Autophagy as a novel insight into mechanism of Danqi pill against post-acute myocardial infarction heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Danqi Pill, composed of the root of Salvia miltiorrhiza Bunge and the root of Panax notoginseng, is effective in the clinical treatment of myocardial ischemia in coronary heart diseases. A number of studies have shown that autophagy plays an essential role in cardiac function and energy metabolism, and disordered autophagy is associated with the progression of heart failure. However, the effect and mechanism of Danqi pill on autophagy have not been reported yet. AIM OF THE STUDY: This study aims to elucidate whether Danqi pill restores autophagy to protect against HF and its potential mechanism. MATERIALS AND METHODS: Left anterior descending ligation was established to induce an HF rat model, H2O2-stimulated H9C2 cells model was conducted to clarify the effects and potential mechanism of Danqi pill. In vivo, Danqi pill (1.5 g/kg) were orally administered for four weeks and Fenofibrate (10 mg/kg) was selected as a positive group. In vitro, Danqi pill (10-200 µg/mL) was pre-cultured for 24 h and co-cultured with H2O2 stimulation for 4 h. Importantly, transmission electron microscopy and fluorescence GPF-mRFP-LC3 reporter system were combined to monitor autophagy flux. Furtherly, we utilized Compound C, a specific AMPK inhibitor, to validate whether the autophagy was mediated by AMPK-TSC2-mTOR pathway. RESULTS: Danqi pill significantly improved cardiac function and myocardial injury in HF rats. Intriguingly, Danqi pill potently regulated autophagy mainly by promoting the formation of autophagosomes in vivo. Further results demonstrated that expressions of p-AMPK (P < 0.001) and p-TSC2 (P < 0.001) in cardiac tissue were upregulated by Danqi pill, accompanied with downregulation of p-mTOR (P < 0.01) and p-ULK1(P < 0.01). In parallel with the vivo experiment, in vitro study indicated that Danqi pill dramatically restored autophagy flux and regulated expressions of critical autophagy-related molecules. Finally, utilization of Compound C abrogated the effects of Danqi pill on autophagy flux and the expressions of p-TSC2 (P < 0.05), p-mTOR (P < 0.01) and p-ULK1 (P < 0.05). CONCLUSION: Danqi pill could improve cardiac function and protect against cardiomyocytes injury by restoring autophagy via regulating the AMPK-TSC2-mTOR signaling pathway.

Pharmacoproteomics reveals the mechanism of Chinese dragon's blood in regulating the RSK/TSC2/mTOR/ribosome pathway in alleviation of DSS-induced acute ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese dragon's blood (CDB), a crude drug extracted from Dracaena cochinchinensis (Lour.) S.C. Chen, has been historically applied for the treatment of various diseases, including ulcerative colitis (UC). Unfortunately, the underlying molecular mechanism remains unclear. MATERIALS AND METHODS: In this paper, the effects of CDB treatment on a mouse model of acute UC and proteomic variation in colonic tissue were investigated. The acute UC model in Balb/c mice was induced by administration of 2.5% (wt/vol) dextran sulfate sodium (DSS) in drinking water for 8 days. After the mice with UC were intragastrically administered CDB and intraperitoneally injected with rapamycin (RAPA, a specific inhibitor of mTORC1), the disease activity index (DAI) and histopathological score were recorded. An isobaric tags for relative and absolute quantification (iTRAQ) based LC-MS/MS proteomic technique was adopted to identify the differentially expressed proteins (DEPs) in colonic tissue. Bioinformatics analysis was used to discover the molecular functions and pathways of the DEPs. Finally, Western blot analysis and immunohistochemistry were used to verify the protein expression. RESULTS: The results showed that CDB treatment significantly ameliorated the symptoms and intestinal damage in acute UC, while RAPA treatment led to severe symptoms and intestinal damage. A total of 489 DEPs were reversed in the control check (CK) group and the CDB group. Most DEPs were enriched in the structural constituents of ribosomes and the ribosome pathway. CDB treatment significantly upregulated the expression of the mTOR, p-mTOR and p70S6K proteins and downregulated the expression of the Akt, p-Akt, and p4EBP1 proteins. However, RAPA treatment, unlike CDB, did not return the levels of mTOR, Akt, and their phosphorylated forms to nearly normal. CONCLUSIONS: In conclusion, the dysfunction of the mTOR/ribosome pathway resulting in the inhibition of ribosome synthesis played an important role in the development of acute UC in mice, and CDB, but not RAPA, was an alternative drug for the treatment of acute UC by enhancing ribosome synthesis via the mTOR/ribosome pathway and further promoting protein synthesis.

