Inhibition Of Akt Phosphorylation Research Articles

The protective effects and underlying mechanisms of dapagliflozin on diabetes-induced testicular dysfunction.

Male diabetic individuals present a marked impairment in fertility; however, knowledge regarding the pathogenic mechanisms and therapeutic strategies is unsatisfactory. The new hypoglycemic drug dapagliflozin has shown certain benefits, such as decreasing the risk of cardiovascular and renal events in patients with diabetes. Even so, until now, the effects and underlying mechanisms of dapagliflozin on diabetic male infertility have awaited clarification. Here, we found that dapagliflozin lowered blood glucose levels, alleviated seminiferous tubule destruction, and increased sperm concentrations and motility in leptin receptor-deficient diabetic db/db mice. Moreover, the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin (9-39) had no effect on glucose levels but reversed the protective effects of dapagliflozin on testicular structure and sperm quality in db/db mice. We also found that dapagliflozin inhibited the testicular apoptotic process by upregulating the expression of the antiapoptotic protein B-cell lymphoma 2 (BCL2) and X-linked inhibitor of apoptosis protein (XIAP) and inhibiting oxidative stress by enhancing the antioxidant status, including total antioxidant capacity, total superoxide dismutase (SOD) activity, and glutathione peroxidase (GPx) activity, as well as decreasing the level of 4-hydroxynonenal (4-HNE). Exendin (9-39) administration partially reversed these effects. Furthermore, dapagliflozin upregulated the glucagon-like peptide-1 (GLP-1) level in plasma and GLP-1R expression by promoting AKT8 virus oncogene cellular homolog (Akt) phosphorylation in testicular tissue. Exendin (9-39) partially inhibited Akt phosphorylation. These results suggest that dapagliflozin protects against diabetes-induced spermatogenic dysfunction via activation of the GLP-1R/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Our results indicate the potential effects of dapagliflozin against diabetes-induced spermatogenic dysfunction.

A network pharmacology-based study on the mechanism of astragaloside IV alleviating renal fibrosis through the AKT1/GSK-3β pathway

Ethnopharmacological revelvanceAstragaloside IV, a glycoside derived from Astragalus membranaceus, has anti-renal fibrosis effects. However, its mechanism of action has not yet been fully elucidated. Aim of the studyThe purpose of this study was to investigate the anti-fibrotic effect of AS-IV and to clarify its underlying mechanism. Materials and methodsThe network pharmacology method, molecular docking and surface plasmon resonance (SPR) was used to identify potential targets and pathways of AS-IV. A unilateral ischemia-reperfusion injury (UIRI) animal model, as well as TGF-β1-induced rat renal tubular epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) were used to investigate and validate the anti-fibrotic activity and pharmacological mechanism of AS-IV. Network pharmacology was performed to construct a drug-target-pathway network. The anti-fibrosis effect of AS-IV was determined using hematoxylin-eosin (H&E) and MASSON staining, as well as immunostaining methods. qRT-PCR and western blotting were used to elucidate and validate the mechanism of AS-IV. ResultsNetwork pharmacology revealed that the PI3K/AKT pathway is an important pathway in AS-IV. AS-IV inhibited the expression of α-SMA, collagen I, and fibronectin in NRK-52E, NRK-49F, and UIRI rats, and reduced serum creatinine and blood urea nitrogen levels in UIRI rats. AS-IV inhibited AKT phosphorylation, blocked GSK-3β phosphorylation, and restored GSK-3β activity, which contributed to the degradation of β-catenin, thereby preventing epithelial-mesenchymal transition (EMT). ConclusionAstragaloside IV alleviated renal fibrosis through the AKT1/GSK-3β pathway. In addition, our findings indicate that the network pharmacology method is a powerful tool for exploring the pharmacological mechanisms of drugs.

The Novel LncRNA WASH5P Inhibits Colorectal Cancer Carcinogenesis via Targeting AKT Signaling Pathway.

