Phosphorylation Of Phosphatidylinositol 3-kinase Research Articles

LINC01060 knockdown inhibits osteosarcoma cell malignant behaviors in vitro and tumor growth and metastasis in vivo through the PI3K/Akt signaling

ABSTRACT Despite its low frequency, osteosarcoma is one of the deadliest malignancies in children and adolescents. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling activation and epithelial-to-mesenchymal transition (EMT) are critical issues during osteosarcoma development. This study found long intergenic non-protein coding RNA 1060 (LINC01060) to be an EMT-related long non-coding RNA (lncRNA) up-regulated in osteosarcoma; higher LINC01060 expression was linked to a worse prognosis in osteosarcoma patients. In vitro, knocking down LINC01060 significantly inhibits osteosarcoma cell malignant behaviors, including hyperproliferation, invasion, migration, and EMT. In vivo, LINC01060 knockdown inhibited tumor growth and metastasis, and suppressed PI3K and Akt phosphorylation. In osteosarcoma cells, Akt agonist SC79 exerted opposite effects to those of LINC01060 knockdown through the promotion of cell viability, cell migration, and cell invasion. Moreover, the Akt agonist SC79 partially eliminated LINC01060 knockdown effects on osteosarcoma cells, suggesting that LINC01060 exerts its effects through the PI3K/Akt signaling. Therefore, it is deduced that LINC01060 is overexpressed in osteosarcoma. In vitro, LINC01060 knockdown inhibits cancer cell malignant behaviors; in vivo, LINC01060 knockdown inhibits tumor development and metastasis. The PI3K/Akt signaling is involved in LINC01060 functions in osteosarcoma.

Auranofin accelerates spermidine-induced apoptosis via reactive oxygen species generation and suppression of PI3K/Akt signaling pathway in hepatocellular carcinoma

Auranofin is a US Food and Drug Administration (FDA)-approved anti-arthritis medication that functions as a thioredoxin reductase inhibitor. Spermidine, a polyamine present in marine algae, can exert various physiological functions. Herein, we examined the synergistic anticancer activity of auranofin and spermidine in hepatocellular carcinoma (HCC). Combined treatment with auranofin and spermidine suppressed cell viability more efficiently than either treatment alone in HCC Hep3B cells. The isobologram plotted by calculating the half maximal inhibitory concentration (IC50) values of each drug indicated that the two drugs exhibited a synergistic effect. Based on the analysis of annexin V and cell cycle distribution, auranofin and spermidine markedly induced apoptosis in Hep3B cells. Moreover, auranofin and spermidine increased mitochondria-mediated apoptosis by promoting mitochondrial membrane potential (Δψm) loss. Auranofin and spermidine significantly increased reactive oxygen species (ROS) production in Hep3B cells, and the blocking ROS suppressed apoptosis induced by spermidine and auranofin. In addition, auranofin and spermidine reduced the expression of phosphorylated phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), and PI3K inhibitor accelerated auranofin- and spermidine-induced apoptosis. Using ROS scavenger and PI3K inhibitor, we revealed that ROS acts upstream of auranofin- and spermidine-induced apoptosis. Collectively, our study suggests that combination treatment with auranofin and spermidine could afford synergistic anticancer activity via ROS overproduction and reduced PI3K/Akt signaling pathway.

Effect of Gubenyiliu formula II and its disassembled prescriptions on cell autophagy in breast cancer through PI3K/AKT/mTOR pathway.

The aim of this study is to reveal the mechanism of Gubenyiliu II (GYII) inhibiting autophagy in breast cancer and the effect of its disassembled prescriptions, Quxie (QX) and Fuzheng (FZ), which cause autophagy difference on tumor growth. After a breast cancer in situ tumor model was established, mice were randomly distributed into different groups: model, GYII, QX, FZ and tamoxifen groups, and treated correspondingly. Then, the tumor volumes and weights were monitored. Immunohistochemistry detected the contents of microtubule-associated protein light chain 3 (LC3), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) in tumor tissues. Furthermore, 4T1 cells were administrated with the 20% contained serum. Cell proliferation, migration and invasion were measured using cell counting kit-8 and transwell assays. Electron microscopy and flow cytometry detected autophagy and apoptosis. The content of LC3 was measured by immunofluorescence. Western blot detected the protein levels of LC3, Beclin1, p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR in tumor tissues and 4T1 cells. GYII, QX and FZ treatment significantly reduced the tumor volumes and weights in breast cancer tumor-bearing mice. The cell proliferation, migration and invasion were restrained, and cell apoptosis and autophagy were promoted in GYII, QX and FZ groups. Moreover, GYII, QX and FZ increased the expression of LC3 in 4T1 cells and tumor tissues and decreased the phosphorylation levels of PI3K, AKT and mTOR in tumor tissues. The protein levels of LC3 and Beclin1 were upregulated, and p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR were downregulated in tumor tissues and 4T1 cells of treatment groups. Our study confirmed that GYII could treat breast cancer by restraining the PI3K/AKT/mTOR signaling pathway-mediated autophagy. While QX focuses on inhibiting tumor growth, FZ acts on inhibiting tumor metastasis.

