The integrin α2β1 agonist,aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production

Background and purpose:

During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin α2β1 agonist.

Experimental approach:

Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques.

Key results:

Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin α2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-β. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by α2β1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-β mAb, or a phosphoinositide-3 kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Aggretin stimulated the phosphorylation of PDGFR-β, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and PD98059.

Conclusions and implications:

Aggretin induces VSMC proliferation and migration mainly through binding to integrin α2β1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.     

Upregulation of Sirt1 by tyrosol suppresses apoptosis and inflammation and modulates extracellular matrix remodeling in interleukin-1β-stimulated human nucleus pulposus cells through activation of PI3K/Akt pathway

Intervertebral disc degeneration (IDD) is the major pathogenesis of lower back pain. Tyrosol is a polyphenolic compound that exhibits anti-oxidant, anti-apoptotic, and anti-inflammatory effects. Herein, we explored the effects and mechanisms of tyrosol on IDD progression in interleukin (IL)-1β-stimulated human nucleus pulposus cells (HNPCs). Cell viability and apoptosis were detected by CCK-8 and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2) was examined to evaluate inflammation. The mRNA expression of matrix metalloproteinases (MMPs) (MMP-3/9/13), collagen type II, SRY-related high mobility group box 9 (SOX-9), and aggrecan was measured by qRT-PCR. Protein levels of silent information regulator 2 homolog 1 (Sirt1), phosphorylated protein kinase B (p-Akt), Akt, collagen type II, SOX-9, and aggrecan were determined by western blot. Results showed that tyrosol attenuated IL-1β-induced viability reduction, apoptosis, and caspase-3/7 activity in HNPCs. The increase in the production of TNF-α, IL-6, NO, and PGE2 in IL-1β-treated HNPCs was abolished by tyrosol treatment. Tyrosol treatment reversed IL-1β-induced upregulation of MMP-3, MMP-9, and MMP-13, and downregulation of collagen II, SOX-9, and aggrecan in HNPCs. Additionally, tyrosol treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in IL-1β-stimulated HNPCs. Sirt1 was upregulated by tyrosol, and Sirt1 silencing inhibited Akt phosphorylation in HNPCs. Sirt1 knockdown attenuated the effects of tyrosol on IL-1β-induced apoptosis, inflammation, and ECM remodeling in HNPCs. In summary, upregulation of Sirt1 by tyrosol suppressed apoptosis and inflammation and regulated ECM remodeling in IL-1β-stimulated HNPCs through activation of PI3K/Akt pathway.     

Recombinant expression in human cells of active integrin α1β1-blocking RTS-disintegrin jerdostatin

Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the α₁β₁ integrin. Jerdostatin selectively blocked the adhesion of α₁β₁-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein’s N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble α₁β₁ integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of α₁β₁ integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC₅₀ 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active α₁β₁-blocking jerdostatin in a mammalian cell system.     

MicroRNA-147b suppresses the proliferation and invasion of non-small-cell lung cancer cells through downregulation of Wnt/β-catenin signalling via targeting of RPS15A

Deregulation of microRNAs (miRNAs) leads to malignant growth and aggressive invasion during cancer occurrence and progression. miR-147b has emerged as one of the cancer-related miRNAs that are dysregulated in multiple cancers. Yet, the relevance of miR-147b in non-small-cell lung cancer (NSCLC) remains unclear. In the present study, we aimed to report the biological function and signalling pathways mediated by miR-147b in NSCLC. Our results demonstrate that miR-147b expression is significantly downregulated in NSCLC tissues and cell lines. Overexpression of miR-147b decreased the proliferative ability, colony-forming capability, and invasive potential of NSCLC cells. Notably, our study identified ribosomal protein S15A (RPS15A), an oncogene in NSCLC, as a target gene of miR-147b. Our results showed that miR-147b negatively modulates RPS15A expression in NSCLC cells. An inverse correlation between miR-147b and RPS15A was evidenced in NSCLC specimens. Moreover, miR-147b overexpression downregulated the activation of Wnt/β-catenin signalling via targeting of RPS15A. Overexpression of RPS15A partially reversed the miR-147b-mediated antitumour effect in NSCLC cells. Collectively, these findings reveal that miR-147b restricts the proliferation and invasion of NSCLC cells by inhibiting RPS15A-induced Wnt/β-catenin signalling and suggest that the miR-147b/RPS15A/Wnt/β-catenin axis is an important regulatory mechanism for malignant progression of NSCLC.     

