title: Role of methotrexate exposure in apoptosis and proliferation during early neurulation.
abstract: Apoptosis and proliferation play important roles in embryonic development and are required for neural tube closure. The antifolate drug methotrexate (MTX) induces folate dysmetabolism by inhibition of dihydrofolate reductase and causes abnormal apoptosis and proliferation. In this study, we established an animal model of neural tube defects (NTDs) using MTX to investigate the role of apoptosis and proliferation in NTDs caused by folate deficiency. Differential gene expressions were studied by microarray and reverse transcription-polymerase chain reaction in the NTD animal model. Results showed that 30.8% of NTDs were caused by using MTX in treatment regimens. Microarray indicated that 166 genes were significantly different between the control and NTD mice, including four apoptosis-related genes (Endog, Trp53, Casp3, Bax) and three proliferation-related genes (Ptch1, Pla2g4a, Foxg1). Levels of Endog, Trp53, Casp3, Bax (fold change>1.5) were upregulated but Ptch1, Pla2g4a, Foxg1 (fold change<0.67) were downregulated (P<0.05). These results were confirmed by reverse transcription-polymerase chain reaction. TUNEL, immunohistochemical assays and Western blot were further used to detect apoptosis and proliferation in the NTD animal model. It was found that apoptosis in neuroepithelial cells was increased as determined by TUNEL (P<0.05). Expressions of caspase-3 were significantly enhanced (P<0.05) but expressions of phosphohistone H3 were greatly decreased (P<0.05). These results concluded that MTX caused a folate and folate-associated dysmetabolism, and further induced abnormal apoptosis and proliferation, which may play a critical role in the occurrence of NTDs caused by folate deficiency.
authors.affiliation: Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing, 100020, China.
authors.forename: Xiuwei
authors.initials: X
authors.lastname: Wang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
authors.forename: Tao
authors.initials: T
authors.lastname: Guan
authors.forename: Qian
authors.initials: Q
authors.lastname: Xiang
authors.forename: Mingsheng
authors.initials: M
authors.lastname: Wang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Guan
authors.forename: Guannan
authors.initials: G
authors.lastname: Li
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Zhu
authors.forename: Qiu
authors.initials: Q
authors.lastname: Xie
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Bo
authors.initials: B
authors.lastname: Niu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D004195:Disease Models, Animal; D015536:Down-Regulation; D047108:Embryonic Development / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D005260:Female; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000633:toxicity*; D005494:Folic Acid Deficiency / Q000150:complications / Q000196:embryology; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008727:Methotrexate / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D046228:Microarray Analysis; D009436:Neural Tube Defects / Q000196:embryology; D054261:Neurulation / Q000187:drug effects*; D011247:Pregnancy; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D005493:Folic Acid Antagonists; D006657:Histones; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; C505409:Casp3 protein, mouse; D053148:Caspase 3; D008727:Methotrexate
keywords: Apoptosis; Methotrexate; Mice; Microarray; Neural tube defects; Proliferation