Kaempferol inhibited invasion and metastasis of gastric cancer cells by targeting AKT/GSK3ß pathway based on network pharmacology and molecular docking.

This study aims to explore the mechanisms of the inhibitory effect of kaempferol on the invasion and metastasis of gastric cancer (GC) cells through network pharmacology prediction and experimental verification. It identifies core targets via PPI network analysis and finds that kaempferol binds to these targets well. In vitro experiments showed that kaempferol could inhibit the proliferation, colony formation, migration and invasion of GC cells. Western blotting indicated kaempferol may reduce AKT and GSK3ß phosphorylation, leading to lower expression of invasion-related genes SRC, MMP9, CXCR4, KDR, and MMP2. Overall, kaempferol may prevent migration and invasion of GC cells via the AKT/GSK3ß signaling pathway.

Apigenin inhibits proliferation and differentiation of cardiac fibroblasts through AKT/GSK3ß signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge (S. miltiorrhiza) is an important Traditional Chinese herbal Medicine (TCM) used to treat cardio-cerebrovascular diseases. Based on the pharmacodynamic substance of S. miltiorrhiza, the aim of present study was to investigate the underlying mechanism of S. miltiorrhiza against cardiac fibrosis (CF) through a systematic network pharmacology approach, molecular docking and dynamics simulation as well as experimental investigation in vitro. MATERIALS AND METHODS: A systematic pharmacological analysis was conducted using the Traditional Chinese Medicine Pharmacology (TCMSP) database to screen the effective chemical components of S. miltiorrhiza, then the corresponding potential target genes of the compounds were obtained by the Swiss Target Prediction and TCMSP databases. Meanwhile, GeneCards, DisGeNET, OMIM, and TTD disease databases were used to screen CF targets, and a protein-protein interaction (PPI) network of drug-disease targets was constructed on S. miltiorrhiza/CF targets by Search Tool for the Retrieval of Interacting Genes/Proteins (STING) database. After that, the component-disease-target network was constructed by software Cytoscape 3.7. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for the intersection targets between drug and disease. The relationship between active ingredient of S. miltiorrhiza and disease targets of CF was assessed via molecular docking and molecular dynamics simulation. Subsequently, the underlying mechanism of the hub compound on CF was experimentally investigated in vitro. RESULTS: 206 corresponding targets to effective chemical components from S. miltiorrhiza were determined, and among them, there were 82 targets that overlapped with targets of CF. Further, through PPI analysis, AKT1 and GSK3ß were the hub targets, and which were both enriched in the PI3K/AKT signaling pathway, it was the sub-pathways of the lipid and atherosclerosis pathway. Subsequently, compound-disease-genes-pathways diagram is constructed, apigenin (APi) was a top ingredients and AKT1 (51) and GSK3ß (22) were the hub genes according to the degree value. The results of molecular docking and dynamics simulation showed that APi has strong affinities with AKT and GSK3ß. The results of cell experiments showed that APi inhibited cells viability, proliferation, proteins expression of α-SMA and collagen I/III, phosphorylation of AKT1 and GSK3ß in MCFs induced by TGFß1. CONCLUSION: Through a systematic network pharmacology approach, molecular docking and dynamics simulation, and confirmed by in vitro cell experiments, these results indicated that APi interacts with AKT and GSK3ß to disrupt the phosphorylation of AKT and GSK3ß, thereby inhibiting the proliferation and differentiation of MCFs induced by TGFß1, which providing new insights into the pharmacological mechanism of S. miltiorrhiza in the treatment of CF.

Pristimerin exhibits anti-cancer activity by inducing ER stress and AKT/GSK3ß pathway through increasing intracellular ROS production in human esophageal cancer cells.

Pristimerin (Pris), a bioactive triterpenoid compound extracted from the Celastraceae and Hippocrateaceae families, has been reported to exhibit an anti-cancer property on various cancers. However, the effects of Pris on esophageal cancer are poorly investigated. This current study sought to explore the activity and underlying mechanism of Pris against human esophageal squamous cell carcinoma (ESCC) cells. We demonstrated that Pris showed cytotoxicity in TE-1 and TE-10 ESCC cell lines, and significantly inhibited cell viability in a concentration dependent manner. Pris induced G0/G1 phase arrest and triggered apoptosis. It was also observed that the intracellular ROS level was remarkedly increased by Pris treatment. Besides, the function of Pris mediating the activation of ER stress and the inhibition of AKT/GSK3ß signaling pathway in TE-1 and TE-10 cells was further confirmed, which resulted in cell growth inhibition. And moreover, we revealed that all of the above pathways were regulated through ROS generation. In conclusion, our findings suggested that Pris might be considered as a novel natural compound for the developing anti-cancer drug candidate for human esophageal cancer.

