Regulation Of AKT Signaling Pathways Research Articles

Targeting lysyl oxidase like 2 attenuates OVA-induced airway remodeling partly via the AKT signaling pathway

BackgroundAirway epithelium is an important component of airway structure and the initiator of airway remodeling in asthma. The changes of extracellular matrix (ECM), such as collagen deposition and structural disturbance, are typical pathological features of airway remodeling. Thus, identifying key mediators that derived from airway epithelium and capable of modulating ECM may provide valuable insights for targeted therapy of asthma.MethodsThe datasets from Gene Expression Omnibus database were analyzed to screen differentially expressed genes in airway epithelium of asthma. We collected bronchoscopic biopsies and serum samples from asthmatic and healthy subjects to assess lysyl oxidase like 2 (LOXL2) expression. RNA sequencing and various experiments were performed to determine the influences of LOXL2 knockdown in ovalbumin (OVA)-induced mouse models. The roles and mechanisms of LOXL2 in bronchial epithelial cells were explored using LOXL2 small interfering RNA, overexpression plasmid and AKT inhibitor.ResultsBoth bioinformatics analysis and further experiments revealed that LOXL2 is highly expressed in airway epithelium of asthmatics. In vivo, LOXL2 knockdown significantly inhibited OVA-induced ECM deposition and epithelial-mesenchymal transition (EMT) in mice. In vitro, the transfection experiments on 16HBE cells demonstrated that LOXL2 overexpression increases the expression of N-cadherin and fibronectin and reduces the expression of E-cadherin. Conversely, after silencing LOXL2, the expression of E-cadherin is up-regulated. In addition, the remodeling and EMT process that induced by transforming growth factor-β1 could be enhanced and weakened after LOXL2 overexpression and silencing in 16HBE cells. Combining the RNA sequencing of mouse lung tissues and experiments in vitro, LOXL2 was involved in the regulation of AKT signaling pathway. Moreover, the treatment with AKT inhibitor in vitro partially alleviated the consequences associated with LOXL2 overexpression.ConclusionsTaken together, the results demonstrated that epithelial LOXL2 plays a role in asthmatic airway remodeling partly via the AKT signaling pathway and highlighted the potential of LOXL2 as a therapeutic target for airway remodeling in asthma.

Licochalcone D Inhibits Skin Epidermal Cells Transformation through the Regulation of AKT Signaling Pathways.

Cell transformation induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is a critical event in cancer initiation and progression, and understanding the underlying mechanisms is essential for the development of new therapeutic strategies. Licorice extract contains various bioactive compounds, which have been reported to have anticancer and anti-inflammatory effects. This study investigated the cancer preventive efficacy of licochalcone D (LicoD), a chalcone derivative in licorice extract, in EGF and TPA-induced transformed skin keratinocyte cells. LicoD effectively suppressed EGF-induced cell proliferation and anchorage-independent colony growth. EGF and TPA promoted the S phase of cell cycle, while LicoD treatment caused G1 phase arrest and down-regulated cyclin D1 and up-regulated p21 expression associated with the G1 phase. LicoD also induced apoptosis and increased apoptosis-related proteins such as cleaved-caspase-3, cleaved-caspase-7, and Bax (Bcl-2-associated X protein). We further investigated the effect of LicoD on the AKT signaling pathway involved in various cellular processes and found decreased p-AKT, p-GSK3β, and p-NFκB expression. Treatment with MK-2206, an AKT pharmacological inhibitor, suppressed EGF-induced cell proliferation and transformed colony growth. In conclusion, this study demonstrated the potential of LicoD as a preventive agent for skin carcinogenesis.

