Background:Inhibitor of NF-κB kinase-interacting protein(IKIP)is known to promote proliferation of glioblastoma(GBM)cells,but how it affects migration and invasion by those cells is unclear.Methods:We compared levels...Background:Inhibitor of NF-κB kinase-interacting protein(IKIP)is known to promote proliferation of glioblastoma(GBM)cells,but how it affects migration and invasion by those cells is unclear.Methods:We compared levels of IKIP between glioma tissues and normal brain tissue in clinical samples and public databases.We examined the effects of IKIP overexpression and knockdown on the migration and invasion of GBM using transwell and wound healing assays,and we compared the transcriptomes under these different conditions to identify the molecular mechanisms involved.Results:Based on data from our clinical samples and from public databases,IKIP was overexpressed in GBM tumors,and its expression level correlated inversely with survival.IKIP overexpression in GBM cells inhibited migration and invasion in transwell and wound healing assays,whereas IKIP knockdown exerted the opposite effects.IKIP overexpression in GBM cells that were injected into mouse brain promoted tumor growth but inhibited tumor invasion of surrounding tissue.The effects of IKIP were associated with downregulation of THBS1 mRNA and concomitant inhibition of THBS1/FAK signaling.Conclusions:IKIP inhibits THBS1/FAK signaling to suppress migration and invasion of GBM cells.展开更多
Toll-like receptor 2(TLR2)mediated macrophages regulate the protective immune response to infectious microorganisms,but the aberrant activation of macrophages often leads to pathological inflammation,including tissue ...Toll-like receptor 2(TLR2)mediated macrophages regulate the protective immune response to infectious microorganisms,but the aberrant activation of macrophages often leads to pathological inflammation,including tissue damage.In this study,we identified antagonists of TLR2 by screening2100 natural products and subsequently identified Taspine,an aporphine alkaloid,as an excellent candidate.Furthermore,analysis of the 10 steps chemical synthesis route and structural optimization yielded the Taspine derivative SMU-Y6,which has higher activity,better solubility,and improved drug-feasible property.Mechanistic studies and seq-RNA analysis revealed that SMU-Y6 inhibited TLR2 over other TLRs,hindered the formation of TLR2/MyD88 complex,and blocked the downstream NF-κB and MAPK signaling pathway,thus suppressing the release of inflammatory cytokines.SMU-Y6 could stabilize TLR2 and bind to TLR2 protein with a Kdof 0.18μmol/L.Additionally,SMU-Y6 could efficiently reverse the M1 phenotype macrophage polarization,reduce the production of cytokines as well as infiltration of neutrophiles and alleviate the local inflammation in mice with acute paw edema and colitis.Collectively,we reported the first aporphine alkaloid derivative that selectively inhibits TLR2 with high binding affinity and superior drug-feasible property,thus providing an urgently-needed molecular probe and potential drug candidate for inflammatory and autoimmune disease therapy.展开更多
AIM： To study the effects of IκBα and its mutants （IκBαM, IκBα3N, IκBαM44C） on NF-κB, p53 and their downstream target genes. The relationship of NF-κB, p53, and IκBα was further discussed. METHODS： pEC...AIM： To study the effects of IκBα and its mutants （IκBαM, IκBα3N, IκBαM44C） on NF-κB, p53 and their downstream target genes. The relationship of NF-κB, p53, and IκBα was further discussed. METHODS： pECFP-IκBα, pECFP-IκBαM （amino acides 1-317, Ser32, 36A）, pECFP-IκBα243N （amino acides 1-243）, pECFP-IκBα244C （amino acides 24±317）, pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-α-1 cells. Cells were transfected with pECFP-Cl as a control. 30 h after the transfection, location patterns of NF-κB, p53 and IκBα（IκBαM, IκBα243N, IκBα224C） were observed by a laser scanning microscope （LSM510/ConfoCor2, Zeiss）. RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IκBα and its mutants on the transprition level of NF-κB, NF-κB downstream target gene TNF-α, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments β-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader. RESULTS： Cells that were transfected with p53- DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IκBα, CFP-IκBαM, and CFP-IκBα243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IκBα244C revealed a predominant nuclear localization. The rnRNA levels of p65, TNF-α, p53 and Bax in CFP-IκBα transfected cells did not change significantly, while in YFP-p65/CFP-IκBα co-transfected cells, IκBα decreased the transcription of p65 downstream gene TNF-α （2.24 ± 0.503） compared with the YFP-p65/ CFP-C1 co-transfected cells （5.08 ± 0.891） （P 〈 0.05）. Phosphorylation defective IκBα （IκB展开更多
基金supported by the National Natural Science Foundation of China(82002638)the National Natural Science Foundation of Sichuan Province(2023NSFSC0734).
