Establishment Of A Drug Screening Method To Screen Chemicals Preferentially Eliminated GSCs

Objective: To provide a strategy for glioblastoma(GBM)chemotherapy chemicals discovery,we establish a high efficient drug screening method.Methods: Patient derived T387 GSC was stably transfected with green fluorescent protein,and then it was applied to establish a high efficient drug screening method in which fluorescence intensity is regarded as a readout.60 chemicals related to tumor stem cell apoptosis signaling pathway were screened using this method,and then were subjected to D456 GSC to reconfirm its efficiency.In which some of the chemicals are further tested to determine its toxicity for normal human astrocyte(NHA)and human microglia(HM).Chemicals,which killed glioma stem cells(GSCs)but had little influence on normal human astrocytes,were chosen as a candidate for induction of the cell apoptosis study using flow cytometry assay.SN38 is chosen to test antitumor activity research on animal.Survive time was evaluated and the effect of SN38 on brain tumor expansion was also evaluated by hematoxylin and eosin(HE)staining.Histopathology analysis method was applied todetermine the ratio of GSCs in tumor tissue and its potential on inducing apoptosis after SN38 treatment.Flow cytometry and western blot analysis was used to determine whether or not apoptosis is the cause of cell death.Results: A high throughput drug screening method was established for the discovery of chemicals with ability against GSCs;SN38 and bortezomib were found to be most lethal toward GSCs.We confirm that sn38 selectively kills GSCs.The half maximal concentration of SN38 on all GSC cell lines we have is lower than 10 nanomole,while the half maximal concentration of SN38 on NHA and HM is higher than 100 nanomole.Animal experimental results show that sn38 extend tumor bearing mouse survival time and inhibit tumor growth.It also implies that SN38 treatment induces glioma stem cell apoptosis and reduces the ratio of glioma stem cells in tumor tissue.Cell immunofluorescence experiment shows that SN38 kills GSCs by inducing apoptosis and inhibiting proliferation.Western-blot experiment results show that GSCS topoisomerase I protein level is lower after SN38 treatment.Inducible knockdown topoisomerase I lead to GSCs less sensitive to SN38.Conclusion: A high throughput screening method based on calculation of cell fluorescence intensity is able to obtain candidate chemicals targeting GSCs efficiently.TopI is the target of sn38 on glioma stem cells.SN38 shows antitumor effect by inducing apoptosis and inhibiting proliferation.