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Chronic kidney disease (CKD), including chronic glomerulonephritis, IgA nephropathy and diabetic nephropathy, are common chronic diseases characterized by structural damage and functional decline of the kidneys. The current treatment of CKD is symptom relief. Several studies have reported that the phosphatidylinositol 3 kinases (PI3K)/protein kinase B (Akt) signaling pathway is a pathway closely related to the pathological process of CKD. It can ameliorate kidney damage by inhibiting this signal pathway which is involved with inflammation, oxidative stress, cell apoptosis, epithelial mesenchymal transformation (EMT) and autophagy. This review highlights the role of activating or inhibiting the PI3K/Akt signaling pathway in CKD-induced inflammatory response, apoptosis, autophagy and EMT. We also summarize the latest evidence on treating CKD by targeting the PI3K/Akt pathway, discuss the shortcomings and deficiencies of PI3K/Akt research in the field of CKD, and identify potential challenges in developing these clinical therapeutic CKD strategies, and provide appropriate solutions., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Renal ischemia-reperfusion injury (RIRI) has become a great concern in clinical practice with high morbidity and mortality rates. Sufentanil has protective effects on IRI-induced organ injury. Herein, the effects of sufentanil on RIRI were investigated., Methods: RIRI cell model was established by hypoxia/reperfusion (H/R) stimulation. The mRNA and protein expressions were assessed using qRT-PCR and western blot. TMCK-1 cell viability and apoptosis were assessed using MTT assay and flow cytometry, respectively. The mitochondrial membrane potential and ROS level were detected by JC-1 mitochondrial membrane potential fluorescent probe and DCFH-DA fluorescent probe, respectively. LDH, SOD, CAT, GSH and MDA levels were determined by the kits. The interaction between FOXO1 and Pin1 promoter was analyzed using dual luciferase reporter gene and ChIP assays., Results: Our results revealed that sufentanil treatment attenuated H/R-induced cell apoptosis, mitochondrial membrane potential (MMP) dysfunction, oxidative stress, inflammation and activated PI3K/AKT/FOXO1 associated proteins, while these effects were reversed by PI3K inhibitor, suggesting that sufentanil attenuated RIRI via activating the PI3K/AKT/FOXO1 signaling pathway. We subsequently found that FOXO1 transcriptionally activated Pin1 in TCMK-1 cells. Pin1 inhibition ameliorated H/R-induced TCMK-1 cell apoptosis, oxidative stress and inflammation. In addition, as expected, the biological effects of sufentanil on H/R-treated TMCK-1 cells were abrogated by Pin1 overexpression., Conclusion: Sufentanil reduced Pin1 expression through activation of the PI3K/AKT/FOXO1 signaling to suppress cell apoptosis, oxidative stress and inflammation in renal tubular epithelial cells during RIRI development., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Oxidative stress can lead to nucleus pulposus cell (NPC) apoptosis, which is considered to be one of the main contributors to intervertebral disc degeneration (IVDD). Procyanidin B2 is a natural antioxidant that protects against oxidative stress. However, whether procyanidin B2 protects NPCs from oxidative stress remains unknown. In this study, we demonstrated that procyanidin B2 could reduce tert-butyl hydroperoxide-induced reactive oxygen species in rat NPCs and attenuate rat NPC apoptosis. Further experiments revealed that procyanidin B2 upregulated the expression of both nuclear factor erythroid 2-related factor 2 (Nrf2) and phosphorylation of protein kinase B (Akt). We then used silencing of Nrf2 and LY294002 to silence Nrf2 expression and block the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, respectively, and found that the protective roles of procyanidin B2 in NPCs were inhibited. Therefore, we demonstrated that procyanidin B2 alleviated rat NPC apoptosis induced by oxidative stress by upregulating Nrf2 via activation of the PI3K/Akt signaling pathway. This study provides a potential therapeutic approach for procyanidin B2 in IVDD, which might help in the development of new drugs for IVDD treatment., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

AXL, a TAM receptor tyrosine kinase (RTK), and its ligand growth arrest-specific 6 (GAS6) are implicated in cancer metastasis and drug resistance, and cellular entry of viruses. Given this, AXL is an attractive therapeutic target, and its inhibitors are being tested in cancer and COVID-19 clinical trials. Still, astonishingly little is known about intracellular mechanisms that control its function. Here, we characterized endocytosis of AXL, a process known to regulate intracellular functions of RTKs. Consistent with the notion that AXL is a primary receptor for GAS6, its depletion was sufficient to block GAS6 internalization. We discovered that upon receptor ligation, GAS6-AXL complexes were rapidly internalized via several endocytic pathways including both clathrin-mediated and clathrin-independent routes, among the latter the CLIC/GEEC pathway and macropinocytosis. The internalization of AXL was strictly dependent on its kinase activity. In comparison to other RTKs, AXL was endocytosed faster and the majority of the internalized receptor was not degraded but rather recycled via SNX1-positive endosomes. This trafficking pattern coincided with sustained AKT activation upon GAS6 stimulation. Specifically, reduced internalization of GAS6-AXL upon the CLIC/GEEC downregulation intensified, whereas impaired recycling due to depletion of SNX1 and SNX2 attenuated AKT signaling. Altogether, our data uncover the coupling between AXL endocytic trafficking and AKT signaling upon GAS6 stimulation. Moreover, our study provides a rationale for pharmacological inhibition of AXL in antiviral therapy as viruses utilize GAS6-AXL-triggered endocytosis to enter cells., (© 2022. The Author(s).)

Background: Gliomas is a major challenge of current medical system, and thousands of people are struggling in the pain of this disease worldwide. In the last decade, the functions of miRNAs have been revealed by many studies, and the intervention on miRNA dysfunctions has been thought as a promising way to counter cancer. MiR-493-5p has been identified as a tumor inhibitor to suppress the progressions of several tumors while its role in gliomas remains unknown. Hence, the study investigated the expression levels of miR-493-5p in glioma tissues and cell lines., Methods: CCK-8 assay, transwell assay and flow cytometry assay were used to observe the effects of miR-493-5p on tumor cells. The downstream targets of miR-493-5p were also searched and verified with online databases and dual-luciferase reporter assay. Moreover, the activities of P53 and PI3K/AKT pathways were also explored by western blot to illustrate the regulation mechanism of miR-493-5p on glioma development., Results: The results showed that miR-493-5p was significantly downregulated in pathological tissues and glioma cell lines, and the increased miR-493-5p effectively inhibited the malignant behavior and promoted the apoptosis of glioma cells., Conclusions: E2F3 was confirmed as a target of miR-493-5p, and the effects of miR-493-5p on the phenotype of glioma cells could be partly reversed by E2F3. Besides, it was also found that miR-493-5p could effectively suppress the expression of E2F3 and then improve the dysfunctions of the P53 and PI3K/AKT pathways., (© 2021. Federación de Sociedades Españolas de Oncología (FESEO).)