The Protective Effects of Ecdysterone on Cognitive Impairment through Regulating Akt/GSK-3ß/Nrf2 Signaling Pathway and Oxidative Stress in Cognitive Mice Model and Aß-Induced Cell Neurotoxicity.

BACKGROUND: Severe neurological condition like Alzheimer's disease (AD) has a significantly negative impact on families and society, wherein there is no proven cure. As one of the principal active constituents of Achyranthes bidentata Blume, ecdysterone (ECR) has demonstrated antioxidant and cognitive dysfunction improvement effects. Nonetheless, the mechanism underlying the improvement of cognitive dysfunction by ECR remains unclear. This study sought to ascertain whether ECR may allebviate cognitive impairment by reducing oxidative stress via activation of the nuclear factor erythroid-2-related factor-2 (Nrf2) antioxidant system through Akt/GSK3ß pathway. METHODS: In terms of the experimental procedure, we determined the neuroprotective benefits of ECR in vivo via a cognitive impairment model of senescence-accelerated mouse prone 8 (SAMP8), we performed procedures such as behavioral testing, biochemical assaying, Nissl and TUNEL stainings, as well as flow cytometry, immunohistochemistry and western blotting. Furthermore, we investigated the underlying mechanistic action of ECR by activating PC12 cells with ß-amyloid peptide fragment 25-35 (Aß25-35). RESULTS: In vivo studies showed that ECR effectively improved cognitive impairment in SAMP8 via enhancement of learning and memory capabilities, but decreased oxidative stress, apoptosis and neuronal damage in the hippocampus. During the in vitro study, we observed that ECR dose-dependently reduced the oxidative stress and apoptosis that were induced in PC12 cells by Aß25-35. Additionally, the use of Akt inhibitors further established the potential of ECR to control Nrf2 through activation of the Akt/GSK3ß pathway and protect the PC12 cells from Aß25-35 induced damage. CONCLUSIONS: These findings offer proof that ECR reduces cognitive impairment by triggering the Nrf2 antioxidant system via the Akt/GSK3ß pathway and offer fresh information on ECR's potential as a promising therapeutic development candidate for AD.

Neurovascular protection of alisol A on cerebral ischemia mice through activating the AKT/GSK3ß pathway.

Alisol A, a triterpene isolated from Alisma Orientale, has been shown to exhibit anti-inflammatory effects and vascular protection. This study was designed to observe the effect of alisol A on cerebral ischemia (CI)-induced neurovascular dysfunction in the hippocampus and to further explore the potential mechanisms. The results showed that alisol A treatment improved the neurological deficits and cognitive impairment of CI mice. Alisol A reduced gliosis and improved neuronal/glial metabolism. Accordingly, alisol A inhibited inflammatory factors IL-6 and IL-1ß induced by overactivation of astrocytes and microglia, thus protecting the neurovasculature. Furthermore, alisol A promoted the survival of neurons by decreasing the ratio of Bax/Bcl-2, and protected brain microvascular endothelial cells (BMECs) by upregulating the expression of ZO-1, Occludin and CD31. The phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3ß (GSK3ß) increased after treatment with alisol A. To explore the underlying mechanism, AKT was inhibited. As expected, the neurovascular protection of alisol A above was eliminated by AKT inhibition. The present study primarily suggested that alisol A could exert neurovascular protection in the hippocampus of CI mice by activating the AKT/GSK3ß pathway and may potentially be used for the treatment of CI.

