Prednisolone induces osteocytes apoptosis by promoting Notum expression and inhibiting PI3K/AKT/GSK3β/β-catenin pathway

Journal of Molecular Histology - The apoptosis of mature osteocytes is the main factor causing damage to the microstructure of cortical bone in glucocorticoid-induced osteoporosis (GIOP). Our...     

Sensitization of glioma cells to tamoxifen-induced apoptosis by Pl3-kinase inhibitor through the GSK-3β/β-catenin signaling pathway

Malignant gliomas represent one of the most aggressive types of cancers and their recurrence is closely linked to acquired therapeutic resistance. A combination of chemotherapy is considered a promising therapeutic model in overcoming therapeutic resistance and enhancing treatment efficacy. Herein, we show by colony formation, Hochest 33342 and TUNEL staining, as well as by flow cytometric analysis, that LY294002, a specific phosphatidylinositide-3-kinase (PI3K) inhibitor, enhanced significantly the sensitization of a traditional cytotoxic chemotherapeutic agent, tamoxifen-induced apoptosis in C6 glioma cells. Activation of PI3K signaling pathway by IGF-1 protected U251 cells from apoptosis induced by combination treatment of LY294002 and tamoxifen. Interference of PI3K signaling pathway by PI3K subunit P85 siRNA enhanced the sensitization of U251 glioma cells to tamoxifen -induced apoptosis. By Western blotting, we found that combination treatment showed lower levels of phosphorylated Akt(Ser473) and GSK-3β(Ser9) than a single treatment of LY294002. Further, we showed a significant decrease of nuclear β-catenin by combination treatment. In response to the inhibition of β-catenin signaling, mRNA and protein levels of Survivin and the other three antiapoptotic genes Bcl-2, Bcl-xL, and Mcl-1 were significantly decreased by combination treatment. Our results indicated that the synergistic cytotoxic effect of LY294002 and tamoxifen is achieved by the inhibition of GSK-3β/β-catenin signaling pathway.     

Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis through modulating GSK3β/β-catenin pathway

In this study, we investigate the anti‐cancer activity of caudatin in carcinomic human alveolar basal epithelial cell line A549 and anti‐angiogenic activity in human umbilical vein endothelial cells (HUVECs). We show that caudatin impairs the cell viability and induces G₀/G₁ phase arrest in A549 cells with a dose dependent manner. A549 cells, not HUVECs, dealing with caudatin exhibited typical characteristics of apoptosis, which were accompanied by activation of caspase‐3, caspase‐9 and Poly(ADP–Ribose) Polymerase (PARP). In addition, caudatin treatment resulted in a decrease of β‐catenin and increase of phosphorylation of β‐catenin, and inhibited phosphorylation levels of GSK3β (Ser 9) in A549 cells. Conditional medium of A549 cells‐induced or growth factors‐induced tube formation of HUVECs was markedly inhibited by caudatin treatment, which was associated with the inhibiting VEGF secretion from A549 cells by caudatin. Our findings suggest that caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis by targeting GSK3β/β‐catenin pathway and suppressing VEGF production. J. Cell. Biochem. 113: 3403–3410, 2012. © 2012 Wiley Periodicals, Inc.     

Neuroprotective effects of linarin through activation of the PI3K/Akt pathway in amyloid-β-induced neuronal cell death