Electroacupuncture Upregulated Ghrelin in Rats with Functional Dyspepsia via AMPK/TSC2/Rheb-Mediated mTOR Inhibition.

BACKGROUND: Gastrointestinal motility disorder is an important pathological basis for functional dyspepsia (FD). Epigastric ache and discomfort are the main symptoms of FD, and ghrelin deficiency is closely related to the occurrence and development of FD. While electroacupuncture (EA) alleviated the symptoms of FD patients and improved their quality of life, there is a lack of sufficient mechanistic evidence to support these beneficial effects. METHODS: An in vivo FD model was established in wild-type and mammalian target of rapamycin (mTOR) knockout (-/-) rats. FD rats were subjected to EA with or without mTOR agonists or inhibitors. Gastric emptying and intestinal propulsion were assessed, and pathological changes in the hypothalamus, gastric antrum, and small intestine were examined histologically. In addition, ghrelin expression and AMPK/TSC2/Rheb/mTOR activation were detected by quantitative reverse transcription polymerase chain reaction and western blot. RESULTS: EA alone or in combination with mTOR inhibitors improved gastrointestinal function in FD rats by increasing the rates of intestinal propulsion and gastric emptying, and pathological changes in the hypothalamus, gastric antrum, and small intestine were alleviated. This may be related to the significant upregulation of ghrelin expression and the effective activation of the AMPK/TSC2/Rheb/mTOR signaling pathway. Interestingly, EA also improved gastrointestinal function and ghrelin expression in mTOR (-/-) KO FD rats. CONCLUSION: Altering the level of ghrelin by regulating AMPK/TSC2/Rheb-mediated mTOR inhibition is an important way through which EA treats FD. The complex EA-mediated regulatory mechanisms of the brain-gut axis still require further exploration.

Dexmedetomidine Protects Against Oxygen-Glucose Deprivation-Induced Injury Through Inducing Astrocytes Autophagy via TSC2/mTOR Pathway.

Although there is an increment in stroke burden in the world, stroke therapeutic strategies are still extremely limited to a minority of patients. We previously demonstrated that dexmedetomidine (DEX) protects against focal cerebral ischemia via inhibiting neurons autophagy. Nevertheless, the role of DEX in regulating astrocytes autophagic status in oxygen-glucose deprivation, a condition that mimics cerebral ischemia, is still unknown. In this study, we have shown that DEX and DEX + RAPA (autophagy inducer) increased viability and reduced apoptosis of primary astrocytes in oxygen-glucose deprivation (OGD) model compared with DEX + 3-methyladenine (3-MA) (autophagy inhibitor). DEX induced the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1, while reduced the expression of p62 in primary cultured astrocytes through induction of autophagy. In addition, DEX enhanced the expression of tuberous sclerosis complex 2 (TSC2) in primary cultured astrocytes, while reduced the expression of mammalian target of rapamycin (mTOR). In conclusion, our study suggests that DEX exerts a neuroprotection against OGD-induced astrocytes injury via activation of astrocytes autophagy by regulating the TSC2/mTOR signaling pathway, which provides a new insight into the mechanisms of DEX treatment for acute ischemic injury.

[Ginsenoside Rg_1 induces leukemia stem cell senescence via SIRT1/TSC_2 signal axis].