Induction of G2/M arrest,caspase activation and apoptosis by α-santonin derivatives in HL-60 cells

Sesquiterpene lactones (SLs) are natural products with a variety of biological activities. Previously, we demonstrated the cytotoxic effects of three new α-santonin derivatives on different tumor cell lines with low toxic effects upon peripheral human leukocytes. Here, we evaluated the mechanism of action triggered by these derivatives. HL-60 cell cycle determined after 24 h treatment revealed a significant inhibition on cell-cycle progression and leading to an increasing of cells in G₂/M [7.6% and 9.0% for compound 3% and 9.0% and 8.6% for compound 4 (1 and 2 μM, respectively)]. However, after 48 h exposure, all compounds caused G₂/M reduction and a significant DNA fragmentation. Compounds 2, 3 and 4 were able to induce apoptosis on leukemia cells, which was corroborated by phosphatidyserine externalization and activation of caspases-3 and -7 after 24 h exposure. None of the derivatives analyzed caused depolarization of mitochondrial membrane within 24 h of incubation, suggesting the involvement of the extrinsic apoptotic pathway in the death process. The antiproliferative action of these compounds is related to the DNA synthesis inhibition and cell cycle arrest, which probably lead to apoptosis activation. Therefore, these santonin derivatives are promising lead candidates for development of new cytotoxic agents.     

Oroxylin A reverses CAM-DR of HepG2 cells by suppressing Integrinβ1 and its related pathway

Oroxylin A, a naturally occurring monoflavonoid extracted from Scutellariae radix, shows effective anticancer activities and low toxicities both in vivo and in vitro in previous studies. In this study, we investigated whether the CAM-DR model of HepG2 cells showed resistance to cytotoxic agents compared with normally cultured HepG2 cells. Furthermore, after the treatment of Paclitaxel, less inhibitory effects and decreased apoptosis rate were detected in the model. Data also revealed increased expression of Integrinβ1 might be responsible for the resistance ability. Moreover, Integrinβ1-siRNA-transfected CAM-DR HepG2 cells exhibited more inhibitory effects and higher levels of apoptosis than the non-transfected CAM-DR cells. The data corroborated that Integrinβ1 played a significant role in CAM-DR. After the treatment of weakly-toxic concentrations of Oroxylin A, the apoptosis induced by Paclitaxel in the CAM-DR model increased dramatically. Western blot assay revealed Oroxylin A markedly down-regulated the expression of Integrinβ1 and the activity of related pathway. As a conclusion, Oroxylin A can reverse the resistance of CAM-DR via inhibition of Integrinβ1 and its related pathway. Oroxylin A may be a potential candidate of a CAM-DR reversal agent.     

Echinacoside stimulates cell proliferation and prevents cell apoptosis in intestinal epithelial MODE-K cells by up-regulation of transforming growth factor-β1 expression

Cistanche deserticola MA (C. deserticola) has been widely used as a laxative herbal in herbal medicine for the treatment of irritable bowel syndrome or constipation, and echinacoside (ECH) is one of the major bioactive ingredients in this herbal. Our aim was to investigate the effect of ECH on intestinal epithelial cell growth and death. MODE-K, an intestinal epithelial cell line, was used as an in vitro model of the intestine. Cell proliferation was measured by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was determined with Annexin-V staining. Here we showed that in cultured MODE-K cells, ECH significantly stimulated cell proliferation and enhanced cell survival by reducing cell apoptosis in the presence of H(2)O(2) or the mixture of pro-inflammatory cytokines, while transforming growth factor (TGF)-β1 expression was up-regulated in a dose-dependent manner. Knockdown of TGF-β1 expression disrupted both the proliferative and cytoprotective activities of ECH, which was further confirmed by neutralization of TGF-β1 activity using anti-TGF-β1 antibody. These data suggest that ECH as one of bioactive ingredients in herbal C. deserticola and others may improve mucosal tissue repair by stimulating intestinal epithelial cell proliferation and preventing cell death via up-regulation of TGF-β.     

Protocatechuic acid inhibits cancer cell metastasis involving the down-regulation of Ras/Akt/NF-��B pathway and MMP-2 production by targeting RhoB activation

BACKGROUND AND PURPOSE

Protocatechuic acid (PCA) is plentiful in edible fruits and vegetables and is thus one anti-oxidative component of normal human diets. However, the molecular mechanisms involved in the chemopreventive activity of PCA are poorly understood. Here, we investigated the mechanism(s) underlying the anti-metastatic potential of PCA.

