Brucine Inhibits Proliferation Of Glioblastoma Cells By Targeting The G-quadruplexes In The C-Myb Promoter

Background:Glioblastomas(GBM)are the most malignant and aggressive type of gliomas which are the most prevalent tumor in neural system.It is eager to find sensitive therapeutic targets and develop effective treating strategies in order to improve the very short average survival time of GBM patients after conventional therapies.The protooncogene c-Myb is a compelling anti-tumor target due to its overexpression in cancers such as leukaemia,breast cancer,colorectal cancer and several types of gliomas.There is a guanine-rich DNA sequence located in the promoter region of c-Myb and can negatively regulate the transcription via forming G-quadruplexes to block the binding of the promoter and its activating transcription factor.Brucine is a ligand of these G-quadruplexes to stable their structure.Therefore,brucine could be an inhibitor of c-Myb to suppress GBM.Studies of c-Myb as a GBM target have been rarely seen up to now.Objective:We used glioblastoma cell lines and tumor xenograft model in nude mice to investigate the therapeutic effects and mechanism of brucine in treating GBM.Methods:Bioinformatics analysis was used to demonstrate the anti-tumor potential of c-Myb as a target to treat glioblastoma.MTT assays,flow cytometry assays,quantitative real-time polymerase chain reaction(qPCR),western blot assays,tumor xenograft model in nude mice and immunohistochemical assessments were used in the present study to assess the cell viability,effects on cell cycle and apoptosis,transcriptions and expressions of specific genes,and anti-tumor effects in vivo of GBM cell lines under treatment of brucine respectively.And dual-luciferase reporter assays and Electrospray ionization mass spectrometry(ESI-MS)were used to investigate the interactivities of brucine with target guanine-rich DNA sequence and G-quadruplexes.Results:1.Bioinformatics analysis showed c-Myb could be an effective target for treating glioblastoma.The result of MTT assays showed that brucine negatively affected the viability of GBM cell lines.The cell cycles of GBM cells were arrested while the apoptosis was not significantly induced by brucine according to the results of flow cytometry assays.In western blot assays,expressions of cyclin D1 and cyclin B1 which were related to cell cycle were suppressed by brucine.However,the apoptosis relevant proteins Bax,Bcl-2 and caspase 3 were not significantly influenced by brucine.2.The result of dual-luciferase reporter assays showed that the expression of luciferase vectors was affected by insertion of the guanine-rich DNA sequences in c-Myb promoter,and brucine could enhance the impact.The binding capacity of brucine and the guanine-rich DNA sequences was proved via ESI-MS.It was suggested that brucine could bind to the G-quadruplexes formed by the guanine-rich sequences to improve their stabilities and inhibit the formation of double strands DNA.The results of qPCR and western-blot assays showed that the expression and transcription of c-Myb in GBM cells were decreased with brucine treatment.3.In our tumor xenograft models,tumor sizes of U87 tumors in nude mice were suppressed by brucine.It was showed that the expressions of c-Myb and Ki-67 in the tumors were negatively affect by brucine via immunohistochemical assessments as well.Conclusion:1.Brucine suppresses GBM both in vitro and in vivo.2.Brucine could improve the formation of G-quadruplexes and inhibit the formation of double strand DNA by binding to the guanine-rich DNA sequence in c-Myb promoter region.3.Brucine could reduce the expression of c-Myb via binding to the G-quadruplexes in c-Myb promoter region.4.Brucine could arrest cell cycle of GBM by reduce the expression of c-Myb.