Penehyclidine hydrochloride protects against lipopolysaccharide-induced acute lung injury by promoting the PI3K/Akt pathway.

Acute lung injury (ALI) attracted attention among physicians because of its high mortality. We aimed to determine whether the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) pathway is involved in the protective effects of penehyclidine hydrochloride (PHC) against lipopolysaccharide (LPS)-induced ALI. H&E staining was used to observed pathological changes in the lung tissues. ELISA was used to evaluate the concentration of inflammatory mediators in the bronchoalveolar lavage fluid (BALF). White-light microscopy was performed to observe the TUNEL-positive nuclei. The viability of NR8383 alveolar macrophages was determined by using CCK-8. The levels of MPO, MDA, SOD, and GSH-Px were analyzed using ELISA kits. Western blotting was used to evaluate the ERS-associated protein levels and the phosphorylation of PI3K and Akt. PHC administration defended against LPS-induced histopathological deterioration and increased pulmonary edema and lung injury scores, while all of these beneficial effects were inhibited by LY. In addition, PHC administration mitigated oxidative stress as indicated by decreases in lung myeloperoxidase (MPO) and malondialdehyde (MDA) concentrations, and increases in glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) concentrations. It also alleviated LPS-induced inflammation. PHC administration attenuated apoptosis-associated protein levels, improved cell viability, and decreased the number of TdT-mediated dUTP Nick-End Labeling (TUNEL)-positive cells. Furthermore, PHC inhibited ERS-associated protein levels. Meanwhile, the protection of PHC against inflammation, oxidative stress, apoptosis, and ERS was inhibited by LY. Moreover, PHC administration increased PI3K and Akt phosphorylation, indicating that the upregulation of the PI3K/Akt pathway, while this pathway was inhibited by LY. PHC significantly activates the PI3K/Akt pathway to ameliorate the extent of damage to pulmonary tissue, inflammation, oxidative stress, apoptosis, and ERS in LPS-induced ALI.

CENPF facilitates endometrial cancer cell progression through PI3K/AKT/mTOR pathway

Centromere protein F (CENPF) has been found to exert vital roles in various cancers, while the detailed effect of CENPF on endometrial cancer (EC) remains unclear. This study indicated that CENPF was up-regulated in EC tissue samples using The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) and Gene Expression Profiling Interactive Analysis (GEPIA) database. Quantitative real time polymerase chain reaction and western blotting assays confirmed the increased CENPF in EC cells. In addition, overexpression of CENPF promoted cell proliferation, whereas CENPF knockdown notably suppressed the proliferation of EC cells, demonstrated by Methyl Thiazolyl Tetrazolium (MTT) as well as colony formation assays. Moreover, CENPF overexpression accelerated cell cycle, migration ad invasion, whereas CENPF knockdown contributed to cell cycle arrest at G2 stage and suppressed migration and invasion in EC cells. Furthermore, western blotting analysis demonstrated that CENPF overexpression up-regulated the content of phosphorylated protein kinase B (p-AKT), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), and phosphorylated mechanistic target of rapamycin kinase (p-mTOR), while CENPF silencing notably decreased their levels, indicating that CENPF positively regulated PI3K/AKT/mTOR signaling in EC cells. In summary, this study verified that CENPF promoted EC cells progression by mediating PI3K/AKT/mTOR cascade signal in EC cell lines.

LncRNA RP11-1100L3.8 Involves in the Pathogenesis of Multiple Myeloma by Regulating NR4A1.