The aim of this paper was to investigate the effect of SIRT1/TSC_2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg_1. CD34~+CD38~- leukemia stem cells(CD34~+CD38~-LSCs) was isolated by magnetic cell sorting(MACS) and divided into two groups. The control group cells were routinely cultured, 40 µmol·L~(-1) ginsenoside Rg_1 was added to the control group for co-culture in Rg_1 group. The effect of Rg_l to induce CD34~+CD38~-LSCs senescence were evaluated by senescence-associated ß-Galactosidase(SA-ß-Gal) staining, cell cycle assay, CCK-8 and Colony-Assay. The expression of senescence associated SIRT1, TSC_2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR) and Western blot. The results showed that the CD34~+CD38~-LSCs could effectively be isolated by MACS, and the purity of CD34~+CD38~-LSCs is up to(95.86±3.04)%. Compared with the control group, the percentage of positive cells expressed SA-ß-Gal in the Rg_1 group is increased, the senescence morphological changes were observed in the CD34~+CD38~-LSCs in the Rg_1 group. The proliferation inhibition rate and the number of cells entered G_0/G_1 phase in the Rg_1 group were increased, but the colony-formed ability was decreased, Rg_1 could significantly inhibit the proliferation and self-renewal ability of CD34~+CD38~-LSCs. The expression of SIRT1 and TSC_2 mRNA and protein were down regulated in the Rg_1 group compared with the control group. Our research implied that Rg_1 may induce the senescence of CD34~+CD38~-LSCs and SIRT1/TSC_2 signal axis plays a significant role in this process.

Comparison of Aristolochic acid I derived DNA adduct levels in human renal toxicity models.

Aristolochic acid (AA) dependent human nephropathy results either from environmental exposure to Aristolochiaceae plant subspecies or their use in traditional phytotherapy. The toxic components are structurally related nitrophenanthrene carboxylic acids, i.e. Aristolochic acid I (AAI) and II (AAII). AAI is considered to be the major cause of Aristolochic acid nephropathy, characterized by severe renal fibrosis and upper urothelial cancer. Following enzymatic activation in kidney and/or liver, AAI metabolites react with genomic DNA to form persistent DNA adducts with purines. To determine whether AAI can be activated in human renal cells to form DNA adducts, we exposed telomerase immortalized renal proximal tubular epithelial cells (RPTEC/TERT1), the human embryonic kidney (HEK293) cell line, as well as primary human kidney cells (pHKC) to AAI in vitro. We modified an isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry (ID-UPLC-MS/MS) based method for the quantification of dA-AAI adducts in genomic DNA. In addition, time dependent accumulation of adducts in renal cortex and bladder tissue from AAI/II treated Eker rats were used to validate the detection method. AAI-induced toxicity in human renal cells was determined by dA-AAI adduct quantification, the impact on cell viability, and NQO1 expression and activity. Our findings demonstrated adduct formation in all cell lines, although only pHKC and RPTEC/TERT1 expressed NQO1. The highest adduct formation was detected in pHKC despite low NQO1 expression, while we observed much lower adduct levels in NQO1-negative HEK293 cells. Adduct formation and decreased cell viability correlated only weakly. Therefore, our data suggested that i.) enzymes other than NQO1 could be at least equally important for AA bioactivation in human renal proximal tubule cells, and ii.) the suggested correlation between adduct levels and viability appears to be questionable.

Potential therapeutic effect of curcumin, a natural mTOR inhibitor, in tuberous sclerosis complex.

BACKGROUND: Curcumin is a polyphenol natural product of the plant Curcuma longa. Recent studies suggest that curcumin inhibit mTOR activity in vitro, which prompts us to investigate curcumin function as a new class of mTOR inhibitor suitable for tuberous sclerosis complex (TSC) treatment. PURPOSE: We aim to investigate the efficacy of curcumin in the treatment of TSC related manifestations in animal model. STUDY DESIGN: Solid lipid curcumin particle (SLCP), a novel curcumin formulation, was used to treat TSC related manifestations in Tsc2 knockout mice. METHODS: The novel object recognition test was used to analyze the recognition memory function. The long-term potentiation was studied using electrophysiological analysis. Western blotting was used to assess the protein expression and activation status. RESULTS: Recognition memory deficit began as early as 4 weeks of age in both male and female Tsc2+/- mice. Oral administration with SLCP activates AMPK activity and inhibits mTOR activity in the brain tissue of Tsc2+/- mice, and can rescue the electrophysiological abnormality and object recognition memory loss in the mice. CONCLUSIONS: Our results suggest that SLCP could be an effective treatment for TSC patients.