Purpose: Increasing evidences suggest dysfunctions of microRNAs (miRNAs) are playing important part in tumors. Therefore, the role of miR-802 in osteosarcoma (OS) was exploited. The object was to evaluate the effect of miR-802 and verify its influence on p27 Kip1 (p27) in OS., Methods: RT-qPCR experiment was used to detect miR-802 and p27 expression in OS tissues and cells. We explored the function of miR-802 through Transwell assays. The phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase pathway and epithelial-mesenchymal transition (EMT) was detected by Western blot assays. Luciferase assay was used to testify the target of miR-802., Results: MiR-802 expression was elevated in OS, which was related to poor clinical outcome in OS patients. MiR-802 overexpression promoted OS migration, invasion and EMT. Further, p27 is a direct target of miR-802. P27 elevation counteracted the promotion effect of OS on EMT, migration and invasion induced by miR-802. In addition, miR-802 overexpression inactivated PI3K/AKT pathway via targeting p27 in OS., Conclusion: MiR-802 promoted the progress of EMT, migration and invasion in OS via targeting p27. This newly identified miR-802/p27/PI3K/AKT axis may represent potential targets for OS., (© 2021. Federación de Sociedades Españolas de Oncología (FESEO).)

Sjögren's syndrome (SS) which could lead to a disorder of our immune system is a chronic autoimmune disease characterized by invading exocrine glands such as salivary glands and lacrimal glands and other exocrine glands. Its common symptom is dry mouth and dry eyes, often accompanied by a large number of lymphocyte infiltrations and can involve other organs to cause complex clinical manifestations. In this study, we aimed at investigating the effect of QZF in SS, identifying the molecular mechanism in modulating autoimmune response, and determining the important roles of these factors' function as a modulator in the pathogenesis of SS. The NOD mice were utilized to establish the rats' model of Sjögren's syndrome. After 10 weeks' hydroxychloroquine and QZF in different dose interference, submandibular gland tissue was collected. The therapeutic effect of QZF on SS rats was identified, and the results suggest the comparable potential to hydroxychloroquine. In submandibular gland tissue, interleukin- (IL-) 17 was significantly lower in high-dose QZF than that in SS rats and the focal lymphocytes were highly attenuated. Moreover, we found that PI3K/Akt signals were activated and the downstream HIF-1 α /VEGF signals were enhanced in SS rats whose protein expression could be inhibited by QZF treatment. In addition, QZF could modulate autophagy in submandibular gland tissue and then inhibit the inflammation response and therefore facilitate the tissue repair., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Ping Zeng et al.)

Background: Hepatocellular carcinoma (HCC) is a substantial health concern worldwide. Increasing studies have suggested that circle RNAs (circRNAs) function as new regulators in HCC progression. The present work explored the role of hsa_circ_0007059 (circ_0007059) in the developing process of hepatocarcinogenesis., Methods: The circ_0007059 level in HCC was determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and northern blot. Its biological role in HCC cells was assessed using 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), colony formation, flow cytometry, Transwell, sphere formation and western blotting analyses. Bioinformatics analysis, luciferase reporter, and RNA immunoprecipitation (RIP) assays were used to test the regulatory mechanisms of circ_0007059., Results: Our results revealed that circ_0007059 expression was downregulated in HCC samples and cells. Moreover, circ_0007059 overexpression inhibited HCC cell proliferation, migration, invasion, and stem cell-like property, and strengthened cell apoptosis. In mechanism, circ_0007059 suppressed AKT/mTOR pathway by positively regulating phosphatase and tensin homolog (PTEN) expression. Additionally, circ_0007059 acted as a positive regulator of PTEN through controlling the availability of miR-421. Rescue assays demonstrated that PTEN knockdown or SC79 (AKT agonist) eliminated the effect of circ_0007059 on HCC cell phenotypes., Conclusion: Circ_0007059 sponges miR-421 to inhibit oncogenic cellular process in HCC by mediating the PTEN-AKT/mTOR pathway., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Extracts from Taiwan's traditional medicinal mushroom, Antrodia cinnamomea , exhibit anti-inflammatory activities in cellular and preclinical studies. However, this paper is the first to report that Antcin K, a triterpenoid isolated from A. cinnamomea , inhibits proinflammatory cytokine production in human rheumatoid synovial fibroblasts (RASFs), which are major players in rheumatoid arthritis (RA) disease. In our analysis of the mechanism of action, Antcin K inhibited the expression of three cytokines (tumor necrosis factor alpha [TNF-α], interleukin 1 beta [IL-1β] and IL-8) in human RASFs; cytokines that are crucial to RA synovial inflammation. Notably, incubation of RASFs with Antcin K reduced the phosphorylation of the focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and nuclear factor-κB (NF-κB) signaling cascades, all of which promote cytokine production in RA. Intraperitoneal injections of Antcin K (10 mg/kg or 30 mg/kg) attenuated paw swelling, cartilage degradation and bone erosion, and decreased serum levels of TNF-α, IL-1β, IL-8 in collagen-induced arthritis (CIA) mice; in further experiments, IL-6 levels were similarly reduced. The inhibitory effects of Antcin K upon TNF-α, IL-1β and IL-8 expression in human RASFs was achieved through the downregulation of the FAK, PI3K, AKT and NF-κB signaling cascades. Our data support clinical investigations using Antcin K in RA disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Achudhan, Liu, Lin, Huang, Tsai, Ko, Chiang, Kuo and Tang.)

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ + NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yao, Yang, Yang, Li, Peng, Wu, Wang, Zhang, Huang, Wang, Xiong, Luo, Li, Jia, Deng and Deng.)

Objectives: Although mulberry fruit possesses some biological activities, it is not known how it protects neuronal cells in neurodegenerative diseases. Here, we examined whether mulberry fruit extract (MFE) protected neuronal cells against oxidative stress-induced neurodegeneration. Methods: In this experiments, glutamate challenged hippocampal neuronal HT-22 cell lines as an in vitro model and scopolamine-induced memoty-impairment mice model were used. Results: MFE improved cell viability and glutathione level as well as reducing reactive oxygen species level in glutamate-treated HT-22 cells. Additionally, MFE suppressed apoptotic bodies and mitochondrial depolarization through regulating expression of apoptosis-related proteins. Furthermore, MFE elevated expression of p-TrkB, p-Akt, p-CREB, BDNF, and antioxidant enzymes as well as nuclear translocation of Nrf2. In contrast, the inclusion of K252a, a TrkB inhibitor, or MK-2206, an Akt selective inhibitor, neutralized the neuroprotective actions of MFE. Separately, MFE attenuated scopolamine-induced amnesia via regulating the activities of enzymes related with cholinergic function and the antioxidant system in mice. Additionally, MFE protected neuronal cells in the hippocampal CA1 and CA3 regions in brain of mice. Conclusions: MFE protects neuronal cells against oxidative stress-induced apoptosis through upregulating the expression of BDNF and antioxidant enzymes by stabilizing the activation of the TrkB/Akt pathway. Such an effect of MFE, which includes rich polyphenols, may provide information for its application as a food supplement for the prevention and treatment of neurodegenerative diseases.

