Exosomal miR-7-25207 Increases Subgroup J Avian Leukosis Virus Titers by Targeting the Akt-CyclinQ1 and PRC1-YAF2 Dual Pathways.

Subgroup J avian leukosis virus (ALV-J) is a major pathogen in poultry, causing substantial economic losses to the poultry industry worldwide. Exosomal small RNAs derived from virus-infected cells or biological fluids can serve as viral transmission vectors. However, the role and mechanism of exosomal miRNA in ALV-J infection are unclear. In this study, we demonstrated that exosomal microRNA-7-25207 (miR-7-25207) could increase the titers of ALV-J. Exosomes isolated from ALV-J-infected DF-1 cells (Exo-ALV-J) contained partial viral proteins from ALV-J and could transmit the infection to uninfected DF-1 cells, leading to productive infection. Additionally, the RNA expression profile of exosomes was altered following ALV-J infection. miRNA analysis revealed that the expression of exosomal miR-7-25207 increased. Overexpression of miR-7-25207 significantly increased the titers of ALV-J in transfected cells. Furthermore, miR-7-25207 directly suppressed the expression of Akt and PRC1. Akt, in turn, directly inhibited CyclinQ1 expression, while PRC1 directly interfered with YAF2 expression. In conclusion, ALV-J infection activates the expression of miR-7-25207, which is subsequently delivered via exosomes to uninfected cells, increasing ALV-J titers by targeting Akt-CyclinQ1 and PRC1-YAF2 dual pathways. These findings suggest that exosomal miR-7-25207 may serve as a potential biomarker for clinical parameters in ALV-J infection.

Empagliflozin demonstrates cytotoxicity and synergy with tamoxifen in ER-positive breast cancer cells: anti-proliferative and anti-survival effects.

Accumulating evidence suggests that sodium-glucose cotransporter 2 (SGLT2) inhibitors may be effective at eliminating tumor cells. While empagliflozin exhibits nearly the highest selectivity for SGLT2 over SGLT1, its specific impact alone and in combination with tamoxifen remains largely unexplored in estrogen receptor α-positive (ERα +) breast cancer. This study investigated the anticancer effects of empagliflozin and its potential synergy with tamoxifen in MCF-7 breast cancer cells. The individual and combined cytotoxic effects of empagliflozin and tamoxifen were assessed using the xCELLigence system. The activities of AMP-activated protein kinase α (AMPKα), p38 mitogen-activated protein kinase (p38 MAPKα), p70-S6 kinase 1 (p70S6K1), and protein kinase B (Akt) were assessed using Western blotting. The gene expression levels of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and Forkhead box O3a (FOXO3a) were assessed via qPCR. Our results revealed time- and concentration-dependent cytotoxic effects of empagliflozin and tamoxifen whether administered separately or in combination. While tamoxifen exhibits potency with an IC50 value of 17 µM, approximately ten times greater than that of empagliflozin (IC50 = 177 µM), synergistic effects are observed when the concentrations of the two agents approach their respective IC50 values. Additionally, empagliflozin significantly increases AMPKα activity while concurrently inhibiting Akt, p70S6K1, and p38 MAPKα, and these effects are significantly enhanced when empagliflozin is combined with tamoxifen. Moreover, empagliflozin modulates the gene expression, downregulating PGC-1α while upregulating FOXO3a. Empagliflozin exerts anti-proliferative and anti-survival effects by inhibiting mTOR, Akt, and PGC-1α, and it exhibits synergy with tamoxifen in MCF-7 breast cancer cells.

KLF15 alleviates oxidative stress and apoptosis of H/R-induced trophoblast cells to improve invasion and migration capacity via the activation of IGF1R.

