The Molecular Mechanism Of Folate Metabolism Pathway Inhibitor Induces Anti-glioma Apoiptosis And Causes Neurotoxicity

Part 1 The molecular mechanism of DHFR/TYMS inhibitor synergize with TMZ to induce glioma cells apoptosisObjective:Glioma is one of the most common primary malignant intracranial tumor with high mortality.Temozolomide(TMZ)is the first-line medicine to treat glioma with less adverse reactions.However,glioma cells frequently exhibit severe drug-resistance.Overcoming TMZ resistance and ameliorating the therapeutic efficacy are of great interest to researchers.Folate signaling pathway inhibitors play a key role in gastric cancer,colorectal cancer and other tumors.Whether it sensitizes glioma cells to TMZ is still unknown.This study is to investigate the anti-glioma activity of DHFR/TYMS(foate signaling pathway key proteins)inhibitor pemetrexed(PTX)synergises with TMZ and further to illustrat the molecular mechanism involved in it.Methods:1.(1)Western blot was performed to detect the expression of DHFR and TYMS in glioma tissues and cells.(2)qRT-PCR,immunohistochemistry and H&E staining were conducted to detect the expression of DHFR and TYMS.2.(1)Cells were transfection with DHFR/TYMS plasmid or siRNA to overexpress or knock down DHFR/TYMS,then detected the effect of DHFR/TYMS to glioma proliferation by western blot and cell count assay.(2)Western blot and cell counts were carried out to detect glioma proliferation after folic acid(FA)treatment.(3)Annexin V-FITC/PI were used to detect the cell apoptosis.(4)Nude mouse xenograft models of human glioma cell U251 was introduced to investigate the in vivo activity of FA(2 ppm,6 ppm and 40 ppm).3.(1)The anti-proliferation activity of drugs on glioma cells was determined using SRB method.(2)Annexin V-FITC/PI staining,western blot and DAPI staining were performed to determine cell apoptosis.(3)Nude mouse xenograft models of human glioma cell U251 was introduced to investigate the in vivo activity of PTX and TMZ.4.(1)Western blot was performed to determine the expression of AMPK.and mTOR signaling(2)Annexin V-FITC/PI staining,western blot were performed to determine cell apoptosis after AMPK inhibition.Results:1.TYMS/DHFR was upregulated in human glioma tissues and cells:(1)The expression of DHFR and TYMS were elevated in U87,U251 cells.(2)DHFR and TYMS were highly expressed in glioma tissues compared with tumor adjacent tissues.2.DHFR/TYMS promoted glioma proliferation:(1)Overexpression of DHFR/TYMS promoted glioma proliferation.(2)Silencing of DHFR/TYMS inhibited cell proliferation and induces cell apoptosis.(3)Overexpressing DHFR/TYMS by FA promoted cell proliferation as well.3.Synergistic anti-glioma activity of PTX and TMZ:(1)DHFR/TYMS inhibition or PTX enhanced the cytotoxicity of and induces significant apoptosis.(2)The synergistic anti-tumor efficacy of PTX and TMZ was validated in a human glioma U251 xenograft model.4.PTX combined with TMZ activated AMPK-mTOR signaling pathway:(1)DHFR/TYMS could upregulate the expression of AMPK,further inhibit the mTOR signaling pathway.(2)AMPK inhibition could reverse the apoptosis induced by DHFR/TYMS inhibition and TMZ.Conclusion:Our data demonstrated that folate signaling pathway key proteins DHFR/TYMS was upregulated in gliomas.Overexpression of DHFR/TYMS promoted glioma proliferation.Meanwhile,DHFR/TYMS inhibitor PTX exhibited anti-glioma activity synergistics with TMZ.We also found that AMPK-mTOR signaling pathway participate in it.Taken together,we emphasized the critical role of DHFR/TYMS in glioma proliferation and TMZ resistance.Targeting DHFR/TYMS may serve as a potential therapeutic regimen for the treatment of glioma.Part 2 The neurotoxicity of TYMS inhibitor 5-FluorouracilObjective:The TYMS inhibitor 5-fluorouracil(5-FU)is a widely used anticancer drug in treating adult solid tumors.In recent years,it has been applied to treat pediatric tumors as well.Although the delayed demyelination induced by 5-FU in adult patients has been reported,the toxicity of 5-FU on oligodendrocyte myelination in adolescence is still unknown.This study is to demonstrate the molecular mechanism of demyelination induced by 5-FU in adolescence and to investigate the key proteins in myelin repair process.Methods:1.(1)Immunofluorescence were used to evaluate the effect of 5-FU on oligodendrocyte(OLs),neuron and astrocytes in adolescent mice.(2)OLs markers was detected after 5-FU treatment by qRT-PCR.(3)Transmission electron microscope was applied to observe myelin sheath integrity.2.(1)Immunofluorescence was used to detect the proliferation and differentiation of OLs after 5-FU injection.(2)Western blot and TUNEL were applied to detect the apoptosis of OLs after 5-FU injection.3.(1)Gene-chip microarray transcriptome and gene ontology(GO)analysis was carried out to detect myelin-associated genes and positive regulators of OLs development,cellular component and biological process,respectively.