Linarin, a natural occurring flavanol glycoside derived from Mentha arvensis and Buddleja davidii is known to have anti-acetylcholinesterase effects. The present study intended to explore the neuroprotective effects of linarin against Aβ(25-35)-induced neurotoxicity with cultured rat pheochromocytoma cells (PC12 cells) and the possible mechanisms involved. For this purpose, PC12 cells were cultured and exposed to 30 μM Aβ(25-35) in the absence or presence of linarin (0.1, 1.0 and 10 μM). In addition, the potential contribution of the PI3K/Akt neuroprotective pathway in linarin-mediated protection against Aβ(25-35)-induced neurotoxicity was also investigated. The results showed that linarin dose-dependently increased cell viability and reduced the number of apoptotic cells as measured by MTT assay, Annexin-V/PI staining, JC-1 staining and caspase-3 activity assay. Linarin could also inhibit acetylcholinesterase activity induced by Aβ(25-35) in PC12 cells. Further study revealed that linarin induced the phosphorylation of Akt dose-dependently. Treatment of PC12 cells with the PI3K inhibitor LY294002 attenuated the protective effects of linarin. Furthermore, linarin also stimulated phosphorylation of glycogen synthase kinase-3β (GSK-3β), a downstream target of PI3K/Akt. Moreover, the expression of the anti-apoptotic protein Bcl-2 was also increased by linarin treatment. These results suggest that linarin prevents Aβ(25-35)-induced neurotoxicity through the activation of PI3K/Akt, which subsequently inhibits GSK-3β and up-regulates Bcl-2. These findings raise the possibility that linarin may be a potent therapeutic compound against Alzheimer's disease acting through both acetylcholinesterase inhibition and neuroprotection.     

Lithium chloride attenuates cell death in oculopharyngeal muscular dystrophy by perturbing Wnt/β-catenin pathway

Expansion of polyalanine tracts causes at least nine inherited human diseases. Among these, a polyalanine tract expansion in the poly (A)-binding protein nuclear 1 (_expPABPN1) causes oculopharyngeal muscular dystrophy (OPMD). So far, there is no treatment for OPMD patients. Developing drugs that efficiently sustain muscle protection by activating key cell survival mechanisms is a major challenge in OPMD research. Proteins that belong to the Wnt family are known for their role in both human development and adult tissue homeostasis. A hallmark of the Wnt signaling pathway is the increased expression of its central effector, beta-catenin (β-catenin) by inhibiting one of its upstream effector, glycogen synthase kinase (GSK)3β. Here, we explored a pharmacological manipulation of a Wnt signaling pathway using lithium chloride (LiCl), a GSK-3β inhibitor, and observed the enhanced expression of β-catenin protein as well as the decreased cell death normally observed in an OPMD cell model of murine myoblast (C2C12) expressing the expanded and pathogenic form of the _expPABPN1. Furthermore, this effect was also observed in primary cultures of mouse myoblasts expressing _expPABPN1. A similar effect on β-catenin was also observed when lymphoblastoid cells lines (LCLs) derived from OPMD patients were treated with LiCl. We believe manipulation of the Wnt/β-catenin signaling pathway may represent an effective route for the development of future therapy for patients with OPMD.     

TMPyP4-regulated cell proliferation and apoptosis through the Wnt/β-catenin signaling pathway in SW480 cells

Background: The aim of this study was to investigate the potential effects of the 5, 10, 15, 20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of SW480 cells and the underlying mechanisms by which TMPyP4 exerted its actions. Methods: After treated with different doses of TMPyP4, cell viability was determined by MTT method, the apoptosis was observed by flow cytometry (FCM) and the expression of Wnt, GSK-3β, β-catenin and cyclinD1 was measured by RT-PCR and Western blot analysis. Results: The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of SW480 cells in a dose-dependent manner. In addition, the downregulation of Wnt, β-catenin and cyclinD1 expression levels was detected in TMPyP4-treated SW480 cells. However, followed by the block of Wnt signaling pathway using siRNA methods, the effects of TMPyP4 on proliferation and apoptosis of SW480 cells were significantly reduced. Conclusion: It indicates that the TMPyP4-inhibited proliferation and -induced apoptosis in SW480 cells was accompanied by the suppression of Wnt/β-catenin signaling pathway. Therefore, TMPyP4 may represent a potential therapeutic method for the treatment of colon carcinoma.     

