BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effect...BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.展开更多
BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stabl...BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.展开更多
The mammalian target of rapamycin （mTOR） pathway plays an important role in neuronal growth, proliferation and differentiation. To better understand the role of mTOR pathway involved in the induction of spinal cord ...The mammalian target of rapamycin （mTOR） pathway plays an important role in neuronal growth, proliferation and differentiation. To better understand the role of mTOR pathway involved in the induction of spinal cord injury, rat models of spinal cord injury were established by modified Allen＇s stall method and interfered for 7 days by intraperitoneal administration of mTOR activator adenosine triphosphate and mTOR kinase inhibitor rapamycin. At 1-4 weeks after spinal cord injury induction, the Basso, Beattie and Bresnahan locomotor rating scale was used to evaluate rat locomotor function, and immunohistochemical staining and western blot analysis were used to detect the expression of nestin （neural stem cell marker）, neuronal nuclei （neuronal marker）, neuron specific enolase, neurofilament protein 200 （axonal marker）, glial fibrillary acidic protein （astrocyte marker）, Akt, mTOR and signal transduction and activator of transcription 3 （STAT3）. Results showed that adenosine triphosphate-mediated Akt/mTOR/STAT3 pathway increased endogenous neural stem cells, induced neurogenesis and axonal growth, inhibited excessive astrogliosis and improved the locomotor function of rats with spinal cord injury.展开更多
Deregulation of the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)- mammalian target of rapamycin(m TOR) signaling pathway is one of the most commonlyinvolved pathways in tumorigenesis. It has also been reporte...Deregulation of the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)- mammalian target of rapamycin(m TOR) signaling pathway is one of the most commonlyinvolved pathways in tumorigenesis. It has also been reported as altered in neuroendocrine tumors(NETs). m TOR inhibitors used in clinical practice are derived from rapamycin,an anti-cancer agent also used as an immunosuppressor after organ transplantation. Everolimus and temsirolimus are the two rapamycin-derived m TOR inhibitors used in NETs. Notably everolimus has been approved in advanced progressive well/moderatelydifferentiated pancreatic NETs(p NETs). It inhibits specifically the m TORC1 subunit of m TOR,not interacting with m TORC2. Although everolimus produced a significant prolongation of progression-free survival a number of patients with p NETs do not benefit from the drug due to early or late progression. Two supposed mechanisms of resistance to m TOR inhibitors are Akt and PI3 K activation,by means of m TORC2 and insulin growth factor(IGF)- IGF receptor signaling,respectively. BEZ235 is a multi-targeted inhibitor binding to PI3 K,m TORC1 and m TORC2,therefore potentially turning off all the supposed molecular targets of resistance to everolimus. The two clinical trials designed in p NETs were stopped early due to unmet statistical endpoint and the global clinical development of BEZ235 was also halted. Tolerability of this drug was challenging and conditioned the feasibility of therapy. The BEZ experience is an example of the huge difference between the preclinical and clinical setting and prompts us to pay more attention to the phase Ⅰ step of clinical development and the design of phase Ⅱ clinical trials.展开更多
Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autop...Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.展开更多
Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. Howe...Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.展开更多
As major fuels for the small intestinal mucosa,dietary amino acids(AA)are catabolized in the mitochondria and serve as sources of energy production.The present study was conducted to investigate AA metabolism that sup...As major fuels for the small intestinal mucosa,dietary amino acids(AA)are catabolized in the mitochondria and serve as sources of energy production.The present study was conducted to investigate AA metabolism that supply cell energy and the underlying signaling pathways in porcine enterocytes.Intestinal porcine epithelial cells(IPEC-J2)were treated with different concentrations of AA,inhibitor,or agonist of mammalian target of rapamycin complex 1(mTORCl)and adenosine monophosphate activated protein kinase(AMPK),and mitochondrial respiration was monitored.The results showed that AA treatments resulted in enhanced mitochondrial respiration,increased intracellular content of pyruvic acid and lactic acid,and increased hormone-sensitive lipase mRNA expression.Meanwhile,decreased citrate synthase,isocitrate dehydrogenase alpha,and carnitine palmitoyltransferase 1 mRNA expression were also observed.We found that AA treatments increased the protein levels of phosphorylated mammalian target of rapamycin(p-mTOR),phosphorylated-p70 ribosomal protein S6 kinase,and phosphorylated-4 E-binding protein 1.What is more,the protein levels of phosphorylated AMPKα(pAMPKa)and nicotinamide adenine dinucleotide(NAD)-dependent protein deacetylase sirtuin-1(SIRT1)were decreased by AA treatments in a time depending manner.Mitochondrial bioenergetics and the production of tricarboxylic acid cycle intermediates were decreased upon inhibition of mTORCl or AMPK.Moreover,AMPK activation could up-regulate the mRNA expressions of inhibitor of nuclear factor kappa-B kinase subunit beta(Ikbk(3),integrin-linked protein kinase(ILK),unconventional myosin-Ic(Myolc),ribosomal protein S6 kinase beta-2(RPS6 Kβ2),and vascular endothelial growth