Objectives:Glioblastoma(GB)is a grade IV glial tumor characterized by high malignancy and dismal prognosis,primarily due to high recurrence rates and therapeutic resistance.The epidermal growth factor receptor(EGFR),a...Objectives:Glioblastoma(GB)is a grade IV glial tumor characterized by high malignancy and dismal prognosis,primarily due to high recurrence rates and therapeutic resistance.The epidermal growth factor receptor(EGFR),a receptor tyrosine kinase(RTK),regulates signaling pathways,including cell growth,proliferation,survival,migration,and cell death.Many cancers utilize immune checkpoints(ICs)to attenuate immune responses.CD73 is an enzyme that functions as an IC by hydrolyzing AMP to adenosine,suppressing immune cells in the tumor microenvironment.However,the role of CD73 in resistance to EGFR inhibitors is poorly understood.This study aims to elucidate the resistance mechanisms induced by anti-EGFR treatment and to evaluate an anti-CD73 approach to overcome resistance mediated by anti-EGFR monotherapy.Methods:The U251 GB cell line was treated with AG1478,an EGFR inhibitor,and the resistance markers MRP-1,PD-L1,and CD73 were evaluated using flow cytometry.Additionally,we assessed the combination effects of AG1478 and APCP(an EGFR and a CD73 inhibitor,respectively)on cell cycle progression,proliferation,apoptosis,and migration in vitro.Results:We observed high EGFR,PD-L1,and CD73 expression in human GB cells.The treatment with AG1478 increased the expression of resistance markers MRP-1,PD-L1,and CD73,whereas it decreased CTLA-4.The combination of AG1478 and APCP did not alter proliferation or apoptosis but interfered with cell cycling,arresting the cells in the G1 phase,decreasing cell motility and partially reversing MRP-1 overexpression.Conclusion:In summary,our findings indicate that CD73 inhibition has a modest effect in overcoming resistance to EGFR monotherapy in vitro.Thus,further in vivo studies are needed,as the inhibition of both EGFR and CD73 affects cells in the tumor microenvironment and could potentially enhance anti-tumor immunity.展开更多
Here we have presented a sensitive and selective LC-MS/MS method for the quantification of tyrphostin A9, which is a selective inhibitor for platelet derived growth factor receptor tyrosine kinase and has been investi...Here we have presented a sensitive and selective LC-MS/MS method for the quantification of tyrphostin A9, which is a selective inhibitor for platelet derived growth factor receptor tyrosine kinase and has been investigated in vitro as a potent oxidative phosphorylation uncoupler. The murine analytical method was developed for three biological matrices: cell culture media, 3T3-L1 cell lysate, and murine plasma. For each matrix the limit of detection and the limit of quantification were found to be 0.5 ng/mL and 1.0 ng/ mL, respectively. The range of standard curve for each matrix was 1.0-100 ng/mL, linearity was >0.99, and the precision and accuracy were within 20%. 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoic acid was found to be the most suitable internal standard. The validated LC-MS/MS method was used to investigate stability and in vitro pharmacokinetics of tyrphostin A9. It was found that tyrphostin A9 is susceptible to hydrolysis, and the degradation product was identified as 3,5-di-tert-butyl-4- hydroxybenzaldehyde. Tyrphostin A9 was not stable in biological matrices, and the rate of its degradation in murine plasma was faster than that in cell culture media. In vitro pharmacokinetic studies revealed that tyrphostin A9 concentrations in the cell culture media declined in a bi-exponential manner and the concentrations inside the adipocytes remained constant, suggesting tyrphostin A9 has an intracellular binding site and is retained within the cell. The LC-MS/MS method presented here paves the way for further quantitative investigations involving tyrphostin A9.展开更多
In the present study we investigated two groups of small molecular tyrosine kinase phosphorylation inhibitors (tyrphostins) with quite different structures (19 compounds of the benzylidene malononitrile famliy and 13 ...In the present study we investigated two groups of small molecular tyrosine kinase phosphorylation inhibitors (tyrphostins) with quite different structures (19 compounds of the benzylidene malononitrile famliy and 13 compounds of the 3-substituted indolin-2-ones family). With the aid of a pharmacophore analysis method (CATALYST). a common three-dimensional pharmacophore model to these two kinds of molecules has been discovered. A better 3D-QSAR analysis based on the generated pharmacophore model was conducted (correlate coeffcient R=0.956) and the model shows very good predictive ability.展开更多
基金supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq,n°406035/2021-0 and PQ n°311580/2021-1,FigueiróF)Instituto Nacional de Ciência e Tecnologia-INCT/CNPq/CAPES/FAPERGS Grant no.465671/2014-4Jean Sévigny received support from the Natural Sciences and Engineering Research Council of Canada(NSERC,RGPIN-2023-05498).