BACKGROUND: Krüppel-like factor 15 (KLF15) has been reported to be involved in ischemia injury of multiple types of diseases. Nevertheless, the roles and underlying mechanisms of KLF15 in preeclampsia (PE) are still unclear. METHODS: In this study, the expression of KLF15 in placenta tissues and hypoxia/reoxygenation (H/R)-induced HTR8/SVneo cells was evaluated by GSE66273 database, qRT-PCR and western blot assay. CCK-8 assay was employed to detect cell proliferation. Wound healing assay and transwell assay were used to detect cell migration and invasion. Cell oxidative stress was measured by DCFH-DA staining and kits. Cell apoptosis was evaluated by TUNEL assay and western blot assay. The JASPAR database was used to analyze the binding site of KLF15 and insulin-like growth factor-1 receptor (IGF1R) promoter region. The luciferase reporter assay was used to detect IGF1R promoter activity and ChIP assay was used to verify the combination of KLF15 and IGF1R promoter. Moreover, western blot was employed to measure the expressions of PI3K/Akt-related proteins. RESULTS: The data showed that the expression of KLF15 was significantly downregulated in GSE66273 database, tissues and HTR8/SVneo cells. KLF15 overexpression increased H/R-induced HTR8/SVneo cell proliferation, invasion and migration, and inhibited oxidative stress and cell apoptosis. In addition, IGF1R was highly expressed in H/R-induced HTR8/SVneo cells after KLF15 overexpression, and the binding of KLF15 and IGF1R promoter was verified. Silencing of IGF1R reversed the effects of KLF15 overexpression on H/R-induced HTR8/SVneo cell proliferation, migration, invasion, oxidative stress and cell apoptosis. Moreover, KLF15 overexpression and IGF1R silencing regulated the expressions of PI3K/Akt-related proteins in H/R-induced HTR8/SVneo cells. CONCLUSION: In conclusion, KLF15 overexpression promoted the proliferation and metastasis, and suppressed oxidative stress and cell apoptosis of H/R-induced HTR8/SVneo cells through mediating the PI3K/Akt pathway, which may provide a promising target for the treatment of preeclampsia.

Isorhapontigenin delays senescence and matrix degradation of nucleus pulposus cells via PI3K/AKT/mTOR-mediated autophagy pathway in vitro and alleviates intervertebral disc degeneration in vivo.

Intervertebral disc degeneration (IVDD), a common degenerative disc disease, is a major etiological factor for back pain, affecting a significant number of middle-aged and elderly individuals worldwide. Thus, IVDD is a major socio-economic burden. The factors contributing to the complex IVDD etiology, which has not been elucidated, include inflammation, oxidative stress, and natural aging. In particular, inflammation and aging of nucleus pulposus cells are considered primary pathogenic factors. Isorhapontigenin (ISO) is a polyphenolic compound commonly found in traditional Chinese herbs and grapes. We have demonstrated that ISO exerts anti-inflammatory and anti-aging effects and mitigates extracellular matrix (ECM) degradation. In this study, in vitro experiments revealed that, ISO delays aging and ECM degradation by promoting PI3K/AKT/mTOR-mediated autophagy. Meanwhile, in vivo experiments affirmed that ISO delays the progression of IVDD.

Validation of Jianpi Qingre Tongluo Recipe in Reducing Inflammation and Dyslipidemia in Osteoarthritis via Lnc RNA HOTAIR/APN/PI3K/AKT.

Objective: Jianpi Qingre Tongluo Recipe (JQP) has been widely used in clinical practice, and its anti-Osteoarthritis (OA) effectiveness and specific mechanism have been concerned. This study aims to explore the clinical effect of JQP in reducing inflammation and dyslipidemia in OA and the molecular mechanism. Methods: The clinical efficacy of JQP in OA treatment was assessed through data mining. Through the network pharmacology technology, the interactive network of "active component-target-disease" was developed, the interaction relationship of the related proteins was analyzed, and enrichment analysis of gene pathway biological process was conducted. Molecular docking was carried out with PyMOL and AutodockTools-1.5.7. Finally, cell experiments were used to verify JQP's delay of immune inflammation in OA. Results: We found that JQP could ameliorate the immune inflammatory and lipid metabolism indicators; reduce VAS and SAS score in OA. A total of 98 genes overlapped between target genes of JQP and OA. TNF, IL-6, IL-1ß, and AKT1 shared the highest centrality among all target genes. KEGG analysis unveiled that 98 intersection genes were predominantly enriched in PI3K/AKT pathway in the anti-OA system. In vitro, after peripheral blood mononuclear cell (PBMC) stimulation, inflammatory cytokines imbalances and the expressions of adiponectin (APN) were decreased in osteoarthritis-chondrocytes (OA-CH). Furthermore, JQP-containing serum protected OA-CHs through down-regulating HOTAIR levels, thereby up-regulating APN and depressing PI3K/AKT pathway. Conclusion: This study suggests that JQP might reduce inflammation and improve lipid metabolism of OA by regulating HOTAIR/APN/PI3K/AKT. Our results bring a new solution for OA.