The study aims to investigate the role of microRNA-149-3p (miR-149-3p) in regulating osteogenic differentiation of human adipose-derived stem cells (hADSCs) by targeting v-akt murine thymoma viral oncogene homolog 1 (AKT1). Bioinformatics websites and a dual luciferase reporter assay were used to predict and verify the targeting relationship between miR-149-3p and AKT1. The hADSCs were divided into the blank, negative control (NC), mimic, control siRNA, AKT1 siRNA, and miR-149-3p inhibitors + AKT1 siRNA groups and then subjected to Alizarin Red staining, Alkaline phosphatase (ALP) staining, ALP activity detections, MTT assay, and EdU cell proliferation assay. Gene or protein expression was quantified using quantitative real-time PCR (qRT-PCR) or Western blotting, respectively. The miR-149-3p expression increased gradually and AKT1 expression decreased gradually during osteogenic differentiation of hADSCs. The prediction of bioinformatics websites miRTarBase and TargetScan and the dual luciferase reporter assay indicated that miR-149-3p can directly target AKT1. After hADSCs were transfected with miR-149-3p mimic, AKT1 expression was significantly downregulated. However, transfection with AKT1 siRNA did not have an impact on miR-149-3p in hADSCs. In comparison with the AKT1 siRNA group, the miR-149-3p inhibitors + AKT1 siRNA group showed decreased miR-149-3p expression but increased AKT1 expression. In addition, AKT1 siRNA enhanced the cell viability and proliferation of hADSCs and increased mineral calcium deposition and ALP activity, resulting in higher expression of osteogenic differentiation-related genes, which was reversed by miR-149-3p inhibition. The miR-149-3p can increase the expression of osteogenic differentiation-related genes by targeting AKT1 and thereby enhance the osteogenic differentiation of hADSCs., (© 2021 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Polycystic ovary syndrome (PCOS) is a typical reproductive and endocrinological disorder of women at child-bearing age. In this study, we used miRNA sequencing technology and verified miR-let-7d-3p as a vital miRNA in PCOS. RT-qPCR confirmed miR-let-7d-3p was significantly increased in granulosa cells (GCs) of PCOS. Cell counting kit-8 (CCK-8) identified the suppression of miR-let-7d-3p mimic in KGN cell proliferation and PI3K/Akt signaling pathway. Dual luciferase reporter assay proved that Toll-like receptor 4 (TLR4) was a target of miR-let-7d-3p, and TLR4 was significantly down-regulated by miR-let-7d-3p. Furthermore, over-expression of TLR4 promoted KGN cell proliferation and rescued the inhibition of miR-let-7d-3p on KGN cells. In conclusion, miR-let-7d-3p was a crucial miRNA up-regulated in GCs of PCOS, and inhibited cell proliferation by targeting TLR4 gene., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The tumorigenic mechanism for pancreatic ductal adenocarcinoma (PDAC) is not clear, although chronic inflammation is implicated. Here, we identified an inflammatory cytokine-regulated transfer RNA-derived (tRNA-derived) fragment, tRF-21-VBY9PYKHD (tRF-21), as a tumor suppressor in PDAC progression. We found that the biogenesis of tRF-21 could be inhibited by leukemia inhibitory factor and IL-6 via the splicing factor SRSF5. Reduced tRF-21 promoted AKT2/1-mediated heterogeneous nuclear ribonucleoprotein L (hnRNP L) phosphorylation, enhancing hnRNP L to interact with dead-box helicase 17 (DDX17) to form an alternative splicing complex. The provoked hnRNP L-DDX17 activity preferentially spliced Caspase 9 and mH2A1 pre-mRNAs to form Caspase 9b and mH2A1.2, promoting PDAC cell malignant phenotypes. The tRF-21 levels were significantly lower in PDACs than in normal tissues, and patients with low tRF-21 levels had a poor prognosis. Treatment of mouse PDAC xenografts or patient-derived xenografts (PDXs) with tRF-21 mimics repressed tumor growth and metastasis. These results demonstrate that tRF-21 has a tumor-suppressive effect and is a potential therapeutic agent for PDAC.

PI3K is a downstream target of multiple cell-surface receptors, which acts as a crucial modulator of both cell polarization and survival. PI3K/AKT signaling pathway is commonly involved in cancer, atherosclerosis, and other diseases. However, its role in cardiovascular diseases, especially in atherosclerosis, remains to be further investigated. To determine the effect of PI3K/AKT signaling pathway on cellular inflammatory response and oxidative stress, PI3K inhibitor (GDC0941) and AKT inhibitor (MK2206) were used. First, THP-1 cells were incubated with ox-LDL (100 µg/ml) to establish an in vitro atherosclerosis model. The inflammatory factors and foam cell formation were then evaluated to ascertain and compare the effects of PI3K and AKT inhibition. ApoE -/- mice fed a high-fat diet were used to assess the roles of PI3K and AKT in aortic plaque formation. Our results showed that the inhibition of PI3K or AKT could suppress the activation of NLRP3, decreased the expression levels of p-p65/p65 and reduced the production of mitochondrial reaction oxygen species (mitoROS) in THP-1 cells. Inhibition of PI3K or AKT could also reduced atherosclerosis lesion and plaque area, and decreased the levels of NLRP3 and IL-1β in ApoE -/- mice. The effect of PI3K inhibition was more significant than AKT. Therefore, PI3K inhibition can retard the progress of atherosclerosis. Besides, there may be other AKT-independent pathways that regulate the formation of atherosclerosis.

Akt signaling has been associated with adult neurogenesis in the hippocampal dentate gyrus (DG). We reported cognitive dysfunction in Akt3 knockout (Akt3-KO) mice with the down-regulation of mTOR activation. However, little is known about the effects of Akt3 signaling on hippocampal neurogenesis. Herein, we show that progenitor cells, neuroblasts, and mature newborn neurons in hippocampal DG expressed Akt3 protein. The Akt3 phosphorylation in hippocampal DG was increased after voluntary wheel running for 7 days in wild-type mice (running WT mice), but not in Akt3-KO mice (running Akt3-KO mice). Subsequently, we observed that the proliferation of progenitor cells was suppressed in Akt3-KO mice and the mTOR inhibitor rapamycin-treated mice, whereas enhanced in running WT mice rather than running Akt3-KO mice. Neurite growth of neuroblasts was impaired in Akt3-KO mice and rapamycin-treated mice. In contrast, neither differentiation of progenitor cells nor migrating of newly generated neurons was altered in Akt3-KO mice or running WT mice. The levels of p70S6K and 4EBP1 phosphorylation were declined in Akt3-KO mice and elevated in running WT mice depending on mTOR activation. Furthermore, telomerase activity, telomere length, and expression of telomerase reverse transcriptase (TERT) were decreased in Akt3-KO mice but increased in running WT mice rather than running Akt3-KO mice, which required the mTOR activation. The study provides in vivo evidence that Akt3-mTOR signaling plays an important role in the proliferation of progenitor cells and neurite growth through positive regulated TERT expression and activation of p70S6K and 4EBP1., (© 2021 International Society for Neurochemistry.)

Maintaining an intact pool of neural progenitor cells (NPCs) is crucial for generating new and functionally active neurons. Methamphetamine (METH) can exacerbate the HIV-induced deficit of adult neurogenesis; however, potential mechanisms of this influence are still poorly understood. In the present study, we present evidence that chronic exposure to METH combined with brain infection by EcoHIV results in enhanced proliferation of NPCs in the subventricular zone (SVZ) in mice. This effect was long-lasting as it was preserved ex vivo in NPCs isolated from the exposed mice over several passages in the absence of additional treatments. Increased proliferation in response to METH plus HIV was associated with dysregulation of cyclin B1 and cyclin D. Transcriptomic studies indicated that 27 out of the top 30 differentially expressed genes in response to METH plus EcoHIV were targets of the forkhead box O transcriptional factor (FOXO) and primarily FOXO3. Additional ex vivo studies and in vitro experiments using human NPCs exposed to METH and infected with HIV revealed upregulation of the CXCL12-CXCR4 axis, leading to activation of downstream pAkt and pErk, the pathways that can phosphorylate FOXO3 and force its exports from the nuclei into the cytoplasm. Indeed, nuclear expulsion of FOXO3 was demonstrated both in mice exposed to METH and infected with EcoHIV and in cell cultures of human NPCs. These results provide novel information that exposure to METH combined with HIV infection can induce aberrant proliferation of SVZ-derived NPCs and identifies CXCL12-CXCR4-Akt-1-mediated phosphorylation of FOXO3 as the mechanism responsible for this effect., (© 2021. The Author(s).)