文摘Objectives:Glioblastoma(GB)is a grade IV glial tumor characterized by high malignancy and dismal prognosis,primarily due to high recurrence rates and therapeutic resistance.The epidermal growth factor receptor(EGFR),a receptor tyrosine kinase(RTK),regulates signaling pathways,including cell growth,proliferation,survival,migration,and cell death.Many cancers utilize immune checkpoints(ICs)to attenuate immune responses.CD73 is an enzyme that functions as an IC by hydrolyzing AMP to adenosine,suppressing immune cells in the tumor microenvironment.However,the role of CD73 in resistance to EGFR inhibitors is poorly understood.This study aims to elucidate the resistance mechanisms induced by anti-EGFR treatment and to evaluate an anti-CD73 approach to overcome resistance mediated by anti-EGFR monotherapy.Methods:The U251 GB cell line was treated with AG1478,an EGFR inhibitor,and the resistance markers MRP-1,PD-L1,and CD73 were evaluated using flow cytometry.Additionally,we assessed the combination effects of AG1478 and APCP(an EGFR and a CD73 inhibitor,respectively)on cell cycle progression,proliferation,apoptosis,and migration in vitro.Results:We observed high EGFR,PD-L1,and CD73 expression in human GB cells.The treatment with AG1478 increased the expression of resistance markers MRP-1,PD-L1,and CD73,whereas it decreased CTLA-4.The combination of AG1478 and APCP did not alter proliferation or apoptosis but interfered with cell cycling,arresting the cells in the G1 phase,decreasing cell motility and partially reversing MRP-1 overexpression.Conclusion:In summary,our findings indicate that CD73 inhibition has a modest effect in overcoming resistance to EGFR monotherapy in vitro.Thus,further in vivo studies are needed,as the inhibition of both EGFR and CD73 affects cells in the tumor microenvironment and could potentially enhance anti-tumor immunity.
基金supported by NIH grant GM114179 and AI138195 to D.K.S.the Centre for Protein Therapeutics at University at BuffaloDonald F. and Edna G. Bishop Scholarship Foundation for their continued support
文摘Here we have presented a sensitive and selective LC-MS/MS method for the quantification of tyrphostin A9, which is a selective inhibitor for platelet derived growth factor receptor tyrosine kinase and has been investigated in vitro as a potent oxidative phosphorylation uncoupler. The murine analytical method was developed for three biological matrices: cell culture media, 3T3-L1 cell lysate, and murine plasma. For each matrix the limit of detection and the limit of quantification were found to be 0.5 ng/mL and 1.0 ng/ mL, respectively. The range of standard curve for each matrix was 1.0-100 ng/mL, linearity was >0.99, and the precision and accuracy were within 20%. 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoic acid was found to be the most suitable internal standard. The validated LC-MS/MS method was used to investigate stability and in vitro pharmacokinetics of tyrphostin A9. It was found that tyrphostin A9 is susceptible to hydrolysis, and the degradation product was identified as 3,5-di-tert-butyl-4- hydroxybenzaldehyde. Tyrphostin A9 was not stable in biological matrices, and the rate of its degradation in murine plasma was faster than that in cell culture media. In vitro pharmacokinetic studies revealed that tyrphostin A9 concentrations in the cell culture media declined in a bi-exponential manner and the concentrations inside the adipocytes remained constant, suggesting tyrphostin A9 has an intracellular binding site and is retained within the cell. The LC-MS/MS method presented here paves the way for further quantitative investigations involving tyrphostin A9.
文摘In the present study we investigated two groups of small molecular tyrosine kinase phosphorylation inhibitors (tyrphostins) with quite different structures (19 compounds of the benzylidene malononitrile famliy and 13 compounds of the 3-substituted indolin-2-ones family). With the aid of a pharmacophore analysis method (CATALYST). a common three-dimensional pharmacophore model to these two kinds of molecules has been discovered. A better 3D-QSAR analysis based on the generated pharmacophore model was conducted (correlate coeffcient R=0.956) and the model shows very good predictive ability.
