Shenmai Injection Alleviates Myocardial Ferroptosis via Activating the AKT1/mTOR Pathway in Rats with Acute Myocardial Infarction.

OBJECTIVE: Acute myocardial infarction (AMI) poses a serious burden on public health. Shenmai Injection (SMI) has been reported to have a cardioprotective effect and is used clinically attributed to its targeting of ferroptosis. This study aims to explore the underlying mechanisms of SMI in treating AMI through the application of network pharmacology analysis. METHODS: This study utilized network pharmacology to identify the bioactive ingredients and potential targets of SMI in treating AMI. A rat model of AMI was created by ligating the coronary arteries of rats, and a cell model was established by subjecting H9c2 cells to oxygen-glucose deprivation (OGD) to reveal the cardioprotective effects of SMI. Western blotting was employed to measure protein expressions, while hematoxylin-eosin staining was used to observe relevant pathological changes. Enzyme linked immunosorbent assay was conducted to measure the levels of biomarkers associated with cardiac injury and oxidative stress. RESULTS: A comprehensive analysis revealed a total of 225 putative targets of SMI in the context of AMI which exerted regulatory effects on numerous pathways and targeted multiple biological processes. AKT1 was identified as a core target mediating the effects of SMI on AMI by topological analysis. In vivo experiments revealed that SMI attenuated myocardial injury, oxidative stress, and ferroptosis in rats with AMI. Furthermore, SMI was found to enhance the expression levels of p-AKT1 and p-mTOR proteins in the myocardial tissues of rats afflicted with AMI. Similar findings were also observed in H9c2 cells subjected to OGD. Of particular interest, the suppression of OGD-induced iron accumulation, oxidative stress, and ferroptosis-associated proteins by SMI in H9c2 cells was reversed upon inhibition of the AKT1/mTOR pathway via MK2206. CONCLUSION: This study revealed that SMI exerts a protective effect against myocardial injury and ferroptosis caused by AMI via the activation of the AKT1/mTOR pathway.

Emodin activates autophagy to suppress oxidative stress and pyroptosis via mTOR-ULK1 signaling pathway and promotes multi-territory perforator flap survival.

BACKGROUND: Multi-territory perforator flap reconstruction has been proven effective in treating large skin and soft tissue defects in clinical settings. However, in view of that the multi-territory perforator flap is prone to partial postoperative necrosis, increasing its survival is the key to the success of reconstruction. In this study, we aimed to clarify the effect of emodin on multi-territory perforator flap survival. METHODS: Flap survival was assessed by viability area analysis, infrared laser imaging detector, HE staining, immunohistochemistry, and angiography. Western blotting, immunofluorescence assays, and real-time fluorescent quantitative PCR were performed to detect the indicators of oxidative stress, pyroptosis and autophagy. RESULTS: After emodin treatment, the multi-territory perforator flap showed a significantly increased survival rate, which was shown to be closely related to the inhibition of oxidative stress and pyroptosis and enhanced autophagy. Meanwhile, the use of autophagy inhibitor 3 MA was found to reverse the inhibitory effects of emodin on oxidative stress and pyroptosis and weaken the improving effect of emodin on flap survival, suggesting that autophagy plays a critical role in emodin-treated flaps. Interestingly, our mechanistic investigations revealed that the positive effect of emodin on multi-territory perforator flap was attributed to the mTOR-ULK1 signaling pathway activation. CONCLUSIONS: Emodin can inhibit oxidative stress and pyroptosis by activating autophagy via the mTOR-ULK1 pathway, thereby improving the multi-territory perforator flap survival.

DT-13 inhibits the proliferation of pancreatic cancer by inducing apoptosis via AMPK-mTOR signaling.