The influence of long-term tacrolimus treatment on cognitive function remains to be elucidated. Using a murine model of chronic tacrolimus neurotoxicity, we evaluated the effects of tacrolimus on cognitive function, synaptic balance, its regulating protein (Klotho), and oxidative stress in the hippocampus. Compared to vehicle-treated mice, tacrolimus-treated mice showed significantly decreased hippocampal-dependent spatial learning and memory function. Furthermore, tacrolimus caused synaptic imbalance, as demonstrated by decreased excitatory synapses and increased inhibitory synapses, and downregulated Klotho in a dose-dependent manner; the downregulation of Klotho was localized to excitatory hippocampal synapses. Moreover, tacrolimus increased oxidative stress and was associated with activation of the PI3K/AKT pathway in the hippocampus. These results indicate that tacrolimus impairs cognitive function via synaptic imbalance, and that these processes are associated with Klotho downregulation at synapses through tacrolimus-induced oxidative stress in the hippocampus., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract: Sepsis is an amplified systemic immune-inflammatory response produced by a microorganism, which involves activation of inflammatory cytokine signaling pathways and oxidative stress. A variety of studies have shown that hydralazine (HDZ) has potent antioxidant and anti-inflammatory proprieties. Therefore, we hypothesize that HDZ can improve the clinical outcome of sepsis. Thus, this work aimed to evaluate therapeutic value of HDZ in reducing inflammatory response, oxidative stress, and mortality in animal sepsis, and to investigate its possible mechanism of action. Sepsis was induced by the cecal ligation and puncture (CLP) method in Wistar rats. After surgery, the animals were randomly divided into three groups: sham, sepsis, and sepsis + HDZ (1 mg/kg, s.c.). All groups were monitored for 48 h to assess survival rate, and clinical, hemodynamic, biochemical, and cellular parameters. After euthanasia, blood, spleen, liver, and kidneys were collected for analysis. Blood serum cytokines, tissue myeloperoxidase (MPO) activity, and oxidative stress parameters were assessed. Involvement of the PI3K/Akt pathway was also investigated. Sepsis was successfully induced by the CLP technique. HDZ treatment increased the survival rate (from 50% to 90%), improved glycemia control, reduced the clinical severity sepsis and mean arterial pressure; and prevented increased MPO activity, TNF-α, IL-1β, IL-10 levels, and oxidative damage markers. Additionally, HDZ significantly prevented the increase of Akt activation in the liver and kidney. HDZ largely mitigated the effects of sepsis by suppressing inflammatory and antioxidant responses via the PI3K/Akt pathway. These findings provide evidence that HDZ can be a new therapeutic alternative for treating sepsis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 by the Shock Society.)

Hyperactivity of serine-threonine kinase AKT is one of the most common molecular abnormalities in cancer, where it contributes to poor outcomes by facilitating the growth and survival of malignant cells. Despite its well-documented anti-apoptotic effects, hyperactivity of AKT is also known to be stressful to a cell. In an attempt to better elucidate this phenomenon, we observed the signs of proteotoxic stress in cells that harbor hyperactive AKT or have lost its principal negative regulator, PTEN. The activity of HSF1 was predictably elevated under these circumstances. However, such cells proved more sensitive to various regimens of heat shock, including the conditions that were well-tolerated by syngeneic cells without AKT hyperactivity. The sensitizing effect of hyperactive AKT was also seen in HSF1-deficient cells, suggesting that the phenomenon does not require the regulation of HSF1 by this kinase. Notably, the elevated activity of AKT was accompanied by increased levels of XBP1, a key component of cell defense against proteotoxic stress. Interestingly, the cells harboring hyperactive AKT were also more dependent on XBP1 for their growth. Our observations suggest that proteotoxic stress conferred by hyperactive AKT represents a targetable vulnerability, which can be exploited by either elevating the stress above the level tolerated by such cells or by eliminating the factors that enable such tolerance.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Acute myeloid leukemia (AML) is associated with a poor prognosis in elderly adults and currently lacks optimal treatment strategies. MicroRNAs (miRNAs or miRs) have increasingly been reported to be associated with AML progression; however, the mechanisms of action of miR‑93 in AML with the involvement of disabled 2 (DAB2) are currently unknown. In the present study, miR‑93 expression was assessed in patients with AML and in AML cell lines. The association between miR‑93 expression and the pathological characteristics of patients with AML was analyzed. AML cells were then transfected to knockdown or overexpress miR‑93 in order to elucidate its function in AML progression. The target gene of miR‑93 was assessed using a dual‑luciferase reporter gene assay. The expression levels of miR‑93, DAB2 and phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) pathway‑related proteins were measured and in vivo experiments were conducted to confirm the results. It was observed that miR‑93 was highly expressed in patients with AML and in AML cells. The knockdown of miR‑93 in HL‑60 cells inhibited AML cell proliferation and resistance to apoptosis, while the overexpression of miR‑93 in THP‑1 cells led to contrasting results. Moreover, miR‑93 targeted DAB2 to inactivate the PI3K/AKT pathway, and the overexpression of DAB2 reversed the effects of miR‑93 on THP‑1 cell growth. Tumor volume, tumor weight, and the positive expression of Ki67, survivin and p53 were increased in THP‑1 cells overexpressing miR‑93. On the whole, the present study demonstrates that miR‑93 is highly expressed in AML cells, and that the suppression of miR‑93 inhibits AML cell growth by targeting DAB2 and inhibiting the PI3K/AKT pathway.

The voltage-sensitive sodium channel Na V 1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate Na V 1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of Na V 1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na + currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing Na V 1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous Na V 1.1 in primary cultures were identified by expressing a fluorescent protein under the Na V 1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3'-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell-specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.

Dendritic cells (DCs) are composed of multiple lineages of hematopoietic cells and orchestrate immune responses upon detecting the danger and inflammatory signals associated with pathogen and damaged tissues. Under steady-state, DCs are maintained at limited numbers and the functionally quiescent status. While it is known that a fine balance in the DC homeostasis and activation status is also important to prevent autoimmune diseases and hyperinflammation, mechanisms that control DC development and activation under stead-state remain not fully understood. Here we show that DC-specific ablation of CBL and CBL-B (CBL -/- CBL-B -/- ) leads to spontaneous liver inflammation and fibrosis and early death of the mice. The mutant mice have a marked expansion of classic CD8α + /CD103 + DCs (cDC1s) in peripheral lymphoid organs and the liver. These DCs exhibit atypical activation phenotypes characterized by an increased production of inflammatory cytokines and chemokines but not the cell surface MHC-II and costimulatory ligands. While the mutant mice also have massive T cell activation, lymphocytes are not required for the disease development. The CBL -/- CBL-B -/- mutation enhances FLT3-mTOR signaling, due to defective FLT3 ubiquitination and degradation. Blockade of FLT3-mTOR signaling normalizes the homeostasis of cDC1s and attenuates liver inflammation. Our result thus reveals a critical role of CBLs in the maintenance of DC homeostasis and immune quiescence. This regulation could be relevant to liver inflammatory diseases and fibrosis in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tong, Li, Zhang, Gong, Sun, Calderon, Zhang, Li, Gadzinski, Langdon, Reizis, Zou and Gu.)