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.

Calcitriol Reverses the Down-Regulation Pattern of Tuberous Sclerosis Complex Genes in an In Vitro Calcification Model.

Tuberous sclerosis complex (TSC) is a neurocutaneous syndrome with autosomal dominant inheritance, and most of the cases are related to loss of function of the TSC1 and TSC2 genes. TSC may occur with a wide range of clinical findings and skin, kidney, brain, and heart are the most commonly affected organs. Brain calcifications in TSC are also described and reported as diffuse and without pattern of symmetry or bilaterality. Recently, a new discovery opened the possibility of using vitamin D (VitD) for treating cerebral calcifications. Calcitriol, the active form of VitD, was able to reduce the calcification in an in vitro model, increasing expression of a gene related to primary familial brain calcification. We show that in the same experimental model, calcitriol was also able to restore and even increase expression of genes related to TSC. This article discusses the use of calcitriol supplementation in patients with TSC, which can be a very interesting strategy due to its low cost and because it is already used in various therapies.

Raspberry Ketone Reduced Lipid Accumulation in 3T3-L1 Cells and Ovariectomy-Induced Obesity in Wistar Rats by Regulating Autophagy Mechanisms.

This study aimed to determine the antiobesity effects of raspberry ketone (RK), one of the major aromatic compounds contained in raspberry, and its underlying mechanisms. During adipogenesis of 3T3-L1 cells, RK (300 µM) significantly reduced lipid accumulation and downregulated the expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferation-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), and fatty acid synthase (FAS). RK also reduced the expression of light chain 3B (LC3B), autophagy-related protein 12 (Atg12), sirtuin 1 (SIRT1), and phosphorylated-tuberous sclerosis complex 2 (TSC2), whereas it increased the level of p62 and phosphorylated-mammalian target of rapamycin (mTOR). Daily administration of RK decreased the body weight (ovariectomy [Ovx] + RK, 352.6 ± 5 vs Ovx, 386 ± 5.8 g; P < 0.05), fat mass (Ovx + RK, 3.2 ± 0.05 vs Ovx, 5.0 ± 0.4 g; P < 0.05), and fat cell size (Ovx + RK, 6.4 ± 0.6 vs Ovx, 11.1 ± 0.7 × 103 µm2; P < 0.05) in Ovx-induced obesity in rats. The expression of PPARγ, C/EBPα, FAS, and FABP4 was significantly reduced in the Ovx + RK group compared with that in the Ovx group. Similar patterns were observed in autophagy-related proteins and endoplasmic reticulum stress proteins. These results suggest that RK inhibited lipid accumulation by regulating autophagy in 3T3-L1 cells and Ovx-induced obese rats.

TSC2 Deficiency Unmasks a Novel Necrosis Pathway That Is Suppressed by the RIP1/RIP3/MLKL Signaling Cascade.