Although numerous studies have revealed that various long-non coding RNA (lncRNA) are implicated in multiple myeloma (MM) regulation, MM lncRNA profile and novel functional lncRNAs in MM need to be elucidated. Herein, lncRNAs and mRNAs (messenger ribonucleic acids) patterns in MM were evaluated using RNA-sequencing (RNAseq). Differentially expressed (DE) genes were defined and a complex regulatory network based on validation and predication was shaped. LncRNA-seq data analysis identified 539 DE lncRNAs and RP11-1100L3.8 was the most up-regulated known lncRNA. Subsequently, the upregulation and clinical RP11-1100L3.8 utilization value was verified in an expanded cohort. Based on the results of Cis nearby-targets and co-expression analysis, 1 correlation pair RP11-1100L3.8-nuclear receptor subfamily 4 group A member 1 (NR4A1) was defined. It is worth noting that NR4A1 is one of the top 5 significantly up-regulated DE mRNAs in MM patients. Moreover, it was found that NR4A1 overexpression is associated with poor prognosis in MM patients, making it suitable as biomarker. Additionally, spearman correlation analysis revealed the positive association between RP11-1100L3.8 and NR4A1 in MM patients. Furthermore, the dominant NR4A1 interacted genes were predicated and it was found that the genes containing NR4A1 were remarkably enriched in phosphatidylinositol 3-kinase (PI3K)-AKT (protein kinase B) signaling pathway. In addition, in vitro experiment suggested that RP11-1100L3.8 downregulation decreased NR4A1 expression in U266 and RPMI 8226 MM cells. RP11-1100L3.8 inhibition declined proliferation and promoted apoptosis in MM cells, which were rescued by NR4A1 overexpression. Moreover, it was found that RP11-1100L3.8 inhibition impeded PI3K and AKT phosphorylation and rapamycin mammalian target in MM cells, which was rescued by NR4A1 overexpression. This study identifies RP11-1100L3.8 as a potential MM biomarker, and it may be involved in MM pathophysiology by regulating NR4A1-mediated PI3K-AKT signaling pathway. This study provides a novel biomarker candidate for MM therapy.

Resveratrol Inhibits Insulin-Induced Vascular Smooth Muscle Cell Proliferation and Migration by Activating SIRT1.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are essential for the development of hypertension. Insulin has been identified to promote VSMC proliferation and migration; resveratrol has been shown to have protective effects against cardiovascular diseases. This study aimed to investigate the effect of resveratrol on insulin-induced VSMC proliferation and migration and its potential mechanism. VSMC proliferation was measured by Cell Counting Kit-8 (CCK-8), cell counting method, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Cell migration was detected by wound healing assay and transwell method. Expression of silent information regulator of transcription 1 (SIRT1) and phosphorylation levels of signaling molecules, such as phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt), in VSMCs were detected by Western blotting. Resveratrol (25-150 μM) was found to inhibit insulin-induced VSMC proliferation. Pretreatment with 100 μM resveratrol reduced insulin (100 nM)-mediated VSMC migration. LY294002, an inhibitor of PI3K, inhibited the stimulatory effect of insulin (100 nM) on the proliferation of VSMCs. Treatment with resveratrol also decreased insulin-induced stimulatory effect on PI3K and Akt phosphorylation levels. Moreover, resveratrol treatment increased SIRT1 protein expression in VSMCs. A SIRT1 inhibitor, EX527, reversed the inhibitory effect of resveratrol on insulin-induced VSMC proliferation and migration and activation of PI3K and Akt phosphorylation levels. In conclusion, our study revealed that treatment with resveratrol inhibited insulin-mediated VSMC proliferation and migration, possibly by activating SIRT1 and downregulating the PI3K/AKT pathway.

Anti-inflammatory Effect of a Limonin Derivative In Vivo and Its Mechanisms in RAW264.7 Cells.

A potential new limonoid derivative, (12S,12aS)-6,6,8a,12a-tetramethyl-12-(5-(4-(piperidin-1-yl)butanoyl)furan-3-yl)decahydro-1H,3H-oxireno[2,3-d]pyrano[4',3':3,3a]isobenzofuro[5,4-f]isochromene-3,8,10(6H,9aH)-trione (I-C-1), has been screened for its anti-inflammatory activity. This study aimed to demonstrate the anti-inflammatory activities of I-C-1 and to further explore the underlying mechanisms of these activities in RAW264.7 macrophages. We verified the anti-inflammatory activity of I-C-1 in vivo by a carrageenan-induced paw edema model in rats and cotton pellet-induced granuloma in mice. Further, we found that I-C-1 significantly inhibited levels of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-induced RAW264.7 cells. I-C-1 demonstrated strong inhibition of the NF-κB activation through repression of the IKKα and IKKβ phosphorylations, as well as a significant suppression of the phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (Akt) pathway, an upstream of the NF-κB pathway. Additionally, we verified the inhibitory effect of I-C-1 on PI3K phosphorylation by immunofluorescence assay and compared the effects of I-C-1 with the PI3K inhibitor LY294002 in IL-1β, IL-6, and TNF-α levels. The data indicated that I-C-1 likely acts as an inhibitor of PI3K, exerting anti-inflammatory effects by inhibiting the PI3K/AKT/NF-κB signaling pathway. Based on these findings, we believe that I-C-1 has the potential to be further developed as a potential therapeutic agent for inflammatory-related diseases.