Background: Gastrointestinal motility disorder is a common gastrointestinal disease, which seriously affects life quality. Traditional Chinese medicine (TCM) has been widely used as an alternative therapy for gastrointestinal motility disorder. Acacetin is a natural flavonoid compound that has antioxidant and anti-inflammatory, antidepressant, and anticancer properties. However, the efficacy of Acacetin in the treatment of gastrointestinal motility disorders has not been studied. Our aim was to investigate the mechanism of Acacetin-alleviated gastrointestinal motility disorder and its efficacy based on network pharmacology., Methods: We performed network pharmacology to predict the active components, match Weishu decoction (WSD) targets in gastrointestinal motility disorders, and investigate its potential pharmacological mechanisms. We performed the GO and KEGG enrichment analysis. In vivo , we investigated the effects of Acacetin in the gastrointestinal motility disorder model., Results: Based on network pharmacological method, the key active ingredient of WSD was identified as Acacetin, and the enrichment signaling pathway was the PI3K-AKT signaling pathway. Acacetin and Mosapride accelerated gastric emptying time, reduced gastric remnant rate, and increased small intestinal propulsion rate. The levels of GAS and MTL were increased after using Acacetin. These results indicated that Acacetin could improve gastrointestinal motility disorders. Among them, high-dose Acacetin showed a better effect. Acacetin could regulate protein and lipid metabolism in mice with gastrointestinal motility disorder. Furthermore, Acacetin could modulate gastrointestinal inflammation and apoptosis. The detection of the PI3K-AKT signaling pathway-related proteins showed that Acacetin improved gastrointestinal motility disorder by inhibiting the activation of the PI3K-AKT signaling pathway., Conclusion: The key ingredient Acacetin in WSD could alleviate gastrointestinal motility disorder by inhibiting the activation of the PI3K-AKT signaling pathway based on network pharmacology analysis. The efficacy and safety of Acacetin treatment provide strong experimental support for the clinical treatment of gastrointestinal motility disorder., Competing Interests: All authors claimed that there was no potential conflict of interest between them., (Copyright © 2021 Xuan Guo et al.)

Aeromonas sobria, a Gram-negative bacterium that can colonize both humans and animals, is found in a variety of environments, including water, seafood, meat, and vegetables (Cahill, 1990; Galindo et al., 2004; Song et al., 2019). Aeromonas spp. are conditionally pathogenic bacteria in aquaculture, which can rapidly proliferate, causing disease and even death in fish, especially when the environment is degraded (Neamat-Allah et al., 2020, 2021a, 2021b). In developing countries, Aeromonas spp. have been associated with a wide spectrum of infections in humans, including gastroenteritis, wound infections, septicemia, and lung infections (San Joaquin and Pickett, 1988; Wang et al., 2009; Su et al., 2013). Infections caused by Aeromonas spp. are usually more severe in immunocompromised individuals (Miyamoto et al., 2017). The presence of a plasmid encoding a β‍-lactamase in A. sobria that confers resistance to β-lactam antibiotics poses a huge challenge to the treatment of diseases caused by this microorganism (Lim and Hong, 2020). Consequently, an in-depth understanding of the interaction between A. sobria and its hosts is urgently required to enable the development of effective strategies for the treatment of A. sobria infections.

The present study evaluated whether epigallocatechin-3-gallate (EGCG) effectively attenuates tumor growth in colon cancer cells and in the xenografts of nude mice and investigated the underlying mechanisms by focusing on the sonic hedgehog (Shh) and phosphoinositide 3-kinase (PI3K) pathways. Three kinds of colon cancer cells and BALB/c nude mice were used to evaluate the antiproliferative effect of EGCG. The apoptosis, migration, and invasion of colon cancer cells were analyzed to explore the toxicity effect of EGCG on colon cancer cells. Western blotting was used to demonstrate the expression levels of related proteins. The results showed that EGCG exhibited an antiproliferative effect against colon cancer cells in a dose-dependent manner with low toxicity against normal colon epithelial cells. Administration of EGCG caused significant apoptosis and inhibited the migration and invasion of colon cancer cells. The toxic effect of EGCG on colon cancer cells was accompanied by downregulation of the Shh and PI3K/Akt pathways. In addition, EGCG reduced tumor volume and weight without affecting the body weight of nude mice and inhibited the activation of the Shh and PI3K/AKT pathways in tumor tissue. Further study showed that purmorphamine (smoothened (Smo) agonist) or insulin like growth factor-1 (IGF-1, PI3K agonist) partly abolished the effect of EGCG on cell proliferation, migration, and apoptosis. Cyclopamine (Smo inhibitor) and LY294002 (PI3K inhibitor) showed the similar toxic effects as EGCG on colon cancer cells. In conclusion, EGCG inhibited colon tumor growth via downregulation of the Shh and PI3K pathways and may be a potential chemotherapeutic agent against colon cancer.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor β1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphor-AKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and -2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: This study aimed to investigate the effects of miR-29b on renal interstitial fibrosis in the obstructed kidney of mouse with unilateral ureteral obstruction (UUO) via inhibiting phosphatidylinositol 3-kinase/protein kinaseB (PI3K/AKT) signaling pathway., Methods: Adult male CD-1 mice were intraperitoneally injected with vehicle or PI3K inhibitor LY294002 (3 mg/kg, 30 mg/kg) daily for 1 or 2 weeks after performing UUO or sham operation. The mice were sacrificed on days 7 and 14 after surgery. The rat proximal tubular epithelial cell (TEC) line NRK-52E was cultured in DMEM and treated with various concentrations angiotensin II (AngII). Obstructed and sham mouse kidneys were analyzed via HE, Masson and immunohistochemistry to assess the degree of renal fibrosis. Real-time quantitative polymerase chain reaction assays (RT-PCR) were performed to investigate changes in the levels of expression of miR-29b and Western blot was used to analyze the activation of PI3K/AKT signaling and expression of E-cadherin, α-smooth muscle actin (α-SMA)., Results: Histologic analyses of obstructed kidney revealed that LY294002 attenuated the degree of renal fibrosis. In this study, loss of miR-29b accompanied with increased epithelial-mesenchymal transition (EMT) was observed in renal tubules of mice after UUO and cultured NRK-52E cells exposed to AngII. LY294002 also prominently decreased phosphorylation of AKT in vivo and vitro. By RT-PCR and Western blot analysis, LY294002 blocked the PI3K/AKT-induced loss of E-cadherin expression and de novo increase of the expression of α-SMA in a time- and dose-dependent manner. The overexpression of miR-29b markedly reversed the phenotype induced by AngII in NRK-52E cells and the downregulation miR-29b expression with an miR-29b inhibitor resulted in enhanced EMT. In addition, the PI3K/AKT signaling pathway was found to be suppressed in the presence of overexpression of miR-29b by direct hybridization with 3'-untranslated region (3'-UTR) of PIK3R2., Conclusion: Our findings suggested that miR-29b significantly prevented tubulointerstitial injury in mouse model of UUO by attenuating renal tubular epithelial cell-mesenchymal transition via repressing PI3K/AKT signaling pathway., (© 2021. The Author(s).)