Moderate-Intensity Treadmill Exercise Regulates GSK3α/ß Activity in the Cortex and Hippocampus of APP/PS1 Transgenic Mice.

BACKGROUND: Physical exercise has been shown to be beneficial for individuals with Alzheimer's disease (AD), although the underlying mechanisms are not fully understood. METHODS: Six-month-old Amyloid precursor protein/Presenilin 1 (APP/PS1) transgenic (Tg) mice and wild-type (Wt) mice were randomly assigned to either a sedentary group (Tg-Sed, Wt-Sed) or an exercise group (Tg-Ex, Wt-Ex) undertaking a 12-week, moderate-intensity treadmill running program. Consequently, all mice were tested for memory function and amyloid ß (Aß) levels and phosphorylation of tau and protein kinase B (Akt)/glycogen synthase kinase-3 (GSK3) were examined in tissues of both the cortex and hippocampus. RESULTS: Tg-Sed mice had severely impaired memory, higher levels of Aß, and increased phosphorylation of tau, GSK3α tyrosine279, and GSK3ß tyrosine216, but less phosphorylation of GSK3α serine21, GSK3ß serine9, and Akt serine473 in both tissues than Wt-Sed mice in respective tissues. Tg-Ex mice showed significant improvement in memory function along with lower levels of Aß and less phosphorylation of tau (both tissues), GSK3α tyrosine279 (both tissues), and GSK3ß tyrosine216 (hippocampus only), but increased phosphorylation of GSK3α serine21 (both tissues), GSK3ß serine9 (hippocampus only), and Akt serine473 (both tissues) compared with Tg-Sed mice in respective tissues. CONCLUSIONS: Moderate-intensity aerobic exercise is highly effective in improving memory function in 9-month-old APP/PS1 mice, most likely through differential modulation of GSK3α/ß phosphorylation in the cortex and hippocampus.

IL-22 activates the PI3K-AKT pathway to promote colorectal cancer cell proliferation and metastasis.

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy with high morbidity and mortality rates. Previous studies have demonstrated that interleukin (IL)-22 is involved in CRC progression; however, the exact mechanism remains unclear. This study aimed to investigate the effects of IL-22 on CRC cell proliferation and metastasis. METHODS: IL-22 levels in the serum and tissues of CRC patients were measured using enzyme-linked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to detect the viability of CRC (HCT116) cells treated with different IL-22 concentrations. Colony formation, Transwell invasion, and scratch assays were employed to assess the effects of IL-22 on cell proliferation, invasion, and migration. Western blotting was performed to measure the expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), p-PI3K, p-AKT, E-cadherin, matrix metalloproteinase (MMP)-2, MMP-9, SNAI1, and TWIST1 in HCT116 cells treated with IL-22 or a PI3K inhibitor. RESULTS: ELISA results showed that the expression of IL-22 was significantly increased in the serum and tissues of CRC patients compared to controls. IL-22 treatment increased cell viability and colony formation in a concentration-dependent manner and enhanced cell invasion and migration. Western blotting analysis revealed that IL-22 stimulation upregulated p-PI3K and p-AKT expression, while total PI3K and AKT levels remained unchanged. Additionally, IL-22 also decreased E-cadherin expression and increased the expression of MMP-2, MMP-9, SNAI1, and TWIST1. CONCLUSIONS: IL-22 activates the PI3K-AKT pathway and promotes HCT116 cell proliferation and metastasis. Targeting the regulation of the PI3K/AKT pathway may be a potential therapeutic strategy for CRC.

Insulin promotes the bone formation capability of human dental pulp stem cells through attenuating the IIS/PI3K/AKT/mTOR pathway axis.