BACKGROUND/OBJECTIVE: DT-13, the principal active component of Mysidium shortscapes from the Liliaceae family, has garnered substantial interest in cancer therapy owing to its potential anticancer properties. This study investigated the effects of DT-13 on the proliferation and apoptosis of human pancreatic cancer cell lines and aimed to elucidate the underlying mechanisms. METHODS: PANC1 and CFPAC1 cells were exposed to DT-13 and their proliferation was assessed using RTCA and clone formation assays. Apoptotic protein expression was analyzed by western blotting, and apoptotic cells were identified by flow cytometry. RNA was extracted from DT-13 treated and untreated PANC1 cells for RNA sequencing. Differentially expressed genes were identified and subjected to GO bioprocess, KEGG pathway analysis, and western blotting. Finally, to evaluate tumor growth, CFPAC1 cells were subcutaneously injected into BALB/c nude mice. RESULTS: DT-13 inhibited proliferation and induced apoptosis of PANC1 and CFPAC1 cells by activating the AMPK/mTOR pathway and suppressing p70 S6K. Moreover, DT-13 hindered the growth of CFPAC1 xenograft tumors in nude mice. CONCLUSIONS: DT-13 effectively inhibited the growth of human pancreatic cancer cells.

Rapamycin promotes hematoma resorption and enhances endothelial cell function by suppressing the mTOR/STAT3 signaling in chronic subdural hematoma.

Chronic subdural hematoma (CSDH) remains a neurosurgical condition and a healthy burden especially in elderly patients. This study focuses on the functions of rapamycin and its related molecular mechanisms in CSDH management. A rat model of CSDH was induced, which developed significant hematoma on day 5 after operation. The rats were treated with rapamycin or atorvastatin, a drug with known effect on hematoma alleviation, or treated with rapamycin and atorvastatin in combination. The atorvastatin or rapamycin treatment reduced the hematoma development, blood-brain barrier permeability, neurological dysfunction in CSDH rats, and the combination treatment showed more pronounced effects. Human brain microvascular endothelial cells hCMEC/D3 were stimulated by hematoma samples to mimic a CSDH condition in vitro. The drug treatments elevated the cell junction-related factors and reduced the pro-inflammatory cytokines both in rat hematoma tissues and in hCMEC/D3 cells. Rapamycin suppressed the mTOR and STAT3 signaling pathways. Overexpression of mTOR or the STAT3 agonist suppressed the alleviating effects of rapamycin on CSDH. In summary, this study demonstrates that rapamycin promotes hematoma resorption and enhances endothelial cell function by suppressing the mTOR/STAT3 signaling.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

Schaftoside improves cerebral ischemia-reperfusion injury by enhancing autophagy and reducing apoptosis and inflammation through the AMPK/mTOR pathway.

BACKGROUND: As a flavonoid compound, schaftoside (SS) possesses a wide range of pharmaceutical activities. Nonetheless, it is unclear whether SS has a neuroprotective effect in cerebral ischemia-reperfusion injury (CI/RI). OBJECTIVES: To examine the neuroprotective effect of SS in CI/RI and explore the underlying mechanism. MATERIAL AND METHODS: An in vivo middle cerebral artery occlusion (MCAO) was used to simulate CI/RI in rats. Oxygen glucose deprivation/reperfusion (OGD/R) of HT22 cells was used to establish a cellular model of CI/RI in vitro. Pathological changes were evaluated with hematoxylin and eosin (H&E) staining, apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry, and inflammatory factors were assessed using enzyme-linked immunosorbent assay (ELISA). Protein expression was detected using western blot or immunofluorescence. RESULTS: Our results indicated that SS attenuated CI/RI by improving neurologic deficits and reducing brain edema. Moreover, SS treatment blocked apoptosis and inflammation and enhanced autophagy in MCAO rats. Schaftoside was found to amplify the activation of adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway induced by MCAO. Similarly, SS pretreatment increased cell viability and autophagy, and reduced apoptosis and inflammation in OGD/R-induced HT22 cells. The OGD/R enlarges the p-AMPK/AMPK ratio while restricting the p-mTOR/mTOR ratio, and it was found that SS further enhanced the effect of OGD/R on the AMPK/mTOR pathway. Rapamycin promoted the effect of SS on OGD/R-induced HT22 cells, while compound C produced the opposite results. Mechanistically, SS promoted autophagy and reduced apoptosis and inflammation through the regulation of the AMPK/mTOR signaling pathway. CONCLUSIONS: The obtained results showed that SS protected against CI/RI through an autophagy-mediated AMPK/mTOR pathway when accessed in vitro and in vivo.

