A Novel Drug Screening Approach For Glioblastoma Targeting Stem-like Cells

Background and aims:Glioblastoma is a brain tumor with very high malignancy and poor prognosis.According to WHO classification in 2007,glioblastoma was divided into astrocytoma population with grade IV malignancy(the highest grade of malignancy in glioma).In China,the annual incidence of glioma is about 5/100,000,and glioblastoma accounts for 60%-70%of glioma.According to the Chinese population of 1.3 billion,the annual number of new cases of glioblastoma is 39,000-45500.At present,the most commonly used therapy for glioblastoma is maximum surgical excision combined with radiotherapy and adjuvant therapy(chemotherapy)with temozolomide.However,the tumor could not be completely cured,partly because glioblastoma showed invasive growth,and glioblastoma occurred under the cerebral cortex,making surgical resection unable to completely remove the tumor.Another important reason is that the tumor itself is resistant to radiation and chemotherapy.After a lot of research,it is found that there is a kind of cell in the tumor,which has the characteristics of stem cells.It is called Cancer Stem Cells(CSCs).Cancer stem cells have the ability of self-renewal,infinite proliferation and multidirectional differentiation potential,which are not only closely related to the tolerance of tumor to radiation and chemotherapy.Meanwhile,CSCs also play a very important role in promoting tumor angiogenesis,hypoxic response,tumor invasion,and tumor recurrence.Plants have always been an important source of anti-tumor drugs.Therefore,the purpose of this study is to screen out compounds that can selectively kill glioma stem cells from a series of compounds isolated from plants and carry on biological research.Methods:Firstly,a series of Extractions and monomer compounds were provided by Kunming institute of botany,Chinese Academy of Sciences,Prof.Xiao-dong Luo research team and Kunming University of Science and Technology,Prof.Rong-tao Li research group,isolated from the plants that have antitumor activity and other medicinal plants,and then diluted by dimethyl sulfoxide(DMSO)to 10 mg/ml.Using high-throughput screening method,preliminary screening showed several components and compounds with high selectively against a type of glioma stem cell line(GSC-3#),and then confirmed by using the other two glioma cell lines(GSC-12#and GSC-18#).To further confirm the compounds selectivity,I screened them against human normal cell lines(293T and HAC),and has no effects on the growth of human normal cell lines.Half-maximal inhibitory concentration(IC50)for different cells treated with the components and compounds measured by MTS method was used to compare the differences in selectivity between the different cell lines.Compounds with more selective killing effect on GSCs were selected for further in vitro studies,mainly to detect the influence of compounds on apoptosis,proliferation,and Carcinogenesis by examining clone formation capacity of GSCs.Results and conclusion:Our laboratory has established a high-throughput drug screening system and successfully isolated glioma stem cells from glioblastoma of tumor patients,named as GSC-3#,GSC-12#and GSC-18#.These three cell lines of glioma stem cells were derived from different tumor patients,providing a stable cellular platform for future research.The samples used were the 2844 plant components provided by Kunming Institute of Botany,Chinese Academy of Sciences,Prof.Xiao-dong Luo research group,and 203 plant components and 301 compounds provide by Kunming University of Science and Technology,Prof.Rong-tao Li research team.According to the screening,the results identified that six compounds and 29 plant components can selectively target glioma stem cells,and 29 plant components need further separation and purification,to get a series of single compounds.The IC50 values of the six compounds were detected by MTS,and the sensitivity to glioma stem cells was 2-3 times higher when compared to human normal cell lines in 4 compounds.According to previous publications,two of these four compounds have been reported to have anti-tumor activity against other tumors.This give me the opportunity to study the other two compounds for the first time.These two compounds respectively are rupesin E and8,9-didehydro-7-hydroxydolichodial.This study mainly focused on determining the in vitro effects for these two candidates in glioma stem cells(GSC-3#and GSC-18#)by studying apoptosis,proliferation and clone formation after the treatment of rupesin E and 8,9-didehydro-7-hydroxydolichodial.The results clearly show that the rupesin E and 8,9-didehydro-7-hydroxydolichodial can induce the apoptosis of glioma stem cells,inhibit the proliferation,and the experiment of clone formation showed that rupesin E and 8,9-didehydro-7-hydroxydolichodial can destroy the formation of clone spheres.