The Correlated Molecular Mechanisms Of Isatin In Inhibiting Invasion And Metastasis Of Neuroblastoma

Objective: Neuroblastoma is one of the most common malignant tumors in children.It has a high degree of malignancy and metastasis in the early stage,and metastasis is the most important cause of death in neuroblastoma.Isatin(2,3-dioxoindoline,ISA)is a natural material that widely presents endogenously in both human and other mammalian tissues and fluids likely as a result of the tryptophan metabolic pathway.Isatin showed significant inhibiting effect on the growth of SH-SY5 Y neuroblastoma cells.The objective of this study was to investigate the possible mechanism of isatin in inhibiting human neuroblastoma SH-SY5 Y cells invasion and metastasis.Methods: SH-SY5 Y cells were treated with isatin at various concentrations(10,25,50,100,150,200,250,300,400,500 and 800 μmol/L),cell viability was evaluated by CCK-8 kit;SH-SY5 Y cells were treated with isatin at various concentrations(50,100,200μmol/L),then the migration ability of SH-SY5 Y cells were assessed by using in vitro wound-healing assays;After treated with isatin at various concentrations(50,100,200μmol/L)for 48 h,cell invasion was tested by Transwell assays;Gene expressions of SHSY5 Y in isatin and possible genes and interaction pathway of invasion and metastasis were examined by Affymetrix gene chip,related gene were analyzed via RT-PCR;the proteins that related were analyzed via Western blotting.Results: The 50% inhibition concentration(IC50)of isatin for human neuroblastoma SHSY5 Y was 279.7 μmol/L,and the growth of tumor was visibly suppressed;After treated with isatin,along with the increase of ISA concentration the migration of SH-SY5 Y cells decreased and the number of invasive cells declined.The gene chip results showed that284 genes were significantly up-regulated and 145 genes were significantly downregulated in isatin-treated cells;mTOR signaling pathway may be related to invasion and metastasis;DDIT4,RHEB,EIF4EBP1 and RPS6KB1 are mTOR signaling pathwayrelated genes,the expression of DDIT4 and EIF4EBP1 raised,and the expression of RHEB and RPS6KB1 decreased,respectively.RT-PCR results were consistent with chip prediction results and Western blotting showed that the expression of HIF-1α(P<0.01)and VEGF(P<0.01)decreased and there was a decline in the phosphorylation level of mTOR(P<0.01)in isatin treated group.The phosphorylation level of AMPK(P<0.01)was significantly raised compared with control group;the phosphorylation level of PI3 K and Akt differences were of no significance between isatin treated group and control group.Conclusions: Compared with the control group(0 μmol/L),isatin could inhibit cell proliferation of SH-SY5 Y cell line;Isatin could inhibit migration and invasion of SH-SY5Y cells.Isatin induced differential gene expressions.Its anti-tumor metastasis effects were complicated and mTOR signal pathway may be one of its mechanisms in inhibiting tumor metastasis,providing a theoretical basis for the clinical use of the drug.