Emerging evidence has shown that long non-coding RNAs (lncRNAs) play an important role in colorectal cancer (CRC) carcinogenesis, so more specific mechanisms of key lncRNAs in CRC initiation and development are needed. Here, we evaluated the expression profiles of lncRNAs in CRC tissues and identified a novel lncRNA generated from the pseudogene Wiskott-Aldrich syndrome protein (WASP) family homolog 5, termed lncRNA WASH5P. However, the role and potential molecular mechanism of this novel lncRNA in diseases, including CRC carcinogenesis, is unknown. Our present study found that WASH5P was significantly downregulated in CRC cell lines and tissues compared with normal controls. The ectopic expression of WASH5P in CRC cells could significantly inhibit CRC cell proliferation, invasion, and migration. In addition, WASH5P could increase the expression of E-cadherin and decrease Vimentin expression. WASH5P-overexpressing CRC cells developed tumors more slowly in different mouse models. Meanwhile, the overexpression of WASH5P could significantly inhibit AKT activation via suppressing AKT phosphorylation. The treatment of PI3K/AKT (phosphatidlinositol 3-kinase /protein kinase B) signaling agonist 740Y-P rescued WASH5P-reduced AKT phosphorylation and abolished the inhibitory effects of WASH5P on cell viability, migration, and invasion. Moreover, 740Y-P restored the WASH5P-induced downregulation of p-AKT and vimentin and the upregulation of E-cadherin via Western blot. In summary, our findings suggested that the novel lncRNA WASH5P might be a potential candidate biomarker and therapeutic target that could inhibit CRC by repressing the AKT signaling pathway.

EGCG regulated osteolytic microenvironment to enhance the antitumor effect of DOX on orthotopic osteosarcoma

Here, we demonstrated that EGCG could enhance the anti-osteosarcoma effect of DOX in vivo and in vitro. The combination of DOX and EGCG could inhibit the activity of osteoclasts at the tumor-bone interface, thereby preventing tumor-induced osteolysis and blocking the vicious circle. Taken together, EGCG may be used as an adjuvant chemotherapy drug to treat osteosarcoma. • EGCG enhanced DOX-induced proliferation inhibition of 143B Cells. • EGCG in synergy with DOX inhibited osteoclast differentiation and bone resorption in vitro. • EGCG in synergy with DOX inhibited AKT phosphorylation in 143B cells and BMMs. • EGCG could enhance the anti-osteosarcoma and anti- osteolysis effect of DOX in vivo. Osteosarcoma is the most common malignant bone tumor with the pathological essence of osteolysis. Doxorubicin (DOX) is common in adjuvant chemotherapy of osteosarcoma. However, it is required to address the conflict between enhancing its dose to improve efficacy and reducing its complications and chemotherapy resistance. Previous studies have demonstrated that EGCG has an antitumor and osteoclastogenesis inhibition effect. However, the combined effect of EGCG and DOX on osteosarcoma is still unclear. Here, EGCG was demonstrated to enhance the 143B cell proliferation inhibition and anti-osteoclastogenesis effect of DOX in vitro. The results suggest that EGCG in synergy with DOX inhibited AKT phosphorylation, which was involved in 143B cells proliferation and osteoclastogenesis. In vivo, the combination of DOX and EGCG could reduce tumor volume and osteolysis destruction compared with the DOX treatment group. This present study raised the possibility that EGCG may be used as an adjuvant chemotherapy drug to treat osteosarcoma.

Abstract LB200: Pdgf/Pi3k/akt axis activation by hey1-ncoa2 fusion and potential target in the treatment of mesenchymal chondrosarcoma

Abstract Mesenchymal chondrosarcoma is a rare and often aggressive cancer mostly affecting children and young adults. Genetically, this tumor is mainly characterized by the recurrent HEY1-NCOA2 fusion. Localized tumor is managed surgically; traditional chemotherapy and radiation therapy, however, did not appear to substantially improve the event-free survival rate of mesenchymal chondrosarcoma patients. It is clear that better therapeutic options are needed for the treatment of this tumor. In our previous study, an in vitro model for studying mesenchymal chondrosarcoma tumorigenesis was developed using stably transduced iPSC-derived mesenchymal stem cells (iPSC-MSCs) with inducible-expression of HEY1-NCOA2. With the in vitro model, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq to identify HEY1-NCOA2-dependent transcriptional regulation and demonstrated the dramatic activation of PDGF-PDGFR and PI3K-AKT signaling pathways (manuscript in review). Our data also suggested that PDGFB is one of the direct targets of HEY1-NCOA2 fusion protein. In the present study, with the in vitro iPSC-MSC model, we verified that HEY1-NCOA2 fusion protein, but not wild-type HEY1 or NCOA2, dramatically increased phospho-AKT (Ser473, a readout of PI3K) expression by western blotting. Further on, we tested the effect of two kinase inhibitors CP-673451 and PF-04691502, a potent and selective PDGFR inhibitor and the mTOR/PI3K inhibitor, respectively. Stably transfused iPSC-MSC were treated with the kinase inhibitors, respectively, at different dosages (0.1 µM, 0.5 µM and 1.0 µM) for one week. At the concentration of 1.0 µM, CP-673451 efficiently suppressed AKT phosphorylation by ~23% as well as suppressed cell growth by ~67% which is significantly higher than that observed in the controls (e.g. ~20% suppression in HEY1- and NCOA2- expression iPSC-MSCs). Similarly, at the concentration of 0.5 µM, PF-04691502 suppressed the HEY1-NCOA2-expression iPSC-MSCs growth by ~24% more than that in the control (p=0.02). In summary, both CP-673451 and PF-04691502 were active against the in vitro mesenchymal chondrosarcoma model tested. Based on these data, the potential benefit of targeting PDGF/PI3K/AKT signaling pathways in the treatment of mesenchymal chondrosarcoma was suggested. Citation Format: Wenqing Qi, Lu Wang. Pdgf/Pi3k/akt axis activation by hey1-ncoa2 fusion and potential target in the treatment of mesenchymal chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB200.