Multi-omics analysis reveals the mechanism of action of ophiopogonin D against pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with limited therapeutic strategies. Therefore, there is an urgent need to search for safe and effective drugs to treat this condition. Ophiopogonin D (OP-D), a steroidal saponin compound extracted from ophiopogon, possesses various pharmacological properties, including anti-inflammatory, antioxidant, and antitumor effects. However, the potential pharmacological effect of OP-D on pulmonary fibrosis remains unknown. PURPOSE: The aim of this study was to investigate whether OP-D can improve pulmonary fibrosis and to explore its mechanism of action. METHODS: The effect of OP-D on pulmonary fibrosis was investigated in vitro and in vivo using a mouse model of IPF induced by bleomycin and an in vitro model of human embryonic lung fibroblasts induced by transforming growth factor-ß1 (TGF-ß1). The mechanism of action of OP-D was determined using multi-omics techniques and bioinformatics. RESULTS: OP-D attenuated epithelial-mesenchymal transition and excessive deposition of extracellular matrix in the lungs, promoted the apoptosis of lung fibroblasts, and blocked the differentiation of lung fibroblasts into myofibroblasts. The multi-omics techniques and bioinformatics analysis revealed that OP-D blocked the AKT/GSK3ß pathway, and the combination of a PI3K/AKT inhibitor and OP-D was effective in alleviating pulmonary fibrosis. CONCLUSION: This study demonstrated for the first time that OP-D can reduce lung inflammation and fibrosis. OP-D is thus a potential new drug for the prevention and treatment of pulmonary fibrosis.

Role of insulin-like growth factor 1 (IGF1) in the regulation of mitochondrial bioenergetics in zebrafish oocytes: lessons from in vivo and in vitro investigations.

Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished reproductive potential and impaired fertility. The role of the intra-ovarian IGF system in ovarian follicular dynamics has been implicated earlier. Although several studies have demonstrated the role of the IGF axis in facilitating mitochondrial function over a multitude of cell lines, its role in oocyte energy metabolism remains largely unexplored. Here using zebrafish, the relative importance of IGF1 in modulating oocyte mitochondrial bioenergetics has been investigated. A dramatic increase in ovarian lhcgr and igf1 expression accompanied heightened ATP levels and mitochondrial polarization in full-grown (FG) oocytes resuming meiotic maturation and ovulation in vivo. Concomitant with elevated igf1 expression and IGF1R phosphorylation, hCG (LH analog) stimulation of FG follicles in vitro prompted a sharp increase in NRF-1 and ATP levels, suggesting a positive influence of gonadotropin action on igf1 expression vis-à-vis oocyte bioenergetics. While recombinant IGF1 administration enhanced mitochondrial function, IGF1R immunodepletion or priming with PI3K inhibitor wortmannin could abrogate NRF-1 immunoreactivity, expression of respiratory chain subunits, ΔΨM, and ATP content. Mechanistically, activation of PI3K/Akt signaling in IGF1-treated follicles corroborated well with the rapid phosphorylation of GSK3ß at Ser9 (inactive) followed by PGC-1ß accumulation. While selective inhibition of GSK3ß promoted PGC-1ß, Akt inhibition could abrogate IGF1-induced p-GSK3ß (Ser9) and PGC-1ß immunoreactive protein indicating Akt-mediated GSK3ß inactivation and PGC-1ß stabilization. The IGF1-depleted follicles showed elevated superoxide anions, subdued steroidogenic potential, and attenuated G2-M1 transition. In summary, this study highlights the importance of IGF1 signaling in oocyte bioenergetics prior to resumption of meiosis.

Homocysteine May Decrease Glucose Uptake and Alter the Akt/GSK3ß/GLUT1 Signaling Pathway in Hippocampal Slices: Neuroprotective Effects of Rivastigmine and Ibuprofen.

Homocysteine (Hcy) is a risk factor for neurodegenerative diseases, such as Alzheimer's Disease, and is related to cellular and tissue damage. In the present study, we verified the effect of Hcy on neurochemical parameters (redox homeostasis, neuronal excitability, glucose, and lactate levels) and the Serine/Threonine kinase B (Akt), Glucose synthase kinase-3ß (GSK3ß) and Glucose transporter 1 (GLUT1) signaling pathway in hippocampal slices, as well as the neuroprotective effects of ibuprofen and rivastigmine alone or in combination in such effects. Male Wistar rats (90 days old) were euthanized and the brains were dissected. The hippocampus slices were pre-treated for 30 min [saline medium or Hcy (30 µM)], then the other treatments were added to the medium for another 30 min [ibuprofen, rivastigmine, or ibuprofen + rivastigmine]. The dichlorofluorescein formed, nitrite and Na+, K+-ATPase activity was increased by Hcy at 30 µM. Ibuprofen reduced dichlorofluorescein formation and attenuated the effect of Hcy. The reduced glutathione content was reduced by Hcy. Treatments with ibuprofen and Hcy + ibuprofen increased reduced glutathione. Hcy at 30 µM caused a decrease in hippocampal glucose uptake and GLUT1 expression, and an increase in Glial Fibrillary Acidic Protein-protein expression. Phosphorylated GSK3ß and Akt levels were reduced by Hcy (30 µM) and co-treatment with Hcy + rivastigmine + ibuprofen reversed these effects. Hcy toxicity on glucose metabolism can promote neurological damage. The combination of treatment with rivastigmine + ibuprofen attenuated such effects, probably by regulating the Akt/GSK3ß/GLUT1 signaling pathway. Reversal of Hcy cellular damage by these compounds may be a potential neuroprotective strategy for brain damage.

