The Inhibition Effect And Mechanism Of Deoxycholic Acid On Human Neuroblastoma SHSY-5Y Cells

Neuroblastoma is a common extracranial tumor in children.Its malignancy and mortality are relatively high.The incidence of children is 10.5/10 million and the proportion of children in all tumors is about 6-10%,but it occupies about 15%of children's tumor mortality.At present,the treatment of neuroblastoma is based on surgery,supplemented by chemotherapy and radiotherapy.It would be great significance to find new effective chemotherapeutic drugs with less side effects.Deoxycholic acid is a secondary bile acid which is produced by the deoxidation of a hydroxyl group on the site of C-7.Some researches have shown that DCA can inhibit some tumors.In this paper,we will study its effect on neuroblastoma,and use SHSY-5Y as an in vitro model to explore the morphological changes and the mechanism of action induced by deoxycholic acid in vitro.Using the method of cell culture in vitro,with different concentrations of deoxycholic acid(DCA)treatment of SHSY-5Y cells.Cell survival rate was detected by MTT method;morphological changes were observed by fluorescence microscopy by AO/EB and Hoechst staining;scratch experiment to explore the impact of DCA on cells transfer;Cell cycle,apoptosis rate and reactive oxygen species(ROS)were detected by flow cytometry.Detection of the expression of related proteins and genes after DCA treatment by Western Blot and RT-PCR to explore the molecular mechanism of DCA action.The results showed that DCA could significantly inhibit the proliferation of SHSY-5Y cell line,promote the release of lactate dehydrogenase,and decrease the cell clone ability.Morphological observation showed that cells showed morphological characteristics of apoptosis,such as cell shrinkage,nuclear chromatin condensation,nuclear fragmentation,cell budding and apoptotic body formation.The scratch test showed that the cell migration ability was reduced under the action of the DCA.The results of flow cytometry showed that after DCA treatment for 48 h,the proportion of cells in G2/M phase increased significantly.Early apoptotic cells and late apoptotic cells increased significantly,and intracellular reactive oxygen species increased.After 0.8 mmol/L DCA treatment of 48 h,SHSY-5 Y was promoted from 10%to 41.5%in G2/M phase,and the apoptosis rate was increased from 1.46%to 51.81%.The results of Western blot and RT-PCR showed that under the action of DCA,the expression levels of related tumor suppressor genes such as p21,p53 and Bax were upregulated,and the expression levels of Bcl-2,Akt and other related oncogenes were down regulated,and the expression of cyclin,such as CDK1,Cylin B1 decreased.So we can come up with a basic conclusion,under the action of DCA,p21,p53,CDK1,Cylin B1 and other related factors were influenced,which make contribution to arrest cell cycle in G2/M phase;the expressions of Bcl-2,Bax and Akt protein were influenced,which make contribution to decreased the mitochondrial membrane potential,increased mitochondrial membrane permeability,Cyt C release from mitochondria to the cytoplasm,combined with cytoplasmic Apafl,cascade reaction between various factors,activate the Caspase induced apoptosis of SHSY-5Y cells.In conclusion,DCA can significantly inhibit the proliferation of human neuroblastoma cell SHSY-5Y in a concentration dependent manner.the cell cycle arrest at G2/M phase after DCA action.And DCA induces apoptosis through mitochondrial pathway.