AXL/CDCP1/SRC axis confers acquired resistance to osimertinib in lung cancer

Osimertinib, a third-generation EGFR-TKI, has nowadays been applied to non-small cell lung cancer harboring activated EGFR mutation with or without T790M, but ultimately develop resistance to this drug. Here we report a novel mechanism of acquired resistance to osimertinib and the reversal of which could improve the clinical outcomes. In osimertinib-resistant lung cancer cell lines harboring T790M mutation that we established, expression of multiple EGFR family proteins and MET was markedly reduced, whereas expression of AXL, CDCP1 and SRC was augmented along with activation of AKT. Surprisingly, AXL or CDCP1 expression was induced by osimertinib in a time-dependent manner up to 3 months. Silencing of CDCP1 or AXL restored the sensitivity to osimertinib with reduced activation of SRC and AKT. Furthermore, silencing of both CDCP1 and AXL increased the sensitivity to osimertinib. Either silencing of SRC or dasatinib, a SRC family kinase (SFK) inhibitor, suppressed AKT phosphorylation and cell growth. Increased expression of AXL and CDCP1 was observed in refractory tumor samples from patients with lung cancer treated with osimertinib. Together, this study suggests that AXL/SFK/AKT and CDCP1/SFK/AKT signaling pathways play some roles in acquired osimertinib resistance of non-small cell lung cancer.

Palmatine Derivatives as Potential Antiplatelet Aggregation Agents via Protein Kinase G/Vasodilator-Stimulated Phosphoprotein and Phosphatidylinositol 3-Kinase/Akt Phosphorylation.

Sixty palmatine (PMT) derivatives were synthesized and evaluated for antiplatelet aggregation taking berberine as the lead, and the structure-activity relationship was first systematically described. Among them, compound 2v showed the best potency in reducing adenosine diphosphate (ADP)-induced platelet aggregation in a dose-dependent manner. It greatly suppressed ADP-induced platelet aggregation, activation, and Akt phosphorylation in vitro and ex vivo after oral administration to mice. It also effectively inhibited carrageenan-induced thrombus formation in the mouse tail and lung, as well as reduced the serum P-selectin level. Compound 2v might simultaneously bind to protein kinase G to improve vasodilator-stimulated phosphoprotein phosphorylation and bind to phosphatidylinositol 3-kinase to inhibit Akt phosphorylation, which synergically reduced platelet aggregation, thereby achieving antithrombotic efficacy. Therefore, PMT derivatives constituted a novel family of antiplatelet aggregation agents with the advantage of a good safety profile, worthy of further investigation.

Free fatty acid-induced miR-181a-5p stimulates apoptosis by targeting XIAP and Bcl2 in hepatic cells