Effects of chitinase-3-like protein 1 on the expression of inflammatory damage-related molecules in mouse skeletal muscle satellite cells induced by lipopolysaccharide

To explore the potential mechanism of chitinase-3-like protein 1 (CHI3L1) involved in skeletal muscle stem cell injury induced by sepsis. Six different concentrations of lipopolysaccharide (LPS) were used to stimulate mouse skeletal muscle satellite cells cultured in vitro. Enzyme linked immunosorbent assay (ELISA) and cell counting kit-8 (CCK-8) were used to determine the optimal concentration. The overexpression and interference vectors of CHI3L1 were constructed to transfect skeletal muscle satellite cells, and the transfection efficiency was verified by polymerase chain reaction (PCR) and Western blotting. The cells were randomly divided into blank control group (cells without any intervention), model group (LPS-stimulated untransfected cells), overexpressing CHI3L1 group (LPS-stimulated cells transfected with CHI3L1 plasmid), overexpressing CHI3L1 control group [LPS-stimulated cells transfected with negative control (NC) plasmid], CHI3L1 interference group [LPS-stimulated cells transfected with CHI3L1 small interfering RNA (siRNA)], CHI3L1 interference control group (LPS-stimulated cells transfected with CHI3L1-siRNA NC). The levels of extracellular caspase-1 and interleukin-1β (IL-1β) were detected by ELISA. The protein expressions of intracellular IL-1β, signal transducters and activator of transcription 3 (STAT3), protein kinase B (Akt) and phosphorylated Akt (p-Akt) were detected by Western blotting. According to the results of CCK-8 and ELISA, the best concentration of 5 mg/L LPS was selected for the subsequent experiment. The transfection was validated by PCR and Western blotting. Compared with the blank control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular Akt, p-Akt, and IL-1β were significantly increased in the model group [IL-1β (ng/L): 11.22±0.55 vs. 8.63±0.63, caspase-1 (pmol/L): 9.47±0.22 vs. 8.65±0.15, Akt/GAPDH: 1.36±0.12 vs. 1.06±0.15, p-Akt/GAPDH: 0.78±0.07 vs. 0.09±0.01, IL-1β/GAPDH: 1.38±0.12 vs. 0.18±0.03, all P < 0.05]. Compared with the model group and the overexpressing CHI3L1 control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly increased in the overexpressing CHI3L1 group [IL-1β (ng/L): 14.93±0.97 vs. 11.22±0.55, 9.38±0.40, caspase-1 (pmol/L): 10.35±0.03 vs. 9.47±0.22, 8.46±0.24, p-Akt/GAPDH: 1.21±0.04 vs. 0.78±0.07, 0.63±0.04, IL-1β/GAPDH: 1.87±0.08 vs. 1.38±0.12, 1.51±0.17, all P < 0.05]. Compared with the model group and the CHI3L1 interference control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly decreased in the CHI3L1 interference group [IL-1β (ng/L): 8.98±0.73 vs. 11.22±0.55, 10.44±0.65, caspase-1 (pmol/L): 7.61±0.63 vs. 9.47±0.22, 8.37±0.38, p-Akt/GAPDH: 0.50±0.04 vs. 0.78±0.07, 0.94±0.06, IL-1β/GAPDH: 0.77±0.02 vs. 1.38±0.12, 1.13±0.07, all P < 0.05]. CHI3L1 may mediate the damage of skeletal muscle stem cells in sepsis by increasing the expression of caspase-1 and IL-1β. CHI3L1 may be involved in the regulation of Akt signaling pathway in skeletal muscle stem cells, but has no significant effect on STAT3 signaling pathway.

Retraction.

Retraction: "TPX2 gene silencing inhibits cell proliferation and promotes apoptosis through negative regulation of AKT signaling pathway in ovarian cancer," by Yuan Tian, Lian-Lian Liu, Dong-Mei Guo, Yan Wang, Wen-Hui Zha, Yang Li, Fu-Ju Wu, J Cell Biochem. 2018; 7540-7555: The above article, published online on 15 June 2018 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.27065) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. A detailed investigation revealed that several image elements of the experimental data were published elsewhere in a different scientific context. Thus, the editors consider the conclusions of this article to be invalid.

Biological Role of AKT and Regulation of AKT Signaling Pathway by Thymoquinone: Perspectives in Cancer Therapeutics

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Biological Role of AKT and Regulation of AKT Signaling Pathway by Thymoquinone: Perspectives in Cancer Therapeutics.