文摘Background:Inhibitor of NF-κB kinase-interacting protein(IKIP)is known to promote proliferation of glioblastoma(GBM)cells,but how it affects migration and invasion by those cells is unclear.Methods:We compared levels of IKIP between glioma tissues and normal brain tissue in clinical samples and public databases.We examined the effects of IKIP overexpression and knockdown on the migration and invasion of GBM using transwell and wound healing assays,and we compared the transcriptomes under these different conditions to identify the molecular mechanisms involved.Results:Based on data from our clinical samples and from public databases,IKIP was overexpressed in GBM tumors,and its expression level correlated inversely with survival.IKIP overexpression in GBM cells inhibited migration and invasion in transwell and wound healing assays,whereas IKIP knockdown exerted the opposite effects.IKIP overexpression in GBM cells that were injected into mouse brain promoted tumor growth but inhibited tumor invasion of surrounding tissue.The effects of IKIP were associated with downregulation of THBS1 mRNA and concomitant inhibition of THBS1/FAK signaling.Conclusions:IKIP inhibits THBS1/FAK signaling to suppress migration and invasion of GBM cells.
基金supported by Foundation National Natural Science Foundation of China(Nos.82073689,82273762)National Key Research and Development Program of China(No.2022YFC2304203)+2 种基金Key Program of National Natural Science Foundation of China(No.82130101)National Natural Science Foundation of Guangdong Province(No.2018B030312010,China)Science and Technology Program of Guangzhou(No.201904010380,China)。
文摘Toll-like receptor 2(TLR2)mediated macrophages regulate the protective immune response to infectious microorganisms,but the aberrant activation of macrophages often leads to pathological inflammation,including tissue damage.In this study,we identified antagonists of TLR2 by screening2100 natural products and subsequently identified Taspine,an aporphine alkaloid,as an excellent candidate.Furthermore,analysis of the 10 steps chemical synthesis route and structural optimization yielded the Taspine derivative SMU-Y6,which has higher activity,better solubility,and improved drug-feasible property.Mechanistic studies and seq-RNA analysis revealed that SMU-Y6 inhibited TLR2 over other TLRs,hindered the formation of TLR2/MyD88 complex,and blocked the downstream NF-κB and MAPK signaling pathway,thus suppressing the release of inflammatory cytokines.SMU-Y6 could stabilize TLR2 and bind to TLR2 protein with a Kdof 0.18μmol/L.Additionally,SMU-Y6 could efficiently reverse the M1 phenotype macrophage polarization,reduce the production of cytokines as well as infiltration of neutrophiles and alleviate the local inflammation in mice with acute paw edema and colitis.Collectively,we reported the first aporphine alkaloid derivative that selectively inhibits TLR2 with high binding affinity and superior drug-feasible property,thus providing an urgently-needed molecular probe and potential drug candidate for inflammatory and autoimmune disease therapy.
基金the National Natural Science Foundation of China No. 60378043 and 30470494)the Natural ScienceFoundation of Guangdong Province No. 015012 and 04010394
文摘AIM： To study the effects of IκBα and its mutants （IκBαM, IκBα3N, IκBαM44C） on NF-κB, p53 and their downstream target genes. The relationship of NF-κB, p53, and IκBα was further discussed. METHODS： pECFP-IκBα, pECFP-IκBαM （amino acides 1-317, Ser32, 36A）, pECFP-IκBα243N （amino acides 1-243）, pECFP-IκBα244C （amino acides 24±317）, pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-α-1 cells. Cells were transfected with pECFP-Cl as a control. 30 h after the transfection, location patterns of NF-κB, p53 and IκBα（IκBαM, IκBα243N, IκBα224C） were observed by a laser scanning microscope （LSM510/ConfoCor2, Zeiss）. RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IκBα and its mutants on the transprition level of NF-κB, NF-κB downstream target gene TNF-α, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments β-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader. RESULTS： Cells that were transfected with p53- DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IκBα, CFP-IκBαM, and CFP-IκBα243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IκBα244C revealed a predominant nuclear localization. The rnRNA levels of p65, TNF-α, p53 and Bax in CFP-IκBα transfected cells did not change significantly, while in YFP-p65/CFP-IκBα co-transfected cells, IκBα decreased the transcription of p65 downstream gene TNF-α （2.24 ± 0.503） compared with the YFP-p65/ CFP-C1 co-transfected cells （5.08 ± 0.891） （P 〈 0.05）. Phosphorylation defective IκBα （IκB