Higenamine mitigates interleukin-1β-induced human nucleus pulposus cell apoptosis by ROS-mediated PI3K/Akt signaling

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. Higenamine exerts multiple pharmacological properties in inflammation-related disorders. Our study aimed to explore the function of higenamine on interleukin (IL)-1β-caused apoptosis of human nucleus pulposus cells (HNPCs). Cell apoptosis was investigated by TUNEL and flow cytometry. Apoptosis-related biomarkers were determined by qRT-PCR or Western blotting. The protein in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling was measured by Western blotting. We found that higenamine showed little effect on cell apoptosis, but mitigated IL-1β-caused apoptosis in a dose-dependent pattern. Higenamine attenuated IL-1β-induced decrease of Bcl-2 and increase of Bax and cleaved caspase-3. Higenamine did not affect the reactive oxygen species (ROS) level and the PI3K/Akt signaling, but attenuated IL-1β-induced ROS production and inhibition of the PI3K/Akt signaling. IL-1β repressed the activation of the PI3K/Akt pathway, but ROS inhibition using N-acetylcysteine (NAC) rescued this pathway. The PI3K/Akt signaling suppression using LY294002 reversed the inhibitive effect of higenamine on IL-1β-caused apoptosis, and this effect was weakened by ROS inhibition. In conclusion, higenamine attenuates IL-1β-caused apoptosis of HNPCs via ROS-mediated PI3K/Akt pathway.

     

LINC00365 promotes colorectal cancer cell progression through the Wnt/β-catenin signaling pathway

In the past decade, substantial evidence established that long noncoding RNAs are serious about mediating the evolution of malignancies. In previous studies, LINC00365, which has not been reported in colorectal cancer (CRC), was selected using the bioinformatics analysis in GSE109454 and GSE41655 data sets. However, the function and mechanism of LINC00365 are still obscure. In our study, LINC00365 was found upregulated in CRC specimens and intimately connected with the prognosis of patients with CRC. In addition, LINC00365 overexpression enhances the cell abilities of proliferation, migration, and invasion in vitro. Meanwhile, mechanistic studies showed that LINC00365 might involve in CRC cell progression by mediating the Wnt/β-catenin pathway. Furthermore, LINC00365 upregulation increased CDK1 protein expression. In conclusion, this study suggests that LINC00365 acts as a vital part in facilitating CRC progression and might play as a therapeutic target for patients with CRC.     

C-X-C motif chemokine receptor 4 aggravates renal fibrosis through activating JAK/STAT/GSK3β/β-catenin pathway

Chronic kidney disease (CKD) has a high prevalence worldwide. Renal fibrosis is the common pathological feature in various types of CKD. However, the underlying mechanisms are not determined. Here, we adopted different CKD mouse models and cultured human proximal tubular cell line (HKC-8) to examine the expression of C-X-C motif chemokine receptor 4 (CXCR4) and β-catenin signalling, as well as their relationship in renal fibrosis. In CKD mice and humans with a variety of nephropathies, CXCR4 was dramatically up-regulated in tubules, with a concomitant activation of β-catenin. CXCR4 expression level was positively correlated with the expression of β-catenin target MMP-7. AMD3100, a CXCR4 receptor blocker, and gene knockdown of CXCR4 significantly inhibited the activation of JAK/STAT and β-catenin signalling, protected against tubular injury and renal fibrosis. CXCR4-induced renal fibrosis was inhibited by treatment with ICG-001, an inhibitor of β-catenin signalling. In HKC-8 cells, overexpression of CXCR4 induced activation of β-catenin and deteriorated cell injury. These effects were inhibited by ICG-001. Stromal cell–derived factor (SDF)-1α, the ligand of CXCR4, stimulated the activation of JAK2/STAT3 and JAK3/STAT6 signalling in HKC-8 cells. Overexpression of STAT3 or STAT6 decreased the abundance of GSK3β mRNA. Silencing of STAT3 or STAT6 significantly blocked SDF-1α-induced activation of β-catenin and fibrotic lesions. These results uncover a novel mechanistic linkage between CXCR4 and β-catenin activation in renal fibrosis in association with JAK/STAT/GSK3β pathway. Our studies also suggest that targeted inhibition of CXCR4 may provide better therapeutic effects on renal fibrosis by inhibiting multiple downstream signalling cascades.     

Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade

SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P(3) and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found that overexpression of Akt enhances formation of an inactive form of GSK3β (phospho-GSK3β) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3β attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3β, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3β attenuates RANKL-induced osteoclast differentiation from SHIP-deficient BMMs. Our data suggest that the PI3K/Akt/GSK3β/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis.     

Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway

Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.     

High FFA-induced proliferation and apoptosis in human umbilical vein endothelial cell partly through Wnt/β-catenin signal pathway

Free fatty acids (FFA)-induced proliferation and apoptosis was studied in human umbilical vein endothelial cells (HUVECs). A recombinant adenovirus containing a RNAi cassette targeting the GSK-3β gene was produced and its silencing effect on GSK-3β gene was detected by Western blot analysis and immunohistochemistry assay in HUVECs. The effect of the RNAi on the protein level of β-catenin was explored by transfecting the RNAi adenovirus to inhibit the expression of GSK-3β protein. The subsequent effect on the Wnt/GSK-3β/β-catenin signal pathway and on proliferation and apoptosis of HUVECs cultured with FFAs, was analyzed by BrdU assay, Annexin V-FITC/PI Apoptosis Detection Kit, and 4′,6-diamidino-2- phenylindole(DAPI) to explore the possible connection between the signaling pathway and FFA-induced proliferation and apoptosis. The Western blot results showed that the expression of GSK-3β protein in HUVECs could be inhibited efficiently by the RNAi adenovirus, and that the protein level of β-catenin was increased by RNAi adenovirus transfection. The results of the BrdU assay suggested that knockdown of GSK-3β with the RNAi adenovirus may stimulate the proliferation of HUVECs. Apoptosis was observed in HUVECs exposed to FFAs (0.75 mmol/L) for 72 h, and this effect could be partly reversed when interfering with the RNAi adenovirus. It may be concluded that the RNAi adenovirus specific to GSK-3β may partly protect HUVECs from apoptosis induced by FFAs, probably through the up-regulation of the Wnt/β-catenin signal pathway.     

LGR5 regulates survival through mitochondria-mediated apoptosis and by targeting the Wnt/β-catenin signaling pathway in colorectal cancer cells

Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly identified surface marker of colorectal cancer stem cells (CSCs). Expression level of LGR5 is commonly elevated in human CRCs. Our previous study demonstrated that the elevated expression of LGR5 is associated with CRC initiation and progression. However, the role of LGR5 in CRC pathogenesis has not been sufficiently established. In this study, we aimed to characterize the role of LGR5 in CRC pathogenesis using the loss-of-function approach. Depletion of LGR5 suppressed the growth of several cultured CRC cells and caused an increase in the fraction of apoptotic cells, which were analyzed using Annexin V/PI staining and DNA fragmentation assay. Furthermore, depleting LGR5 induced apoptosis through the loss of mitochondrial membrane potential. Additionally, depletion of LGR5 suppressed β-catenin nuclear translocation and blocked the activity of Wnt/β-catenin signaling as manifested in the reduced expression of c-myc and cyclin D, two Wnt/β-catenin targets in CRC cells. Treatment with Wnt3a considerably alleviated the growth inhibition and apoptotic cell death induced by LGR5 depletion in CRC cells. These data suggested that LGR5 regulates cell proliferation and survival by targeting the Wnt/β-catenin signaling pathway. Thus, the findings of this study suggest that LGR5 plays a vital role in CRC pathogenesis and has the potential to serve as a diagnostic marker and a therapeutic target for CRC patients.