AKT/PKB is an important enzyme with numerous biological functions, and its overexpression is related to carcinogenesis. AKT stimulates different signaling pathways that are downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase, hence functions as an important target for anti-cancer drugs. In this review article, we have interpreted the role of AKT signaling pathway in cancer and the natural inhibitory effect of Thymoquinone (TQ) in AKT and its possible mechanisms. We have collected the updated information and data on AKT, its role in cancer and the inhibitory effect of TQ in AKT signaling pathway from Google Scholar, PubMed, Web of Science, Elsevier, Scopus, and many more. Many drugs are already developed, which can target AKT, but very few among them have passed clinical trials. TQ is a natural compound, mainly found in black cumin, which has been found to have potential anti-cancer activities. TQ targets numerous signaling pathways, including AKT, in different cancers. In fact, many studies revealed that AKT is one of the major targets of TQ. The preclinical success of TQ suggests its clinical studies on cancer. This review article summarizes the role of AKT in carcinogenesis, its potent inhibitors in clinical trials, and how TQ acts as an inhibitor of AKT and TQ's future as a cancer therapeutic drug.

Knockdown of Rab9 Suppresses the Progression of Gastric Cancer Through Regulation of Akt Signaling Pathway.

Rabs have been reported to be involved in the carcinogenesis process and in the progression of cancer. However, it is unclear whether or not Rab9 is associated with the development of cancer. In the present study, we aimed to investigate the role of Rab9 in the biological functions of gastric cancer cells. The gastric cancer cell lines AGS and MKN45 were transfected with siRNA-Rab9 to block the expression of Rab9. The cell viability, proliferation, migration, invasion, and apoptosis were examined using Cell Count Kit-8, colony formation, wound healing, Transwell, and flow cytometry assays, respectively. Our data showed that silencing of Rab9 significantly inhibited the viability, proliferation, migration, and invasion abilities of AGS and MKN45 cells. Moreover, transfection with siRab9 promoted the rate of apoptosis in AGS and MKN45 cells through regulating the Bcl-2–Bax axis and the Caspase cascade. We also found that silencing of Rab9 inhibited activation of the Akt signaling pathway by downregulating the phosphorylation level of Akt. In conclusion, our data suggest that Rab9 plays an oncogenic role in the progression of gastric cancer, providing a potential target for the treatment of gastric cancer.

Fyn stimulates the progression of pancreatic cancer via Fyn-GluN2b-AKT axis.

To assess the expression levels of Fyn in human tissue samples and pancreatic cancer cells and explore the potential mechanisms of Fyn in pancreatic cancer progression. Quantitative PCR and immunohistochemical (IHC) assays were performed to detect the expression of Fyn in 30 cancer tissue samples from pancreatic cancer patients and corresponding adjacent normal tissues. In addition, the potential correlations between Fyn expression levels and clinical pathological features were assessed. We further detected the effects of Fyn on the proliferation, apoptosis, migration, and invasion of the pancreatic cancer cells through colony formation assay, flow cytometry (FCM) assay, wound healing assay, and transwell assay, respectively. The potential effects of Fyn on tumor growth were assessed using an animal model. We demonstrated the possible involvement of Fyn in the progression of pancreatic cancer. We found that Fyn was upregulated in human pancreatic cancer tissues and cells, and we analyzed the correlations between Fyn expression and the clinicopathological features, including metastasis staging (p=0.010*) and tumor size (p=0.025*) of patients with pancreatic cancer. Our data further confirmed that Fyn affects cell proliferation, apoptosis, migration, and invasion of pancreatic cancer cells via the phosphorylation of GluN2b and regulation of AKT signaling pathway. We also demonstrated that Fyn promoted tumor growth of pancreatic cancer cells in vivo. We investigated the potential involvement of Fyn in the progression of pancreatic cancer, and therefore indicated Fyn as a possible therapeutic target for pancreatic cancer.

