Inhibition Of 2,3-indolequinone On Neuroblastoma And Its Mechanism

Objective: Neuroblastoma(NB)is a common tumor in children.It originates from any nerve ridge of the sympathetic nervous system.It usually originates from the sympathetic nerve chain and adrenal medulla.NB has the characteristics of low cure rate,easy metastasis,high mortality and poor prognosis.Mainly used in clinical treatment for NB include surgery,chemotherapy and radiotherapy,but there is high risk of surgery,radiotherapy and chemotherapy have toxic and side effects as so too,and chemotherapy is easily to happen drug resistance,which increases the difficulty of treatment for NB and seriously threatens the health of children.Therefore,it is urgent to find new safe and effective drugs to treat NB.In recent years,the development and application of natural drugs in clinical tumor treatment are increasing,developing the safe and effective natural antitumor drugs becomea a popular research.2,3-indole-2-3-dione(isatin)is a natural product with variety of biological activities,which the antitumor effect is more famous,but the mechanism of isatin on tumor is still unclear,which needs to be further explored.Isatin,which exists in a small number of plants and human body fluids,is the precursor of indirubine,an original anticancer drug with independent intellectual property rights,and its monomer has been synthesized.Isatin is also a monoamine oxidase inhibitor with anti-tumor activity,with small molecular weight,and is a fat-soluble substance,which can pass the blood-brain barrier.it has the potential to be developed as a drug for the treatment of nervous system tumors because of the characteristics and adwantages.This study mainly discussed the inhibition of isatin on SH-SY5 Y cells in neuroblastoma include proliferation,invasion and migration,also its molecular mechanism.The aim is to support a theoretical foundation for the development of isatin as a clinical antitumor drug.Methods: 1.After SH-SY5 Y cells treatment with different concentrations of isatin at different time,the changes of cell proliferation ability were detected by CCK8 assay,and to confirm the optimal time and concentration for the anti-tumor effect of isatin.2.SH-SY5 Y cells were treated with concentration gradient isatin,flow cytometry and DAPI staining to detect the change of apoptosis.The cell cycle changes of SH-SY5 Y cells detect by flow cytometry.3.Transwell and scratch tests were used to detect the effects of different concentrations of 2,3-indolequinone on the invasion and migration of SH-SY5 Y cells.4.The colony cloning formation of SH-SY5 Y cells after continuous culture with different concentrations of isatin was detected by plate cloning experiment.5.qPCR and Western Blot assay were used to detect changes in relative mRNA expression levels and protein expression levels of p53,LSD1,SESN2,mTOR,LC3,P62,Beclin1.To investigate the molecular mechanism of isatin inhibiting the proliferation and invasion and migration of SH-SY5 Y cells.Res?lts: 1.CCK8 experimental results showed that,compared with the control group,the higher concentration of isatin was,the greater inhibition rate was after SH-SY5 Y was added to the neuroblastoma cells with different concentrations of isatin,and the most obvious inhibition effect was found after 48 h treatment.2.The results of flow cytometry and DAPI staining showed that with the increase of concentration,apoptosis increased and nuclear apoptosis was obvious.Flow cytometry was used to detect the cell cycle distribution of SH-SY5 Y cells.The results showed that,with the increase of isatin concentration,the G1 phase ratio of SH-SY5 Y cells increased and the S phase ratio decreased,that is,G1 phase cycle arrest occurred and DNA replication was inhibited.3.Transwell and scratch test results showed that,compared with the control group,the invasion and migration ability of SH-SY5 Y cells treated with isatin for 48 h was significantly reduced.4.The results of plate cloning experiments showed that,compared with the control group,with the increase of isatin concentration,the formation of colony clones in the experimental group was significantly reduced.5.q-PCR and Western Blot results showed that after SH-SY5 Y cells were treated with isatin,the higher the concentration of isatin,the lower the relative expression levels of LSD1,mTOR and P62 mRNA and protein expression levels.Relative mRNA expression levels and protein expression levels of SESN2,LC3,and Beclin1 were increased.Changes in relative mRNA expression levels and protein expression levels of p53,m TOR,P62,LC3,and Beclin1 suggest that autophagy and apoptosis were induced after SH-SY5 Y cells were treated with isatin for 48 h.Therefore,the mechanism of action of isatin in inhibiting proliferation and invasion and migration of SH-SY5 Y cells in neuroblastoma may be related to autophagy.Conclusion: 2,3-indoleone can inhibit the proliferation,invasion and migration of SH-SY5 Y cells in neuroblastoma,and promote apoptosis and autophagy.The molecular mechanism of its role may be related to inhibition of LSD1-induced SESN2-mTOR-dependent autophagy signaling pathway.