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.

The role of folate metabolism in orofacial development and clefting

“…We observed these apoptotic features when DHFR function was blocked in Xenopus embryos. This is consistent with reports in other vertebrates where defects in folate metabolism also resulted in increased apoptosis during development (Lee et al, 2012; Sun et al, 2014; Tang et al, 2005; Wang et al, 2014). Moreover, we demonstrated that DHFR inhibition induced apoptosis is in part due to increased DNA damage.…”

“…We show that DHFR deficiency specifically during early head and facial development causes a narrower midface and malformed mouth shape, due to loss of jaw muscle and cartilage elements. This is consistent with reported effects in mammals where folate deficiencies or mutations to major pathway components can also result in craniofacial defects and smaller head size (Burgoon et al, 2002; Momb et al, 2013; Tang et al, 2005; Wang et al, 2014; Zhao et al, 2013). Further, DHFR mutations are associated with cleft lip in humans (Martinelli et al, 2014).…”

“…There is considerable evidence to support the idea that decreased folate metabolism can result in increased apoptosis in animal models and cell lines (for some examples see (Tang et al, 2005; Wang et al, 2014; Zhang et al, 2009))(Li et al, 2003). To determine if decreased DHFR function also increased cell death in Xenopus orofacial development we; i) performed immunohistochemistry for cleaved caspase-3 in the face as well as ii) examined and quantified cell morphology by FACS analysis in embryos deficient for DHFR function.…”

“…A reduction in SAM can thus greatly impact a number of processes, notably epigenetic changes such as DNA and histone methylation, integral to the developing embryo. Certainly, changes in folate metabolism have been shown to affect both DNA synthesis and histone methylation during neurulation, heart development, and in diseases such as cancer (Beaudin and Stover, 2009; Momb et al, 2013; Sun et al, 2011; Tang et al, 2005; Wang et al, 2014). Folate deficiency and mutations to genes involved in the folate pathway cause orofacial defects in model vertebrates and humans (Li et al, 2011; Momb et al, 2013; Tang et al, 2005; Wehby and Murray, 2010; Wilcox et al, 2007) (Burgoon et al, 2002; Kao et al, 2014; Lee et al, 2012).…”

The effect of inhibiting glycinamide ribonucleotide formyl transferase on the development of neural tube in mice

“…Reports have shown that the occurrence of NTDs is associated with imbalance between cell proliferation and apoptosis. Studies have proved that disordered proliferation and apoptosis in animal models of NTDs were induced by hyperthermia [12], hyperglycemia [13], cyclophosphamide [14], or methotrexate (MTX) [15]. Therefore, cell proliferation and apoptosis have to be kept in dynamic balance during the formation of neural tube.…”