P ‐Synephrine exhibits anti‐adipogenic activity by activating the Akt/GSK3β signaling pathway in 3T3‐L1 adipocytes

This work aimed to investigate the effects of p-synephrine on the differentiation of adipocyte and explore the underlying mechanism. We found that p-synephrine suppressed the 3T3-L1 cell adipogenesis by reducing the expression level of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), which subsequently led to a reduction in the fatty acid-binding protein 4 (aP2) expression. p-Synephrine treatment markedly activated the protein kinase B (PKB/Akt)pathway and sequentially inhibited glycogen synthase kinase 3β (GSK3β) activity. Inhibition of GSK3β activity by LiCl was found to partially ameliorate the above-mentioned effects. All these data suggested that p-synephrine exhibited the anti-adipogenic effects via the regulation of Akt signaling pathway and the suppression of adipogenesis-related proteins. PRACTICAL APPLICATIONS: Citrus aurantium often uses as herbal or dietary supplement in various countries around the world, including in Seville, Spain and South Africa. In traditional Chinese herbs, it is referred to as "Fructus aurantii immaturus," "Zhi shi," or "Zhi ke," and has been used for hundreds of years for various digestive problems. Its primary protoalkaloid, p-synephrine, exhibited lipolytic effects and energy expenditure, which has rapidly replaced ephedrine as an "ephedra-free" alternative dietary supplement. The current study firstly demonstrated the anti-adipogenic effects of p-synephrine in 3T3-L1 preadipocytes, which was due to the regulation of Akt signaling pathway and the subsequent suppression of adipogenesis-related proteins. The present study may offer invaluable opinions into the mechanisms of body weight/fat-losing activities of p-synephrine intheory, and scientificexperimentalevidence on dietary supplement in practice. p-Synephrine could be utilized for the preventive and therapeutic uses against metabolic syndrome.

Murrangatin suppresses angiogenesis induced by tumor cell-derived media and inhibits AKT activation in zebrafish and endothelial cells.

IntroductionLung cancer is a major cancer type and a leading cause of cancer-related death. Angiogenesis plays a crucial role in lung cancer pathogenesis and its inhibition is beneficial to patients.Materials and methodsMurrangatin, a natural product, can inhibit the proliferation of lung cancer cells, so herein we investigated its anti-angiogenic effects in transgenic zebrafish TG (fli1: EGFP) and in lung cancer cell-induced angiogenesis in human umbilical vein endothelial cells.ResultsWe found that murrangatin strongly inhibited the growth of subintestinal vessels in zebrafish embryos and tumor conditioned media-induced angiogenic phenotypes including cell proliferation, cell invasion, cell migration, and tube formation. Additionally, murrangatin greatly attenuated conditioned medium-induced AKT phosphorylation, but not extracellular signal-regulated kinase 1/2 phosphorylation.Discussion and conclusionThese findings indicate that murrangatin can inhibit tumor-induced angiogensis, at least in part through the regulation of AKT signaling pathways. Murrangatin may, therefore, be a potential candidate for the development of new anti-lung-cancer drugs.

TPX2 gene silencing inhibits cell proliferation and promotes apoptosis through negative regulation of AKT signaling pathway in ovarian cancer.

Ovarian cancer (OC) is the leading cause of death from gynecological malignancy. Accumulated studies have revealed that targeting protein for Xklp2 (TPX2) was tightly associated with the development and progression of OC. The present study further determined a novel mechanism of TPX2 in OC via the AKT signaling pathway. The differentially expressed genes were screened in GEO database for gene expression microarray of OC. Bioinformatics was used to analyze the key differentially expressed genes in OC. We prepared CD133/1+ OC stem cells. Then cells were treated with TPX2-1 siRNA and perifcsine to explore the correlation of TPX2 and the AKT signaling pathway. We determined the expression of TPX2, AKT, Pl3 K, PTEN, caspase-3, Bax and Bcl-2 in OC cells. Cell proliferation, migration, invasion, and apoptosis rate were respectively measured using MTT and EdU assays, Transwell assay, Scratch test, and flow cytometry. Xenograft tumor in nude mice was used to determine the effect of TPX2 in OC cells in vitro. Initially, TPX2 overexpression was observed in OC, and TPX2 mediated the effect of the AKT signaling pathway in OC. TPX2 knockdown decreased expression of AKT, Pl3 K, and Bcl-2, and the extent of AKT phosphorylation, but increased expression of PTEN, Caspase-3, and Bax. Furthermore, TPX2 knockdown suppressed OC cell proliferation, migration and invasion, but promoted OC cell apoptosis. Taken together, TPX2 silencing negatively regulates the AKT signaling pathway by which OC cell proliferation was inhibited yet cell apoptosis was accelerated, suggesting a potential therapeutic approach to OC.