BACKGROUND: Insulin has been known to regulate bone metabolism, yet its specific molecular mechanisms during the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs) remain poorly understood. This study aimed to explore the effects of insulin on the bone formation capability of human DPSCs and to elucidate the underlying mechanisms. METHODS: Cell proliferation was assessed using a CCK-8 assay. Cell phenotype was analyzed by flow cytometry. Colony-forming unit-fibroblast ability and multilineage differentiation potential were evaluated using Toluidine blue, Oil red O, Alizarin red, and Alcian blue staining. Gene and protein expressions were quantified by real-time quantitative polymerase chain reaction and Western blotting, respectively. Bone metabolism and biochemical markers were analyzed using electrochemical luminescence and chemical colorimetry. Cell adhesion and growth on nano-hydroxyapatite/collagen (nHAC) were observed with a scanning electron microscope. Bone regeneration was assessed using micro-CT, fluorescent labeling, immunohistochemical and hematoxylin and eosin staining. RESULTS: Insulin enhanced the proliferation of human DPSCs as well as promoted mineralized matrix formation in a concentration-dependent manner. 10- 6 M insulin significantly up-regulated osteogenic differentiation-related genes and proteins markedly increased the secretion of bone metabolism and biochemical markers, and obviously stimulated mineralized matrix formation. However, it also significantly inhibited the expression of genes and proteins of receptors and receptor substrates associated with insulin/insulin-like growth factor-1 signaling (IIS) pathway, obviously reduced the expression of the phosphorylated PI3K and the ratios of the phosphorylated PI3K/total PI3K, and notably increased the expression of the total PI3K, phosphorylated AKT, total AKT and mTOR. The inhibitor LY294002 attenuated the responsiveness of 10- 6 M insulin to IIS/PI3K/AKT/mTOR pathway axis, suppressing the promoting effect of insulin on cell proliferation, osteogenic differentiation and bone formation. Implantation of 10- 6 M insulin treated DPSCs into the backs of severe combined immunodeficient mice and the rabbit jawbone defects resulted in enhanced bone formation. CONCLUSIONS: Insulin induces insulin resistance in human DPSCs and effectively promotes their proliferation, osteogenic differentiation and bone formation capability through gradually inducing the down-regulation of IIS/PI3K/AKT/mTOR pathway axis under insulin resistant states.

Transcription factor 7 like 2 promotes metastasis in hepatocellular carcinoma via NEDD9-mediated activation of AKT/mTOR signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors of the digestive system, and the exact mechanism of HCC is still unclear. Transcription factor 7 like 2 (TCF7L2) plays a pivotal role in cell proliferation and stemness maintenance. However, the exact mechanism of TCF7L2 in HCC remains unclear. METHODS: Clinical samples and public databases were used to analyze the expression and prognosis of TCF7L2 in HCC. The function of TCF7L2 in HCC was studied in vitro and in vivo. ChIP and luciferase assays were used to explore the molecular mechanism of TCF7L2. The relationship between TCF7L2 and NEDD9 was verified in HCC clinical samples by tissue microarrays. RESULTS: The expression of TCF7L2 was upregulated in HCC, and high expression of TCF7L2 was associated with poor prognosis of HCC patients. Overexpression of TCF7L2 promoted the metastasis of HCC in vitro and in vivo, while Knockdown of TCF7L2 showed the opposite effect. Mechanically, TCF7L2 activated neural precursor cell expressed developmentally downregulated protein 9 (NEDD9) transcription by binding to the -1522/-1509 site of the NEDD9 promoter region, thereby increasing the phosphorylation levels of AKT and mTOR. The combination of TCF7L2 and NEDD9 could distinguish the survival of HCC patients. CONCLUSIONS: This study demonstrated that TCF7L2 promotes HCC metastasis by activating AKT/mTOR pathway in a NEDD9-dependent manner, suggesting that potential of TCF7L2 and NEDD9 as prognostic markers and therapeutic targets for HCC.

Immunohistochemical Expression of PTEN in Canine Gliomas.