Purpose: To clarify how ZCCHC14 affects the development of hepatocellular carcinoma (HCC)., Methods: Differential levels of ZCCHC14 in HCC tissues and cells were examined. Proliferative and migratory changes in HCC cells with overexpression or knockdown of ZCCHC14 were detected using 5-Ethynyl-2'- deoxyuridine (EdU) and Transwell assay, respectively. Expression changes of p-Akt/Akt, p-GSK3β/GSK3β and β-catenin in HCC cells mediated by ZCCHC14 were determined. Intervened by the p-Akt activator SC79 or overexpression of β-catenin, further validated the involvement of the Akt/GSK3β/β-catenin signaling in HCC cell phenotypes mediated by ZCCHC14., Results: Upregulated ZCCHC14 in HCC accelerated in vitro proliferative potential of HCC cells. Knockdown of ZCCHC14 inactivated the Akt/GSK3β/β-catenin signaling and inhibited malignant phenotypes of HCC, which were partially reversed by SC79 induction or overexpression of β-catenin., Conclusions: By activating the Akt/GSK3β/β-catenin signaling, ZCCHC14 accelerates HCC cells proliferation.

Purpose: The extent to which routine genomic sequencing can identify relevant secondary genomic alterations among BRAF V600E -mutant papillary thyroid carcinoma (PTC) is unknown. Such markers would prove highly valuable for prognostic purposes., Experimental Design: We reviewed clinicopathologic data of 225 patients with BRAF V600E -mutant PTC and integrated them with genomic data derived from targeted next-generation sequencing (NGS) on tumor specimens. We defined patient subgroups based on bona fide secondary oncogenic events (separate from BRAF V600E ) and compared their clinical features and outcomes with those without additional oncogenic alterations., Results: Additional oncogenic alterations were identified in 16% of tumors. Patients in the " BRAF +additional mutations" group were more likely to be at high American Thyroid Association (ATA) risk of recurrence (48.6% vs. 17.6%; P = 0.0009), had larger baseline tumor (2.7 vs. 1.9 cm; P = 0.0005) and more advanced stage at presentation (14.3% vs. 1.1% stage 4; P < 0.0001). Importantly, over a 65-month follow-up, disease-specific mortality (DSM) was increased when additional mutations were identified (13.8% vs. 1.4% in the BRAF -only group; P = 0.005). Separately, we identified a subcluster of patients harboring oncogenic mutations in key effectors of the PI3K/AKT/mTOR pathway, which were independently associated with DSM (OR = 47.9; 95% confidence interval, 3.5-1,246.5; P = 0.0043)., Conclusions: Identification of additional PIK3/AKT/mTOR alterations in patients with BRAF V600E -mutant PTC provides important and actionable prognostic risk stratification. These data support genomic profiling of PTC tumors to inform prognosis and clinical strategy., (©2021 American Association for Cancer Research.)

Background: Huai Hua San (HHS), a famous Traditional Chinese Medicine (TCM) formula, has been widely applied in treating ulcerative colitis (UC). However, the interaction of bioactives from HHS with the targets involved in UC has not been elucidated yet., Aim: A network pharmacology-based approach combined with molecular docking and in vitro validation was performed to determine the bioactives, key targets, and potential pharmacological mechanism of HHS against UC., Materials and Methods: Bioactives and potential targets of HHS, as well as UC-related targets, were retrieved from public databases. Crucial bioactive ingredients, potential targets, and signaling pathways were acquired through bioinformatics analysis, including protein-protein interaction (PPI), as well as the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Subsequently, molecular docking was carried out to predict the combination of active compounds with core targets. Lastly, in vitro experiments were conducted to further verify the findings., Results: A total of 28 bioactive ingredients of HHS and 421 HHS-UC-related targets were screened. Bioinformatics analysis revealed that quercetin, luteolin, and nobiletin may be potential candidate agents. JUN, TP53, and ESR1 could become potential therapeutic targets. PI3K-AKT signaling pathway might play an important role in HHS against UC. Moreover, molecular docking suggested that quercetin, luteolin, and nobiletin combined well with JUN, TP53, and ESR1, respectively. Cell experiments showed that the most important ingredient of HHS, quercetin, could inhibit the levels of inflammatory factors and phosphorylated c-Jun, as well as PI3K-Akt signaling pathway in LPS-induced RAW264.7 cells, which further confirmed the prediction by network pharmacology strategy and molecular docking., Conclusion: Our results comprehensively illustrated the bioactives, potential targets, and molecular mechanism of HHS against UC. It also provided a promising strategy to uncover the scientific basis and therapeutic mechanism of TCM formulae in treating diseases., Competing Interests: The authors declare no conflicts of interest for this work., (© 2021 Liu et al.)

The coiled-coil domain containing protein members have been well documented for their roles in many diseases including cancers. However, the function of the coiled-coil domain containing 65 (CCDC65) remains unknown in tumorigenesis including gastric cancer. Methods: CCDC65 expression and its correlation with clinical features and prognosis of gastric cancer were analyzed in tissue. The biological role and molecular basis of CCDC65 were performed via in vitro and in vivo assays and a various of experimental methods including co-immunoprecipitation (Co-IP), GST-pull down and ubiquitination analysis et al. Finally, whether metformin affects the pathogenesis of gastric cancer by regulating CCDC65 and its-mediated signaling was investigated. Results : Here, we found that downregulated CCDC65 level was showed as an unfavourable factor in gastric cancer patients. Subsequently, CCDC65 or its domain (a.a. 130-484) was identified as a significant suppressor in GC growth and metastasis in vitro and in vivo. Molecular basis showed that CCDC65 bound to ENO1, an oncogenic factor has been widely reported to promote the tumor pathogenesis, by its domain (a.a. 130-484) and further promoted ubiquitylation and degradation of ENO1 by recruiting E3 ubiquitin ligase FBXW7. The downregulated ENO1 decreased the binding with AKT1 and further inactivated AKT1, which led to the loss of cell proliferation and EMT signal. Finally, we observed that metformin, a new anti-cancer drug, can significantly induce CCDC65 to suppress ENO1-AKT1 complex-mediated cell proliferation and EMT signals and finally suppresses the malignant phenotypes of gastric cancer cells. Conclusion: These results firstly highlight a critical role of CCDC65 in suppressing ENO1-AKT1 pathway to reduce the progression of gastric cancer and reveals a new molecular mechanism for metformin in suppressing gastric cancer. Our present study provides a new insight into the mechanism and therapy for gastric cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3β (GSK-3β), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.

Purpose: Endometrial cancer is the most common malignant tumor of female genital system worldwide. Homeobox A11 (HOXA11) is an evolutionarily conserved Homeobox gene closely implicated in carcinogenesis. However, the mechanisms of HOXA11 in the progression and cisplatin resistance of endometrial cancer remain unclear., Methods: The expression of HOXA11 was analyzed based on 548 endometrial cancer and 35 control tissues from The Cancer Genome Atlas (TCGA) database. Transwell assay was performed to investigate the effect of HOXA11 on endometrial cell migration and invasion. TUNEL staining was carried out to assay the role of HOXA11 in endometrial cell apoptosis. Western blot was employed to detect the protein levels of B cell lymphoma-2 (Bcl-2), Bcl-2 associated X (Bax), cleaved caspase-3, matrix metalloproteinase-2/9 (MMP/9), phosphatase and tensin homolog (PTEN), protein kinase B (AKT) and p-AKT., Results: TCGA data showed that HOXA11 expression was significantly down-regulated in endometrial cancer tissue samples. The overexpression of HOXA11 promoted the apoptosis, but inhibited the proliferation, migration and invasion of endometrial cancer cells. HOXA11 knockdown with small interfering RNA (siRNA) considerably repressed cell apoptosis, while promoted cell proliferation, migration, and invasion through PTEN/AKT signaling pathway. Interestingly, HOXA11 was lowly expressed in Ishikawa cells treated with cisplatin. In addition, HOXA11 knockdown increased the resistance of endometrial cancer to cisplatin through activating PTEN/AKT signaling pathway., Conclusion: Low HOXA11 expression may promote the proliferation, migration, invasion of endometrial cancer cells, and increase their resistance to cisplatin through activating PTEN/AKT pathway.