EXPERIMENTAL APPROACH

We used AGS cells in a wound healing model and Boyden chamber assays in vitro and injection of B16/F10 melanoma cells in mice (metastasis model in vivo) to analyse the effect of PCA on cancer cell invasion and metastasis. The activities and expression of molecular proteins were measured by zymographic assay, real-time RT-PCR and Western blotting.

KEY RESULTS

PCA inhibited cell migration and invasion at non-cytotoxic concentrations. Decreased expression of matrix metalloproteinase (MMP)-2 and a coincident increase in tissue inhibitor of MMP followed treatment with PCA. The PCA-inhibited MMP-2 activity and expression was accompanied by inactivation of NF-κB. All these effects of PCA could be mediated via the RhoB/ protein kinase Cε (PKCε) and Ras/Akt cascade pathways, as demonstrated by inhibition of PKCε and transfection of PKCε siRNA and ras overexpression vector. Finally, PCA inhibited metastasis of B16/F10 melanoma cells to the liver in mice.

CONCLUSION AND IMPLICATIONS

Our data imply that PCA down-regulated the Ras/Akt/NF-κB pathway by targeting RhoB activation, which in turn led to a reduction of MMP-mediated cellular events in cancer cells and provides a new mechanism for the anti-cancer activity of PCA.     

Stable expression of amloid protein precursor gene in SH-EP1 cells transfected with nicotinic acetylcholine receptor α4β2 subtype gene

Alzheimer＇s disease （AD） is one of the most common neurodegenerative diseases that affects the elderly. Neuropathologically, AD brains are characterized by the accumulation of extracellular amyloid plaques, mainly formed by a small peptide called amyloid-β （Aβ） .     

MicroRNA-520e targets AEG-1 to suppress the proliferation and invasion of colorectal cancer cells through Wnt/GSK-3β/β-catenin signalling

MicroRNA-520e (miR-520e) is increasingly being recognized as a cancer-related miRNA in multiple cancer types; however, little is known about its role in colorectal cancer. In this study, we determined the specific role of miR-520e in colorectal cancer. Expression of miR-520e was lower in colorectal cancer tissues compared to normal tissues. Overexpression of miR-520e significantly decreased the proliferation, colony formation and invasion of colorectal cancer cells, while inhibition of miR-520e exhibited the opposite effect. Moreover, miR-520e was found to target the 3′-untranslated region of astrocyte elevated gene-1 (AEG-1) and inhibit AEG-1 expression in colorectal cancer cells. An inverse correlation between miR-520e and AEG-1 expression was confirmed in colorectal cancer tissues. Notably, miR-520e suppressed the phosphorylation of glycogen synthase kinase-3β and decreased the expression of β-catenin, leading to inactivation of Wnt/β-catenin signalling in colorectal cells. A rescue assay confirmed that miR-520e regulates cell proliferation, invasion and Wnt/β-catenin signalling through targeting AEG-1. Taken together, these results indicate that miR-520e plays a critical role in regulating colorectal cancer cell proliferation and invasion by inhibiting Wnt/β-catenin signalling via AEG-1. Our study highlights the importance of the miR-520e/AEG-1/Wnt/β-catenin signalling axis in colorectal cancer, thus targeting miR-520e may represent an effective therapeutic strategy.     

Nephroblastoma overexpressed gene (NOV) enhances cell motility and COX-2 upregulation of human osteosarcoma involves αvβ5 integrin, ILK and AP-1-dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Nephroblastoma overexpressed gene (NOV), also called CCN3, was regulated proliferation and differentiation of cancer cells. However, the effect of NOV on migration activity and COX-2 expression in human osteosarcoma cells is mostly unknown. Here we found that NOV increased the migration and expression of COX-2 in human osteosarcoma cells. αvβ5 monoclonal antibody (mAb), integrin-linked kinase (ILK) and Akt inhibitor reduced the NOV-enhanced the migration and COX-2 up-regulation of osteosarcoma cells. NOV stimulation increased the ILK kinase activity and phosphorylation of Akt. In addition, c-Jun siRNA also antagonized the NOV-mediated migration and COX-2 expression. Moreover, NOV enhanced the AP-1 binding activity and promoter activity. Taken together, these results suggest that the NOV acts through αvβ5 integrin to activate ILK and Akt, which in turn activates c-Jun and AP-1, resulting in the activations of COX-2 and contributing the migration of human osteosarcoma cells.     