Metformin inhibits the development and metastasis of colorectal cancer.

Metformin is a commonly used drug for the treatment of diabetes. Accumulating evidence suggests that it exerts anti-cancer effects in many cancers, including colorectal cancer. However, the underlying molecular mechanisms of colorectal cancer metastasis remain unclear. Colorectal cancer cell lines were treated with metformin, and cell proliferation, invasion, and migration were analyzed in vitro. The relationship between metformin and the AMPK-mTOR axis was assessed by Western blot analysis and transfection with small interfering RNA. A colorectal cancer xenograft mouse model was used to observe the effects of metformin on liver metastasis. Immunohistochemical analysis was performed on liver metastatic tumors. In in vitro experiments, metformin significantly inhibited the proliferation, migration, and invasion only in HCT116 and SW837 cells, but not in HCT8 and Lovo cells. Only in HCT116 and SW837, a change in AMPK-mTOR expression was observed in a dose-dependent manner. In colorectal cancer xenograft mice, the liver metastatic rate (10% vs. 50%, p = 0.05) and the number of liver metastatic nodules (0.1/body vs. 1.2/body, p = 0.04) were significantly lower in the metformin group. Tumor proliferation and EMT were decreased and apoptosis was promoted only in metastatic liver tumors of mice treated with metformin. The molecular mechanism of the anti-cancer effects of metformin involves repression of mTOR pathways via AMPK activation. Moreover, the differences in metformin sensitivity depend on the response of the AMPK-mTOR pathway to metformin. Our study provides a theoretical basis for the anti-metastatic treatment of colorectal cancer using metformin.

Zexie Tang targeting FKBP38/mTOR/SREBPs pathway improves hyperlipidemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Zexie Tang (ZXT), only two consists with Alismatis Rhizoma (AR) and Atractylodes macrocephala Rhizoma (AM), a classical Chinese medicine formula from Synopsis of the Golden Chamber with a history of 2000 years. Clinical observation in recent years has found that ZXT has excellent lipid-lowering effect. AIM OF THE STUDY: To explore the potential mechanism of ZXT ameliorates hyperlipidemia based on FKBP38/mTOR/SREBPs pathway. MATERIALS AND METHODS: WD-induced hyperlipidemia mice and oleic acid induced cell lipid accumulation model were used to investigate pharmacodynamic. The effect of ZXT on the transcriptional activity of SREBPs was detected by reporter gene assay. Proteins and downstream genes of mTOR/SREBPs pathway were detected in vivo and in vitro. Combined with network pharmacology and HPLC-Q-TOF/MS, the active ingredients were screened and identified. The interaction between active compounds of ZXT and FKBP38 protein were analyzed by docking analysis. RESULTS: ZXT decreased TC, TG and LDL-c levels in blood of WD-induced hyperlipidemia mouse model, and improved insulin resistance in vivo. ZXT also reduced TC, TG and lipid accumulation in cells line, and inhibited SREBPs luciferase activity, protein and its target genes expression such as FASN, HMGCR, etc. Meanwhile, ZXT inhibited protein expression levels of p-mTOR, p-S6K, etc in vitro and in vivo. Combined with network pharmacology and HPLC-Q-TOF/MS, 16 active ingredients were screened and identified. Docking results showed that active compounds of ZXT binding to FKBP38 and formed hydrogen bond. CONCLUSION: Our findings highlighted that ZXT ameliorates hyperlipidemia, in which FKBP/mTOR/SREBPs pathway might be the potential regulatory mechanism.

Modulation of mTOR signaling by radiation and rapamycin treatment in canine mast cell cancer cells.

Rapamycin has been reported to reduce cancer cell survival in certain tumors following radiation therapy, but the mechanisms driving this phenomenon are unclear. Rapamycin inhibits mTOR signaling, a pathway responsible for several essential cell functions. The objective of this study was to investigate the effects of rapamycin and radiation on the activation and inhibition of mTOR signaling and the relationship between mTOR signaling and DNA damage response in vitro using canine mast cell tumor (MCT) cancer cell lines. Rapamycin rapidly inhibited S6K phosphorylation in a dose-dependent manner. Ionizing radiation (3, 6, or 10 Gy) was able to activate mTOR signalling, but the combination of radiation and rapamycin maintained mTOR inhibition. The comet assay revealed that co-treatment with rapamycin induced modest increases in the severity of DNA damage to MCT cells, but that these differences were not statistically significant. Although the relationship between mTOR and DNA damage response in MCT cancer cell lines remains unclear, our findings suggest the possibility of interaction, leading to enhancement of radiation response.