Cancer stem cells (CSCs) are regarded as essential targets to overcome tumor progression and therapeutic resistance; however, practical targeting approaches are limited. Here, we identify testis-specific Y-like protein 5 (TSPYL5) as an upstream regulator of CSC-associated genes in non-small cell lung cancer cells, and suggest as a therapeutic target for CSC elimination. TSPYL5 elevation is driven by AKT-dependent TSPYL5 phosphorylation at threonine-120 and stabilization via inhibiting its ubiquitination. TSPYL5-pT120 also induces nuclear translocation and functions as a transcriptional activator of CSC-associated genes, ALDH1 and CD44. Also, nuclear TSPYL5 suppresses the transcription of PTEN, a negative regulator of PI3K signaling. TSPYL5-pT120 maintains persistent CSC-like characteristics via transcriptional activation of CSC-associated genes and a positive feedback loop consisting of AKT/TSPYL5/PTEN signaling pathway. Accordingly, elimination of TSPYL5 by inhibiting TSPYL5-pT120 can block aberrant AKT/TSPYL5/PTEN cyclic signaling and TSPYL5-mediated cancer stemness regulation. Our study suggests TSPYL5 be an effective target for therapy-resistant cancer.

The loss of auditory sensory hair cells (HCs) is the most common cause of sensorineural hearing loss (SNHL). As the main sound transmission structure in the cochlea, it is necessary to maintain the normal shape and survival of HCs. In this review, we described and summarized the signaling pathways that regulate the development and survival of auditory HCs in SNHL. The role of the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channels, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant. The molecular interactions of these signaling pathways play an important role in the survival of HCs, which may provide a theoretical basis and possible therapeutic interventions for the treatment of hearing loss., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Yao Liu et al.)

Gene expression profiling has been broadly performed in the field of cancer research. This study aims to explore the key gene regulatory network and focuses on the functions of microRNA (miR)-216a in pancreatic cancer (PC). PC datasets GSE15471, GSE16515, and GSE32676 were used to screen the differentially expressed genes (DEGs) in PC. A miRNA microarray analysis and gene oncology analysis suggested miR-216a as an important differentially expressed miRNA in PC. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that miR-216a and the DEGs are largely enriched on the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. miR-216a targeted Wilms Tumor 1 (WT1), while WT1 promoted transcription activity of keratin 7 (KRT7). Upregulation of miR-216a reduced proliferation and invasiveness of PC cells, while further upregulation of WT1 blocked the functions of miR-216a. Silencing of KRT7 diminished the oncogenic role of WT1. The in vitro results were reproduced in vivo. High expression of miR-216a while poor expression of WT1 indicated better prognosis of PC patients. The miR-216a/WT1/KRT7 axis influenced the activity of the PI3K/AKT pathway. To conclude, this study evidenced that miR-216a suppressed WT1 expression and blocked KRT7 transcription, which inactivated the PI3K/AKT signaling and reduced PC progression., (© 2021 International Union of Biochemistry and Molecular Biology.)

Intervertebral discs are important for maintaining mobility and offer support to the body trunk. If these discs lose their biomechanical features, lower back pain can occur. We previously reported that hepatocyte growth factor (HGF) promotes cell proliferation and suppresses apoptosis, inflammation, and matrix degradation in nucleus pulposus (NP) cells. In the present study, we investigated the molecular mechanisms of how HGF promotes the proliferation of NP cells in hypoxic conditions. Hypoxic stimulation promoted modest cell proliferation, which was further upregulated by HGF. Expression of hypoxia-inducible factor (HIF-1α) protein, which contributes to the maintenance of homeostasis in NP cells, was also upregulated in hypoxia-treated cell groups; HGF further increased HIF-1α expression in NP cells. Additionally, knockdown of HIF-1α expression significantly reduced the proliferation of NP cells. An MAPK inhibitor inhibited the expression of HIF-1α and pERK, as well as cell proliferation in a dose-dependent manner. Similarly, inhibiting the PI3K/Akt and STAT3 pathways also decreased the expression of HIF-1α and cell proliferation. These results show that under hypoxic conditions, HGF promotes NP cell proliferation via HIF-1α-, MAPK-, PI3K/Akt-, and STAT3-mediated signaling which is involved in this pathway. The control of these signaling pathways may be a target for potential therapeutic strategies for the treatment of disc degeneration in hypoxic conditions., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background: Arteriovenous fistulae (AVF) are the preferred mode of vascular access for hemodialysis. Before use, AVF remodel by thickening and dilating to achieve a functional conduit via an adaptive process characterized by expression of molecular markers characteristic of both venous and arterial identity. Although signaling via EphB4, a determinant of venous identity, mediates AVF maturation, the role of its counterpart EphrinB2, a determinant of arterial identity, remains unclear. We hypothesize that EphrinB2 signaling is active during AVF maturation and may be a mechanism of venous remodeling., Methods: Aortocaval fistulae were created or sham laparotomy was performed in C57Bl/6 mice, and specimens were examined on Days 7 or 21. EphrinB2 reverse signaling was activated with EphB4-Fc applied periadventitially in vivo and in endothelial cell culture medium in vitro. Downstream signaling was assessed using immunoblotting and immunofluorescence., Results: Venous remodeling during AVF maturation was characterized by increased expression of EphrinB2 as well as Akt1, extracellular signal-regulated kinases 1/2 (ERK1/2), and p38. Activation of EphrinB2 with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and was associated with increased diameter and wall thickness in the AVF. Both mouse and human endothelial cells treated with EphB4-Fc increased phosphorylation of EphrinB2, endothelial nitric oxide synthase, Akt1, ERK1/2, and p38 and increased endothelial cell tube formation and migration., Conclusions: Activation of EphrinB2 signaling by EphB4-Fc was associated with adaptive venous remodeling in vivo while activating endothelial cell function in vitro. Regulation of EphrinB2 signaling may be a new strategy to improve AVF maturation and patency., (Published by Elsevier Inc.)

Purpose: This study aims to elucidate the biological functions of CDCA2 (cell division cycle associated 2) in hepatocellular carcinoma (HCC) progression and the potential mechanism., Methods: CDCA2 levels in HCC tissues and cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between CDCA2 and clinical characteristics in HCC patients was analyzed. Cox proportional-hazards model was applied for assessing the potential factors influencing overall survival in HCC. Three CDCA2 siRNAs were generated and the most effective one was used in the following experiments. After knockdown of CDCA2 in HCC-LM3 cells, clonality and viability were examined. Meanwhile, cell cycle progression was detected by flow cytometry. Relative levels of CDCA2, p21, p27, CDK2, CCND1, CCNE1 and CCNB1 in HCC-LM3 cells were determined by qRT-PCR. The activation of the protein kinase B (Akt) signaling was examined by Western blot. Subsequently, we constructed HCC xenograft model in nude mice. Tumor volume and tumor weight of xenografted HCC were recorded., Results: CDCA2 was upregulated in HCC tissues than that of para-tumor ones, especially HCC tissues with larger than 5 cm in tumor size or vascular invasion. CDCA2 level was related to tumor size, vascular invasion and tumor differentiation in HCC. Knockdown of CDCA2 inhibited clonality and viability in HCC-LM3 cells, and arrested cell cycle progression in G1 phase via downregulating CCND1. The phosphatidilinositol 3-kinase (PI3K)/Akt was activated by CDCA2 during the progression of HCC. Tumor volume and tumor weight of xenografted HCC decreased in nude mice with in vivo knockdown of CDCA2., Conclusions: CDCA2 triggers proliferative potential in HCC by targeting CCND1 via activating the PI3K/Akt signaling.