GPRC6A is a key mediator of palmitic acid regulation of lipid synthesis in bovine mammary epithelial cells.

Fatty acids (FAs) can promote lipid synthesis in the mammary gland via stimulating lipogenic gene expression, but the underlying molecular mechanism is still not fully understood. Here, we showed the dose-dependent effects of palmitic acid (PA) on lipid synthesis in primary bovine mammary epithelial cells (BMECs) and explored the corresponding molecular mechanism. BMECs were treated with PA (0, 50, 100, 150, and 200 μM), and the 100 μM treatment had the best stimulatory effect on lipid synthesis and expression and maturation of sterol regulatory element-binding protein 1c (SREBP-1c) in cells. Inhibition of phosphatidylinositol 3-kinase (PI3K) almost totally blocked the stimulation of PA on SREBP-1c expression, whereas protein kinase Cα (PKCα) knockdown only partially decreased the stimulation of PA on SREBP-1c expression but abolished the stimulation of PA on its maturation. Knockdown of GPR120 did not change the stimulation of PA on the SREBP-1c signaling. G protein-coupled receptor family C group 6 member A (GPRC6A) knockdown almost totally blocked the stimulation of FA on PI3K and PKCα phosphorylation as well as SREBP-1c expression and maturation. Furthermore, PA dose-dependently promoted GPRC6A expression and plasma membrane localization. Together, these above results reveal that GPRC6A is a key mediator of PA signaling to lipid synthesis in BMECs via the PI3K/PKCα-SREBP-1c pathways.

An Emerging Role of Proanthocyanidins on Psoriasis: Evidence from a Psoriasis-Like Mouse Model.

Background Psoriasis is an immune-mediated, chronic inflammatory disease, and genetic, immune, oxidative stress (OS), and environmental factors are all thought to contribute to its occurrence. Proanthocyanidins (PCs) are natural flavonoids consisting of catechins and epicatechins which have anti-inflammatory and anti-OS activities. PCs have been widely used to treat various diseases, but reports regarding psoriasis are rare. Objective To investigate the therapeutic effect and potential mechanisms of action of PCs in a psoriasis-like mouse model. Methods Thirty male BALB/c hairless mice were assigned to six groups (n = 5): normal, model, low-dose PCs, medium-dose PCs, high-dose PCs, and control groups. The final five groups were dorsally exposed to 5% imiquimod (IMQ) cream once a day for 6 consecutive days, while the normal group received no intervention. Following the first day of IMQ application, mice in the PC-treated group were dosed with different amounts of PCs daily by oral gavage for six days, whereas mice in the control group received normal saline in the same way. One week later, skin lesions were evaluated by the severity of scoring system based on psoriasis area and severity index (PASI), and pathological alterations were assessed by hematoxylin-eosin (HE) staining. Indicators of inflammation or OS, such as interleukin- (IL-) 17, IL-23, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), catalase (CAT), vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), and heme oxygenase-1 (HO-1), were determined by ELISA, RT-PCR, western blot, and immunohistochemistry (IHC) analysis. Results IMQ administration induced the formation of large dark red plaques with thickly layered scales on the dorsal skin of mice; nevertheless, the lesions were substantially alleviated by PC administration. Histopathological alterations were observed in both model and control groups with epidermal hyperkeratosis, granulosa layer thinning, acanthosis, downward extension of rete ridges, dermal papillae expansion, capillary hyperplasia, and infiltration by inflammatory cells around blood vessels. These pathological changes, however, were restored by a range of doses of PCs, high-dose PCs in particular. Different doses of PCs significantly lowered the spleen index, levels of inflammatory or oxidative proteins (IL-17, IL-23, MDA, ROS, p-PI3K, and p-STAT3), and the mRNA expression of Il-17, Il-23, Vegf, and iNos. Protein and mRNA levels of anti-OS and anti-inflammatory biomarkers, including SOD, CAT, GSH, and HO-1, greatly increased after PC treatment, especially at the highest dose. Conclusions Our findings reveal that PCs ameliorate psoriasis-like symptoms, suppressing the inflammatory response and mitigating OS damage in an IMQ-induced psoriasis-like mouse model. These effects are probably related to the inactivation of STAT3 and PI3K and activation of HO-1 signaling.