Phosphatase and tensin homolog (PTEN) is a critical tumor suppressor gene with a vital role in regulating cell proliferation, migration, and survival. The loss of PTEN function, either by genetic alterations or decreased protein expression, is frequent in human gliomas and has been correlated with tumor progression, grade, therapeutic resistance, and decreased overall survival in patients with glioma. While different genetic mutations in PTEN gene have been occasionally reported in canine gliomas, no alterations in protein expression have been reported. This study investigates the immunohistochemical expression of PTEN in canine gliomas to evaluate possible alterations, as those reported in human gliomas. Immunohistochemical PTEN expression and pattern distribution were analyzed in 37 spontaneous canine gliomas. Among gliomas, 52.6% cases showed high PTEN expression and 48.6% displayed reduced (13.5%) or highly reduced (35.1%) immunopositivity. Most oligodendrogliomas showed high expression (73.7%), while the majority of astrocytomas (69.2%) showed a reduced or highly reduced expression. A reduced PTEN expression was mostly associated with a heterogeneous loss of PTEN immunopositivity. These observations are in line with those reported in human gliomas and provide a rationale for future studies regarding abnormalities in PTEN expression and PI3K/Akt/mTor pathway in canine gliomas, to evaluate its prognostic and therapeutic implications.

Parecoxib and 5-Fluorouracil Synergistically Inhibit EMT and Subsequent Metastasis in Colorectal Cancer by Targeting PI3K/Akt/NF-κB Signaling.

Colorectal cancer is one of the most common causes of cancer mortality worldwide, and innovative drugs for the treatment of colorectal cancer are continually being developed. 5-Fluorouracil (5-FU) is a common clinical chemotherapeutic drug. Acquired resistance to 5-FU is a clinical challenge in colorectal cancer treatment. Parecoxib is a selective COX-2-specific inhibitor that was demonstrated to inhibit metastasis in colorectal cancers in our previous study. This study aimed to investigate the synergistic antimetastatic activities of parecoxib to 5-FU in human colorectal cancer cells and determine the underlying mechanisms. Parecoxib and 5-FU synergistically suppressed metastasis in colorectal cancer cells. Treatment with the parecoxib/5-FU combination induced an increase in E-cadherin and decrease in ß-catenin expression. The parecoxib/5-FU combination inhibited MMP-9 activity, and the NF-κB pathway was suppressed as well. Mechanistic analysis denoted that the parecoxib/5-FU combination hindered the essential molecules of the PI3K/Akt route to obstruct metastatic colorectal cancer. Furthermore, the parecoxib/5-FU combination could inhibit reactive oxygen species. Our work showed the antimetastatic capacity of the parecoxib/5-FU combination for treating colorectal cancers via the targeting of the PI3K/Akt/NF-κB pathway.

Rasagiline Exerts Neuroprotection towards Oxygen-Glucose-Deprivation/Reoxygenation-Induced GAPDH-Mediated Cell Death by Activating Akt/Nrf2 Signaling.

Rasagiline (Azilect®) is a selective monoamine oxidase B (MAO-B) inhibitor that provides symptomatic benefits in Parkinson's disease (PD) treatment and has been found to exert preclinical neuroprotective effects. Here, we investigated the neuroprotective signaling pathways of acute rasagiline treatment for 22 h in PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) for 4 h, followed by 18 h of reoxygenation (R), causing 40% aponecrotic cell death. In this study, 3-10 µM rasagiline induced dose-dependent neuroprotection of 20-80%, reduced the production of the neurotoxic reactive oxygen species by 15%, and reduced the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by 75-90%. In addition, 10 µM rasagiline increased protein kinase B (Akt) phosphorylation by 50% and decreased the protein expression of the ischemia-induced α-synuclein protein by 50% in correlation with the neuroprotective effect. Treatment with 1-5 µM rasagiline induced nuclear shuttling of transcription factor Nrf2 by 40-90% and increased the mRNA levels of the antioxidant enzymes heme oxygenase-1, (NAD (P) H- quinone dehydrogenase, and catalase by 1.8-2.0-fold compared to OGD/R insult. These results indicate that rasagiline provides neuroprotection to the ischemic neuronal cultures through the inhibition of α-synuclein and GAPDH-mediated aponecrotic cell death, as well as via mitochondrial protection, by increasing mitochondria-specific antioxidant enzymes through a mechanism involving the Akt/Nrf2 redox-signaling pathway. These findings may be exploited for neuroprotective drug development in PD and stroke therapy.

