Investigation Of The Role Of RhBMP2-induced Differentiation In Glioblastoma Stem Cells

Background:Glioblastoma （GB） is the most common primary brain tumor in adults. The incidencerate is12%¹5%in brain tumors. GB is very aggressive due to its poorcellular differentiation and invasiveness, which makes complete surgical resectionvirtually impossible. Current treatment strategies in patients are surgical resection withpost-operative radiotherapy. However, the median survival time of GB patients is less than1year. GB’s infiltrative nature and intrinsic resistance to current treatment modalitiesmake it a unique therapeutic challenge.According to the cancer stem cell concept, resistance to conventional therapy mayreside in a small subset of glioblastoma stem cells （GSCs）, which comprise asubpopulation of tumor cells, capable of differentiating into the actively expanding tumor bulk, they have the ability to proliferate, exhibit self-maintenance, self-renew to generatenew cells, generate a large number of progeny, and retain their multilineage potential overtime.Currently, therapeutic, drug-induced differentiation is considered as a promisingapproach to eradicate this GSCs population. The differentiation-inducing drugs, which areknown as potent modulator of cellular differentiation and proliferation, proved to be apowerful antitumor agent in the treatment of certain cancers. The role of the drugs aspromising therapeutics agents is receiving increasing attention, some of these substancesare being tested in clinical trials or are already in clinical use. However, previous studieshave reported limited efficacy in GB patients, hardly any anticancer drugs affect GSCsdifferentiation.Bone morphogenetic proteins （BMPs） are members of the transforming growthfactor-β （TGF-β） superfamily serving multiple functions in many cell and tissue types, themature active protein with a molecular mass in the20–38kDa range. Over40members ofBMPs have been identified in a wide variety of organisms. BMP2，concentrations of0.3–3nM promote neural differentiation. When telencephalic neural progenitors are brieflyexposed to BMP2in culture, their developmental fate is changed from neuronal cells toastrocytic cells. In recent years，BMP2-mediated growth regulation has been extensivelystudied in carcinogenesis. The findings are diverse. But the specific role and molecularmechanism of BMP2-induced GSCs differentiation are not clear. It remains unknownwhether and how tumor growth–relevant features of these cells are affected. Also needin-depth study that BMP2is expected to be applied to the comprehensive treatment ofclinic.Objective:The purpose of this study was to determine the role of rhBMP2-induced GSCsdifferentiation, clarify the effect of GSCs differentiation impair proliferation, and disruptsself-renewal.The following experiment simulated clinical radiotherapy, investigate the roleof GSCs differentiation by co-treatment with rhBMP2and⁶⁰Co-γ, clarify the effect of radiosensitivity of GSCs after treatment with rhBMP2. Finally, we hope this study maycontribute to understand the mechanism, and knowledge about the efficacy ofrhBMP2-induced differentiation to target the GSCs could have therapeutic implications.Methods:1. GSCs could be obtained from primary culture of GB samples and glioma cell lineU251screening, and GSC clone spheres could be identified and cultured.2. Induced GSCs differentiation by rhBMP2protein adding, detected the expressionand morphological changes of GFAP protein, the marker for mature astrocytes, observedthe effect of proliferation and self-renewal, analyze the mechanism rhBMP2-induceddifferentiation by indirect immunofluoresence staining and western blot assays.3. The experiment simulated clinical radiotherapy, co-treatment with rhBMP2andvarious doses60Co-γ, observed the effect of GSCs differentiation. By the analysis of cellcycle and self-renewal capacity, forecasted the effect of radiosensitivity of GSCs aftertreatment with rhBMP2.Results:1. GSC spheres from the glioma cell line U251were successfully cultured.This studyprovided a new and efficient screening method.By the cell cultural technology, based on the cell’s growth state, collected thesuspended cells and cultured in neurobasal medium supplemented with growth factors,such as epidermal growth factor or basic fibroblast growth factor, the cells could bepropagated and expanded indefinitely, grown into clone spheres. Indirectimmunofluoresence staining, clonogenic assays, proliferation assays and cell cycle assayswere performed. The results demonstrated that the suspended cells had characteristics ofCSCs, including higher proliferation rates, self-maintenance and self-renewal, and theystained positively for markers of brain CSCs and multilineage potential.2. rhBMP2protein could induce GSCs differentiation, so as to impair proliferation and disrupts self-renewal.By western blot assaysand indirect immunofluoresence staining, rhBMP2proteinadding could induce GSCs differentiation. We found that the GFAP expression wassignificantly higher than that in control group, and morphological changes of GFAP incytoplasm, the protein likes filamentous. The clone spheres easily adherent on the dish. Todetermine the apoptotic rate of cells accurately, quantitative analysis of apoptosis by FCMof AV/PI dual staining was performed. The result indicated that the apoptotic rate of cellswas unchangeableness. To study the biological changes after differentiation byrhBMP2-inducing, carboxyfluorescein succinimidyl ester （CFSE）-labelling, MTT assays,and cell cycle assays were performed. The results demonstrated that rhBMP2-inducinggroup cells had benign characteristics, including lower proliferation rates and self-renewal.By western blot assays, the key differentiation-related genes, we found the expression ofEZH2was decreased, the expression of Smad4, Wnt5a and β-catenin wereunchangeableness, which suggested that EZH2play roles in rhBMP2-induceddifferentiation.3. Co-treatment with rhBMP2and60Co-γ, efficiently induced GSCs differentiation,so as to enhance radiosensitivity and disrupts self-renewal of GSCs.Co-treatment with rhBMP2and various doses （0.25gy and20gy）60Co-γ, the GFAPexpression was significantly higher than that in control group, and morphological changesof GFAP protein in cytoplasm, it likes filamentous. The cell cycle stage was analysed witha FACSCalibur flow cytometer and CellQuest software, co-treatment with rhBMP2grouphad significantly fewer cells in the G0and G1phases and significantly more in the Sphase than60Co-γ group. The result indicated that co-treatment with rhBMP2couldenhance radiosensitivity of GSCs. Afterwards, clonogenic assay was carried out, and itwas found that28.33±5.13%of co-treatment with rhBMP2group cells could formsecondary clones, which is a significantly lower percentage than0.25gy60Co-γ group cells（41±6.08%）, and the number of living cells in rhBMP2group was less in the20gy60Co-γ group,which suggested that rhBMP2could efficiently impair GSCs self-renewal capacitywith60Co-γ stimulation.Conclusion:In this study, we succeeded in the GSC spheres culture from the glioma cell lineU251, it provided a new and efficient screening method. We demonstrated the role ofrhBMP2-induced GSCs differentiation, and clarified the effect of the biological changesafter differentiation by multi-analysis. We firstly simulated the clinical radiotherapycondition in vitro and investigate the role of GSCs differentiation by co-treatment withrhBMP2and60Co-γ. We demonstrated that co-treatment with rhBMP2could enhanceradiosensitivity of GSCs and efficiently impair the GB recurrence with radiotherapy.