Ganoderma lucidum polysaccharide enhances radiosensitivity of hepatocellular carcinoma cell line HepG2 through Akt signaling pathway.

Ganoderma lucidum polysaccharide (GLP) is a well-known traditional Chinese medicine, known for its anti-cancer and immunomodulatory properties. The present study aims to investigate whether GLP has a therapeutic effect on hepatocellular carcinoma (HCC) cells exposed to radiation. Immunofluorescence was used to detect the nuclei, the protein expression was measured by western blot analysis and flow cytometry was used to detect the rate of cell apoptosis. GLP treatment was demonstrated to enhance radiation-induced growth inhibition and apoptotic death of HCC cells. At a molecular level, GLP suppressed the activities of DNA repair-associated proteins including ataxia-telangiectasia mutated (ATM) and DNA dependent-protein kinase (DNA-PK) in liver cancer cells under radiation conditions. Furthermore, the addition of an Akt inhibitor elevated the activities of DNA-PK and ATM and attenuated the GLP-induced HepG2 cell injury under the radiation condition. In conclusion, the present study demonstrates that GLP enhances the radiosensitivity of HCC cells via the regulation of Akt signaling pathways, implying a potential therapeutic effect of GLP as a radiation sensitizer in HCC treatment.

LHD-Modified Mechanism-Based Liposome Coencapsulation of Mitoxantrone and Prednisolone Using Novel Lipid Bilayer Fusion for Tissue-Specific Colocalization and Synergistic Antitumor Effects.

Coencapsulation liposomes are of interest to researchers because they maximize the synergistic effect of loaded drugs. A combination regimen of mitoxantrone (MTO) and prednisolone (PLP) has been ideal for tumor therapy. MTO and PLP offer synergistic antitumor effects confirmed by several experiments in this research. The deduced synergistic mechanism is regulation of Akt signaling pathway including the targets of p-Akt, p-GSK-3β, p-s6 ribosomal protein, and p-AMPK by MTO reactivating PLP-induced apoptosis. The liposome fusion method is adopted to create coencapsulation liposomes (PLP-MTO-YM). Low molecular weight heparin-sodium deoxycholate conjugate (LHD) then is used as a targeting ligand to prove target binding and inhibition of angiogenesis. LHD-modified liposomes (PLP-MTO-HM) have a high entrapment efficiency around 95% for both MTO and PLP. DSC results indicate that both drugs interacted with liposomes to prevent drug leak during liposome fusion. DiD-C6-HM dyes colocalize well to tumor tissue, and coadministration of DiD-HM and C6-CM did not achieve dye colocalization until 24 h after administration. In both CT26 and B16F10 mouse model, PLP-MTO-HM shows a significantly higher tumor inhibition rate relative to the coadministration of MTO-HM and PLP-CM (p < 0.05 or p < 0.01). Thus, the coencapsulation system (PLP-MTO-HM) offers ideal antitumor effects relative to coadministration therapy due to enhanced synergistic effect, and this suggests a promising future for the tumor targeting vectors.

Calycosin and Genistein Induce Apoptosis by Inactivation of HOTAIR/p-Akt Signaling Pathway in Human Breast Cancer MCF-7 Cells

Background: Calycosin and genistein are the two main components of isoflavones. Previously, we reported that these compounds display antitumor activities in the breast cancer cell lines MCF-7 and T47D. In the present study, we investigated the mechanism of action of calycosin and genistein, and their respective efficacies as potential therapies for the treatment of breast carcinoma in the clinic. Methods: MCF-7 cells were treated with calycosin or genistein. Cell proliferation and apoptosis were measured using CCK8 assay and Hoechst 33258. The expression level of phosphorylated Akt protein was determined by western blotting. Expression level of HOTAIR was quantified by real-time PCR. Results: Both calycosin and genistein inhibited proliferation and induced apoptosis in MCF-7 breast cancer cells, especially after treatment with calycosin. Treatment of MCF-7 cells with calycosin or genistein resulted in decreased phosphorylation of Akt, and decreased expression of its downstream target, HOTAIR. Conclusion: Calycosin is more effective in inhibiting breast cancer growth in comparison with genistein, through its regulation of Akt signaling pathways and HOTAIR expression.