AimsNon-alcoholic fatty liver disease is one of the major health concerns in the World. The dietary free fatty acids (FFAs) affect the metabolic status of the hepatocytes by modulating cellular pathways. In this study, we showed that free fatty acids stimulate apoptosis by upregulating miR-181a-5p expression, which in turn targets XIAP and Bcl2. MethodsHuh7 cells were incubated with FFAs for 72 h and the expression of XIAP, Bcl2, bax, pAkt, Akt, PTEN and β-actin were determined by Western blots, and miR-181a-5p expression was determined using real-time RT-PCR. The Huh7 cells were transfected with either miR-181a-5p pre-miRs or anti-miR-181a-5p and the regulation of apoptosis and proliferation was studied. Three groups of C57BL/6 mice (n = 6 per group) were fed with standard diet, CSAA or CDAA diet for 6, 18, 32 and 54 weeks. Total protein and RNA were isolated from the liver tissues and used for Western blots and real-time RT-PCR respectively. Key findingsFFAs inhibited Akt phosphorylation, expression of XIAP and Bcl2, while upregulating the expression of PTEN, bax, and miR-181a-5p in Huh7 cells. Similar results were observed when the Huh7 cells were transfected with miR-181a-5p premiRs, while these changes were reversed in anti-miR-181a-5p-transfected, FFA-treated Huh7 cells. The CDAA-fed mice showed a significant inhibition of Akt phosphorylation, XIAP and Bcl2, whereas PTEN and bax expression were upregulated. The expression of miR-181a-5p was also significantly higher in CDAA-fed mice. SignificanceThese findings showed that free fatty acids induced apoptosis via upregulating miR-181a-5p in hepatic cells.

ACT001 inhibits the proliferation of non-small cell lung cancer cells by upregulating NKTR expression.

BackgroundLung cancer, the primary cause of cancer‐related deaths worldwide, is diagnosed at an advanced stage and has a poor prognosis. Non‐small cell lung cancer (NSCLC) is a major histological type of lung malignancy. This study investigated the effect of ACT001, a novel sesquiterpene lactone derivative, on the proliferation of NSCLC cells and explored the underlying mechanism.MethodsThe effect of ACT001 on cell proliferation was examined by clone formation and MTT assay. Differentially expressed genes and enrichment pathways were analyzed by RNA‐seq. Flow cytometry and cell cycle‐related protein expression analysis were performed to study the cell cycle. Phosphorylated AKT was detected to explore the mechanism in natural killer cell triggering receptor (NKTR) KD cells with AKT activator and/or inhibitor. The therapeutic effect of ACT001 in vivo was studied in the xenograft tumor model.ResultsACT001 inhibited the proliferation and G1/S transition in NSCLC cell lines. By RNA‐seq analysis, NKTR may be the target of ACT001. Moreover, knockdown NKTR promoted cell proliferation and reversed the effects of ACT001. In addition, ACT001 inhibited AKT phosphorylation, but NKTR knockdown promoted AKT phosphorylation.ConclusionOur results suggested NKTR may be the target of ACT001 in NSCLC. ACT001 holds promise as a novel method for the treatment of NSCLC.

Regorafenib Induces the Apoptosis of Gastrointestinal Cancer-Associated Fibroblasts by Inhibiting AKT Phosphorylation.

Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and are essential for tumorigenesis and development. Regorafenib is a multikinase inhibitor that targets CAFs and suppresses tumor growth. In this study, we investigated the effects of regorafenib on gastrointestinal CAFs and the underlying molecular mechanisms. First, we established two in vivo tumor models, the cancer cell line HCT116 with and without mesenchymal stem cells (MSCs), and treated them with regorafenib. We found that application of regorafenib potently impaired tumor growth, an effect that was more pronounced in tumors with a high stromal ratio, thus demonstrating that regorafenib can inhibit CAF proliferation and induce CAF apoptosis in vivo. Moreover, we showed that regorafenib affected macrophage infiltration by reducing the proportion of CAFs in tumors. Second, we induced MSCs into CAFs with exosomes to establish an in vitro model. Then, we used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt and flow cytometry to detect the effects of regorafenib on proliferation and apoptosis of CAFs and western blot to determine the expression level of apoptosis-related proteins. We found that regorafenib inhibited proliferation of CAFs and induced apoptosis in CAFs in vitro. Furthermore, western blot results showed that regorafenib downregulated the expression of B cell lymphoma-2 (Bcl-2) and concurrently upregulated the expression of Bcl-2-associated X (Bax), and regorafenib inhibited the phosphorylation pathway of AKT in CAFs. In conclusion, our results provide a model in which regorafenib induces CAF apoptosis by inhibiting the phosphorylation of AKT and regorafenib affects macrophage infiltration by reducing the proportion of CAFs in tumor tissues.