Evaluation of antidiabetic, antioxidant and antilipidemic potential of natural dietary product prepared from Cyphostemma digitatum in rats’ model of diabetes

Introduction: Cyphostemma digitatum has a high content of antioxidant constituents and has been employed by the traditional healers and local people of Yemen for diabetes treatment. However, scientific evidence regarding its antidiabetic efficacy is largely unknown. Accordingly, the present study aimed to confirm the treatment effects of a dietary natural product prepared from Cyphostemma digitatum (PCD) in diabetic rats.Methods: Diabetes was induced by a high-fat diet and streptozotocin (HF-STZ). PCD (1 g/kg) was given by gavage administration once a day continuously for 30 days. At the end of treatment, blood and skeletal muscle samples were collected for further analysis.Results: The antidiabetic effects of PCD were demonstrated by significant reduction (P ≤ 0.05) in the levels of serum glucose (40%), triglyceride (32%), cholesterol (53%), low-density lipoprotein (LDL) (44%), malondialdehyde (MDA) (61%) in PCD treated groups compared to the diabetic control group. Additionally, PCD treatment significantly (P ≤ 0.05) restored the decreased levels of insulin (70%) and the activities of superoxide dismutase (SOD) (57%) and reduced glutathione (GSH) (544%) when compared to that of diabetic control rats. We found that treatment with PCD for 30 days fully restored the plasmalemmal glucose transporter type 4 (GLUT4) contents, as well as the phosphorylation of phosphatidylinositol 3-kinase (PI3K) (P ≤ 0.05).Conclusion: Thus, PCD treatment can be considered a potential drug candidate for diabetes.

Human umbilical vein endothelial cells-derived exosomes enhance cardiac function after acute myocardial infarction by activating the PI3K/AKT signaling pathway

ABSTRACT Currently, acute myocardial infarction (AMI) is one of the leading causes of human health issues worldwide. The sudden and continuous occlusion of the coronary artery results in myocardial hypoxic-ischemic necrosis, which is accompanied by inflammatory infiltration and fibrosis, leading to pathological cardiac remodeling. Exosome-based therapy is a promising cell-free approach for repairing the ischemic myocardium. This study aimed to explore the effects and mechanism of human umbilical vein endothelial cells (HUVECs)-derived exosomes on AMI. The results indicated that the localized injection of HUVECs-derived exosomes in the infarcted area could significantly improve cardiac function in AMI mouse models. It could also ameliorate myocardial fibrosis and decrease infarct size after AMI. Additionally, HUVECs-derived exosomes had cardioprotective effects on the H9C2 cells in hypoxic culture conditions, including increased cell viability and decreased lactate dehydrogenase (LDH) release. In both the in-vivo and in-vitro experiments, HUVECs-derived exosomes could effectively inhibit cardiomyocyte apoptosis. The low expression levels of Bcl-2–associated X protein (Bax) and cleaved caspase-3, high expression levels of B-cell lymphoma 2 (Bcl-2), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) were detected in AMI mouse models treated with HUVECs-derived exosomes in-vivo. In conclusion, HUVECs-derived exosomes effectively enhanced cardiac function after AMI and inhibited cardiomyocyte apoptosis, which might be regulated through the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) signaling pathway.

MiR-186-5p inhibits the progression of oral squamous cell carcinoma by targeting ITGA6 to impair the activity of the PI3K/AKT pathway.

microRNAs (miRNAs) are pivotal regulators of multiple biological processes. miR-186-5p functions as a tumor suppressor in a variety of cancers and promotes the malignant proliferation of oral squamous cell carcinoma (OSCC). This study aimed to clarify the role and regulatory mechanism of miR-186-5p in OSCC. The levels of miR-186-5p and integrin subunit alpha 6 (ITGA6) were investigated in clinical specimens and OSCC cell lines by reverse transcription-quantitative polymerase chain reaction. The effects of miR-186-5p and ITGA6 on the cell migration, proliferation, and phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (AKT) pathway activity were evaluated by transwell assay, cell counting kit 8 assay, and western blotting, respectively. A xenograft model was used to analyze the effect of miR-186-5p on tumor growth. Bioinformatic analyses were conducted to identify the putative targets of miR-186-5p in OSCC. Decreased miR-186-5p expression levels were observed in OSCC tumor tissues and cell lines. The overexpression of miR-186-5p suppressed the proliferation and migration of OSCC cells, and weakened the phosphorylation of PI3K and AKT. Moreover, the overexpression of miR-186-5p in xenograft tumor models impedes tumor growth. miR-186-5p is bound to ITGA6 and negatively related to ITGA6 expression in tumor tissues. The forced expression of ITGA6 promoted OSCC cell proliferation and migration and enhanced the phosphorylation levels of PI3K and AKT, while additional miR-186-5p enrichment partly abolished these effects. miR-186-5p binds to ITGA6 to impair the activity of the PI3K/AKT signaling pathway, thereby blocking the development of OSCC. This study provides insight to understand the pathogenesis of OSCC.