Effect of tacolimus postconditioning on serine-threonine protein kinase signaling pathway via regulating generation of reactive oxygen species after spinal cord ischemia-reperfusion injury

Objective To explore the influence of tacrolimus postconditioning (TP) on serine-threonine protein kinase (Akt) signaling pathway afier spinal cord ischemia-reperfusion injury (SCIRI)and the role of reactive oxygen species (ROS) in the mechanism.Methods The rat model of SCIRI was prepared on the basis of Taira' s method.Eighty male SD rats were randomly divided into 4 groups:sham operation (SO) group,ischemia-reperfusion (IR) group,TP group,and TP plus N-Acetylcysteine (TP +N) group.IR group underwent reperfusion for 20 min after spinal cord ischemia.TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery right at the onset of reperfusion,with remaining procedures the same as IR group.TP + N group was injected through the left common carotid artery with N-Acetylcysteine (150 mg/kg) 5 min before spinal cord ischemia,with remai-ning procedures the same as TP group.SO group received exclusively femoral artery catheterization.Fluo-rospectrophotometry was employed to detect the level of ROS in injured spinal cord segments,and immuno-blotting was used to determine the expression of phosphorylated Akt (p-Akt) at 15 min after reperfusion.Flow cytometry was applied to assess apoptosis of neural cells at 24 h after reperfusion.Nissl's staining was utilized to observe pathological changes of injured spinal cord segments at 7th day after reperfusion.Re-sults The ROS levels in spinal cord tissue were 49.0 ± 3.5,145.3±11.7,79.0±6.3 and12.8 ± 0.9in SO group,IR group,TP group,and TP + N group at 15 rain after reperfusion,respectively.The ROSlevel in TP group was significantly lower than that in IR group at 15 min after reperfusion (P ＜0.01),and that in TP + N group was significantly lower than the other three groups (P ＜ 0.01).The p-Akt expression in spinal cord tissue was 0.54 ± 0.06,0.34 ± 0.03,0.42 ± 0.07,and 0.28 ± 0.05 in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The p-Akt expression was sig-nificantly lower in IR group than in SO group (P ＜ 0.01),higher in TP group than in IR group (P ＜0.05),and lower in TP + N group than in SO group (P ＜0.01) or in TP group (P ＜0.01).The percentages of apoptotic cells in spinal cord tissue were (7.5 ± 1.8) ％,(31.4 ± 5.5) ％,(22.3-± 4.0) ％,and (33.3 ± 3.1) ％ in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The percentage of apoptotic cells was significantly higher in IR or TP + N group than in TP group (P ＜0.01),while there was no significant difference between IR group and TP + N group (P ＞0.05).The spinal cord sections revealed that pathological changes could hardly be observed in SO group,and mild in TP group but serious in IR and TP + N groups.Conclusion TP produces protective effect on ischemic spinal cord via positive regulation of Akt signaling pathway,and ROS,acting as a signaling molecule,plays an indispensable role in initiating the mechanism. Key words: Spinal cord ischemia; Tacrolimus ; Reactive oxygen species ; Serine-threonine protein kinase