Mutations in Plasmodium falciparum PfK13 dysregulates PI3K/AKT signalling pathway in Artemisinin Resistance.

BACKGROUND: PfK13 protein mutations are associated with the emergence of artemisinin resistance in Plasmodium falciparum. PfK13 protein is essential for mediating ubiquitination and controlling the PI3K/AKT pathway. Mutant PfK13 variations can interfere with substrate binding, especially with PfPI3K, which raises PfPI3K levels. METHODS: DUET, DynaMut2, mCSM, iStable 2.0, I-Mutant 2.0, and MuPro were utilized to study the protein stability and protein-substrate binding was studied using HADDOCK 2.4 docking algorithm between Wild-type and mutant PfK13 with the helical and catalytic domain of PfPI3K. RESULTS: i-Stable server analysis predicted that seven, out of the nine mutations associated with artemisinin resistance (F446I, Y493H, R539T, I543T, P553L, R561H, C580Y) reduced the protein stability. HADDOCK scores of the catalytic domain underscores the significant impact of the reported mutations on the binding affinity of the PfK13 protein. Further validation through the MM_GBSA technique, the binding free energy (ddG) between the wild-type and the mutant PfK13 protein analysis revealed a loss of interactions resulting from mutations in PfK13. CONCLUSION: The study finding suggest that mutations in the PfK13 cause destabilization in the protein structure and affects the binding of PfPI3K. Although the findings remain preliminary and require further validation, it provides the basis for further research considering the importance of the interaction of PfK13 and PfPI3K to overcome the impact of artemisinin resistance.

MiR-3571 modulates traumatic brain injury by regulating the PI3K/AKT signaling pathway via Fbxo31.

To investigate the effect of miR-3571 on traumatic brain injury (TBI) via the regulation of neuronal apoptosis through F-box-only protein 31/phosphoinositide 3-kinase/protein kinase B (Fbxo31/PI3K/AKT). We established TBI rat and cell models. Hematoxylin‒eosin (HE) and Nissl staining were used to observe brain injury and the number of Nissl bodies, respectively. Cell proliferation and apoptosis were assessed by 5-ethynyl-2'-deoxyuridine (EdU), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and flow cytometry. Gene and protein expression was measured via reverse transcription quantitative polymerase chain reaction (RT‒qPCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). In this study, miR-3571 was highly expressed in TBI models. Inhibition of miR-3571 expression can suppress autophagy, reduce the expression of proinflammatory cytokines, and reduce neuronal apoptosis, thus alleviating the pathological conditions of tissue congestion, edema and structural damage after TBI. These experiments demonstrated that miR-3571 could target and regulate the level of Fbxo31. Knockdown of Fbxo31 weakened the remission effect of the miR-3571 inhibitor on TBI and promoted neurological damage; moreover, overexpression of Fbxo31 enhanced the protective effect on neural function, whereas the PI3K/AKT pathway inhibitor LY294002 increased the damage caused by miR-3571 on neural function and weakened the protective effect of Fbxo31. In conclusion, miR-3571 regulates the PI3K/AKT signaling pathway by reducing Fbxo31 expression, promotes neuronal apoptosis and exacerbates TBI.

Calcium-sensing receptor regulates the angiogenic differentiation of LPS-treated human dental pulp cells via the phosphoinositide 3-kinase/Akt pathway in vitro.