Retraction for Su et al., SHP-1 aggravates obesity-related glomerulopathy and palmitic acid-induced podocyte injury via regulating the PI3K/Nrf2/SIRT1 axis.

Obesity-related glomerulopathy (ORG) is described as obesity-driven glomerular hypertrophy. SHP-1 is associated with glomerular pathology. This study is designed to investigate the molecular mechanism of Src homology region 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) in ORG and palmitic acid (PA)-induced podocyte injury. After analysis of clinical samples, an ORG mouse model was established via high-fat diet feeding, followed by detection of physiological and biochemical indexes, glomerular area, and inflammatory factor levels. In vitro cell model was established using (PA). Cell viability, apoptosis, and lipid deposition were determined. SHP-1, nuclear factor-erythroid 2-related factor 2 (Nrf2), phosphatidylinositol 3-kinase (PI3K), and sirtuin 1 (SIRT1) expressions were measured, and their binding relationships were testified. SHP-1 was highly expressed in ORG patients and was associated with renal function indexes. Silencing SHP-1 alleviated symptoms of ORG mice and PA-induced podocyte injury in vitro. SHP-1 bound to PI3K to inhibit PI3K phosphorylation and Nrf2 expression. Nrf2 bound to the SIRT1 promoter to promote SIRT1 transcription. Inhibition of Nrf2 or SIRT1 reversed the role of silencing SHP-1 in podocyte injury. Overall, SHP-1 was highly expressed in ORG and inhibited PI3K phosphorylation to block activation of Nrf2/SIRT1, thereby aggravating ORG-induced renal injury in vivo and PA-induced podocyte injury in vitro.

Bupivacaine modulates the apoptosis and ferroptosis in bladder cancer via phosphatidylinositol 3-kinase (PI3K)/AKT pathway

ABSTRACT The study aimed to explore the effects of local anesthetic bupivacaine on bladder cancer cells in vivo and in vitro. The cytotoxicity was detected by MTT assay. Apoptosis was measured by Hoechst 33342 staining and TUNEL. The contents of Fe2+, Malondialdehyde (MDA), Glutathione (GSH) and reactive oxygen species (ROS) were evaluated by the corresponding kit. Mitochondrial membrane potential was assessed by JC-1 kit. HE staining, TUNEL and immunohistochemistry were used to detect the xenografted tumors. Protein expression was estimated by Western blot. Bupivacaine significantly inhibited the activity of T24 cells and 5637 cells at 0.25–16 mM. Bupivacaine promoted cell apoptosis with increased concentration. bupivacaine inhibited the expression of Bcl-2 and increased the expression of Bax and cytochrome C. Moreover, bupivacaine amplified the level of Fe2+ and ROS, and restrained the expression of cystine/glutamic acid reverse transporter (xCT) and glutathione peroxidase 4 (GPX4). Further results showed that bupivacaine decreased mitochondrial membrane potential, reduced GSH, and increased MDA levels. Besides, bupivacaine attenuated the phosphorylation of PI3K, Akt, and mTOR. In addition, bupivacaine suppressed the growth of xenografted tumors, induced apoptosis and ferroptosis, and inhibited the activity of PI3K/AKT signaling pathway in xenografted tumors. Bupivacaine could induce apoptosis and ferroptosis by inhibiting PI3K/Akt signaling pathway in bladder cancer cells.

Effect of electroacupuncture on GLP-1R/PI3K/Akt protein pathway in mice with Parkinson's disease