Inhibitory Effect of Isopanduratin A on Adipogenesis: A Study of Possible Mechanisms.

The root of Boesenbergia rotunda, a culinary plant commonly known as fingerroot, has previously been reported to possess anti-obesity activity, with four flavonoids identified as active principles, including pinostrobin, panduratin A, cardamonin, and isopanduratin A. However, the molecular mechanisms underlying the antiadipogenic potential of isopanduratin A remain unknown. In this study, isopanduratin A at non-cytotoxic concentrations (1-10 µM) significantly suppressed lipid accumulation in murine (3T3-L1) and human (PCS-210-010) adipocytes in a dose-dependent manner. Downregulation of adipogenic effectors (FAS, PLIN1, LPL, and adiponectin) and adipogenic transcription factors (SREBP-1c, PPARγ, and C/EBPα) occurred in differentiated 3T3-L1 cells treated with varying concentrations of isopanduratin A. The compound deactivated the upstream regulatory signals of AKT/GSK3ß and MAPKs (ERK, JNK, and p38) but stimulated the AMPK-ACC pathway. The inhibitory trend of isopanduratin A was also observed with the proliferation of 3T3-L1 cells. The compound also paused the passage of 3T3-L1 cells by inducing cell cycle arrest at the G0/G1 phase, supported by altered levels of cyclins D1 and D3 and CDK2. Impaired p-ERK/ERK signaling might be responsible for the delay in mitotic clonal expansion. These findings revealed that isopanduratin A is a strong adipogenic suppressor with multi-target mechanisms and contributes significantly to anti-obesogenic activity. These results suggest the potential of fingerroot as a functional food for weight control and obesity prevention.

Kaempferol ameliorates pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension rats via regulating Akt-GSK3ß-cyclin axis.

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is considered a major contributor to elevated pulmonary vascular resistance and a key mechanism of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Kaempferol is a natural flavonoid compound and can be derived from numerous common medicinal herbs and vegetables, which exhibit antiproliferative and proapoptotic properties, however, the effects of kaempferol on vascular remodeling in HPH remain unexplored. In this study, SD rats were placed in a hypobaric hypoxia chamber for four weeks to establish a pulmonary hypertension model and given either kaempferol or sildenafil (an inhibitor of PDE-5) during days 1-28, after which the hemodynamic parameter and pulmonary vascular morphometry were assessed. Furthermore, primary rat PASMCs were exposed to hypoxic conditions to generate a cell proliferation model, then incubated with either kaempferol or LY294002 (an inhibitor of PI3K). Immunoblotting and real-time quantitative PCR assessed the protein and mRNA expression levels in HPH rat lungs and PASMCs. We found that kaempferol reduced pulmonary artery pressure and pulmonary vascular remodeling, and alleviated right ventricular hypertrophy in HPH rats. The mechanistic analysis demonstrated that kaempferol reduced the protein levels of phosphorylation of Akt and GSK3ß, leading to decreased expression of pro-proliferation (CDK2, CDK4, Cyclin D1, and PCNA) and anti-apoptotic related proteins (Bcl-2) and increased expression of pro-apoptosis proteins (Bax and cleaved caspase 3). These results collectively demonstrate that kaempferol ameliorates HPH in rats by inhibiting PASMC proliferation and pro-apoptosis via modulation of the Akt/GSK3ß/CyclinD axis.

Curcumol suppresses endothelial-to-mesenchymal transition via inhibiting the AKT/GSK3ß signaling pathway and alleviates pulmonary arterial hypertension in rats.