Il a été rapporté que la rapamycine réduisait la survie des cellules cancéreuses dans certaines tumeurs après une radiothérapie, mais les mécanismes à l'origine de ce phénomène ne sont pas clairs. La rapamycine inhibe la signalisation mTOR, une voie responsable de plusieurs fonctions cellulaires essentielles. L'objectif de cette étude était d'étudier les effets de la rapamycine et des radiations sur l'activation et l'inhibition de la signalisation mTOR et la relation entre la signalisation mTOR et la réponse aux dommages à l'ADN in vitro à l'aide de lignées cellulaires cancéreuses de tumeurs mastocytaires canines (MCT). La rapamycine a rapidement inhibé la phosphorylation de S6K de manière dose-dépendante. Le rayonnement ionisant (3, 6 ou 10 Gy) a pu activer la signalisation mTOR, mais la combinaison de rayonnement et de rapamycine a maintenu l'inhibition de mTOR. Le test des comètes a révélé que le co-traitement avec la rapamycine induisait des augmentations modestes de la gravité des dommages à l'ADN des cellules MCT, mais que ces différences n'étaient pas statistiquement significatives. Bien que la relation entre mTOR et la réponse aux dommages à l'ADN dans les lignées cellulaires cancéreuses MCT reste incertaine, nos résultats suggèrent la possibilité d'une interaction, conduisant à une amélioration de la réponse aux radiations.(Traduit par Docteur Serge Messier).

The novel role of circular RNA ST3GAL6 on blocking gastric cancer malignant behaviours through autophagy regulated by the FOXP2/MET/mTOR axis.

Gastric cancer (GC) ranks third in mortality among all cancers worldwide. Circular RNAs (circRNAs) play an important role in the occurrence and development of gastric cancer. Forkhead box P2 (FOXP2), as a transcription factor, is closely associated with the development of many types of tumours. However, the regulatory network between FOXP2 and circRNAs remains to be explored. In our study, circST3GAL6 was significantly downregulated in GC and was associated with poor prognosis in GC patients. Overexpression of circST3GAL6 inhibited the malignant behaviours of GC cells, which was mediated by inducing apoptosis and autophagy. In addition, we demonstrated that circST3GAL6 regulated FOXP2 through the mir-300 sponge. We further found that FOXP2 inhibited MET Proto-Oncogene (MET), which was the initiating factor that regulated the classic AKT/mTOR pathway of autophagy. In conclusion, our results suggested that circST3GAL6 played a tumour suppressive role in gastric cancer through miR-300/FOXP2 axis and regulated apoptosis and autophagy through FOXP2-mediated transcriptional inhibition of the MET axis, which may become a potential target for GC therapy.

Quercetin for managing type 2 diabetes and its complications, an insight into multitarget therapy.

BACKGROUND: Quercetin, a bioflavonoid abundant in grapefruit, onion, berries, etc., has vast therapeutic potential, especially against Type 2 diabetes and its complications. Quercetin showed similar effects as that of metformin, (widely prescribed antidiabetic drug) in cell lines models (Sajan et al., 2010; Dhanya et al., 2017). In vivo findings also showcase it as a promising agent against diabetes and its pathophysiological complications. SCOPE AND APPROACH: Quercetin can be produced on a large scale through a novel fermentation-based glycosylation strategy from cheap substrates and can be utilized as a dietary supplement. The review focuses on the mounting evidence pointing to Quercetin as a promising candidate for managing type 2 diabetes and its oxidative stress mediated pathophysiological complications. CONCLUSION: Quercetin acts on multiple targets of diabetes and regulates key signalling pathways which improve the symptoms as well as the complications of Type 2 diabetes. However further studies are needed to improve the bioavailability and to establish a dosing regimen for Quercetin.