Either apigenin or chrysin alone has been found to exert anti-inflammatory and tumor suppressive effect. However, the combined effect of apigenin and chrysin on colorectal cancer (CRC) has not been fully clarified. We attempted to explore the effect of chrysin and apigenin on CRC and its related mechanism. SW480 and HCT-116 cells were treated with either apigenin or chrysin alone or two-drug combination at different doses of 5, 25, 50, 100 μM for optimal concentration determination. Then, we focused on the individual and combined effect of apigenin and chrysin on clonogenicity, apoptosis, metastasis-related behaviors of CRC cells by colony formation assay, cell scratch assay, flow cytometry, and transwell assay. The changes of the activation of P38-MAPK/AKT pathway were evaluated underlying apigenin and chrysin intervention, further after co-treated with P38-MAPK agonist anisomycin. Apigenin (25 μM) combined with chrysin (25 μM) were determined to be optimal. Treatment with the combination of apigenin (25 μM) and chrysin (25 μM) significantly reduced cell clone numbers, migration, and invasion ability, while increased the cell apoptosis in both CRC cell lines. The combined effect was higher than chrysin or apigenin alone. Meanwhile, p-P38 and p-AKT were significantly downregulated by chrysin and apigenin treatment. The tumor inhibitive effect of apigenin combined with chrysin was obviously reversed by adding P38 agonist, anisomycin. Apigenin (25 μM) combined with chrysin (25 μM) showed synergetic effect in inhibiting the growth and metastasis of CRC cells by suppressing the activity of P38-MAPK/AKT pathway., (© 2021 International Union of Biochemistry and Molecular Biology.)

The aim of this work was to investigate whether Caveolin-1 (Cav-1), a membrane scaffolding protein widely implicated in cancer, may play a role in radiation response in rhabdomyosarcoma (RMS), a pediatric soft tissue tumor. For this purpose, we employed human RD cells in which Cav-1 expression was stably increased via gene transfection. After radiation treatment, we observed that Cav-1 limited cell cycle arrest in the G2/M phase and enhanced resistance to cell senescence and apoptosis via reduction of p21 Cip1/Waf1 , p16 INK4a and Caspase-3 cleavage. After radiotherapy, Cav-1-mediated cell radioresistance was characterized by low accumulation of H2AX foci, as confirmed by Comet assay, marked neutralization of reactive oxygen species (ROS) and enhanced DNA repair via activation of ATM, Ku70/80 complex and DNA-PK. We found that Cav-1-overexpressing RD cells, already under basal conditions, had higher glutathione (GSH) content and greater catalase expression, which conferred protection against acute treatment with hydrogen peroxide. Furthermore, pre-treatment of Cav-1-overexpressing cells with PP2 or LY294002 compounds restored the sensitivity to radiation treatment, indicating a role for Src-kinases and Akt pathways in Cav-1-mediated radioresistance. These findings were confirmed using radioresistant RD and RH30 lines generated by hypofractionated radiotherapy protocol, which showed marked increase of Cav-1, catalase and Akt, and sensitivity to PP2 and LY294002 treatment. In conclusion, these data suggest that concerted activity of Cav-1 and catalase, in cooperation with activation of Src-kinase and Akt pathways, may represent a network of vital mechanisms that allow irradiated RMS cells to evade cell death induced by oxidative stress and DNA damage., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Previous work showed a link between Tie2 + nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% ( p = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% ( p = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 ( p = 0.01). However, the addition of 1 μM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression ( p = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.

Background: Fluorochloridone (FLC), a selective pyrrolidone herbicide, has been recognized as a potential endocrine disruptor and reported to induce male reproductive toxicity, but the underlying mechanism is unclear. The aim of this study was to investigate the mechanism of FLC-induced reproductive toxicity on male mice with particular emphasis on the role of autophagy in mice' TM4 Sertoli cells., Methods: Adult C57BL/6 mice were divided into one control group (0.5% sodium carboxymethyl cellulose), and four FLC-treated groups (3,15,75,375 mg/kg). The animals (ten mice per group) received gavage for 28 days. After treatment, histological analysis, sperm parameters, the microstructure of autophagy and the expression of autophagy-associated proteins in testis were evaluated. Furthermore, to explore the autophagy mechanism, TM4 Sertoli cells were treated with FLC (0,40,80,160 μM) in vitro for 24 h. Cell activity and cytoskeletal changes were measured by MTT assay and F-actin immunofluorescence staining. The formation of autophagosome, accumulation of reactive oxygen species (ROS), expression of autophagy marker proteins (LC3, Beclin-1 and P62) and AKT-related pathway proteins (AKT, mTOR) were observed. The ROS scavenger N-acetylcysteine (NAC) and AKT agonist (SC79) were used to treat TM4 cells to observe the changes of AKT-mTOR pathway and autophagy., Results: In vivo, it showed that FLC exposure caused testicular injuries, abnormality in epididymal sperm. Moreover, FLC increased the formation of autophagosomes, the accumulation of LC3II/LC3I, Beclin-1 and P62 protein, which is related to the degradation of autophagy. In vitro, FLC triggered TM4 cell autophagy by increasing the formation of autophagosomes and upregulating of LC3II/LC3I, Beclin-1 and P62 levels. In addition, FLC induced ROS production and inhibited the activities of AKT and mTOR kinases. The Inhibition of AKT/mTOR signaling pathways and the activation of autophagy induced by FLC could be efficiently reversed by pretreatment of NAC. Additionally, decreased autophagy and increased cell viability were observed in TM4 cells treated with SC79 and FLC, compared with FLC alone, indicating that FLC-induced autophagy may be pro-death., Conclusion: Taken together, our study provided the evidence that FLC promoted autophagy in TM4 Sertoli cells and that this process may involve ROS-mediated AKT/mTOR signaling pathways.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.

The pathological characteristics of osteoarthritis are cartilage matrix degradation, chondrocytes apoptosis, and low-grade inflammation of the joint. Recent studies have shown that blood vessels grow from the subchondral bone to the articular cartilage. However, the relationship among inflammation, angiogenesis, and chondrocyte apoptosis is still unclear. We found that chondrocytes could secrete chemokines and VEGF to promote the migration of vascular endothelial cells in response to TNF-α stimulation. The invasion of blood vessels leads to increased oxygen tension in the local environment, which increased the expression of SETD7 in chondrocytes by activating the JAK-STAT5 pathway. The bond of phosphorylated STAT5 and the specific locus in the promoter of SETD7 directly increased the transcription of SETD7. On the one hand, SETD7-regulated chemokine expression by forming a positive loop; on the other hand, SETD7-mediated chondrocyte apoptosis by inhibiting the nuclear localization of HIF-1α. In this study, we discovered a novel function of chondrocytes as mediators of inflammation and angiogenesis. Our study demonstrates that SETD7 is a potential molecular target to prevent OA development and progression., (© 2021 Federation of American Societies for Experimental Biology.)

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.