Endothelial dysfunction is essential in pulmonary arterial hypertension (PAH) pathogenesis and is considered to be a therapeutic target of PAH. Curcumol is a bioactive sesquiterpenoid with pharmacological properties including restoring endothelial cells damage. This study aimed to evaluate the effect of curcumol on PAH rats and investigate its possible mechanisms. PAH was induced by subcutaneous injection of 60 mg/kg monocrotaline (MCT) in male Sprague Dawley rats. Curcumol (12.5, 25, and 50 mg/kg/day) were administered by intragastric administration for 3 weeks. The results demonstrated that curcumol dose-dependently alleviated MCT-induced right ventricular hypertrophy and pulmonary arterial wall thickness. In addition, endothelial-to-mesenchymal transition (EndMT) in the pulmonary arteries of MCT-challenged rats was inhibited after curcumol treatment, as evidenced by the restored expressions of endothelial and myofibroblast markers. The possible pharmacological mechanisms of curcumol were analyzed using network pharmacology. After screening the common therapeutic targets of PAH and curcumol by searching related databases and comparison, pathway enrichment was performed and AKT/GSK3ß was screened out as a possible signaling pathway which was relevant to the therapeutic mechanism of curcumol on PAH. Western blot analysis verified this in lung tissues. Moreover, combination of TNF-α, TGF-ß1 and IL-1ß-induced EndMT in primary rat pulmonary arterial endothelial cells were blocked by curcumol, and this effect was resembled by PI3K/AKT inhibitor LY294002. Above all, our study suggested that curcumol inhibited EndMT via inhibiting the AKT/GSK3ß signaling pathway, which may contribute to its alleviated effect on PAH. Curcumol may be developed as a therapeutic for PAH in the future.

Ferrostatin-1 alleviates cerebral ischemia/reperfusion injury through activation of the AKT/GSK3ß signaling pathway.

Ischemic stroke is the major cause of disability and death worldwide, but post-stroke neuronal death and related mechanisms remain unclear. Ferroptosis, a newly identified type of regulated cell death, has been shown to be associated with neurological disorders, yet the exact relationship between ferroptosis and ischemic stroke has not been elucidated. The purpose of this study is to investigate the effects of ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) on neuronal injury after cerebral ischemia/reperfusion (I/R) and the underlying mechanism. In this study, we demonstrated that ferroptosis does occur in the stroke model. We found that Fer-1 reduced the levels of iron and malondialdehyde, and increased the content of glutathione and the expression of solute carrier family 7 member 11 and glutathione peroxidase 4 in cerebral I/R models. Additionally, Fer-1 significantly reduced the infarct volume and improved neurobehavioral outcomes. Moreover, we found that Fer-1 increased the levels of phosphorylated AKT and GSK3ß following cerebral I/R. To further investigate the functional role of the AKT in the neuroprotective effects of Fer-1, MCAO models and oxygen-glucose deprivation-induced HT22 cells were pretreated with the AKT inhibitor MK-2206 before treatment with Fer-1 and the protective effects of Fer-1 were reversed. In conclusion, Fer-1 has protective effects on cerebral I/R injury by activating the AKT/GSK3ß pathway, indicating that ferroptosis may become a novel target in the treatment of ischemic stroke.

Knockdown of Bcl-3 alleviates psoriasis and dyslipidemia comorbidity by regulating Akt pathway.

BACKGROUND: Psoriasis is considered as an inflammatory skin disease accompanied by dyslipidemia comorbidity. B-cell leukemia-3 (Bcl-3) belongs to IκB (inhibitor of nuclear factor kappa B [NF-κB]) family, and regulates inflammatory response through associating with NF-κB. The role of Bcl-3 in psoriasis was investigated in this study. METHODS: Apolipoprotein E (ApoE)-deficient mice were treated with imiquimod to induce psoriasis and dyslipidemia. Mice were injected intradermally in the back with lentiviral particles encoding Bcl-3 small hairpin RNA (shRNA). Hematoxylin and eosin were used to detect pathological characteristics. The blood lipid levels were determined by automatic biochemical analyzer, and inflammation was assessed by enzyme-linked-immunosorbent serologic assay and real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Bcl-3 was elevated in imiquimod-induced ApoE-deficient mice. Injection with lentiviral particles encoding Bcl-3 shRNA reduced Psoriasis area and severity index (PASI) score in ApoE-deficient psoriatic mice. Knockdown of Bcl-3 also ameliorated imiquimod-induced psoriasiform skin lesions in ApoE-deficient mice. Moreover, loss of Bcl-3 enhanced expression of loricrin, an epidermal barrier protein, reduced expression of proliferating cell nuclear antigen (PCNA) and lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) in imiquimod-induced ApoE-deficient mice. The enhanced levels of blood lipid in ApoE-deficient mice were attenuated by silencing of Bcl-3 with increase of high-density lipoprotein, and reduction of total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Knockdown of Bcl-3 attenuated imiquimod-induced decrease of transforming growth factor beta (TGF-ß), and increase of Interleukin (IL)-17A, IL-23, IL-6, and tumor necrosis factor-α (TNF-α) in ApoE-deficient mice. Protein expression of phospho-Akt (p-Akt) and p-GSK3ß in ApoE-deficient psoriatic mice was decreased by silencing of Bcl-3. CONCLUSION: Loss of Bcl-3 exerted anti-inflammatory effect on psoriasis and dyslipidemia comorbidity through inactivation of Akt/GSK3ß pathway.