Valsartan- and melatonin-supported adipose-derived mesenchymal stem cells preserve renal function in chronic kidney disease rat through upregulation of prion protein participated in promoting PI3K-Akt-mTOR signaling and cell proliferation.

This study tested the hypothesis that valsartan (Val) and melatonin (Mel)-assisted adipose-derived mesenchymal stem cells (ADMSCs) preserved the residual renal function in chronic kidney disease (CKD) rat through promoting cellular-prior-protein (PrPC) to upregulate PI3K/Akt/mTOR signaling and cell proliferation. In vitro study demonstrated that as compared with CKD-derived-ADMSCs, Val/Mel/overexpression of PrPC-treated CKD derived-ADMSCs significantly upregulated cell proliferation and protein expressions of PrPC and phosphorylated (p)-PI3K/p-Akt/p-mTOR, and downregulated oxidative stress (all p < 0.001). Rats (n = 42) were categorized into group 1 (sham-operated-control), group 2 (CKD), group 3 (CKD + ADMSCs/1.2 ×106 cells) + Mel/20 mg/kg/day), group 4 (CKD + siRNA-PrPC-ADMSCs/1.2 ×106 cells), group 5 (CKD + ADMSCs/1.2 ×106 cells + Val/20 mg/kg/day) and group 6 (CKD + Val + Mel). By day 35, the kidney specimens were harvested and the result showed that the protein expression of PrPC was highest in group 1, lowest in groups 2/4 and significantly lower in group 6 than in groups 3/5, but it was similar in groups 3/5 (all p < 0.0001). The protein expressions of cell-stress-signaling (p-PI3K/p-Akt/p-mTOR) and cell-cycle activity (cyclin-D1/clyclin-E2/Cdk2/Cdk4) exhibited an identical pattern, whereas the protein expressions of oxidative-stress (NOX-1/NOX-2)/mitochondrial fission (PINK1/DRP1)/apoptosis (cleaved-capsase3/cleaved-PARP) and fibrosis (TFG-ß/Smad3) as well as creatinine/BUN levels, ratio of urine-protein to urine-creatine and kidney-injured score exhibited an opposite pattern of PrPC among the groups (all p < 0.0001). In conclusion, Mel/Val facilitated-ADMSCs preserved renal architecture and function in CKD rat through promoting PrPC to regulate the cell proliferation/oxidative-stress/cell-stress signalings.

Nimbolide-encapsulated PLGA nanoparticles induces Mesenchymal-to-Epithelial Transition by dual inhibition of AKT and mTOR in pancreatic cancer stem cells.

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis and remains highly aggressive despite current advancements in therapies. Chemoresistance and high metastatic nature of PDAC is attributed to a small subset of stem-like cells within the tumor known as Cancer Stem Cells (CSCs). Here, we developed a strategy for targeting pancreatic CSCs through forceful induction of mesenchymal-to-epithelial transition driven by encapsulating a phytochemical Nimbolide in nanoparticles. Binding of Nimbolide with the key regulator proteins of CSCs were studied through molecular docking and molecular dynamic simulation studies, which revealed that it binds to AKT and mTOR with high affinity. Further, in vitro studies revealed that Nim NPs are capable of inducing forceful mesenchymal-to-epithelial transition of pancreatospheres that leads to loss of multidrug resistance and self-renewal properties of pancreatospheres. Our study gives a proof of concept that encapsulation of Nim in PLGA nanoparticles increases its therapeutic effect on pancreatospheres. Further, binding of Nim to AKT and mTOR negatively regulates their activity that ultimately leads to mesenchymal-to-epithelial transition of pancreatic CSCs.

NLRC5 enhances autophagy via inactivation of AKT/mTOR pathway and ameliorates cardiac hypertrophy.

