WNT-LRP5 Signaling Induces Warburg Effect through mTORC2 Activation during Osteoblast Differentiation

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Flouride Promotes Viability and Differentiation of Osteoblast-Like Saos-2 Cells Via BMP/Smads Signaling Pathway

The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.     

Berberine Inhibits Invasion and Metastasis of Colorectal Cancer Cells via COX-2/PGE2 Mediated JAK2/STAT3 Signaling Pathway

Berberin, extracted from Chinese herbal medicine Coptis chinensis, has been found to have anti-tumor activities. However, the underlying mechanisms have not been fully elucidated. Our current study demonstrated that berberin inhibited the in vitro and in vivo growth, migration/invasion of CRC cells, via attenuating the expression levels of COX-2/PGE₂, following by reducing the phosphorylation of JAK2 and STAT3, as well as the MMP-2/-9 expression. We further clarified that an increase of COX-2/PGE₂ expression offset the repressive activity of Berberin on JAK2/STAT3 signaling, and a JAK2 inhibitor AZD1480 blocked the effect of COX-2/PGE₂ on MMP-2/-9 expression. In summary, Berberin inhibited CRC invasion and metastasis via down-regulation of COX-2/PGE₂- JAK2/STAT3 signaling pathway.     

毒蝇碱型乙酰胆碱受体经Ras—ERK—1／2信号通路上调Bcl—2和磷酸化Bad表达

毒蝇碱型乙酰胆碱受体 (muscarinicacetylcholinereceptor,mAChR)和Bcl 2家族蛋白均具有调控神经细胞凋亡和生存的作用 ,然而mAChR和Bcl 2家族蛋白之间的内在联系即信号转导通路仍然不清楚。为此 ,对mAChR调控神经母细胞瘤SH SY5Y细胞生存蛋白Bcl 2和磷酸化Bad的信号转导通路进行了研究。结果显示 :(1)mAChR激动剂卡巴可 (carbachol)不仅活化SH SY5Y细胞的MEK/ERK 1/ 2 ,而且上调Bcl 2和磷酸化Bad的表达 ;(2 )mAChR拮抗剂阿托品、MEK抑制剂PD980 5 9、PKC抑制剂bisindolymaleimide I和Src抑制剂PP1均能完全阻断或显著减弱卡巴可的上述作用 ,但G蛋白脱偶联剂百日咳毒素和PI 3激酶抑制剂wortmannin对卡巴可的上述作用无明显影响 ;(3)显性负突变Ras和Raf均能阻断卡巴可上调转染至SH SY5Y细胞内的Bcl 2启动子的转录调控活性。结果表明 :mAChR通过Gq/ 11、PKC和Src依赖的Ras ERK 1/ 2信号转导通路上调SH SY5Y细胞Bcl 2和磷酸化Bad蛋白表达。这一研究将有助于揭示神经递质、神经营养因子和神经营养药物等抑制神经细胞凋亡、促进神经细胞生存的分子机制。     

ERK1/2信号通路活化对Tamoxifen所致胶质瘤细胞凋亡的影响

为了探讨ERK1/2信号通路在他莫昔芬(tamoxifen, TAM)所致胶质瘤细胞凋亡中的作用,以C6和U87MG胶质瘤细胞为研究对象,经TAM处理后,采用MTT法检测细胞的存活率;倒置显微镜和DAPI染色观察细胞的形态;流式细胞术检测细胞凋亡; Western-blot法检测细胞内ERK1/2磷酸化水平。最后应用ERK1/2抑制剂(PD98059)与TAM共同作用,观察其对胶质瘤细胞内ERK1/2磷酸化水平和细胞凋亡的影响。实验结果显示:TAM可呈浓度和时间依赖性地抑制胶质瘤细胞生长; TAM处理组的细胞凋亡明显增加且呈浓度依赖性;TAM能增加细胞内ERK1/2磷酸化水平;以PD98059阻断ERK1/2的激活,能增强TAM诱导细胞凋亡的作用。实验结果表明TAM能够抑制胶质瘤细胞生长和促进其细胞凋亡, ERK1/2信号通路的激活参与调控TAM所致胶质瘤细胞凋亡。     

Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway

Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

     

Quinoline Compound KM11073 Enhances BMP-2-Dependent Osteogenic Differentiation of C2C12 Cells via Activation of p38 Signaling and Exhibits In Vivo Bone Forming Activity

Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl) piperazino][1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]methanone (KM11073) strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the in vivo osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.     