To observe the effect of electroacupuncture(EA) on glucagon-like peptide-1 receptor (GLP-1R)/ phosphatidylinositol 3-kinase (PI3K)/ protein kinase B(Akt) protein pathway in the substantia nigra of mice with Parkinson's di-sease (PD)，so as to explore its underlying mechanisms in treatment of PD. Forty-eight C57BL/6 male mice were randomly divided into normal, model, EA and inhibitor groups, with 12 mice in each group. PD mouse model was established by intragastrical administration of rotenone for 4 weeks. In the EA group, EA was applied at "Fengfu"(GV16), "Taichong"(LR3) and"Zusanli"(ST36) for 30 min, once daily, for 2 weeks. The mice of the inhibitor group received gavage of dipeptidyl peptidase-4 inhibitor ligliptin (10 mg·kg-1·d-1) once a day for 2 weeks. The behavioral scores of mice in each group were observed. The levels of tyrosine hydroxylase (TH) in serum and substantia nigra were detected by ELISA, and the protein relative expression levels of GLP-1R, phosphorylation of PI3K (p-PI3K) and phosphorylation of Akt (p-Akt) in substantia nigra of midbrain of mice were detected by Western blot. Compared with the normal group, the behavioral scores were significantly increased (P<0.01), TH levels in serum and substantia nigra, protein expression levels of GLP-1R, p-PI3K and p-Akt of the substantia nigra in the model group were significantly decreased (all P<0.01). After intervention and in comparison with the model group, the behavioral scores were significantly decreased (P<0.01), TH levels and the protein expression levels of GLP-1R, p-PI3K and p-Akt in both EA and inhibitor groups were significantly increased (all P<0.01). There were no significant differences in the abovementioned indexes between EA group and inhibitor group (all P>0.05), except for TH levels which were considerably down-regulated in the EA group relative to the inhibitor group (P<0.01, P<0.05). EA at GV16, LR3 and ST36 may increase the level of TH in serum and substantia nigra by up-regulating the activity of GLP-1R/PI3K/Akt protein pathway, and improve the behavioral performance of PD induced by rotenone.

Piperine Alkaloid Induces Anti-Cancer Effects in Human Breast Carcinoma Cells by Targeting Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway

Piperine is a dietary alkaloid with huge pharmacological importance and was first isolated from pepper extract. It has been reported with anti-cancer, anti-amoebic, anti-mycobacterial, anti-mutagenic, anticonvulsant, anti-asthmatic, immunomodulatory, anti-inflammatory and anti-oxidant activity. Therefore, the current investigation was designed to unveil the anticancer effects of piperine molecule against human breast carcinoma. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was executed for determination of effects of piperine on breast cancer cell viability. Clonogenic assay was implemented for checking the effect of piperine on clonogenic potency of breast cancer cells. To monitor morphology of cancerous breast cells, phase contrast microscopy was performed after piperine exposure. Apoptosis was checked by acridine orange/ethidium bromine staining assay and by execution of Western blotting analysis. Cell migration and invasion was monitored by transwell chambers assay. Muse flow cytometric analysis was performed for assessing of different cell cycle phases. Western blotting assay was implemented to check the levels of phosphatidylinositol 3-kinase and protein kinase B. Results revealed that piperine induced dose reliant cytotoxicity in MCF-7 human breast cancer cells. Further, application of piperine on cancer cell colonies remarkably reduced the number of cell colonies to minimum. Phase contrast microscopy evidenced some specific morphological modifications indicating apoptosis allied cytotoxicity of piperine like apoptotic bodies, condensed nucleus and membrane blabbing. Acridine orange/ethidium bromine staining assay revealed that piperine induced apoptosis in MCF-7 cells which was further investigated by Western blotting. This revealed increased expression of B-cell lymphoma 2 associated X and reduced expression of B-cell lymphoma 2, indicating apoptosis induction by piperine. Cell migration as well as cell invasive ability of MCF-7 cells was reduced to minimum by the application of piperine in dose dependent-manner. Flow cytometric analysis evidenced that piperine arrested the cell cycle at growth 2/mitosis phase, hence suppressed the breast cancer progression. Finally, Western blotting assay predicted constant expression of phosphatidylinositol 3-kinase and protein kinase B and reduced expression of phosphorylated phosphatidylinositol 3-kinase. Hence, evidenced the blocking of phosphatidylinositol 3-kinase/protein kinase B signaling pathway. In conclusion, the current investigation regarding anticancer effects of piperine against breast cancer revealed remarkable suppression of cancer cells. It induced apoptosis, suppressed cell migration and invasion, blocked cell cycle and phosphatidylinositol 3-kinase/ protein kinase B signaling pathway.

Estrogenic Activity of Mycoestrogen (3β,5α,22E)-Ergost-22-en-3-ol via Estrogen Receptor α-Dependent Signaling Pathways in MCF-7 Cells.

