in G1 phase of the cell cycle. Curcumin also induced apoptosis and inhibited the basal or type I insulin-like growth factor-induced motility of the cells. At physiological concentrations (2.5 microM), curcumin rapidly inhibited phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream effector molecules, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), in a panel of cell lines (Rh1, Rh30, DU145, MCF-7 and Hela). Curcumin also inhibited phosphorylation of Akt in the cells, but only at high concentrations (>40 microM). The data suggest that curcumin may execute its anticancer activity primarily by blocking mTOR-mediated signaling pathways in the tumor cells.