factor(VEGF)-β,which are downstream effectors of mammalian target of rapamycin(mTOR).The mRNA expressions of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform(PIK3 CD)and5’-AMP-activated protein kinase subunit gamma-1(PRKAG1),which are upstream regulators of mTOR,were also up-regulated by AMPK activation.On the other hand,AMPK activation also down-regulated FK506-binding protein 1 A(FKBP1 A),serine/threonine-protein phosphatase 2 A 55 kDa regulatory subunit B beta isoform,phosphatase and tensin homolog(PTEN),and unc-51 like autophagy activating kinase 1(Ulkl),which are up-stream regulators of mTORCl.Taken together,these data indicated that AA regulated cellular energy metabolism through mTOR and AMPK pathway in porcine enterocytes.These results demonstrated interactions of AMPK and mTORCl pathways in AA catabolism and energy metabolism in intestinal mucosa cells of piglets,and also provided reference for using AA to remedy human intestinal diseases.展开更多
Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current st...Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.展开更多
Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson’s disease has been ...Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson’s disease has been shown to have a positive effect, but the underlying mechanism remains unknown. Based on this, we explored whether moxibustion could protect dopaminergic neurons by promoting autophagy mediated by mammalian target of rapamycin （mTOR）, with subsequent elimination of α-syn. A Parkinson’s disease model was induced in rats by subcutaneous injection of rotenone at the back of their necks, and they received moxibustion at Zusanli （ST36）, Guanyuan （CV4）and Fengfu （GV16）, for 10 minutes at every point, once per day, for 14 consecutive days. Model rats without any treatment were used as a sham control. Compared with the Parkinson’s disease group, the moxibustion group showed significantly greater tyrosine hydroxylase immunoreactivity and expression of light chain 3-II protein in the substantia nigra, and their behavioral score, α-synuclein immunoreactivity,the expression of phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase （p-p70S6K） in the substantia nigra were significantly lower. These results suggest that moxibustion can promote the autophagic clearance of α-syn and improve behavioral performance in Parkinson’s disease model rats. The protective mechanism may be associated with suppression of the mTOR/p70S6K pathway.展开更多
文摘BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.
基金Health and Family Planning Committee Joint Fund Project of Hubei Province,No.WJ2018H0020Fundamental Research Funds for the Central Universities,No.2042016kf0187 and No.2042017kf0068Zhongnan Hospital of Wuhan University Science,Technology and Innovation Seed Fund,No.znpy2016022.
文摘BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.
文摘The mammalian target of rapamycin （mTOR） pathway plays an important role in neuronal growth, proliferation and differentiation. To better understand the role of mTOR pathway involved in the induction of spinal cord injury, rat models of spinal cord injury were established by modified Allen＇s stall method and interfered for 7 days by intraperitoneal administration of mTOR activator adenosine triphosphate and mTOR kinase inhibitor rapamycin. At 1-4 weeks after spinal cord injury induction, the Basso, Beattie and Bresnahan locomotor rating scale was used to evaluate rat locomotor function, and immunohistochemical staining and western blot analysis were used to detect the expression of nestin （neural stem cell marker）, neuronal nuclei （neuronal marker）, neuron specific enolase, neurofilament protein 200 （axonal marker）, glial fibrillary acidic protein （astrocyte marker）, Akt, mTOR and signal transduction and activator of transcription 3 （STAT3）. Results showed that adenosine triphosphate-mediated Akt/mTOR/STAT3 pathway increased endogenous neural stem cells, induced neurogenesis and axonal growth, inhibited excessive astrogliosis and improved the locomotor function of rats with spinal cord injury.
文摘Deregulation of the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)- mammalian target of rapamycin(m TOR) signaling pathway is one of the most commonlyinvolved pathways in tumorigenesis. It has also been reported as altered in neuroendocrine tumors(NETs). m TOR inhibitors used in clinical practice are derived from rapamycin,an anti-cancer agent also used as an immunosuppressor after organ transplantation. Everolimus and temsirolimus are the two rapamycin-derived m TOR inhibitors used in NETs. Notably everolimus has been approved in advanced progressive well/moderatelydifferentiated pancreatic NETs(p NETs). It inhibits specifically the m TORC1 subunit of m TOR,not interacting with m TORC2. Although everolimus produced a significant prolongation of progression-free survival a number of patients with p NETs do not benefit from the drug due to early or late progression. Two supposed mechanisms of resistance to m TOR inhibitors are Akt and PI3 K activation,by means of m TORC2 and insulin growth factor(IGF)- IGF receptor signaling,respectively. BEZ235 is a multi-targeted inhibitor binding to PI3 K,m TORC1 and m TORC2,therefore potentially turning off all the supposed molecular targets of resistance to everolimus. The two clinical trials designed in p NETs were stopped early due to unmet statistical endpoint and the global clinical development of BEZ235 was also halted. Tolerability of this drug was challenging and conditioned the feasibility of therapy. The BEZ experience is an example of the huge difference between the preclinical and clinical setting and prompts us to pay more attention to the phase Ⅰ step of clinical development and the design of phase Ⅱ clinical trials.