Tuberous sclerosis complex (TSC) is a genetic multiorgan disorder characterized by the development of neoplastic lesions in kidney, lung, brain, heart, and skin. It is caused by an inactivating mutation in tumor suppressor genes coding the TSC1/TSC2 complex, resulting in the hyperactivation of mTOR- and Raf/MEK/MAPK-dependent signaling that stimulates tumor cell proliferation and metastasis. Despite its oncogenic effect, cells with TSC deficiency were more sensitive to oxidative stress and dependent on mitochondrial metabolism, providing a rationale for a new therapeutic approach. The current study shows that simultaneous inhibition of two major pathways regulating redox homeostasis using l-buthionine-sulfoximine (BSO, glutathione synthesis inhibitor) and auranofin (thioredoxin reductase inhibitor) induces oxidative burst, mitochondrial damage, and necrotic cell death in TSC-deficient cells in a highly synergistic and cell context-specific manner. Furthermore, blocking RIP1/RIP3/MLKL-dependent signaling using chemical inhibitors necrostatin-1 (Nec-1) and necrosulfonamide (NSA) synergizes with BSO and auranofin in killing TSC-deficient cells. Expression analysis demonstrated that RIP1, RIP3, and MLKL protein levels are elevated in cells with TSC2 deficiency, and their inactivation enhances mitochondrial dysfunction in a glutaminolysis-dependent and autophagy-independent manner. Finally, supplementation with the mitochondrial metabolite α-ketoglutarate, whose synthesis is regulated by RIP1/RIP3/MLKL, rescues cells from the sensitizing effect of Nec-1 and NSA. Together, this study identifies a previously unrecognized novel regulated necrotic death pathway that involves mitochondrial homeostasis, is suppressed by the RIP1/RIP3/MLKL signaling in TSC-deficient cells, and could be a promising therapeutic target for TSC-associated tumors. Cancer Res; 76(24); 7130-9. ©2016 AACR.

Next-generation sequencing reveals somatic mutations that confer exceptional response to everolimus.

BACKGROUND: Given the modest responses to everolimus, a mTOR inhibitor, in multiple tumor types, there is a pressing need to identify predictive biomarkers for this drug. Using targeted ultra-deep sequencing, we aimed to explore genomic alterations that confer extreme sensitivity to everolimus. RESULTS: We collected formalin-fixed paraffin-embedded tumor/normal pairs from 39 patients (22 with exceptional clinical benefit, 17 with no clinical benefit) who were treated with everolimus across various tumor types (13 gastric cancers, 15 renal cell carcinomas, 2 thyroid cancers, 2 head and neck cancer, and 7 sarcomas). Ion AmpliSeqTM Comprehensive Cancer Panel was used to identify alterations across all exons of 409 target genes. Tumors were sequenced to a median coverage of 552x. Cancer genomes are characterized by 219 somatic single-nucleotide variants (181 missense, 9 nonsense, 7 splice-site) and 22 frameshift insertions/deletions, with a median of 2.1 mutations per Mb (0 to 12.4 mutations per Mb). Overall, genomic alterations with activating effect on mTOR signaling were identified in 10 of 22 (45%) patients with clinical benefit and these include MTOR, TSC1, TSC2, NF1, PIK3CA and PIK3CG mutations. Recurrently mutated genes in chromatin remodeling genes (BAP1; n = 2, 12%) and receptor tyrosine kinase signaling (FGFR4; n = 2, 12%) were noted only in patients without clinical benefit. CONCLUSIONS: Regardless of different cancer types, mTOR-pathway-activating mutations confer sensitivity to everolimus. Targeted sequencing of mTOR pathway genes facilitates identification of potential candidates for mTOR inhibitors.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Lycopene in the prevention of renal cell cancer in the TSC2 mutant Eker rat model.

Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80-85% of malignant renal tumors. Eker rat represents a unique animal model to study RCC since these rats develop spontaneous renal tumors and leiomyoma, which may be due to tuberous sclerosis 2 (TSC2) mutation resulting in the activation of the mammalian target of rapamycin (mTOR) pathway. This study examines the role of a lycopene-rich diet in the development of RCC in the TSC2 mutant Eker rat model. Ten-week old female Eker rats (n=90) were assigned in equal numbers to receive 0, 100 or 200mg/kg of lycopene as part of their daily diet. After 18 months the rats were sacrificed and the kidneys were removed. Immunohistochemical staining with antibodies against mTOR, phospho-S6 and EGFR were performed, as well as hematoxylin-eosin staining for histologic examination of the tumors. Tumors were counted and measured in individual kidneys. Presence of tumor decreased from 94% in control animals to 65% in the experimental group, but the difference was not statistically significant (P<0.12). However, mean numbers of renal carcinomas were statistically significantly decreased in the lycopene-treated rats (P<0.008) when compared to untreated controls. In the lycopene group, tumor numbers decreased (P<0.002) and the numbers tended to decrease linearly (P<0.003) as supplemental lycopene increased from 0 to 200. Control rats fed only basal diet had a greater length of tumors (23.98 mm) than rats fed lycopene supplement groups (12.90 mm and 11.07 mm) (P<0.05). Moreover tumor length decreased (P<0.02) and tumor length tended to decrease linearly (P<0.03) as supplemental lycopene increased from 0 to 200mg/kg. All tumors showed strong staining with antibodies against mTOR, phospho-S6 and EGFR. In conclusion, dietary supplementation with lycopene attenuates the development of renal cell cancers in the predisposed TSC2 mutant Eker rat model. These results suggest that lycopene may play a role in the prevention of RCC.