The aim of this study was to investigate the effect of nucleotide-binding oligomerization domain (NOD)-like receptor family CARD domain containing 5 (NLRC5) in cardiac hypertrophy, and to explore the mechanism implicated in this effect Cardiac hypertrophy was induced in neonatal rat cardiac myocytes using 1 µM of angiotensin II (Ang II) for 12, 24 and 48 h. Overexpression of NLRC5 was induced in H9C2 cells, and the NLRC5 + Ang II-treated cells were exposed to SC9 and 3-methyladenine (3MA). An immunofluorescence assay was used for α-actinin staining, and quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for NLRC5, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) determination. Western blot analysis was applied to measure the levels of NLRC5, microtubule-associated protein 1A/1B-light chain 3 type I (LC3I), LC3II, sequestosome 1 (p62), protein kinase B (AKT), phosphorylated Akt (pAKT), mammalian target of rapamycin (mTOR) and phosphorylated mTOR (pmTOR). The level of NLRC5 was significantly decreased after Ang II treatment in cardiomyocytes, but the levels of ANP and BNP were increased. Overexpression of NLRC5 reduced the cell size, downregulated the levels of ANP and BNP, increased LC3II / LC3I, but decreased p62 in Ang II-induced cardiomyocyte hypertrophy. In addition, the results from Western blot showed that overexpression of NLRC5 distinctly decreased the ratios of pAKT/AKT and pmTOR/mTOR in cardiomyocyte hypertrophy. SC79 and 3MA significantly downregulated the ratio of LC3I/LC3II but increased the level of p62 in NLRC5 + Ang II-treated cells. These results provide a possible novel therapeutic strategy for cardiac hypertrophy that might be useful in a clinical setting.

Neuregulin-1ß Alleviates Sepsis-Induced Skeletal Muscle Atrophy by Inhibiting Autophagy via AKT/mTOR Signaling Pathway in Rats.

BACKGROUND: Several studies have shown that excessive protein degradation is a major cause of skeletal muscle atrophy induced by sepsis, and autophagy is the main pathway participating in protein degradation. However, the role of autophagy in sepsis is still controversial. Previously, we found that neuregulin-1ß (NRG-1ß) alleviated sepsis-induced diaphragm atrophy through the phosphatidylinositol-3 kinase signaling pathway. Akt/mechanistic target of rapamycin (mTOR) is a classic signaling pathway to regulate autophagy, which maintains intracellular homeostasis. This study aimed to investigate whether NRG-1ß could alleviate sepsis-induced skeletal muscle atrophy by regulating autophagy. METHODS: L6 rat myoblast cells were differentiated using 2% fetal bovine serum into myotubes, which were divided into four groups: Con group treated with normal serum; Sep group treated with septic serum to form a sepsis cell model; septic serum + NRG-1ß (SN) group treated with septic serum for 24 h followed by injection with NRG-1ß and incubation for another 48 h; and serum+NRG-1ß+LY294002 group, in which the PI3K inhibitor LY294002 was added 30 min before NRG-1ß, and other treatments were similar to those in SN group. Effects of NRG-1ß were also evaluated in vivo using Sprague-Dawley (SD) rats, in which sepsis was induced by cecal ligation and puncture (CLP). RESULTS: In L6 myotubes treated with septic serum, the expression of autophagy-related proteins UNC-51 like kinase 1, p-Beclin-1, and Beclin-1, and the ratio of LC3B II/I were highly increased, while protein p62 expression was decreased, indicating that autophagy was excessively activated. Moreover, NRG-1 expression was decreased, as detected by confocal immunofluorescence and western blotting. Upon exogenous addition of NRG-1ß, autophagy was inhibited by the activation of Akt/mTOR signaling pathway, and cell viability was also increased. These effects disappeared in the presence of LY294002. In SD rats, sepsis was induced by CLP. NRG-1ß was shown to inhibit autophagy in these rats via the Akt/mTOR pathway, leading to increased body weight of the septic SD rats and alleviation of atrophy of the tibialis anterior muscle. CONCLUSION: NRG-1ß could alleviate sepsis-induced skeletal muscle atrophy by inhibiting autophagy via the AKT/mTOR signaling pathway.

Cell-Penetrating Peptide-Modified Graphene Oxide Nanoparticles Loaded with Rictor siRNA for the Treatment of Triple-Negative Breast Cancer.