基金supported by grants from the Natural Science Foundation of China(91949205,31730035,81721005)the Science and Technology Committee of China(2016YFC1305800)+1 种基金the Special Project of Technological Innovation of Hubei Province(2018ACA142)Guangdong Provincial Key S&T Program(2018B030336001)。
文摘Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.
文摘Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.
基金funded by the National Natural Science Foundation of China(31672433,31560640),ChinaSpecial fund for scientific innovation strategy-construction of high level Academy of Agriculture Science-the Outstanding Talents Training Program of Guangdong Academy of Agricultural Sciences(R2018PY-JC001),ChinaSpecial fund for scientific innovation strategy-construction of high level Academy Of Agriculture Science-The Talents Training Program Of Guangdong Academy Of Agricultural Sciences-Young Associate Researcher(R2018PY-QF001),China.
文摘As major fuels for the small intestinal mucosa,dietary amino acids(AA)are catabolized in the mitochondria and serve as sources of energy production.The present study was conducted to investigate AA metabolism that supply cell energy and the underlying signaling pathways in porcine enterocytes.Intestinal porcine epithelial cells(IPEC-J2)were treated with different concentrations of AA,inhibitor,or agonist of mammalian target of rapamycin complex 1(mTORCl)and adenosine monophosphate activated protein kinase(AMPK),and mitochondrial respiration was monitored.The results showed that AA treatments resulted in enhanced mitochondrial respiration,increased intracellular content of pyruvic acid and lactic acid,and increased hormone-sensitive lipase mRNA expression.Meanwhile,decreased citrate synthase,isocitrate dehydrogenase alpha,and carnitine palmitoyltransferase 1 mRNA expression were also observed.We found that AA treatments increased the protein levels of phosphorylated mammalian target of rapamycin(p-mTOR),phosphorylated-p70 ribosomal protein S6 kinase,and phosphorylated-4 E-binding protein 1.What is more,the protein levels of phosphorylated AMPKα(pAMPKa)and nicotinamide adenine dinucleotide(NAD)-dependent protein deacetylase sirtuin-1(SIRT1)were decreased by AA treatments in a time depending manner.Mitochondrial bioenergetics and the production of tricarboxylic acid cycle intermediates were decreased upon inhibition of mTORCl or AMPK.Moreover,AMPK activation could up-regulate the mRNA expressions of inhibitor of nuclear factor kappa-B kinase subunit beta(Ikbk(3),integrin-linked protein kinase(ILK),unconventional myosin-Ic(Myolc),ribosomal protein S6 kinase beta-2(RPS6 Kβ2),and vascular endothelial growth factor(VEGF)-β,which are downstream effectors of mammalian target of rapamycin(mTOR).The mRNA expressions of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform(PIK3 CD)and5’-AMP-activated protein kinase subunit gamma-1(PRKAG1),which are upstream regulators of mTOR,were also up-regulated by AMPK activation.On the other hand,AMPK activation also down-regulated FK506-binding protein 1 A(FKBP1 A),serine/threonine-protein phosphatase 2 A 55 kDa regulatory subunit B beta isoform,phosphatase and tensin homolog(PTEN),and unc-51 like autophagy activating kinase 1(Ulkl),which are up-stream regulators of mTORCl.Taken together,these data indicated that AA regulated cellular energy metabolism through mTOR and AMPK pathway in porcine enterocytes.These results demonstrated interactions of AMPK and mTORCl pathways in AA catabolism and energy metabolism in intestinal mucosa cells of piglets,and also provided reference for using AA to remedy human intestinal diseases.
文摘Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.
基金supported by the National Natural Science Foundation of China,No.81403456,81473788a grant from the Hubei Provincial Collaborative Innovation Center of Preventive Treatment by Acupuncture and Moxibustion,No.HBPCIC-2016-003
文摘Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson’s disease has been shown to have a positive effect, but the underlying mechanism remains unknown. Based on this, we explored whether moxibustion could protect dopaminergic neurons by promoting autophagy mediated by mammalian target of rapamycin （mTOR）, with subsequent elimination of α-syn. A Parkinson’s disease model was induced in rats by subcutaneous injection of rotenone at the back of their necks, and they received moxibustion at Zusanli （ST36）, Guanyuan （CV4）and Fengfu （GV16）, for 10 minutes at every point, once per day, for 14 consecutive days. Model rats without any treatment were used as a sham control. Compared with the Parkinson’s disease group, the moxibustion group showed significantly greater tyrosine hydroxylase immunoreactivity and expression of light chain 3-II protein in the substantia nigra, and their behavioral score, α-synuclein immunoreactivity,the expression of phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase （p-p70S6K） in the substantia nigra were significantly lower. These results suggest that moxibustion can promote the autophagic clearance of α-syn and improve behavioral performance in Parkinson’s disease model rats. The protective mechanism may be associated with suppression of the mTOR/p70S6K pathway.