Armillariella tabescens (Scop.) Sing., a mushroom of the family Tricholomataceae, has been used in traditional oriental medicine to treat cholecystitis, improve bile secretion, and regulate bile-duct pressure. The present study evaluated the estrogen-like effects of A. tabescens using a cell-proliferation assay in an estrogen-receptor-positive breast cancer cell line (MCF-7). We found that the methanol extract of A. tabescens fruiting bodies promoted cell proliferation in MCF-7 cells. Using bioassay-guided fractionation of the methanol extract and chemical investigation, we isolated and identified four steroids and four fatty acids from the active fraction. All eight compounds were evaluated by E-screen assay for their estrogen-like effects in MCF-7 cells. Among the tested isolates, only (3β,5α,22E)-ergost-22-en-3-ol promoted cell proliferation in MCF-7 cells; this effect was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of (3β,5α,22E)-ergost-22-en-3-ol was evaluated using Western blot analysis to detect the expression of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, and estrogen receptor α (ERα). We found that (3β,5α,22E)-ergost-22-en-3-ol induced an increase in phosphorylation of ERK, PI3K, Akt, and ERα. Together, these experimental results suggest that (3β,5α,22E)-ergost-22-en-3-ol is responsible for the estrogen-like effects of A. tabescens and may potentially aid control of estrogenic activity in menopause.

MrgprX2 regulates mast cell degranulation through PI3K/AKT and PLCγ signaling in pseudo-allergic reactions

The G protein-coupled receptor MrgprX2 in mast cells is known to be a crucial receptor for pseudo-allergic reactions. MrgprX2 activation leads to elevated intracellular calcium levels and mast cell degranulation, but the underlying mechanism remains to be elucidated. Herein, we investigated the role of the phosphatidylinositol 3 kinase (PI3K)/serum-threonine kinase (AKT) signaling pathway and phospholipase C gamma (PLCγ) in mast cell degranulation mediated by MrgprX2 in LAD2 human-derived mast cells. The results showed that phosphorylated AKT (p-AKT) and PLCγ up-regulation were accompanied by an increase in intracellular calcium following activation of MrgprX2 by Compound 48/80, an inducer of mast cell degranulation. In contrast, p-AKT and PLCγ were down-regulated and intracellular calcium levels decreased after MrgprX2 knockdown. Mast cell degranulation was clearly suppressed; however, inhibiting PI3K and PLCγ phosphorylation did not influence MrgprX2 expression. The increase in calcium concentration was suppressed and mast cell degranulation was weakened. Furthermore, by inhibiting PI3K and PLCγ phosphorylation in animals, the allergic symptoms caused by C48/80 were obviously reduced. We deduced that during the mast cell degranulation observed in pseudoallergic reactions, MrgprX2 regulated intracellular calcium levels via the PI3K/AKT and PLCγ pathways.

Exploring the Mechanism of the miRNA-145/Paxillin Axis in Cell Metabolism During VEGF-A-Induced Corneal Angiogenesis.

PurposePaxillin (PXN) is a key component of focal adhesions and plays an important role in angiogenesis. The aim of the present study was to investigate the effect of PXN in vascular endothelial growth factor A (VEGF-A)–induced angiogenesis in human umbilical vein endothelial cells (HUVECs).MethodsHUVECs were transfected with PXN overexpression and PXN interference vectors. Biochemical detection was used to detect adenosine triphosphate and lactic acid production. The morphology of mitochondria was observed under an electron microscope, and flow cytometry was conducted to measure mitochondrial membrane potential. Transwell experiments were used to detect the migration and tube formation ability of each group of cells. The expression of hexokinase (HK)1, HK2, glucose transporter 1 (GLUT1), phosphorylated phosphatidylinositol 3-kinase (PI3K), phosphorylated AKT, and phosphorylated mechanistic target of rapamycin (mTOR) was evaluated by western blot.ResultsPXN silencing reduced the levels of lactic acid and adenosine triphosphate, downregulated HK1, HK2, and GLUT1, suppressed PI3K/AKT/mTOR signaling activation, and inhibited VEGF-A–induced mitochondria injury in VEGF-A–induced HUVECs. We also determined that miR-145-5p decreased the VEGF-A–induced expression of PXN and inhibited the invasion and angiogenesis of HUVECs. Also, miR-145-5p inhibition blocked the protective effect of PXN interference on VEGF-A–induced HUVEC injury. Furthermore, PXN interference significantly decreased lactic acid and adenosine triphosphate levels, inhibited PI3K/AKT/mTOR activation, and decreased the levels of HK1, HK2, and GLUT1 in VEGF-A-treated mouse corneal.ConclusionsThe results indicate that PXN silencing inhibited the VEGF-A–induced invasion and angiogenesis of HUVECs via regulation of cell metabolism and mitochondrial damage, suggesting that PXN may be a potential target for antiangiogenic therapies.