INTRODUCTION: Breast cancer is a malignant tumor that seriously threatens women's life and health. METHODS: In this study, we proposed to use graphene nanoparticles loaded with siRNA that can silence Rictor molecules essential for the mammalian target of rapamycin (mTOR) complex 2 (mTORC2) complex to enhance gene delivery to tumor cells through modification of cell-penetrating peptide (CPP) for the treatment of breast cancer. RESULTS: Remarkably, we successfully synthesized graphene oxide (GO)/polyethyleneimine (PEI)/polyethylene glycol (PEG)/CPP/small interfering RNA (siRNA) system, and the results were observed by atomic force microscopy (AFM) and ultraviolet visible (UV-Vis) absorption spectra. The optimum mass ratio of siRNA to GO-PEI-PEG-CPP was 1:0.5. We screened out Rictor siRNA-2 from 9 candidates, which presented the highest inhibition rate, and this siRNA was selected for the subsequent experiments. We validated that Rictor siRNA-2 significantly reduced the Rictor expression in triple negative breast cancer (TNBC) cells. Confocal fluorescence microscope and flow cytometry analysis showed that GO-PEI-PEG-CPP/siRNA was able to be effectively uptake by TNBC cells. GO-PEI-PEG-CPP/siRNA improved the effect of siRNA on the inhibition of TNBC cell viability and the induction of TNBC cell apoptosis. The expression of Rictor and the phosphorylation of Akt and p70s6k were inhibited by GO-PEI-PEG-CPP/siRNA. Tumorigenicity analysis in nude mice showed that GO-PEI-PEG-CPP/siRNA significantly repressed the tumor growth of TNBC cells in vivo. The levels of ki-67 were repressed by GO-PEI-PEG-CPP/siRNA, and the apoptosis was induced by GO-PEI-PEG-CPP/siRNA in the system. DISCUSSION: Therefore, we concluded that CPP-modified GO nanoparticles loaded with Rictor siRNA significantly repressed TNBC progression by the inhibition of PI3K/Akt/mTOR signaling. Our finding provides a promising therapeutic strategy for the treatment of TNBC.

Pongamol Inhibits Epithelial to Mesenchymal Transition Through Suppression of FAK/Akt-mTOR Signaling.

BACKGROUND/AIM: Cancer metastasis is the main cause of mortality in cancer patients. As lung cancer patients are mostly detected at metastatic stages, strategies that inhibit cancer metastasis may offer effective therapies. Activation of FAK and Akt/mTOR pathways promotes the highly metastatic phenotypes of epithelial to mesenchymal transition (EMT). We unraveled EMT inhibitory action of pongamol and the mechanism controlling cell dissemination in lung cancer cells. MATERIALS AND METHODS: Cytotoxic and antiproliferative effects of pongamol were determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis and necrosis induction in response to pongamol treatment was observed and visualized by nuclei staining assay. Wound healing migration, invasion, and anchorage-dependent growth assay were conducted to evaluate metastatic behaviors. EMT protein expression and FAK pathway were detected by western blot analysis. RESULTS: Pongamol at 0-100 µM exhibited significant inhibition on migration, and invasion of cancer cells. Regarding anoikis resistance potential, the compound significantly inhibited survival and growth of cancer cells in an anchorage-independent manner, as indicated by the depletion of growing colonies in pongamol-pretreated cells. Protein level analysis further showed that pongamol exerted its anti-metastasis effect by inhibiting EMT, as indicated by a decrease of several mesenchymal proteins (N-cadherin, vimentin, Snail, and Slug). Regarding the up-stream mechanisms, we found that pongamol inhibited activation of FAK and Akt/mTOR signaling pathways. CONCLUSION: Pongamol exhibits potent anti-metastatic activity through suppressing key potentiating factors of cancer metastasis EMT and FAK.

Berberine exerts its antineoplastic effects by reversing the Warburg effect via downregulation of the Akt/mTOR/GLUT1 signaling pathway.