Prolonging the survival of Tsc2 conditional knockout mice by glutamine supplementation.

The genetic disease tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by loss of function mutations in either TSC1 (hamartin) or TSC2 (tuberin), which serve as negative regulators of mechanistic target of rapamycin complex 1 (mTORC1) activity. TSC patients exhibit developmental brain abnormalities and tuber formations that are associated with neuropsychological and neurocognitive impairments, seizures and premature death. Mechanistically, TSC1 and TSC2 loss of function mutations result in abnormally high mTORC1 activity. Thus, the development of a strategy to inhibit abnormally high mTORC1 activity may have therapeutic value in the treatment of TSC. mTORC1 is a master regulator of growth processes, and its activity can be reduced by withdrawal of growth factors, decreased energy availability, and by the immunosuppressant rapamycin. Recently, glutamine has been shown to alter mTORC1 activity in a TSC1-TSC2 independent manner in cells cultured under amino acid- and serum-deprived conditions. Since starvation culture conditions are not physiologically relevant, we examined if glutamine can regulate mTORC1 in non-deprived cells and in a murine model of TSC. Our results show that glutamine can reduce phosphorylation of S6 and S6 kinase, surrogate indicators of mTORC1 activity, in both deprived and non-deprived cells, although higher concentrations were required for non-deprived cultures. When administered orally to TSC2 knockout mice, glutamine reduced S6 phosphorylation in the brain and significantly prolonged their lifespan. Taken together, these results suggest that glutamine supplementation can be used as a potential treatment for TSC.

Ursolic acid inhibits leucine-stimulated mTORC1 signaling by suppressing mTOR localization to lysosome.

Ursolic acid (UA), a pentacyclic triterpenoid widely found in medicinal herbs and fruits, has been reported to possess a wide range of beneficial properties including anti-hyperglycemia, anti-obesity, and anti-cancer. However, the molecular mechanisms underlying the action of UA remain largely unknown. Here we show that UA inhibits leucine-induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in C2C12 myotubes. The UA-mediated inhibition of mTORC1 is independent of Akt, tuberous sclerosis complex 1/2 (TSC1/2), and Ras homolog enriched in brain (Rheb), suggesting that UA negatively regulates mTORC1 signaling by targeting at a site downstream of these mTOR regulators. UA treatment had no effect on the interaction between mTOR and its activator Raptor or inhibitor Deptor, but suppressed the binding of RagB to Raptor and inhibited leucine-induced mTOR lysosomal localization. Taken together, our study identifies UA as a direct negative regulator of the mTORC1 signaling pathway and suggests a novel mechanism by which UA exerts its beneficial function.

Statins in lymphangioleiomyomatosis. Simvastatin and atorvastatin induce differential effects on tuberous sclerosis complex 2-null cell growth and signaling.

