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Beijing Da Xue Xue Bao. Yi Xue Ban =... Feb 2018To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism.
[Decreased phosphorylation of mitogen activated protein kinase and protein kinase B contribute to the inhibition of osteogenic differentiation mediated by activation of Toll like receptor in human periodontal ligament stem cells].
OBJECTIVE
To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism.
METHODS
Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium.
RESULTS
Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 μg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125,SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression.
CONCLUSION
Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.
Topics: Cell Differentiation; Cells, Cultured; Humans; Ligaments; Mitogen-Activated Protein Kinases; Osteogenesis; Periodontal Ligament; Phosphorylation; Proto-Oncogene Proteins c-akt; Stem Cells; Toll-Like Receptors
PubMed: 29483719
DOI: No ID Found -
Oncology Research May 2019Hypoxia-induced chemoresistance is a major obstacle in the development of effective cancer therapy. In our study, the reversal abilities of NADPH oxidase 4 (NOX4)...
Hypoxia-induced chemoresistance is a major obstacle in the development of effective cancer therapy. In our study, the reversal abilities of NADPH oxidase 4 (NOX4) silence on hypoxia resistance and the potential mechanism were investigated. Our data showed that the expression of NOX4 was upregulated in human neuroblastoma cells SH-SY5Y under hypoxia condition time dependently. Knockdown of NOX4 expression by siRNA inhibited glycolysis induced by hypoxia through decreasing the expression of glycolysis-related proteins (HIF-1α, LDHA, and PDK1), decreasing glucose uptake, lactate production, and ROS production, while increasing mitochondria membrane potential. Moreover, NOX4 silence inhibited cell growth under hypoxia condition through suppressing cell proliferation and proliferation-related proteins (Ki-67 and PCNA) compared with the hypoxia 24 h + siRNA NC group. Further, Western blot experiments exhibited that NOX4 siRNA could downregulate the rate of p-Akt/Akt. Treatment with PI3K/Akt signaling activator IGF-1 blocked, while treatment with Akt inhibitor perifosine enhanced the inhibitory effect of si-NOX4 on glycolysis and cell growth. In summary, knockdown of NOX4 had the ability of reversing hypoxia resistance, and the major mechanism is considered to be the inhibition of glycolysis and cell growth via the PI3K/Akt signaling pathway. Therefore, NOX4 could be a novel target against hypoxia resistance in neuroblastoma.
Topics: Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Gene Silencing; Glycolysis; Humans; Hypoxia; Insulin-Like Growth Factor I; Membrane Potential, Mitochondrial; NADPH Oxidase 4; Neuroblastoma; Neurons; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction
PubMed: 29426376
DOI: 10.3727/096504018X15179668157803 -
Cell Discovery 2018The mammalian target of rapamycin (mTOR) pathway is commonly activated in human cancers. The activity of mTOR complex 1 (mTORC1) signaling is supported by the...
The mammalian target of rapamycin (mTOR) pathway is commonly activated in human cancers. The activity of mTOR complex 1 (mTORC1) signaling is supported by the intracellular positioning of cellular compartments and vesicle trafficking, regulated by Rab GTPases. Here we showed that tuftelin 1 (TUFT1) was involved in the activation of mTORC1 through modulating the Rab GTPase-regulated process. TUFT1 promoted tumor growth and metastasis. Consistently, the expression of TUFT1 correlated with poor prognosis in lung, breast and gastric cancers. Mechanistically, TUFT1 physically interacted with RABGAP1, thereby modulating intracellular lysosomal positioning and vesicular trafficking, and promoted mTORC1 signaling. In addition, expression of predicted sensitivity to perifosine, an alkylphospholipid that alters the composition of lipid rafts. Perifosine treatment altered the positioning and trafficking of cellular compartments to inhibit mTORC1. Our observations indicate that TUFT1 is a key regulator of the mTORC1 pathway and suggest that it is a promising therapeutic target or a biomarker for tumor progression.
PubMed: 29423269
DOI: 10.1038/s41421-017-0001-2 -
Oncogene Apr 2018Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal...
Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to "leader bleb-based" migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors. Elevated Src activity was found to change actin and focal adhesion dynamics, whereas inhibiting Src triggered focal adhesion disassembly and blebbing. On non-adherent substrates and in collagen matrices, amoeboid-like, blebbing cells having high Src activity formed protrusions of the plasma membrane. To evaluate the role of Src in confined cells, we use a novel approach that places cells under a slab of polydimethylsiloxane (PDMS), which is held at a defined height. Using this method, we find that leader bleb-based migration is resistant to Src inhibition. High Src activity was found to markedly change the architecture of cortical actomyosin, reduce cell mechanical properties, and the percentage of cells that undergo leader bleb-based migration. Thus, Src is a signal transducer that can potently influence transitions between migration modes with implications for the rational development of metastasis inhibitors.