CXCR7 Promotes Adipogenesis and Insulin Resistance in Human Visceral Adipocytes

Obesity is a multifactorial chronic condition that increases the risk of cardiovascular diseases, type II diabetes, and certain types of cancer. It has been known that some chemokines and their receptors facilitate the development of obesity, leading to other related comorbidities. However, the exact role of atypical chemokine receptor CXCR7 in obesity and obesity‐induced type II diabetes remains unknown. We aimed to study whether CXCR7 promotes adipogenesis and insulin resistance in human visceral adipocytes in vitro. Using RT‐PCR and flow cytometry studies, we found that both CXCR7 mRNA and its protein expressions were significantly upregulated during the differentiation of human visceral adipocytes. Stimulation of CXCR7 by SDF‐1 or LIH383 (a CXCR7‐selective peptide ligand) increased lipid droplets formation during adipocytes differentiation. In addition, we found that SDF‐1 or LIH383 treatment inhibited AKT phosphorylation in a dose‐dependent manner with no effect on ERK1/2 and JNK signaling pathways, which indicates a selective suppression of the AKT signaling axis by CXCR7. Furthermore, activation of CXCR7 by SDF‐1 inhibited insulin signaling to the AKT pathway and insulin‐induced glucose uptake. In contrast, ablation of CXCR7 by CRISPR/Cas9 system restored AKT signaling and enhanced insulin sensitivity in human visceral adipocytes. We concluded that CXCR7 plays a critical role in human visceral adipogenesis and insulin resistance by suppressing the AKT signaling pathway, highlighting CXCR7 as a potential new drug target against obesity and type II diabetes.

Interactions between β3‐adrenergic receptors and bFGF‐induced effects in rat aortic smooth muscle cells

BackgroundBasic fibroblast growth factor (bFGF)‐mediated vascular smooth muscle cell (VSMC) proliferation and migration play an important role in vascular injury‐induced neointima formation and subsequent vascular restenosis, a major event that hinders the long‐term success of angioplasty. The function of β3‐adrenergic receptors (β3‐ARs) in vascular injury‐induced neointima formation has not yet been defined.ObjectivesOur current study explored the possible role of β3‐ARs in vascular injury‐induced neointima formation by testing its effects on bFGF‐induced VSMC migration and proliferation.Methodsβ3‐AR expression in rat carotid arteries was examined at 14 days following a balloon catheter‐induced injury. The effects of β3‐AR activation on bFGF‐induced rat aortic smooth muscle cell proliferation, migration, and signaling transduction (including extracellular‐signal‐regulated kinase/ mitogen activated protein kinase, ERK/MAPK and Protein kinase B, AKT) were tested.ResultsWe found that vascular injury induced upregulation of β3‐ARs in neointima. Pretreatment of VSMCs with a selective β3‐AR agonist, CL316,243 significantly potentiated bFGF‐induced cell migration and proliferation, and ERK and AKT phosphorylation. Our results also revealed that suppressing phosphorylation of ERK and AKT blocked bFGF‐induced cell migration and that inhibiting AKT phosphorylation reduced bFGF‐mediated cell proliferation.ConclusionOur results suggest that activation of β3‐ARs enhances bFGF‐mediated effects on VSMCs by interacting with bFGF‐mediated ERK and AKT phosphorylation and β3‐ARs may play a role in vascular injury‐induced neointima formation.

Mammalian D‐cysteine: A novel regulator of neural progenitor cell proliferation

D-amino acids are being recognized as functionally important molecules in mammals. We recently identified endogenous D-cysteine in mammalian brain. D-cysteine is present in neonatal brain in substantial amounts (mM) and decreases with postnatal development. D-cysteine binds to MARCKS and a host of proteins implicated in cell division and neurodevelopmental disorders. D-cysteine decreases phosphorylation of MARCKS in neural progenitor cells (NPCs) affecting its translocation. D-cysteine controls NPC proliferation by inhibiting AKT signaling. Exogenous D-cysteine inhibits AKT phosphorylation at Thr 308 and Ser 473 in NPCs. D-cysteine treatment of NPCs led to 50% reduction in phosphorylation of Foxo1 at Ser 256 and Foxo3a at Ser 253. We hypothesize that in the developing brain endogenous D-cysteine is as a physiologic regulator of NPC proliferation by inhibiting AKT signaling mediated by Foxo1 and Foxo3a. Endogenous D-cysteine may regulate mammalian neurodevelopment with roles in schizophrenia and Alzheimer's disease (AD).

Evening Primrose Extracts Inhibit PDGF-BB-Induced Vascular Smooth Muscle Cell Proliferation and Migration by Regulating Cell-Cycle-Related Proteins.