Low-Intensity Pulsed Ultrasound Accelerates Tooth Movement via Activation of the BMP-2 Signaling Pathway

The present study was designed to determine the underlying mechanism of low-intensity pulsed ultrasound (LIPUS) induced alveolar bone remodeling and the role of BMP-2 expression in a rat orthodontic tooth movement model. Orthodontic appliances were placed between the homonymy upper first molars and the upper central incisors in rats under general anesthesia, followed by daily 20-min LIPUS or sham LIPUS treatment beginning at day 0. Tooth movement distances and molecular changes were evaluated at each observation point. In vitro and in vivo studies were conducted to detect HGF (Hepatocyte growth factor)/Runx2/BMP-2 signaling pathways and receptor activator of NFκB ligand (RANKL) expression by quantitative real time PCR (qRT-PCR), Western blot and immunohistochemistry. At day 3, LIPUS had no effect on the rat orthodontic tooth movement distance and BMP-2-induced alveolar bone remodeling. However, beginning at day 5 and for the following time points, LIPUS significantly increased orthodontic tooth movement distance and BMP-2 signaling pathway and RANKL expression compared with the control group. The qRT-PCR and Western blot data in vitro and in vivo to study BMP-2 expression were consistent with the immunohistochemistry observations. The present study demonstrates that LIPUS promotes alveolar bone remodeling by stimulating the HGF/Runx2/BMP-2 signaling pathway and RANKL expression in a rat orthodontic tooth movement model, and LIPUS increased BMP-2 expression via Runx2 regulation.     

Human and Simian T-Cell Leukemia Viruses Type 2 (HTLV-2 and STLV-2pan-p) Transform T Cells Independently of Jak/STAT Activation

Human T-lymphotropic virus type 1 (HTLV-1) and HTLV-2 differ in pathogenicity in vivo. HTLV-1 causes leukemia and neurologic and inflammatory diseases, whereas HTLV-2 is less clearly associated with human disease. Both retroviruses transform human T cells in vitro, and transformation by HTLV-1 was found to be associated with the constitutive activation of the Jak/STAT pathway. To assess whether HTLV-2 transformation may also result in constitutive activation of the Jak/STAT pathway, six interleukin-2-independent, HTLV-2-transformed T-cell lines were analyzed for the presence of activated Jak and STAT proteins by electrophoretic mobility shift assay. In addition, the phosphorylation status of Jak and STAT proteins was assessed directly by immunoprecipitation and immunoblotting with an antiphosphotyrosine antibody. Jak/STAT proteins were not found to be constitutively activated in any of the T-cell lines infected by the type 2 human and nonhuman primate viruses, suggesting that HTLV-2 and the cognate virus simian T-lymphotropic virus type 2 from Pan paniscus transform T cells in vitro by mechanisms at least partially different from those used by HTLV-1.     

Dihydromyricetin Enhances the Chemo-Sensitivity of Nedaplatin via Regulation of the p53/Bcl-2 Pathway in Hepatocellular Carcinoma Cells

Chemotherapy is an effective weapon in the battle against cancer. Nedaplatin (NDP) is an improved platinum-containing drug with lower cytotoxicity than other similar drugs. However, the repeated use of NDP results in substantial hepatocyte damage as well as drug resistance in hepatocellular carcinoma (HCC) cases. Therefore, the development of effective chemotherapy strategies that enhance tumor sensitivity to chemotherapeutics and reduce the secondary damage to liver cells is urgently needed. Dihydromyricetin (DHM), a natural flavonoid compound, has been shown to have antitumor activity with no obvious toxicity to normal cells in vitro and in vivo. In this study, DHM and NDP were combined to treat liver cancer cells; we found that DHM functions as a protector of normal cells compared with the use of NDP alone. In addition, the synergy of DHM with NDP enhanced the effect of NDP on the induction of HCC cell apoptosis. We found that the combination caused clear changes in the level of reactive oxygen species (ROS). Furthermore, we demonstrated that the combination of DHM and NDP activated the p53/Bcl-2 signaling pathway, which resulted in mitochondrial dysfunction and induced cell death and growth inhibition in HCC cells.     

Hyperglycemia Enhances the Proliferation of Non-Tumorigenic and Malignant Mammary Epithelial Cells through Increased leptin/IGF1R Signaling and Activation of AKT/mTOR

Obesity and diabetes are associated with increased breast cancer risk and worse disease progression once cancer is diagnosed; however, the exact etiology behind these observations remains to be fully elucidated. Due to the global obesity/diabetes pandemic, it is imperative to understand how these diseases promote and enhance breast cancer and other common cancers. In this study we demonstrate that hyperglycemia promotes breast cancer by altering leptin/IGF1R and AKT/mTOR signaling. To our knowledge, we show for the first time that in breast epithelial cells, hyperglycemia alone directly impacts leptin signaling. Hyperglycemia increased proliferation of both non-tumorigenic and malignant mammary epithelial cells. These observations coincided with increased leptin receptor and IGF1R receptor, as well as, increased levels of GRB2, pJAK2, pSTAT3, pIRS1/2, pAKT, and p-mTOR. Moreover, pJAK2 was almost completely colocalized with leptin receptor under high glucose conditions. These results demonstrate how hyperglycemia can potentially increase the risk of breast cancer in premalignant lesions and enhance cancer progression in malignant cells.