Study On Antitumor Role Of Indol 2,3-dione On Human Neuroblastoma Cell Line (SHSY-5Y) In Vitro

ObjectiveIndol 2, 3-dione (Mr147, isatine, ISA) is a new type of natural marine compound. It exists in the traditional Chinese medicine natural indigo and is prosoma of category antitumor new drug indirubin, which is created originally by our country. We have reported that isatine in the dose of 60, 180 mg-kg-1 can markedly inhibit the growth of implanted tumor in mice and the inhibition show dose-effect relationship. In order to deplore its antitumor value, we studied its antitumor activity in vitro with human neuroblastoma cell line (SHSY-5Y) and investigated its mechanism.Methods(1)Nuclear staining with Hoechst 33258, human neuroblastoma cells (SHSY-5Y) were divided into four groups and given different treatment (DMSO negative control, 50,100,200μM isatin). After 80 hours, the cells were stained with Hoechest33258.And the morphological changes of nuclear were examined under a fluorescent microscope.(2)MTT assay, human neuroblastoma cells (SHSY-5Y) were treated with isatin (50,100,200,300μM) and DMSO (as control group). After 48 hours, proliferations of the cells were detected by using MTT assay. Then calculate the inhibitory rate of distinct dose.(3)Measurement of the amount of VEGF protein, human neuroblastoma cells (SHSY-5Y) were divided into four groups and given the distinct treatment (DMSO negative control, 50,100,200uM isatin). After 24 hours cell supernatants were collected and cell pellets were harvested .Then cell numbers were determined. The amount of VEGF protein in the supernatants was determined with ELISA kits. A linear equation was evaluated according to the data of standards. Then the amount of the samples was converted. The secretions of VEGF were expressed as pg protein/ml/10~5cells/24 h. (3)Western-blot, human neuroblastoma cells (SHSY-5Y) were divided into four groups and given the different treatment (DMSO negative control, 50,100,200μM isatin).After we get proteins, protein concentrations were measured by Lowery assay. Then proteins were applied to 10% SDS-polyacrylamide gel. The gels were blotted onto PVDF filters. After blocking, the filter was incubated with the primary antibody, the secondary antibody and DAB reagents in order. The relative expressions of phosphorylated mitogen-activated protein kinase (pp42/pp44) were examined.