Suppression Of mTOR Signaling Pathway Research Articles

Resveratrol attenuates staphylococcal enterotoxin B-activated immune cell metabolism via upregulation of miR-100 and suppression of mTOR signaling pathway.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of insults, such as bacterial and viral infections, including SARS-CoV-2, leading to high mortality. In the murine model of ARDS induced by Staphylococcal enterotoxin-B (SEB), our previous studies showed that while SEB triggered 100% mortality, treatment with Resveratrol (RES) completely prevented such mortality by attenuating inflammation in the lungs. In the current study, we investigated the metabolic profile of SEB-activated immune cells in the lungs following treatment with RES. RES-treated mice had higher expression of miR-100 in the lung mononuclear cells (MNCs), which targeted mTOR, leading to its decreased expression. Also, Single-cell RNA-seq (scRNA seq) unveiled the decreased expression of mTOR in a variety of immune cells in the lungs. There was also an increase in glycolytic and mitochondrial respiration in the cells from SEB + VEH group in comparison with SEB + RES group. Together these data suggested that RES alters the metabolic reprogramming of SEB-activated immune cells, through suppression of mTOR activation and its down- and upstream effects on energy metabolism. Also, miR-100 could serve as novel potential therapeutic molecule in the amelioration of ARDS.

Glutathione peroxidase 4 inhibition induces ferroptosis and mTOR pathway suppression in thyroid cancer

Papillary thyroid carcinoma (PTC) demonstrates significantly reduced patient survival with metastatic progression. Tumor progression can be influenced by metabolism, including antioxidant glutathione (GSH). Glutathione peroxidase 4 (GPX4) is a selenoenzyme that uses GSH as a co-factor to regulate lipid peroxidation of cell membranes during increased oxidative stress. GPX4 suppression in tumor cells can induce ferroptosis. This study aims to examine ferroptosis as a potentially critical pathway in effective targeting of thyroid cancer (TC) cells. We treated human TC cells (K1, MDA-T68, MDA-T32, TPC1) with (1S,3R)-RSL3 (RSL3), a small-molecule inhibitor of GPX4 and examined the effects on ferroptosis, tumor cell survival and migration, spheroid formation, oxidative stress, DNA damage repair response, and mTOR signaling pathway in vitro. GPX4 inhibition activated ferroptosis, inducing TC cell death, rapid rise in reactive oxygen species and effectively arrested cell migration in vitro. Suppression of mTOR signaling pathway triggered autophagy. GPX4 genetic knockdown mirrored RSL3 effect on mTOR pathway suppression. RSL3 subdued DNA damage repair response by suppressing phosphorylation of nucleophosmin 1 (NPM1). Thus, observed potent induction of ferroptosis, GPX4-dependent novel suppression of mTOR pathway and DNA damage repair response in preclinical in vitro model of TC supports GPX4 targeting for therapeutic benefit in advanced therapy-resistant thyroid cancers.

Theobromine ameliorates nonalcoholic fatty liver disease by regulating hepatic lipid metabolism via mTOR signaling pathway in vivo and in vitro.

Theobromine, a methylxanthine present in cocoa, has been shown to possess many beneficial pharmacological properties such as anti-oxidative stress, anti-inflammatory property, and anti-microbial activity. In this study, we investigated the effects of theobromine on nonalcoholic fatty liver disease (NAFLD) and the possible underlying mechanisms in vivo and in vitro. The results showed that theobromine reduced body weight and fat mass and improved dyslipidemia. Theobromine mitigated liver injury and significantly reduced hepatic triglyceride level in mice with obesity. Histological examinations also showed hepatic steatosis was alleviated after theobromine treatment. Furthermore, theobromine reversed the elevated mRNA and protein expression of SREBP-1c, FASN, CD36, FABP4, and the suppressed expression of PPARα and CPT1a in the liver of mice with obesity, which were responsible for lipogenesis, fatty acid uptake, and fatty acid oxidation respectively. In vitro, theobromine also downregulated SREBP-1c, FASN, CD36, FABP4 and upregulated PPARα and CPT1a mRNA and protein levels in hepatocytes in a dose-dependent manner, while these changes were reversed by L-leucine, a mammalian target of rapamycin (mTOR) agonist. The present study demonstrated that theobromine improved NAFLD by inhibiting lipogenesis and fatty acid uptake and promoting fatty acid oxidation in the liver and hepatocytes, which might be associated with its suppression of mTOR signaling pathway. Novelty: Theobromine protects against high-fat diet - induced NAFLD. Theobromine inhibits lipogenesis and fatty acid uptake and promotes fatty acid oxidation in the liver and hepatocytes via inhibiting mTOR signaling pathway.