The role of hypoxia inducible factor-1α in the increased MMP-2 and MMP-9 production by human monocytes exposed to nickel nanoparticles

Abstract

Nickel is an important economic commodity, but it can cause skin sensitization and may cause lung diseases such as lung fibrosis, pneumonitis, bronchial asthma and lung cancer. With development of nanotechnology, nano-sized nickel (Nano-Ni) and nano-sized titanium dioxide (Nano-TiO₂) particles have been developed and produced for many years with new formulations and surface properties to meet novel demands. Our previous studies have shown that Nano-Ni instilled into rat lungs caused a greater inflammatory response as compared with standard-sized nickel (5 μm) at equivalent mass concentrations. Nano-Ni caused a persistent high level of inflammation in lungs even at low doses. Recently, several studies have shown that nanoparticles can translocate from the lungs to the circulatory system. To evaluate the potential systemic effects of metal nanoparticles, we compared the effects of Nano-Ni and Nano-TiO₂ on matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) gene expression and activity. Our results showed that exposure of human monocyte U937 to Nano-Ni caused dose- and time- dependent increase in MMP-2 and MMP-9 mRNA expression and pro-MMP-2 and pro-MMP-9 activity, but Nano-TiO₂ did not. Nano-Ni also caused dose- and time- related increase in tissue inhibitor of metalloproteinases 1 (TIMP-1), but Nano-TiO₂ did not. To determine the potential mechanisms involved, we measured the expression of hypoxia inducible factor 1α (HIF-1α) in U937 cells exposed to Nano-Ni and Nano-TiO₂. Our results showed that exposure to Nano-Ni caused HIF-1α accumulation in the nucleus. Furthermore, pre-treatment of U937 cells with heat shock protein 90 (Hsp90) inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), prior to exposure to Nano-Ni significantly abolished Nano-Ni-induced MMP-2 and MMP-9 mRNA upregulation and increased pro-MMP-2 and pro-MMP-9 activity. Our results suggest that HIF-1α accumulation may be involved in the increased MMP-2 and MMP-9 production in U937 cells exposed to Nano-Ni.     

The role of hypoxia inducible factor-1α in the increased MMP-2 and MMP-9 production by human monocytes exposed to nickel nanoparticles

Nickel is an important economic commodity, but it can cause skin sensitization and may cause lung diseases such as lung fibrosis, pneumonitis, bronchial asthma and lung cancer. With development of nanotechnology, nano-sized nickel (Nano-Ni) and nano-sized titanium dioxide (Nano-TiO?) particles have been developed and produced for many years with new formulations and surface properties to meet novel demands. Our previous studies have shown that Nano-Ni instilled into rat lungs caused a greater inflammatory response as compared with standard-sized nickel (5 μm) at equivalent mass concentrations. Nano-Ni caused a persistent high level of inflammation in lungs even at low doses. Recently, several studies have shown that nanoparticles can translocate from the lungs to the circulatory system. To evaluate the potential systemic effects of metal nanoparticles, we compared the effects of Nano-Ni and Nano-TiO? on matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) gene expression and activity. Our results showed that exposure of human monocyte U937 to Nano-Ni caused dose- and time- dependent increase in MMP-2 and MMP-9 mRNA expression and pro-MMP-2 and pro-MMP-9 activity, but Nano-TiO? did not. Nano-Ni also caused dose- and time- related increase in tissue inhibitor of metalloproteinases 1 (TIMP-1), but Nano-TiO? did not. To determine the potential mechanisms involved, we measured the expression of hypoxia inducible factor 1α (HIF-1α) in U937 cells exposed to Nano-Ni and Nano-TiO?. Our results showed that exposure to Nano-Ni caused HIF-1α accumulation in the nucleus. Furthermore, pre-treatment of U937 cells with heat shock protein 90 (Hsp90) inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), prior to exposure to Nano-Ni significantly abolished Nano-Ni-induced MMP-2 and MMP-9 mRNA upregulation and increased pro-MMP-2 and pro-MMP-9 activity. Our results suggest that HIF-1α accumulation may be involved in the increased MMP-2 and MMP-9 production in U937 cells exposed to Nano-Ni.     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