The proliferation and migration of vascular smooth muscle cells (VSMCs) are important factors in the occurrence of cardiovascular diseases, such as blood flow abnormalities, stroke and atherosclerosis. Evening primrose, known as Oenothera biennis, is a plant native to Korea that exerts physiological activities, such as antioxidant effects, the inhibition of lipid accumulation and the prevention of muscle atrophy. However, the function of evening primrose stem (EVP) in the regulation of VSMC proliferation and migration and the underlying mechanisms have not been identified. In this study, the effect of EVP on the platelet-derived growth factor (PDGF)-induced proliferation and migration of VSMCs was investigated. The results show that PDGF-BB-induced proliferation of VSMCs was inhibited by EVP at concentrations of 25, 50 or 100 μg/mL in a concentration-dependent manner, and a migration assay showed that EVP inhibited cell migration. Cell cycle analysis was performed to confirm the mechanism by which cell proliferation and migration was inhibited. The results indicate that proteins involved in the cell cycle, such as cyclin, CDK and phosphorylated Rb, were downregulated by EVP at concentrations of 100 μg/mL, thereby increasing the proportion of cells in the G0/G1 phase and inhibiting cell cycle progression. In the PDGF receptor (PDGFR) signaling pathway, phosphorylation of the PDGFR was inhibited by EVP at concentrations of 100 μg/mL, and PLCγ phosphorylation was also decreased. The PDGF-BB-induced effect of EVP on the proliferation of VSMCs involved the inhibition of Akt phosphorylation and the reduction in the phosphorylation of MAPK proteins such as ERK, P38 and JNK. In conclusion, the results demonstrate that EVP inhibited PDGF-BB-induced VSMC proliferation and migration by regulating cell-cycle-related proteins.

ACT001 attenuates microglia-mediated neuroinflammation after traumatic brain injury via inhibiting AKT/NF\u03baB/NLRP3 pathway

BackgroundMicroglia-mediated neuroinflammatory response following traumatic brain injury (TBI) is considered as a vital secondary injury factor, which drives trauma-induced neurodegeneration and is lack of efficient treatment. ACT001, a sesquiterpene lactone derivative, is reportedly involved in alleviation of inflammatory response. However, little is known regarding its function in regulating innate immune response of central nervous system (CNS) after TBI. This study aimed to investigate the role and underlying mechanism of ACT001 in TBI.MethodsControlled cortical impact (CCI) models were used to establish model of TBI. Cresyl violet staining, evans blue extravasation, neurobehavioral function assessments, immunofluorescence and transmission electron microscopy were used to evaluate therapeutic effects of ACT001 in vivo. Microglial depletion was induced by administering mice with colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Cell-cell interaction models were established as co-culture system to simulate TBI conditions in vitro. Cytotoxic effect of ACT001 on cell viability was assessed by cell counting kit-8 and activation of microglia cells were induced by Lipopolysaccharides (LPS). Pro-inflammatory cytokines expression was determined by Real-time PCR and nitric oxide production. Apoptotic cells were detected by TUNEL and flow cytometry assays. Tube formation was performed to evaluate cellular angiogenic ability. ELISA and western blot experiments were used to determine proteins expression. Pull-down assay was used to analyze proteins that bound ACT001.ResultsACT001 relieved the extent of blood-brain barrier integrity damage and alleviated motor function deficits after TBI via reducing trauma-induced activation of microglia cells. Delayed depletion of microglia with PLX5622 hindered therapeutic effect of ACT001. Furthermore, ACT001 alleviated LPS-induced activation in mouse and rat primary microglia cells. Besides, ACT001 was effective in suppressing LPS-induced pro-inflammatory cytokines production in BV2 cells, resulting in reduction of neuronal apoptosis in HT22 cells and improvement of tube formation in bEnd.3 cells. Mechanism by which ACT001 functioned was related to AKT/NFκB/NLRP3 pathway. ACT001 restrained NFκB nuclear translocation in microglia cells through inhibiting AKT phosphorylation, resulting in decrease of NLRP3 inflammasome activation, and finally down-regulated microglial neuroinflammatory response.ConclusionsOur study indicated that ACT001 played critical role in microglia-mediated neuroinflammatory response and might be a novel potential chemotherapeutic drug for TBI.FQdKbSYTeaAq3ny7ETc-jcVideo

The zinc transporter ZIP12 regulates monocrotaline-induced proliferation and migration of pulmonary arterial smooth muscle cells via the AKT/ERK signaling pathways