1β–OH–arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway

Ethnopharmacological relevanceChansu, dried secretions from Bufonidae, has long been used for cancer treatment as a traditional Chinese medicine. In searching for effective anti-hepatoma agents from Chansu, our preliminary drug screening found that a bufadienolide, namely 1β-hydroxyl-arenobufagin (1β–OH–ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1β–OH–ABF have not been defined. Aim of the studyTo evaluate the anti-hepatoma activity of 1β–OH–ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo, as well as explore the underlying mechanisms. Materials and methodsThe anti-proliferative effects of 1β–OH–ABF on liver cancer Hep3B, HepG2, HuH7, SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1β–OH–ABF. The collapse of the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining assay. Western blotting was used to examine the expression levels of targeted proteins. The role of mTOR in 1β–OH–ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1β–OH–ABF in vivo. ResultsWe found that 1β–OH–ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 1β–OH–ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway, which is accompanied by the loss of ΔΨm, upregulation and translocation of Bax, as well as cleavages of caspase-9, caspase-3 and PARP. Mechanistically, 1β–OH–ABF markedly decreases the expression level of p-AKT/AKT and p-mTOR (Ser2248 and Ser2481)/mTOR in a time-dependent manner. Inhibition of mTOR by siRNA strengthens 1β–OH–ABF-mediated apoptosis. Critically, 1β–OH–ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. Conclusion1β–OH–ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1β–OH–ABF may serve as a potential agent for the treatment of liver cancer.

Suppression of mTOR signaling pathway promotes bone marrow mesenchymal stem cells differentiation into osteoblast in degenerative scoliosis: in vivo and in vitro.

To investigate the role of mTOR signaling pathway in bone marrow mesenchymal stem cells (BMSCs) differentiation into osteoblast in degenerative scoliosis (DS). The rat model of DS was established. Thirty-two Sprague-Dawley (SD) rats were selected and divided into the normal control group, the positive control group (normal rats injected with rapamycin), the negative control group (DS rats injected with PBS) and the experiment group (DS rats injected with rapamycin). H&E staining was performed to observe the osteogenesis of scoliosis. The BMSCs were obtained and assigned into seven groups: the normal control group, the positive control group, the negative control group and 1.0/10.0/100.0/1000.0nmol/L experiment groups. Flow cytometry was conducted to testify cell cycle. The mRNA and protein expressions of mTOR and osteoblastic differentiation markers were measured by qRT-PCR and western blotting. In vivo, compared with the negative control group, bone trabecular area and the number of differentiated bone cells were significantly increased in the experiment groups. In vitro, at 24 and 48h after rapamycin treatment, compared with the negative control group, BMSCs at G0/G1 stage increased, but BMSCs at S stage decreased in the 1.0/10.0/100.0/1000.0nmol/L experiment groups; the expressions of mTOR and p70-S6K1 proteins were reduced in the 1.0/10.0/100.0/1000.0nmol/L experiment groups, while ALP activity, OC levels, calcium deposition, Co1-I protein expression and the mRNA expressions of OC and Co1-I were significantly increased. Suppression of mTOR signaling pathway by rapamycin could promote BMSCs differentiation into osteoblast in DS.

1, 25(OH)2D3 protects β cell against high glucose-induced apoptosis through mTOR suppressing

Diabetes mellitus is a leading cause of death and disability worldwide, which presents a serious public health crisis in China nowadays. It has been well recognized that excessive β-cell apoptosis is the key pathogenesis of diabetes, of which the mammalian target of rapamycin (mTOR) serves as the critical signaling pathway. Emerging evidence indicates that vitamin D deficiency acts as a potential risk factor for diabetes. The present study aims to test the hypothesis that 1 alpha, 25-dihydroxyvitamin D(3) [1, 25(OH)2D3] can inhibit β-cell apoptosis via the suppression of mTOR signaling pathway. β-cells (INS-1) were cultured in the context of normal glucose or high glucose media with or without 1, 25(OH)2D3 treatment. β-cell apoptosis was evaluated by inverted fluorescence microscope, flow cytometry and electron microscope, respectively. Quantitative RT-PCR and Western blotting were performed to assess the possible perturbations in mTOR signaling pathway. High glucose significantly increased β-cell apoptosis. Of importance, RT-PCR and Western blotting demonstrated that high glucose inhibited DNA-damage-inducible transcript 4 (DDIT4) and TSC1/TSC2, up-regulated Rheb/mTOR/p70S6K and enhanced expression of the apoptosis regulating proteins, such as phospho-Bcl-2, cytochrome C and cleaved caspase. Interestingly, 1, 25(OH)2D3 treatment reversed high glucose induced pathological changes in mTOR signaling pathway, restored expression of DDIT4 and TSC1/TSC2, blocked aberrant up-regulation of Rheb/mTOR/p70S6K and the apoptosis regulating proteins, and effectively inhibited β-cell apoptosis. Therefore, 1, 25(OH)2D3 treatment can effectively protects β cell against high glucose-induced apoptosis mainly via the suppression of mTOR signaling pathway, which may be considered as a potential therapy for patients with diabetes.