Mutations of the tumor suppressor genes tuberous sclerosis complex (TSC)1 and TSC2 cause pulmonary lymphangioleiomyomatosis (LAM) and tuberous sclerosis (TS). Current rapamycin-based therapies for TS and LAM have a predominantly cytostatic effect, and disease progression resumes with therapy cessation. Evidence of RhoA GTPase activation in LAM-derived and human TSC2-null cells suggests that 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor statins can be used as potential adjuvant agents. The goal of this study was to determine which statin (simvastatin or atorvastatin) is more effective in suppressing TSC2-null cell growth and signaling. Simvastatin, but not atorvastatin, showed a concentration-dependent (0.5-10 µM) inhibitory effect on mouse TSC2-null and human LAM-derived cell growth. Treatment with 10 µM simvastatin induced dramatic disruption of TSC2-null cell monolayer and cell rounding; in contrast, few changes were observed in cells treated with the same concentration of atorvastatin. Combined treatment of rapamycin with simvastatin but not with atorvastatin showed a synergistic growth-inhibitory effect on TSC2-null cells. Simvastatin, but not atorvastatin, inhibited the activity of prosurvival serine-threonine kinase Akt and induced marked up-regulation of cleaved caspase-3, a marker of cell apoptosis. Simvastatin, but not atorvastatin, also induced concentration-dependent inhibition of p42/p44 Erk and mTORC1. Thus, our data show growth-inhibitory and proapoptotic effects of simvastatin on TSC2-null cells compared with atorvastatin. These findings have translational significance for combinatorial therapeutic strategies of simvastatin to inhibit TSC2-null cell survival in TS and LAM.

Novel mechanism of regulation of fibrosis in kidney tumor with tuberous sclerosis.

BACKGROUND: Deficiency in tuberin results in activation the mTOR pathway and leads to accumulation of cell matrix proteins. The mechanisms by which tuberin regulates fibrosis in kidney angiomyolipomas (AMLs) of tuberous sclerosis patients are not fully known. METHOD: In the present study, we investigated the potential role of tuberin/mTOR pathway in the regulation of cell fibrosis in AML cells and kidney tumor tissue from tuberous sclerosis complex (TSC) patients. RESULTS: AML cells treated with rapamycin shows a significant decrease in mRNA and protein expression as well as in promoter transcriptional activity of alpha-smooth muscle actin (α-SMA) compared to untreated cells. In addition, cells treated with rapamycin significantly decreased the protein expression of the transcription factor YY1. Rapamycin treatment also results in the redistribution of YY1 from the nucleus to cytoplasm in AML cells. Moreover, cells treated with rapamycin resulted in a significant reduce of binding of YY1 to the αSMA promoter element in nuclear extracts of AML cells. Kidney angiomyolipoma tissues from TSC patients showed lower levels of tuberin and higher levels of phospho-p70S6K that resulted in higher levels of mRNA and protein of αSMA expression compared to control kidney tissues. In addition, most of the α-SMA staining was identified in the smooth muscle cells of AML tissues. YY1 was also significantly increased in tumor tissue of AMLs compared to control kidney tissue suggesting that YY1 plays a major role in the regulation of αSMA. CONCLUSIONS: These data comprise the first report to provide one mechanism whereby rapamycin might inhibit the cell fibrosis in kidney tumor of TSC patients.

Faslodex inhibits estradiol-induced extracellular matrix dynamics and lung metastasis in a model of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.

A drug screening method based on the autophagy pathway and studies of the mechanism of evodiamine against influenza A virus.

In this research, we have established a drug screening method based on the autophagy signal pathway using the bimolecular fluorescence complementation-fluorescence resonance energy transfer (BiFC-FRET) technique to develop novel anti-influenza A virus (IAV) drugs. We selected Evodia rutaecarpa Benth out of 83 examples of traditional Chinese medicine and explored the mechanisms of evodiamine, the major active component of Evodia rutaecarpa Benth, on anti-IAV activity. Our results showed that evodiamine could significantly inhibit IAV replication, as determined by a plaque inhibition assay, an IAV vRNA promoter luciferase reporter assay and the Sulforhodamine B method using cytopathic effect (CPE) reduction. Additionally, evodiamine could significantly inhibit the accumulation of LC3-II and p62, and the dot-like aggregation of EGFP-LC3. This compound also inhibited the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-α, IL-1ß, IL-6 and IL-8 after IAV infection. Evodiamine inhibited IAV-induced autophagy was also dependent on its action on the AMPK/TSC2/mTOR signal pathway. In conclusion, we have established a new drug screening method, and selected evodiamine as a promising anti-IAV compound.