Hypoxia is fine-tuned by Hif-1α and regulates mesendoderm differentiation through the Wnt/ß-Catenin pathway.

BACKGROUND: Hypoxia naturally happens in embryogenesis and thus serves as an important environmental factor affecting embryo development. Hif-1α, an essential hypoxia response factor, was mostly considered to mediate or synergistically regulate the effect of hypoxia on stem cells. However, the function and relationship of hypoxia and Hif-1α in regulating mesendoderm differentiation remains controversial. RESULTS: We here discovered that hypoxia dramatically suppressed the mesendoderm differentiation and promoted the ectoderm differentiation of mouse embryonic stem cells (mESCs). However, hypoxia treatment after mesendoderm was established promoted the downstream differentiation of mesendoderm-derived lineages. These effects of hypoxia were mediated by the repression of the Wnt/ß-Catenin pathway and the Wnt/ß-Catenin pathway was at least partially regulated by the Akt/Gsk3ß axis. Blocking the Wnt/ß-Catenin pathway under normoxia using IWP2 mimicked the effects of hypoxia while activating the Wnt/ß-Catenin pathway with CHIR99021 fully rescued the mesendoderm differentiation suppression caused by hypoxia. Unexpectedly, Hif-1α overexpression, in contrast to hypoxia, promoted mesendoderm differentiation and suppressed ectoderm differentiation. Knockdown of Hif-1α under normoxia and hypoxia both inhibited the mesendoderm differentiation. Moreover, hypoxia even suppressed the mesendoderm differentiation of Hif-1α knockdown mESCs, further implying that the effects of hypoxia on the mesendoderm differentiation were Hif-1α independent. Consistently, the Wnt/ß-Catenin pathway was enhanced by Hif-1α overexpression and inhibited by Hif-1α knockdown. As shown by RNA-seq, unlike hypoxia, the effect of Hif-1α was relatively mild and selectively regulated part of hypoxia response genes, which fine-tuned the effect of hypoxia on mESC differentiation. CONCLUSIONS: This study revealed that hypoxia is fine-tuned by Hif-1α and regulates the mesendoderm and ectoderm differentiation by manipulating the Wnt/ß-Catenin pathway, which contributed to the understanding of hypoxia-mediated regulation of development.

Monotropein Protects against Inflammatory Bone Loss and Suppresses Osteoclast Formation and Bone Resorption by Inhibiting NFATc1 via NF-κB and Akt/GSK-3ß Pathway.

Monotropein (Mon) is a kind of iridoid glycoside plant secondary metabolite primarily present in some edible and medicinal plants. The aim of this study was to investigate the effect of Mon on lipopolysaccharide (LPS)-induced inflammatory bone loss in mice and osteoclasts (OCs) derived from bone marrow-derived macrophages (BMMs), and explore the mechanisms underlying the effect of Mon on LPS-induced osteoclastogenesis. It was found that Mon markedly attenuated deterioration of the bone micro-architecture, enhanced tissue mineral content (TMC) and bone volume/total volume (BV/TV), reduced structure model index (SMI) and trabecular separation/spacing (Tb.Sp) in the bone tissue and decreased the activities of tartrate resistant acid phosphatase-5b (TRACP-5b), receptor activator NF-κB (RANK), and receptor activator NF-κB ligand (RANKL) as well as the serum levels of interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) in LPS-treated mice. In addition, Mon treatment reduced the number of TRAP positive OCs in the bone tissue of LPS-treated mice and also exerted a stronger inhibitory effect on formation, differentiation, and F-actin ring construction of OCs derived from BMMs. Mon significantly inhibited the expression of the nuclear factor of activated T-cells c1 (NFATc1) and the immediate early gene (C-Fos) and nuclear translocation of NFATc1 in LPS-treated OCs, thereby inhibiting the expression of matrix metalloproteinase-9 (MMP-9), cathepsin K (CtsK), and TRAP. Mon significantly inhibited the expression of TRAF6, phosphorylation of P65, and degradation of IKBα, thus inhibiting the activation of NF-κB pathway in LPS-induced inflammatory mice and OCs derived from BMMs, and also inhibited LPS-induced phosphorylation of protein kinase B (Akt) and Glycogen synthase kinase 3ß (GSK-3ß) in OCs derived from BMMs. In conclusion, these results suggested that Mon could effectively inhibit osteoclastogenesis both in vitro and in vivo and therefore may prove to be potential option for prevention and treatment of osteoclastic bone resorption-related diseases.