Topics: Actins; Amino Acid Substitution; Butadienes; Cell Adhesion; Cell Movement; Dasatinib; Humans; Imidazoles; Mutant Proteins; Neoplasms; Nitriles; Phosphorylcholine; Protein Kinase Inhibitors; Proto-Oncogene Proteins pp60(c-src); Quinolines; Tumor Cells, Cultured
PubMed: 29379163
DOI: 10.1038/s41388-017-0071-5 -
Food and Chemical Toxicology : An... Jan 2018Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have...
The sp-iminosugar glycolipid 1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO-ONJ) as selective anti-inflammatory agent by modulation of hemeoxygenase-1 in Bv.2 microglial cells and retinal explants.
Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp-iminosugar glycolipid (sp-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO-ONJ in microglia.
Topics: Animals; Anti-Inflammatory Agents; Cell Line; Gene Expression Regulation, Enzymologic; Glycolipids; Heme Oxygenase-1; Lipopolysaccharides; Mice; Mice, Inbred NOD; Microglia; Retina; Tissue Culture Techniques
PubMed: 29191728
DOI: 10.1016/j.fct.2017.11.050 -
Medical Science Monitor : International... Nov 2017BACKGROUND Chronic mountain sickness (CMS) has a higher incidence in the plateau region. The one of its principal characters is excessive erythrocytosis. The PI3K-Akt...
BACKGROUND Chronic mountain sickness (CMS) has a higher incidence in the plateau region. The one of its principal characters is excessive erythrocytosis. The PI3K-Akt pathway plays an important role in the process of erythropoiesis, and could downregulate apoptosis by regulating apoptosis-related molecules. In this paper, we explored the change in apoptosis of erythroblasts and the effect of the PI3K-Akt signal pathway on erythroblasts apoptosis in CMS. MATERIAL AND METHODS A total of 22 CMS and 20 non-CMS participants were involved in this study. Bone marrow mononuclear cells were cultured and treated with celecoxib and perifosine in vitro for 72 hours. The apoptotic rate, the mRNA expressions of Akt, Bcl-xl, and caspase-9, and the protein expressions of Akt, p-Akt, Bcl-xl, and caspase-9 were determined by flow cytometry, quantitative RT-PCR, and western-blot technique. RESULTS The apoptotic rate of cultured erythroblasts was lower in the CMS group than in the non-CMS group. It was increased after perifosine intervention. The mRNA and protein expressions of Akt and Bcl-xl were higher and caspase-9 was lower in the CMS group than the non-CMS group. Perifosine induced decreased Bcl-xl mRNA and proteins and p-Akt proteins, and increased caspase-9 mRNA and proteins in vitro. In the CMS group, the hemoglobin concentration was correlated with apoptotic rate negatively and with Bcl-xl mRNA positively in erythroblasts; the erythroblasts apoptotic rate was negatively associated with the Akt mRNA and Bcl-xl mRNA. CONCLUSIONS The erythroblasts apoptosis was downregulated and the PI3K-Akt signal pathway appeared to be involved in the mechanism of decreased erythroblasts apoptosis in CMS.
Topics: Adult; Altitude Sickness; Apoptosis; Bone Marrow Cells; Caspase 9; Celecoxib; China; Chronic Disease; Down-Regulation; Erythroblasts; Humans; Male; Middle Aged; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Signal Transduction; bcl-X Protein
PubMed: 29176544
DOI: 10.12659/msm.905739 -
Oncotarget Sep 2017Tripchlorolide (T4) has been shown to induce A549 lung cancer cell death predominantly by activating an autophagy pathway. However, the underlying mechanism remains...