Glucose transporter 1 (GLUT1) plays a primary role in the glucose metabolism of cancer cells. However, to the best of our knowledge, there are currently no anticancer drugs that inhibit GLUT1 function. The present study aimed to investigate the antineoplastic activity of berberine (BBR), the main active ingredient in numerous Traditional Chinese medicinal herbs, on HepG2 and MCF7 cells. The results of Cell Counting Kit­8 assay, colony formation assay and flow cytometry revealed that BBR effectively inhibited the proliferation of tumor cells, and induced G2/M cell cycle arrest and apoptosis. Notably, the results of luminescence ATP detection assay and glucose uptake assay showed that BBR also significantly inhibited ATP synthesis and markedly decreased the glucose uptake ability, which suggested that the antitumor effect of BBR may occur via reversal of the Warburg effect. In addition, the results of reverse transcription­quantitative PCR, western blotting and immunofluorescence staining indicated that BBR downregulated the protein expression levels of GLUT1, maintained the cytoplasmic internalization of GLUT1 and suppressed the Akt/mTOR signaling pathway in both HepG2 and MCF7 cell lines. Augmentation of Akt phosphorylation levels by the Akt activator, SC79, abolished the BBR­induced decrease in ATP synthesis, glucose uptake, GLUT1 expression and cell proliferation, and reversed the proapoptotic effect of BBR. These findings indicated that the antineoplastic effect of BBR may involve the reversal of the Warburg effect by downregulating the Akt/mTOR/GLUT1 signaling pathway. Furthermore, the results of the co­immunoprecipitation assay demonstrated that BBR increased the interaction between ubiquitin conjugating enzyme E2 I (Ubc9) and GLUT1, which suggested that Ubc9 may mediate the proteasomal degradation of GLUT1. On the other hand, BBR decreased the interaction between Gα­interacting protein­interacting protein at the C­terminus (GIPC) and GLUT1, which suggested that the retention of GLUT1 in the cytoplasm may be achieved by inhibiting the interaction between GLUT1 and GIPC, thereby suppressing the glucose transporter function of GLUT1. The results of the present study provided a theoretical basis for the application of the Traditional Chinese medicine component, BBR, for cancer treatment.

Identification of Clinical Candidate M2698, a Dual p70S6K and Akt Inhibitor, for Treatment of PAM Pathway-Altered Cancers.

Herein, we report the discovery of a novel class of quinazoline carboxamides as dual p70S6k/Akt inhibitors for the treatment of tumors driven by alterations to the PI3K/Akt/mTOR (PAM) pathway. Through the screening of in-house proprietary kinase library, 4-benzylamino-quinazoline-8-carboxylic acid amide 1 stood out, with sub-micromolar p70S6k biochemical activity, as the starting point for a structurally enabled p70S6K/Akt dual inhibitor program that led to the discovery of M2698, a dual p70S6k/Akt inhibitor. M2698 is kinase selective, possesses favorable physical, chemical, and DMPK profiles, is orally available and well tolerated, and displayed tumor control in multiple in vivo studies of PAM pathway-driven tumors.

Effects of ß-carboline alkaloids from Peganum harmala on the FAK/PI3K/AKT/Mtor pathway in human gastric cancer cell line SGC-7901 and tumor-bearing mice.

This study investigates the effects of ß-carboline alkaloids from Peganum harmala on FAK/PI3K/AKT/mTOR pathway in gastric cancer cell line SGC-7901 and tumor-bearing mice. Western blot, immunohistochemistry and RT-PCR were performed to detect protein and mRNA expressions of BCL-2, Bax, FAK, PI3K, AKT and mTOR. Mice model of gastric tumor was established with SGC-7901 cells. TUNEL assay was used to detect apoptosis. HE staining was used to observe morphological changes. In vitro, the protein and mRNA expressions of FAK, PI3K, AKT and mTOR in ß-carboline alkaloids groups were significantly lower than those in control and fluorouracil groups (P<0.05). BCL-2 decreased while Bax increased. In vivo, the tumor weights of ß-carboline alkaloids and fluorouracil groups were significantly lower than those of control group (P<0.05). FAK, PI3K, AKT and mTOR proteins in tumor tissues of ß-carboline alkaloids and fluorouracil groups were significantly lower than control group (P<0.05). Additionally, ß-carboline alkaloids treatment in vivo caused obvious cell necrosis and apoptosis. Conclusively, ß-carboline alkaloids can reduce FAK, PI3K, AKT and mTOR expressions at both protein and mRNA levels in SGC-7901 cells and tumor tissues formed by SGC-7901 cells. They may be targets of ß-carboline in FAK/PI3K/AKT/mTOR pathway.