Antitumor Effects Of Isatin And The Related Mechanism In Vitro And In Vivo

BACKGROUND & OBJECTIVE:Isatin(ISA) is a natural material that exists in mammalian body fluids and tissues.ISA could inhibit the growth of tumors,but the mechanism remains unclear.This study aimed to explore the antitumor effects and the related mechanism of ISA in vitro and in vivo with neuroblastoma cell line SH-SY5Y as target cell.METHODS:in vitro experiment:MTT,Fluorescent staining,argerose electrophoresis, flow cytometry,RT-PCR and Western blot were performed to analyze the cell cycle arrest and apoptosis of SH-SY5Y cells after treatment of ISA at different concentrations(0,50, 100,200μmol/L).In vivo experiment:Establish the murine exnograft model,the growth of tumor was messured in vivo.RT-PCR and Western blot were performed to analyze Bcl-2 mRNA and proteinRESULTS:in vitro experiment:When treated with 200μmol/L ISA for 48 h,SH-SY5Y cells showed typical apoptotic morphologic changes including chromatin condensation and DNA fragment.Along with the increase of ISA concentration,Bcl-2 expression was decreased,the ratio of Bcl-2 to Bax was significantly decreased(P＜0.05).Dym Depolarization in SH-SY5YCells was decreased when treated with ISA(P＜0.05). Cytosyl Cyt-c Proteins increased in SH-SY5Y cells after 48-hour treatment of ISA detected by Western blot.When treated with ISA(50,100,200μmol/L) for 48 h,the positive rates of activated Caspase-3 in SH-SY5Y cells were significantly higher than that in control SH-SY5Y cells(19.28%,25.88%,and 33.43%vs.10.58%,P＜0.05). Moreover,inhibitor of caspase-activated DNAse(ICAD),the substrate of Caspase-3,was degraded.In addition,the proportion of SH-SY5Y cells at G₁ phase was significantly increased with an apparent G₁ phase arrest when treated with ISA(50,100,200μmol/L) for 48 h.In the progress of cell cycle arrest induced by ISA,phosphorylated ERK and CDK1 expression were down-regulated(P＜0.05).In vivo experiment:The growth of tumor was visibly suppressed in vivo.RT-PCR and Western blot results indicated expression reduced in excised tumors.In contrast,there were no obvious changes in negative control.CONCLUSION:ISA can induce apoptosis and G₁ phase arrest in SH-SY5Y cells, possibly by decreasing Bcl-2/Bax,activating Caspase-3,down-regulating the expression of phosphorylated ERK and CDK1.