BackgroundThe zinc transporter ZIP12 is a membrane-spanning protein that transports zinc ions into the cytoplasm from the extracellular space. Recent studies demonstrated that upregulation of ZIP12 is involved in elevation of cytosolic free zinc and excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia. However, the expression of ZIP12 and its role in pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) in rats have not been evaluated previously. The aim of this study was to investigate the effect of ZIP12 on the proliferation and migration of PASMCs and its underlying mechanisms in MCT-induced PAH.MethodsA PAH rat model was generated by intraperitoneal injection of 20 mg/kg MCT twice at one-week intervals. PASMCs were isolated from the pulmonary arteries of rats with MCT-induced PAH or control rats. The expression of ZIP12 and related molecules was detected in the lung tissues and cells. A ZIP12 knockdown lentivirus and an overexpressing lentivirus were constructed and transfected into PASMCs derived from PAH and control rats, respectively. EdU assays, wound healing assays and Western blotting were carried out to explore the function of ZIP12 in PASMCs.ResultsIncreased ZIP12 expression was observed in PASMCs derived from MCT-induced PAH rats. The proliferation and migration of PASMCs from PAH rats were significantly increased compared with those from control rats. These results were corroborated by Western blot analysis of PCNA and cyclin D1. All these effects were significantly reversed by silencing ZIP12. Comparatively, ZIP12 overexpression resulted in the opposite effects as shown in PASMCs from control rats. Furthermore, selective inhibition of AKT phosphorylation by LY294002 abolished the effect of ZIP12 overexpression on enhancing cell proliferation and migration and partially suppressed the increase in ERK1/2 phosphorylation induced by ZIP12 overexpression. However, inhibition of ERK activity by U0126 resulted in partial reversal of this effect and did not influence an increase in AKT phosphorylation induced by ZIP12 overexpression.ConclusionsZIP12 is involved in MCT-induced pulmonary vascular remodeling and enhances the proliferation and migration of PASMCs. The mechanism of these effects was partially mediated by enhancing the AKT/ERK signaling pathways.

Altersolanol B, a fungal tetrahydroanthraquinone, inhibits the proliferation of estrogen receptor-expressing (ER+) human breast adenocarcinoma by modulating PI3K/AKT, p38/ERK MAPK and associated signaling pathways

The present study focused on the apoptosis-inducing effects and cellular signal-modulating properties of altersolanol B (AB), a minor fungal tetrahydroanthraquinone (THAQ) metabolite, in the estrogen receptor positive (ER+) human breast adenocarcinoma cell line, MCF-7. AB demonstrated approximately 4-fold greater antiproliferative activity in ER+ MCF-7 cells (IC50 5.5 μM) compared to the ER-negative (triple-negative) MDA-MB-231 (IC50 21.3 μM). The viability of normal breast fibrocystic epithelial cells, MCF-10A, was unaffected. AB induced intrinsic apoptosis in MCF-7 cells; it triggered the activation of caspase 9 and poly (ADP-ribose) polymerase (PARP), upregulated the expression of pro-apoptotic Bax, and downregulated the expression of anti-apoptotic Bcl-2. AB induced cell cycle arrest at G0/G1, as indicated by the downregulation of key checkpoint proteins operating at the G0/G1 phase of the cell cycle (cyclin D1, CDK4 and CDK2). The observed increase in p21Waf1/Cip1 and p53 expression may facilitate cell cycle arrest, and the subsequent induction of apoptosis. AB lacked significant effects on intracellular ROS levels, while it down-regulated nuclear factor erythroid 2-related factor 2 (Nrf2), and the Nrf2-dependent antioxidant enzyme, heme oxygenase-1. The compound disrupted AKT signaling through the downregulation of phospho-AKT and phospho-FOXO1, and the upregulation of PTEN, a phosphatase and tumor suppressor that negatively regulates the PI3K/AKT pathway. AB also disrupted the phosphorylation of AKT-controlled eukaryotic translation initiation factor, 4E-BP1, and GSK-3β, both of which are aberrantly regulated in human cancer. The AB-dependent downregulation of NF-κB was corroborated by the inhibition of TNFα-induced NF-κB activity as monitored in a luciferase reporter. The NF-κB inhibitory activity of AB was 3-fold more potent than that of the standard inhibitor, N-p-Tosyl-l-phenylalanine chloromethyl ketone. In addition to reducing the pro-survival effects of NF-кB, the inhibition of AKT phosphorylation by AB may also lead to FOXO1-mediated growth arrest and apoptosis. AB upregulated the expression of phospho-MKK4 and phospho-p38, and downregulated the expression of phospho-MEK1/2 and phospho-ERK1/2 indicating opposing effects on the two important oncogenic signaling cascades that are aberrantly activated in many cancers. AB disrupted both the AKT and ERK1/2 signaling pathways leading to apoptosis in ER+ MCF-7 cells through mitochondria-associated mechanisms coupled with the potent inhibition of NF-кB activation. The clinical limitations of multi-agent combination therapy that targets multiple pathways in cancer may potentially be circumvented by using a single molecule, such as AB, that inhibits both AKT and ERK1/2 signaling. Our preliminary study suggested that the THAQ pharmacophore, with its disrupted conjugated ring system and relative redox inactivity, may possess greater mechanistic advantage against ER+ breast cancer when compared to the fully conjugated ring systems of the anthraquinone that possess intrinsic redox activity and DNA interacting ability. This study supports the continued investigation of THAQs as lead molecules in anticancer drug discovery and development.