(2)TCF7L2 expression in oligodendrocytes was determined by immunostaining.(3)Detection of TCF7L2 expression after 5-FU treatment in vivo by western blot,qRT-PCR and immunostaining.4.(1)HDAC1/2 expression in OLs was determined by immunostaining in OPC.(2)Co-immunoprecipitation was carried out to detect the relationship between TCF7L2 and HDAC1/2.(3)Overexpression TCF7L2,HDAC1/2 by transfection with TCF7L2,HDAC1/2 plasmid,qRT-PCR was used to test the OLs markers.5.(1)Immunofluorescence were used to evaluate the expression of MBP.(2)Transmission electron microscope was applied to observe myelin sheath integrity after 5-FU withdrawal.6.(1)TCF7L2 expression in CC was determined by immunostaining.(2)Overexpression of TCF7L2 by transfection with TCF7L2 plasmid into Oli-Neu cells,immunostaining was used to test the effect of TCF7L2 on proliferation.(3)qRT-PCR was used to test the OLs marker expression in Oli-Neu transfected with TCF7L2 or TCF7L2-EN plasmids.(4)Immunostaining was carried out to detect the differentiation in OPC transfected with TCF7L2.(5)qRT-PCR was used to test the OLs marker expression in Oli-Neu transfected with TCF7L2 plasmids.Results:1.5-FU treatment led to CNS myelination defects:(1)The expression of myelin basic protein(MBP,a mature marker of OLs)is downregulated in 5-FU injected mice,but Neuronal nuclei(NeuN,a neuron marker)and Glial fibrillary acid protein(GFAP,an astrocyte marker)in cortex and white matter(WM)of adolescent mice treated with 5-FU were comparable with control mice.(2)The myelinate axons were mainly reduced in the optic nerve and spinal cord of 5-FU-injected mice by EM detection.2.5-FU induced oligodendrocytes death:(1)5-FU may not block the proliferation and differentiation of OLs,but jmmunostaining results indicated that the PDGFRa+ or NG2+ cells were markedly decreased in the spinal cord of 5-FU-damaged mice.(2)Western blot and TUNEL assay suggested that 5-FU injection significantly decreased the expression of procaspase-3 and total poly(ADP-ribose)polymerase(PARP),increased TUNEL+ cells in the corpus callosum,respectively.3.5-FU repressed the expression of transcription factor TCF7L2:(1)In contrast to control mice,a significant downregulation of myelin associated genes and positive regulators of OLs development,especially a transcription factor TCF7L2,was observed in 5-FU-treated mice by microarray analysis.(2)Immunostaining revealed that high TCF7L2 protein levels were detected in Olig2+ cells in corpus callosum.Furthermore,a remarkably decrease of TCF7L2 mRNA and protein level was detected by qRT-PCR and western blot after 5-FU administration both in cortex and spinal cord,respectively,4.5-FU disrupted the interaction of TCF7L2-HDAC1/2:(1)Immunostaining results indicated that HD AC 1/2 were expressed in OLs.(2)Endogenous HDAC1 or HDAC2 was found to interact with TCF7L2 in cortical tissues,and co-IP results showed that 5-FU obviously decreased TCF7L2 and HD AC 1/2 expression in OPC and disrupted the interaction between TCF7L2 and HD AC 1/2.5.Remyelination occurred spontaneously after 5-FU withdrawal in adolescent and adult mice:(1)The expression of MBP was slightly decreased in 5-FU-treated adult mice on the first day post lesion(1 dpl),subsequently,a dramatic reduction of MBP was observed in the 5-FU-injected cerebral white matter at 8 dpl.(2)The expression of MBP was immediately and significantly decreased in the 5-FU-treated cerebral white matter of adolescent mice at 1 dpl,subsequently,MBP-positive cells remarkably increased at 5 dpl compared with the acute injury of white matter at 1 dpl.After long-term recovery(22 dpl),high MBP protein levels were detected in adolescent mice with 5-FU injection in contrast to low levels in 5 dpl mice.6.TCF7L2 was activated in remyelinating lesions:(1)TCF7L2 was re-activated at 5 dpl and 8 dpl in the white matter tracts of adolescent and adult mice,respectively.(2)TCF7L2-expressing promotes the proliferation of Oli-Neu cells,and increases PDGFRa and Olig2 expression by qRT-PCR.(3)TCF7L2 overexpression resulted in a drastic increase of MBP+ mature oligodendrocytes.Conclusion:The current study confirmed that 5-FU caused CNS toxicity in adolescent mice and spontaneously remyelination occurred after 5-FU withdrawl in adolescent and adult mice.Remarkably,demyelination in 5-FU-treated adolescent mice recovered more quickly than that in adult mice.We also identified that the transcription factor TCF7L2 participated in de-/re-myelination caused by 5-FU.Further,we found that 5-FU disrupted the interaction between TCF7L2 and HDAC1/2 leading to OLs death.This study provides the indispensable basis for 5-FU application in adolescent patients,indicates that TCF7L2 may play a positive role in remyelination after 5-FU withdrawal,and uncovers the potentially therapeutic target for demyelinating desease.