Similarity Neurotoxicity Mechanism Of Anti-Tumoral Drug Carmofur And Mutation In ASAH1

ASAH1 encode acid ceramidase,a lipid hydrolase,which can hydrolyze ceramide into sphingosine and free fatty acids in lysosome.Acid ceramidase is related to many human diseases:ASAH1 gene mutation will cause Farber disease and spinal muscular atrophy;ASAH1 gene is overexpressed in prostate cancer,breast cancer,colorectal cancer and other malignant tumors.In the serum from melanoma patients,acid ceramidase is also increased.Hence,the compounds capable of inhibiting acid ceramidase activity is becoming a hotspot:such as Carmofur for colon cancer treatment.This article comparatively study acid ceramidase inhibitor Carmofur and ASAH1 gene silencing SH-SY5Y cell,including morphology and metabolic pathway,and try to find their similarity of neurotoxic mechanism.This paper firstly established a co-cultured model of SH-SY5Y cell and ceramidase inhibitor Carmofur.Through observing cell morphology,the expression of 12 key enzymes in the ceramide metabolism pathway and the amount of protein biosynthesis from control group,low concentration and high concentration of Carmofur drug group.The result found that Carmofur can inhibit the growth of neuron cells,cells are losing their adherent property to a suspension state.The amount of ceramidase protein biosynthesis is increased.Secondly,ASAH1 gene was silenced by siRNA interference,and the biosynthesis of ceramidase was successfully blocked after 48 hours.Cells lost their axonal and dendritic branches,and most of cells became black apoptotic cells.ASHA1 gene increased significantly during the first 24 hours,but declined after 48 hours.Whereas CERS gene decreased which encode ceramide synthase,and the substrate ceramide was decreased.It is found that two kinds of models caused neuron cells apoptosis,cells lost their axons and dendrites into globular form,and cells were floating in the culture medium.After comparison of main metabolic enzymes in the ceramide metabolic pathway,whether acid ceramidase biosynthesis was blocking or enzyme activity was inhibited,ASHA1 gene was up-regulated.At the same time,SMPD1 gene had an excessive expression and activation,and a lot of degradation of sphingomyelin(mainly composed of biological axon dendritic biomolecule),resulting an accumulation of ceramide which induced apoptosis.Hence,the activation of the sphingomyelin phospholipase may be the main common mechanism of neuronal apoptosis.