PARP Inhibitor Decreases Akt Phosphorylation and Induces Centrosome Amplification and Chromosomal Aneuploidy in CHO-K1 Cells

Cancer cells are known to have chromosomal number abnormalities (aneuploidy), a hallmark of malignant tumors. Cancer cells also have an increased number of centrosomes (centrosome amplification). Paradoxically, cancer therapies, including γ-irradiation and some anticancer drugs, are carcinogenic and can induce centrosome amplification and chromosomal aneuploidy. Thus, the processes of carcinogenesis and killing cancer cells might have some mechanisms in common. Previously, we found that the inhibitors of polyADP-ribosylation, a post-translational modification of proteins, caused centrosome amplification. However, the mechanism of action of the inhibitors of polyADP-ribosylation is not fully understood. In this study, we found that an inhibitor of polyADP-ribosylation, 3-aminobenzamide, caused centrosome amplification, as well as aneuploidy of chromosomes in CHO-K1 cells. Moreover, inhibitors of polyADP-ribosylation inhibited AKT phosphorylation, and inhibitors of AKT phosphorylation inhibited polyADP-ribosylation, suggesting the involvement of polyADP-ribosylation in the PI3K/Akt/mTOR signaling pathway for controlling cell proliferation. Our data suggest a possibility for developing drugs that induce centrosome amplification and aneuploidy for therapeutic applications to clinical cancer.

Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation

DEPTOR is a 48 kDa protein upregulated in multiple myeloma (MM) cells. DEPTOR inhibits mTOR and, by repressing a negative feedback loop, promotes AKT activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here, we screened for drug-induced posttranslational modifications and identified reduced phosphorylation of DEPTOR on serine 235 (S235). We show that an S235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this posttranslational modification. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S235 could only be expressed upon concurrent proteasome inhibition. Thus, S235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown, and an in vitro kinase assay. Inhibition of ERK1 also downregulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this crosstalk, MM cells were transfected with WT or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on the inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose that DEPTOR-dependent crosstalk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).

Angiopoietin-like 4 promotes glucose metabolism by regulating glucose transporter expression in colorectal cancer.

Angiopoietin-like 4 (ANGPTL4) was recently shown to be associated with cancer progression but little is known about its contribution to cancer metabolism. The purpose of this study was to elucidate the role of ANGPTL4 in glucose metabolism in colorectal cancer (CRC). Immunohistochemical staining of CRC specimens classified 84 patients into two groups according to ANGPTL4 expression. Clinicopathological characteristics, gene mutation status obtained by next-generation sequencing, and fluorodeoxyglucose (FDG) uptake measured by positron emission tomography/computed tomography (PET/CT) were compared between the two groups. Furthermore, the impact of ANGPTL4 expression on cancer metabolism was investigated by a subcutaneous xenograft mouse model using the ANGPTL4 knockout CRC cell line, and glucose transporter (GLUT) expression was evaluated. There were significantly more cases of T3/4 tumours (94.3% vs. 57.1%, P < 0.001) and perineural invasion (42.9% vs. 22.4%, P = 0.046) in the ANGPTL4-high group than in the low group. Genetic exploration revealed a higher frequency of KRAS mutation (54.3% vs. 22.4%, P = 0.003) in the ANGPTL4-high tumours. All the FDG uptake parameters were significantly higher in ANGPTL4-high tumours. In vivo analysis showed a significant reduction in tumour size due to ANGPTL4 knockout with lower expression of GLUT1 and GLUT3, and suppression of AKT phosphorylation. ANGPTL4 regulates the expression of GLUTs by activating the PI3K-AKT pathway and thereby promoting glucose metabolism in CRC. These findings establish a new functional role of ANGPTL4 in cancer progression and lay the foundation for developing a novel therapeutic target.