AIM: The purpose of this study was to investigate the role of calcium-sensing receptor (CaSR) in the angiogenic differentiation of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). METHODOLOGY: The LPS-induced hDPCs were cultured in the medium with different combinations of CaSR agonist R568 and antagonist Calhex231. The cell proliferation, migration, and angiogenic capacity were measured by Cell Counting Kit-8 (CCK-8), scratch wound healing, and tube formation assays, respectively. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were conducted to determine the gene/protein expression of CaSR, inflammatory mediators, and angiogenic-associated markers. The activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) was assessed by western blot analysis. RESULTS: The cell proliferation was elevated in response to R568 or Calhex231 exposure, but an enhanced cell migration was only found in cultures supplemented with Calhex231. Furthermore, R568 was found to potentiate the formation of vessel-like structure, up-regulated the protein expression of tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF)-1; comparable influences were also observed in R568-stimulated cells in the presence of PI3K inhibitor LY294002. In contrast, Calhex231 obviously inhibited the tube formation and VEGF protein level, whereas promoted the production of IL-6, TNF-α, and eNOS; however, in the presence of LY294002, Calhex231 showed a significant promotion on the protein expression of CaSR, VEGF, and SDF-1. In addition, R568 exhibited a promotive action on the Akt phosphorylation, which can be reversed by LY294002. CONCLUSIONS: Our results demonstrated that CaSR can regulate the angiogenic differentiation of LPS-treated hDPCs with an involvement of the PI3K/Akt signalling pathway.

Uncovering the Molecular Pathways Implicated in the Anti-Cancer Activity of the Imidazoquinoxaline Derivative EAPB02303 Using a Caenorhabditis elegans Model.

Imiqualines are analogues of the immunomodulatory drug imiquimod. EAPB02303, the lead of the second-generation imiqualines, is characterized by significant anti-tumor effects with IC50s in the nanomolar range. We used Caenorhabditis elegans transgenic and mutant strains of two key signaling pathways (PI3K-Akt and Ras-MAPK) disrupted in human cancers to investigate the mode of action of EAPB02303. The ability of this imiqualine to inhibit the insulin/IGF1 signaling (IIS) pathway via the PI3K-Akt kinase cascade was explored through assessing the lifespan of wild-type worms. Micromolar doses of EAPB02303 significantly enhanced longevity of N2 strain and led to the nuclear translocation and subsequent activation of transcription factor DAF-16, the only forkhead box transcription factor class O (Fox O) homolog in C. elegans. Moreover, EAPB02303 significantly reduced the multivulva phenotype in let-60/Ras mutant strains MT2124 and MT4698, indicative of its mode of action through the Ras pathway. In summary, we showed that EAPB02303 potently reduced the activity of IIS and Ras-MAPK signaling in C. elegans. Our results revealed the mechanism of action of EAPB02303 against human cancers associated with hyperactivated IIS pathway and oncogenic Ras mutations.

Molecular Mechanisms Underlying the Anticancer Properties of Pitavastatin against Cervical Cancer Cells.

Cervical cancer ranks as the fourth most prevalent form of cancer and is a significant contributor to female mortality on a global scale. Pitavastatin is an anti-hyperlipidemic medication and has been demonstrated to exert anticancer and anti-inflammatory effects. Thus, the purpose of this study was to evaluate the anticancer effect of pitavastatin on cervical cancer and the underlying molecular mechanisms involved. The results showed that pitavastatin significantly inhibited cell viability by targeting cell-cycle arrest and apoptosis in Ca Ski, HeLa and C-33 A cells. Pitavastatin caused sub-G1- and G0/G1-phase arrest in Ca Ski and HeLa cells and sub-G1- and G2/M-phase arrest in C-33 A cells. Moreover, pitavastatin induced apoptosis via the activation of poly-ADP-ribose polymerase (PARP), Bax and cleaved caspase 3; inactivated the expression of Bcl-2; and increased mitochondrial membrane depolarization. Furthermore, pitavastatin induced apoptosis and slowed the migration of all three cervical cell lines, mediated by the PI3K/AKT and MAPK (JNK, p38 and ERK1/2) pathways. Pitavastatin markedly inhibited tumor growth in vivo in a cancer cell-originated xenograft mouse model. Overall, our results identified pitavastatin as an anticancer agent for cervical cancer, which might be expanded to clinical use in the future.

Scutellarin alleviates microglia-mediated neuroinflammation and apoptosis after ischemic stroke through the PI3K/AKT/GSK3ß signaling pathway.