Lidocaine enhances the efficacy of palbociclib in triple-negative breast cancer.

The use of anesthetics in the surgical resection of tumors may influence the prognosis of cancer patients. Lidocaine, a local anesthetic, is known to act as a chemosensitizer and relieve pain in some cancers. In addition, palbociclib, a potent cyclin-dependent kinase (CDK) 4/6 inhibitor, has been approved for chemotherapy of advanced breast cancer. However, recent studies have revealed the acquired resistance of breast cancer cells to palbociclib. Therefore, the development of combination therapies that can extend the efficacy of palbociclib or delay resistance is crucial. This study investigated whether lidocaine would enhance the efficacy of palbociclib in breast cancer. Lidocaine synergistically suppressed the growth and proliferation of breast cancer cells by palbociclib. The combination treatment showed an increased cell cycle arrest in the G0/G1 phase by decreasing retinoblastoma protein (Rb) and E2F1 expression. In addition, it increased apoptosis by loss of mitochondrial membrane potential as observed by increases in cytochrome c release and inhibition of mitochondria-mediated protein expression. Additionally, it significantly reduced epithelial-mesenchymal transition and PI3K/AKT/GSK3ß signaling. In orthotopic breast cancer models, this combination treatment significantly inhibited tumor growth and increased tumor cell apoptosis compared to those treated with a single drug. Taken together, this study demonstrates that the combination of palbociclib and lidocaine has a synergistic anti-cancer effect on breast cancer cells by the inhibition of the PI3K/AKT/GSK3ß pathway, suggesting that this combination could potentially be an effective therapy for breast cancer.

EZH2 promotes the progression of osteosarcoma through the activation of the AKT/GSK3ß pathway.

Enhancer of zeste homologue 2 (EZH2) is a clarified promoter in a list of tumours, including osteosarcoma (OS). Our research was projected to define the mechanism involved in EZH2-mediated OS progression through the protein kinase B (AKT)/glycogen synthase kinase 3ß (GSK3ß) pathway. EZH2 expression was tested in 66 OS tissues and five osteosarcoma cell lines (143B, SJSA-1, HOS, MG63, and U2OS). In HOS and U2OS cells, cellular malignant characteristics, and the markers of the AKT/GSK3ß signalling pathway were measured when EZH2 was silenced or overexpressed. Meanwhile, rescue assays were implemented to observe whether the AKT/GSK3ß signalling pathway inhibitor (MK-2206) could affect the role of overexpressed EZH2 in OS cells. EZH2 was up-regulated in tumour tissues of OS patients. OS cell lines (HOS and U2OS) showed impairments of proliferative, migratory, invasive and anti-apoptotic properties when EZH2 was silenced. Downregulated EZH2 inhibited the activation of the AKT/GSK3 signalling pathway. However, the situation in HOS and U2OS cells over-expressing EZH2 was opposite. MK-2206 erased EZH2 up-regulation-induced promotion of OS cell growth. It is demonstrated that EZH2 promotes the progression of OS via inducing the activation of the AKT/GSK3ß pathway, offering a therapeutic direction for OS treatment.

Acacetin Prevents Bone Loss by Disrupting Osteoclast Formation and Promoting Type H Vessel Formation in Ovariectomy-Induced Osteoporosis.