Tripchlorolide (T4) has been shown to induce A549 lung cancer cell death predominantly by activating an autophagy pathway. However, the underlying mechanism remains unclear. Herein, we demonstrated that compared with T4 treatment alone, pretreatment with wortmannin (an inhibitor of phosphatidylinositol 3-kinase), perifosine (an inhibitor of AKT) or rapamycin (an inhibitor of mTOR) combined with a subsequent T4 treatment significantly impaired the cell viability of A549 and A549/DDP lung cancer cells. We found that either treatment scheme markedly reduced the activity of P13K and AKT. Expression of LC3II increased in parallel to the increase of the T4 concentration in both A549 and A549/DDP cells and was repressed by overexpression of AKT. The expression levels of PI3-K, PI3-P, AKT, TSC2, mTOR, p70S6K and 4E-BP1 were minimally affected by the wortmannin, perifosine, or rapamycin plus T4 treatments, but their phosphorylated products were greatly affected in A549 lung cancer cells and slightly affected in A549/DDP lung cancer cells. These results indicate that T4 induces autophagy in lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway. We further found that T4 decreased expression of MDR1 and improved cisplatin sensitivity of A549/DDP cells. Altogether, these results have meaningful implications for tumor therapy in the future.
PubMed: 28969040
DOI: 10.18632/oncotarget.19201 -
Cancer Medicine Nov 2017Malignant pleural mesothelioma (MPM), an asbestos-related occupational disease, is an aggressive and incurable tumor of the thoracic cavity. Despite recent advances in...
Malignant pleural mesothelioma (MPM), an asbestos-related occupational disease, is an aggressive and incurable tumor of the thoracic cavity. Despite recent advances in MPM treatment, overall survival of patients with MPM is very low. Recent studies have implicated that PI3K/Akt signaling is involved in MPM cell survival and development. To investigate the effects of Akt inhibitors on MPM cell survival, we examined the effects of nine selective Akt inhibitors, namely, afuresertib, Akti-1/2, AZD5363, GSK690693, ipatasertib, MK-2206, perifosine, PHT-427, and TIC10, on six MPM cell lines, namely, ACC-MESO-4, Y-MESO-8A, MSTO-211H, NCI-H28, NCI-H290, and NCI-H2052, and a normal mesothelial cell line MeT-5A. Comparison of IC values of the Akt inhibitors showed that afuresertib, an ATP-competitive specific Akt inhibitor, exerted tumor-specific effects on MPM cells. Afuresertib significantly increased caspase-3 and caspase-7 activities and apoptotic cell number among ACC-MESO-4 and MSTO-211H cells. Moreover, afuresertib strongly arrested the cell cycle in the G phase. Western blotting analysis showed that afuresertib increased the expression of p21 and decreased the phosphorylation of Akt substrates, including GSK-3β and FOXO family proteins. These results suggest that afuresertib-induced p21 expression promotes G phase arrest by inducing FOXO activity. Furthermore, afuresertib significantly enhanced cisplatin-induced cytotoxicity. Interestingly, results of gene set enrichment analysis showed that afuresertib modulated the expression E2F1 and MYC, which are associated with fibroblast core serum response. Together, these results suggest that afuresertib is a useful anticancer drug for treating patients with MPM.
Topics: Antineoplastic Agents; Apoptosis; Benzylamines; Caspase 3; Caspase 7; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Forkhead Box Protein O1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Heterocyclic Compounds, 3-Ring; Heterocyclic Compounds, 4 or More Rings; Humans; Imidazoles; Inhibitory Concentration 50; Mesothelioma; Oxadiazoles; Phosphorylation; Phosphorylcholine; Pleural Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridines; Pyrimidines; Pyrroles; Quinoxalines; Sulfonamides; Thiadiazoles; Thiophenes
PubMed: 28960945
DOI: 10.1002/cam4.1179 -
PLoS Neglected Tropical Diseases Aug 2017Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory....
BACKGROUND
Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.
METHODOLOGY/PRINCIPAL FINDINGS
Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.
CONCLUSIONS/SIGNIFICANCE
The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis.
Topics: Animals; Antineoplastic Agents; Antiprotozoal Agents; Cell Survival; Cells, Cultured; Disease Models, Animal; Gene Deletion; Humans; Leishmania; Leishmaniasis; Macrophages; Membrane Microdomains; Membrane Potential, Mitochondrial; Mice; Mitochondria; Phospholipid Ethers; Proton-Translocating ATPases; Saccharomyces cerevisiae; Treatment Outcome
PubMed: 28829771
DOI: 10.1371/journal.pntd.0005805 -
Neuroscience Bulletin Apr 2018Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by...
Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.
Topics: Animals; Anticonvulsants; Brain; Convulsants; Disease Models, Animal; Epilepsy, Temporal Lobe; Kainic Acid; Male; Neurons; Phosphorylcholine; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Status Epilepticus
PubMed: 28786074
DOI: 10.1007/s12264-017-0165-7