Abstract A18: Geranylgeranoic acid (GGA) induced autophagic cell death through induction of unfolded protein response (UPR) and suppression of mTOR signaling pathway

Abstract Background and Aim: Geranylgeranoic acid (GGA) is a natural polyprenoic acid that is expected to prevent a second primary hepatoma. In human hepatoma-derived HuH-7 cells, GGA induces cell death as well as the initial phase of autophagy, but blocks the maturation process of autolysosomes, which may be linked to cell death. However we have not yet revealed the molecular mechanism of GGA-induced initial autophagy. In this meeting, we present a possible mechanism of GGA-induced initial autophagy, which may involve mTOR signaling pathway and unfolded protein response (UPR). Methods: To analyze the cellular process of autophagy, we observed multicolor fluorescent puncta by live-cell imaging in HuH-7/mRFP-GFP-LC3 cells after GGA (10 μM) treatment. We also examined cellular protein levels of phospho-mTOR (Ser2448 or Ser2481), mTOR, Raptor, Rictor, phospho-AKT, Beclin1, LC3β and FoxO1 by western blotting. The cellular levels of XBP1 mRNA splicing, CHOP and PDIA4 mRNAs were measured by RT-qPCR to estimate UPR. Results: In HuH-7 cells, phospho-mTOR (Ser2448) protein expression level was high under normal culture condition. GGA treatment further upregulated phospho-mTOR (Ser2448) and induced a transient surge of phospho-mTOR (Ser2481) at 30 min. However from 1 h to 8 h after GGA treatment, phosphorylation of mTOR was suppressed down to sub-basal levels. The similar up-and-down movements were observed also in the cellular levels of mTOR, Raptor and Rictor, and at 4 h these proteins became undetectable. However phospho-AKT levels did not show significant change after GGA treatment. Rapid and dramatic induction of XBP1 splicing was observed after GGA treatment. In other words, after 15 min of GGA treatment, the upregulation of XBP1s mRNA (spliced form) was observed and the upregulation continued until 4 h. In contrast, XBP1u mRNA (unspliced form) was downregulated in a time dependent manner until 4 h. However at 24 h, these changes were reversed and XBP1u became dominant. CHOP mRNA was upregulated after 1 h GGA treatment and the upregulation continued to 4 h. The cellular level of PDIA4 mRNA was not changed by GGA. The cellular protein levels of Beclin1 and FoxO1 were upregulated by GGA treatment. The accumulations of LC3β-II and autophagosome were also induced with GGA. GGA-induced accumulation of LC3β-II was partially attenuated by co-treatment with 4μ8C, a specific inhibitor of IRE1 ribonuclease activity. Discussion: In the literature, it has been repeatedly reported that signaling pathway of mTOR is activated at a basal level in HuH-7 cells. Therefore, we were interested what would happen on mTOR signaling when GGA induced initial autophagy in HuH-7 cells. As a result, signaling pathway of mTOR, either mTORC1 or mTORC2, was unexpectedly further upregulated by GGA treatment, but this upregulation at 30 min was transient. After 1-h treatment, GGA induced rapid unfolded protein response (UPR), which may trigger suppression of mTOR signaling. Hence, we speculate that UPR-mediated suppression of mTOR signaling may provide permissive effects on GGA-induced initial autophagy. Citation Format: Chieko Iwao, Yoshihiro Shidoji. Geranylgeranoic acid (GGA) induced autophagic cell death through induction of unfolded protein response (UPR) and suppression of mTOR signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A18.

Fisetin induces autophagic cell death through suppression of mTOR signaling pathway in prostate cancer cells

The mammalian target of rapamycin (mTOR) kinase is an important component of PTEN/PI3K/Akt signaling pathway, which is frequently deregulated in prostate cancer (CaP). Recent studies suggest that targeting PTEN/PI3K/Akt and mTOR signaling pathway could be an effective strategy for the treatment of hormone refractory CaP. Here, we show that the treatment of androgen-independent and PTEN-negative human CaP PC3 cells with fisetin, a dietary flavonoid, resulted in inhibition of mTOR kinase signaling pathway. Treatment of cells with fisetin inhibited mTOR activity and downregulated Raptor, Rictor, PRAS40 and GbetaL that resulted in loss of mTOR complexes (mTORC)1/2 formation. Fisetin also activated the mTOR repressor TSC2 through inhibition of Akt and activation of AMPK. Fisetin-mediated inhibition of mTOR resulted in hypophosphorylation of 4EBP1 and suppression of Cap-dependent translation. We also found that fisetin treatment leads to induction of autophagic-programmed cell death rather than cytoprotective autophagy as shown by small interfering RNA Beclin1-knockdown and autophagy inhibitor. Taken together, we provide evidence that fisetin functions as a dual inhibitor of mTORC1/2 signaling leading to inhibition of Cap-dependent translation and induction of autophagic cell death in PC3 cells. These results suggest that fisetin could be a useful chemotherapeutic agent in treatment of hormone refractory CaP.