Microglia are resident immune cells in the central nervous system that are rapidly activated to mediate neuroinflammation and apoptosis, thereby aggravating brain tissue damage after ischemic stroke (IS). Although scutellarin has a specific therapeutic effect on IS, the potential target mechanism of its treatment has not been fully elucidated. In this study, we explored the potential mechanism of scutellarin in treating IS using network pharmacology. Lipopolysaccharide (LPS) was used to induce an in vitro BV-2 microglial cell model, while middle cerebral artery occlusion (MCAO) was used to induce an in vivo animal model. Our findings indicated that scutellarin promoted the recovery of cerebral blood flow in MCAO rats at 3 days, significantly different from that in the MCAO group. Western blotting and immunofluorescence revealed that scutellarin treatment of BV-2 microglial cells resulted in a significant reduction in the protein expression levels and incidence of cells immunopositive for p-NF-κB, TNF-α, IL-1ß, Bax, and C-caspase-3. In contrast, the expression levels of p-PI3K, p-AKT, p-GSK3ß, and Bcl-2 were further increased, significantly different from those in the LPS group. The PI3K inhibitor LY294002 had similar effects to scutellarin by inhibiting neuroinflammation and apoptosis in activated microglia. The results of the PI3K/AKT/GSK3ß signaling pathway and NF-κB pathway in vivo in MCAO models induced microglia at 3 days were consistent with those obtained from in vitro cells. These findings indicate that scutellarin plays a neuroprotective role by reducing microglial neuroinflammation and apoptosis mediated by the activated PI3K/AKT/GSK3ß/NF-κB signaling pathway.

ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling.

Objective: This study aimed to investigate the role of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in triple-negative breast cancer (TNBC). Methods: ROR2 expression in primary TNBC and metastatic TNBC tissues was analyzed by immunohistochemical staining and PCR. ROR2 expression in TNBC cell lines was detected by PCR and Western blot analysis. The migration, invasion and chemosensitivity of TNBC cells with overexpression or knockdown of ROR2 were examined. Results: ROR2 expression was high in metastatic TNBC tissues. ROR2 knockdown suppressed the migration, invasion and chemoresistance of TNBC cells. ROR2 overexpression in MDA-MB-435 cells promoted the migration, invasion, and chemoresistance. Moreover, ROR2 knockdown in HC1599 and MDA-MB-435 adriamycin-resistant cells enhanced chemosensitivity to adriamycin. ROR2 could activate PI3K/AKT/mTOR signaling in TNBC cells. Conclusion: ROR2 is upregulated and promotes metastatic phenotypes of TNBC by activating PI3K/AKT/mTOR signaling.

PLAUR facilitates the progression of clear cell renal cell carcinoma by activating the PI3K/AKT/mTOR signaling pathway.

Background: PLAUR has been found upregulated in various tumors and closely correlated with the malignant phenotype of tumor cells. The aim of this study was to investigate the relationship between PLAUR and clear cell renal cell carcinoma (ccRCC) and its potential mechanism of promoting tumor progression. Methods: The expression levels and clinical significance of PLAUR, along with the associated signaling pathways, were extensively investigated in ccRCC samples obtained from The Cancer Genome Atlas (TCGA). PLAUR expression in 20 pairs of ccRCC tumor tissues and the adjacent tissues was assessed using qRT-PCR and IHC staining. Additionally, a series of in vitro experiments were conducted to investigate the impact of PLAUR suppression on cellular proliferation, migration, invasion, cell cycle progression, and apoptosis in ccRCC. The Western blot analysis was employed to investigate the expression levels of pivotal genes associated with the PI3K/AKT/mTOR signaling pathway. Results: The expression of PLAUR was significantly upregulated in ccRCC compared to normal renal tissues, and higher PLAUR expression in ccRCC was associated with a poorer prognosis than low expression. The in-vitro functional investigations demonstrated that knockdown of PLAUR significantly attenuated the proliferation, migration, and invasion capabilities of ccRCC cells. Concurrently, PLAUR knockdown effectively induced cellular apoptosis, modulated the cell cycle, inhibited the EMT process, and attenuated the activation of the PI3K/AKT/mTOR signaling pathway. PLAUR may represent a key mechanism underlying ccRCC progression. Conclusions: The involvement of PLAUR in ccRCC progression may be achieved through the activation of the PI3K/AKT/mTOR signaling pathway, making it a reliable biomarker for the identification and prediction of ccRCC.