A Marketed Drug Screening Approach Targeting Stem-like Cells In Glioblastoma

Objectives:Glioblastoma is a kind of brain tumor with very high malignant degree and poor prediction.the malignant degree of glioblastoma is mainly reflected in uncontrollable cell proliferation,high invasiveness,anti-apoptosis,a large number of angiogenesis and so on.The tumors are located under the cortex and most of them grow in the supratentorial hemisphere.The frontal lobe was the most common site of occurrence.It is infiltrative growth,often invading several lobes,and invading the deep structure,but also through the corpus callosum to the contralateral cerebral hemisphere.It is precisely because of the high invasiveness of glioblastoma that its prognosis is very poor.In the 2007 WHO classification,glioblastoma was classified as astrocytoma,The malignant grade was IV(the most malignant grade in glioma),in which the median survival time was about 12-15 months,15%had a survival time of more than two years,and only 3%had a survival time of more than 3 years.The most commonly used treatment for glioblastoma.The treatment is maximum surgical resection combined with radiotherapy,And adjuvant therapy(chemotherapy)with temozolomide.However,there is still no complete cure for the tumor,in part because of the invasive grorwth of glioblastoma,and glioblastoma occurs in the cerebral cortex,so that surgical resection can not completely remove the tumor.Another important reason is that the tumor itself is resistant to radiotherapy 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 was named "tumor stem cell"(Cancer stem cells,CSCs).CSCs stem cells have the ability of self-renewal,unlimited proliferation and multidirectional differentiation potential,which are not only closely related to the tolerance of tumor to radiotherapy and chemotherapy.At the same time,CSCs also plays a very important role in promoting tumor angiogenesis,hypoxic response,tumor invasion and tumor recurrence.The sources of antitumor drugs are scarce,mainly rely on drug screening,and the re-screening and use of listed drugs are often ignored.Therefore,the purpose of this study is to screen out the listed drugs which can selectively kill glioma stem cells and inhibit their proliferation from the listed drugs,and carry out biological research.Methods:Firstly,the monomer compound was diluted to 10mg/ml by dimethyl sulfoxide(dimethyl sulfoxide,DMSO),relying on the monomer library of Shanghai Institute of Medicine,Chinese Academy of Sciences.Using the high-throughput screening platform of Zhao Xudong research group of Kunming Institute of Zoology,Chinese Academy of Sciences,the components and compounds that can selectively kill a glioma stem cell line(glioma stem cells,GSC-3#)were screened out.Then two other glioma stem cell lines(GSC-18#)and human normal cell lines(293T and HAC),were used to screen the components and compounds that could kill GSC,but had no effect on the growth of human normal cell lines.The 50%inhibitory concentration(half maximal inhibitory concentration,IC50)of the compound on different cells was measured by MTS method,and then the difference of the value was compared.The sensitivity of compounds to different cells was determined.Select the compounds that have more selective killing effect on GSC,compare with the chemical structure of the listed drugs,select the drugs with similar structure to carry on the next step research,mainly detect the effect of the compounds on the apoptosis and proliferation of GSC.Results:Kunming Institute of Zoology,Chinese Academy of Sciences,has established a high-throughput drug screening platform,and successfully isolated glioma stem cells from glioblastoma of tumor patients,named GSC-3#,GSC-18#.These two glioma stem cells come from different tumor patients and provide a stable platform for subsequent research.In this paper,800 compounds were selected from the compound library to selectively kill glioma stem cells.by consulting the literature.74 compounds have been reported to have antitumor activity against other tumors,and the chemical structure comparison has been carried out.It was found that the remaining two drugs had selective killing effect on glioma stem cells,so the next study of the other two compounds was carried out.The two compounds are amlodipine and loperamide,respectively.The effects of compounds on apoptosis and proliferation of glioma stem cells(GSC-3#and GSC-18#)were studied.The results showed that amlodipine and loperamide could promote the apoptosis and inhibit the proliferation of glioma stem cells.Conclusions:Glioblastoma multiforme(Glioblastoma multiform,GBM)is a highly invasive brain tumor with low life expectancy,and glioma stem cells(gli..cells,GSCs)are a few tumor cells present in GBM.GSCs responds to the recurrence,invasion and anticancer activity of GBM.GSCs has been identified and developed as a therapeutic target for GBM and can be used for drug screening.Amlodipine is a low-cost drug for the treatment of hypertension,which is widely used in clinical treatment,but the anticancer effect of amlodipine on GSC has not been reported.The antitumor effect of amlodipine on GSCs was studied.the results showed that amlodipine significantly inhibited the growth of GSCs.IC50 were 4.048?g/ml,4.8461?g/ml and 7.397?g/ml,respectively.In the further study of the mechanism,amlodipine inhibited the proliferation of GSCs cells,induced GSCs apoptosis and increased the proportion of caspase-3 cleavage.The results showed that amlodipine induced apoptosis of GSCs through Caspase-3-dependent apoptosis pathway.In addition,amlodipine can still inhibit tumor growth and improve survival time in vivo.These suggest that amlodipine is expected to be a new anti-glioma drug.