Alpha-Mangostin suppresses interleukin-1β-induced apoptosis in rat chondrocytes by inhibiting the NF-κB signaling pathway and delays the progression of osteoarthritis in a rat model

Osteoarthritis (OA) is a chronic degenerative joint disease that is characterized by progressive joint dysfunction and pain. Apoptosis and catabolism in chondrocytes play critical roles in the development of OA. Alpha-Mangostin (α-MG), one of the main components of the mangosteen, has been reported to have anti-apoptotic, anti-inflammatory and antioxidant effects. We investigated the therapeutic effects of α-MG on OA through experiments on rat chondrocytes in vitro and in a rat model of OA induced by destabilization of the medial meniscus (DMM). In vitro, we provided experimental evidence that α-MG inhibits the expression of MMP-13 and ADAMTs-5, and promotes the expression of SOX-9 in rat chondrocytes stimulated with interleukin-1β (IL-1β). In addition, we also found that α-MG can inhibit the expression of pro-apoptotic proteins such as Bax, Cyto-c, and C-caspase3, and increase the expression of the anti-apoptotic protein Bcl-2. These changes may be related to an α-MG induced inhibition of the IL-1β-induced activation of the NF-kB signaling pathway. In vivo, we also found that α-MG can limit the development of OA in rat models. The above results show that α-MG has a potential therapeutic effect on OA, and that this effect may be achieved by inhibiting the mitochondrial apoptosis of chondrocytes induced by an activation of the NF-kB pathway.     

Induction of p27<Superscript>kip1</Superscript> by 2,4,3′,5′-tetramethoxystilbene is regulated by protein phosphatase 2A-dependent Akt dephosphorylation in PC-3 prostate cancer cells

trans-Stilbenes induce cytochrome P450 1B1 (CYP1B1) inhibition and cell death. 2,4,3',5' tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, induced apoptotic cell death in PC-3 prostate cancer cells, as evidenced by a decrease in the mitochondrial membrane potential. TMS-induced apoptosis was associated with an increase in the level of cell cycle inhibitor, p27(kip1), through reduction of Akt-mediated Skp2 expression. TMS-induced activation of protein phosphatase 2A (PP2A) inhibited Akt phosphorylation and p27(kip1) expression, indicating that PP2A is involved in the induction of p27(kip1) via Akt inhibition. These results suggest that TMS may inhibit the cell cycle through induction of p27(kip1), leading to apoptotic cell death in PC-3 prostate cancer cells.     

Withanolide B promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via ERK1/2 and Wnt/β-catenin signaling pathways

BackgroundThe treatment of bone defects has always been a problem for clinicians. In recent years, research on human bone mesenchymal stem cells (hBMSCs) has found that promoting their osteogenic differentiation could be a useful therapeutic strategy for bone healing. Previous studies have been reported that Withania somnifera Dunal inhibits osteoclastogenesis by inhibiting the NF-κB signaling pathway. Withanolide B is an active component of W. somnifera Dunal, but its role in osteogenic differentiation of hBMSCs remains unknown. Here, we performed a preliminary study on the role of Withanolide B in promoting osteogenic differentiation and its possible mechanism.MethodsWe investigated the effect of Withanolide B on osteogenic differentiation of hBMSCs in vitro and in vivo. The effect of Withanolide B on the activity of hBMSCs was verified by CCK-8 assay and quantitative Real-time polymerase chain reaction (qPCR) and Western blotting analysis were used to verify the effect of Withanolide B on osteogenic differentiation-specific genes and proteins. The effect of Withanolide B on ALP activity and mineral deposition was verified by ALP and ARS staining. We then used a rat tibial osteotomy model to observe the effect of Withanolide B on bone healing.ResultsWithanolide B is noncytotoxic to hBMSCs and can effectively promote their osteogenic differentiation. Moreover, we found that Withanolide B can regulate the osteogenic differentiation of hBMSCs through the ERK1/2 and Wnt/β-catenin signaling pathways. When inhibitors of the ERK1/2 and Wnt/β-catenin signaling pathways were used, the enhancement of osteogenic differentiation induced by Withanolide B was attenuated. Withanolide B also effectively promoted bone healing in the rat tibial osteotomy model.ConclusionsOur results suggest that Withanolide B can promote the osteogenic differentiation of hBMSCs through the ERK1/2 and Wnt/β-catenin signaling pathways and can effectively promote bone defect healing.