183 ENDOTHELIAL LINEAGE DIFFERENTIATION FROM IPS CELLS IS REGULATED BY MIRNA-21/AKT AND TGF-Β2 PATHWAYS

Background Endothelial death/dysfunction is a critical process in the development of cardiovascular diseases. Finding a source of endothelial cells (ECs) for regenerative medicine is a challenging yet fundamental issue. Induced pluripotent stem cells (iPSCs) constitute an attractive source of cells for transplantation because of their high proliferation and differentiation potential. Methods and Results In this study, we investigated the mechanisms regulating iPSC differentiation into ECs, focusing on miRNAs and their targets. Firstly, we established a differentiation protocol using collagen and VEGF to drive the functional differentiation of iPSCs into ECs. After 7 days, the cells strongly expressed EC markers such as VE-cadherin, Flk-1, vWF and eNOS and formed tubes on Matrigel culture. A parallel study was conducted on embryonic stem cells (ESCs) and iPSCs to analyze the synergistic effect of VEGF and shear stress in improving EC differentiation. Next, we compared the miRNA signature of undifferentiated and differentiated cells with VEGF for 3 days. Amongst the 5 validated miRNAs we focused on miR-21, previously shown to be involved in angiogenesis. Overexpression of miR-21 (pre-21) in pre-differentiated iPSCs induced EC marker upregulation, while inhibition miR-21 (LNA-21) produced the opposite effect. Accordingly, in vitro and in vivo capillary formation was increased by pre-21 and reduced by LNA-21. Interestingly, miR-21 overexpression increased TGFβ2 mRNA and secreted protein level, which was consistent with the strong upregulation of TGFβ-2 during VEGF-induced iPSC differentiation. In addition treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation through induction of VEGF secretion. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression, indicating the importance of the TGFβ-2 pathway in the differentiation process. Furthermore, TGFβ-2 neutralizing antibody inhibited miR-21 induced EC marker upregulation, indicating that TGFβ-2 is a required downstream effecter for miR-21-mediated EC differentiation. In silico target screening revealed PTEN as one of the predicted targets of miR-21. Our results confirmed that pre-21 significantly inhibited PTEN expression whereas LNA-21 induced it. Luciferase assay confirmed that miR-21directly binds to the 3’ UTR of PTEN, which is inhibited by a site-specific mutation. Since PTEN is the upstream negative regulator of AKT signaling pathway, we then confirmed that pre-21 induced Akt phosphorylation, while LNA-21 decreased it. Functionally, it was found that inhibition of miR-21 in vivo Matrigel assay revealed the significant influence on angiogenesis in animal models. Conclusion These data demonstrate that miR-21 directly targets the PTEN/Akt pathway, in which TGFβ-2 pathway regulated by miR-21 is also involved. Thus, the molecular mechanisms elucidated in this work might provide the basic information for stem cell therapy for vascular disease, e.g. tissue engineering and endothelial repair in damaged vessels.

Protein Kinase B/AKT and Focal Adhesion Kinase: Two Close Signaling Partners in Cancer

AKT (or protein kinase B) and focal adhesion kinase (FAK) are two important kinases that regulate various cellular functions. Each is overexpressed and/or aberrantly activated in diverse cancers. Several small molecular inhibitors targeting either AKT or FAK are in development or in clinical trials. It is well established that FAK is an upstream regulator of AKT signaling pathway in various cancer cell lines and in xenograft tumor models. However, very recent reports from our laboratory and others demonstrate that AKT can also directly regulate FAK through direct association and serine phosphorylation. This indicates that AKT and FAK may be dual therapeutic targets for pharmacologic intervention in the treatment of primary and metastatic cancer. FAK-AKT interaction is particularly critical for metastatic adhesion. We review recent developments in AKT and FAK signaling in cancer with the particular emphasis on the novel signaling pathways in which FAK is downstream of AKT. We also provide an update on inhibitors targeting AKT or FAK currently in clinical trials.

Regulation of akt pathway in apoptosis of pancreatic cancer cells induced by polyphenols

Objective To investigate the regulation of Akt signaling pathway involved in apeptosis of Panc-I cells induced by polyphenol ( - )-epigallocatechin-3-gallate. Methods Pane-1 cells were maintained in DMEM with 10% fetal calf serum in a 95% air,5% CO_2 atmosphere,then incubated in different concentrations of EGCG (6.3,12.5,25.0,50.0 μmol/L). The growth arrest effect was determined by MTT assay, and apoptosis was detected by agarose gel electrophoresis. The levels of Akt (60×10~3 ) and Bad (23×10~3) were determined by Western blot. Results EGCG time and dose dependently repressed the growth of Panc-1 cells and induced apeptosis in Panc-1 cells ( P <0.05 ). The expression of p-Akt (set473) and p-Bad (ser136) was reduced in a dose-dependent way,whereas the pan-Akt and pan-Bad were unchanged (P < 0.05). Conclusion EGCG can induce apoptosis in pancreatic cancer cells in a dose-and time-dependent fashion by attenuating the blocking effects of Akt signaling pathway on Bad proteins. Key words: Pancreatic carcinoma; EGCG; Apotosis