Osteoporosis, characterized by the destruction of bone resorption and bone formation, is a serious disease that endangers human health. Osteoporosis prevention and treatment has become one of the important research contents in the field of medicine. Acacetin, a natural flavonoid compound, could promote osteoblast differentiation, and inhibit osteoclast formation in vitro. However, the mechanisms of acacetin on osteoclast differentiation and type H vessel formation, as well as the effect of preventing bone loss, remain unclear. Here, we firstly used primary bone marrow derived macrophages (BMMs), endothelial progenitor cells (EPCs), and ovariectomized (OVX) mice to explore the function of acacetin on bone remodeling and H type vessel formation. In this study, we found that acacetin inhibits osteoclast formation and bone resorption of BMMs induced by the macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) in a concentration of 20 µM without exerting cytotoxic effects. It was accompanied by downregulation of osteoclast differentiation marker genes (Ctsk, Acp5, and Mmp9) and cell fusion genes (CD9, CD47, Atp6v0d2, Dc-stamp, and Oc-stamp). Moreover, acacetin disrupted actin ring formation and extracellular acidification in osteoclasts. Mechanistic analysis revealed that acacetin not only inhibits the expression of the major transcription factor NFATc1 and NF-κB during RANKL-induced osteoclast formation, but also suppresses RANKL-induced the phosphorylation of Akt, GSK3ß, IκBα, and p65. Additionally, acacetin enhanced the ability of M-CSF and RANKL-stimulated BMMs to promote angiogenesis and migration of EPCs. We further established that, in vivo, acacetin increased trabecular bone mass, decreased the number of osteoclasts, and showed more type H vessels in OVX mice. These data demonstrate that acacetin prevents OVX-induced bone loss in mice through inhibition of osteoclast function and promotion of type H vessel formation via Akt/GSK3ß and NF-κB signalling pathway, suggesting that acacetin may be a novel therapeutic agent for the treatment of osteoporosis.

Stearoyl-CoA desaturase 1 regulates malignant progression of cervical cancer cells.

The primary regulatory gene for fatty acid synthesis, stearoyl-CoA desaturase 1 (SCD1), has been linked to the progression of several malignancies. Its role in cervical cancer remains unclear till now. This paper aimed to explore the role and mechanism of SCD1 in cervical cancer. The GEPIA database was used to perform a bioinformatics analysis of the role of SCD1 in cervical cancer staging and prognosis. The influences of SCD1 knockdown on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) progress were then investigated. Following transcription factor Kruppel like factor 9 (KLF9) was discovered to be negatively correlated with SCD1, the regulatory role of KLF9 in the effects of SCD1 on cervical cancer cells and the signaling pathway was evaluated. According to the GEPIA database, SCD1 level was associated with the cervical cancer stage, the overall survival level, and the disease-free survival level. Cell proliferation, migration, invasion, and EMT progress were all hindered when its expression was knocked down. Novelty, KLF9 reversed the effects of SCD1 on cells, as well as the Akt/glycogen synthase kinase 3ß (GSK3ß) signaling pathway. Together, SCD1 was negatively regulated by KLF9 and it activated the Akt/GSK3ß signaling pathway to promote the malignant progression of cervical cancer cells. Developing SCD1 inhibitors offers novel ideas for the biological treatment of cervical cancer.

Plantamajoside promotes metformin-induced apoptosis, autophagy and proliferation arrest of liver cancer cells via suppressing Akt/GSK3ß signaling.

Metformin, a well-known antidiabetic drug, exhibits anticancer effect in a variety of cancers, including liver cancer. Plantamajoside (PMS), a phenylethanoid glycoside compound isolated from Plantago asiatica, is proved to possess anticancer effects, too. In our study, we hypothesized that PMS might promote metformin mediated anticancer effects on liver cancer. The half maximal inhibitory concentration (IC50) of metformin was evaluated by cell viability assay. The influence of PMS on proliferation, migration, invasion and apoptosis of metformin-treated cells was evaluated by BrdU incorporation assay, flow cytometry, western blot, wound scratch healing assay, transwell cell migration assay and immunofluorescence. A fasting/feeding mouse model was built to evaluate the influence of PMS on metformin sensitivity in vivo. PMS (2.5, 10 or 40 µg/mL) treatment reduced the IC50 of metformin under different glucose concentrations. PMS (10 µg/mL) promoted metformin (5 mm) induced apoptosis and autophagy, and inhibition on proliferation, migration and invasion of HepG2 and HuH-7 cells. In the fasting/feeding mouse model, PMS (50 mg/kg) promoted metformin (200 mg/kg) induced proliferation arrest and apoptosis in vivo. Meanwhile, PMS reduced the level of pAkt(ser473) and GSK3ß(ser9) in HepG2 and HuH-7 cells. Restoration of Akt/GSK3ß signaling by a constitutively activated myr-Akt1 abrogated the effects of PMS on metformin-treated liver cancer cells. Our results demonstrated that PMS promoted the anticancer effects of metformin on liver cancer in vitro and in vivo.