Beclin-1 Overexpression Induced Autophagy On Inhibitory Effect Of Bcr-Abl And Its Molecular Mechanism

Background:Chronic myeloid leukemia(CML)is a hematologic malignancy.Tyrosine kinase inhibitor imatinib has achieved good efficacy as a first-line treatment,but 30%patients still occur drug resistance due to mutation of Bcr-Abl gene or mechanism with independent of Bcr-Abl.Recently,some studies have shown that there are multiple pathways that can degrade or inhibit Bcr-Abl gene expression,which may be new methods to treat patients of CML.It is showed that autophagy induced death is a new strategy for treatment of tumor.Our team's previous researchs have showed that oncolytic virus infection with Beclin-1 could significantly increase the killing effect of oncolytic virus on CML cells by inducing autophagy death,but whether it could kill drug resistant cells or CML stem cells and its molecular mechanism remained unknow.This study was divided into two parts.In the first part,bioinformatics analysis is applied to study the interaction between Beclin-1 and Bcr-Abl in vitro.The interactions between the two proteins in K562 cells were tested by Gst-Pull down,immunoprecipitation and Western blot.The effect of Beclin-1 overexpression on Bcr-Abl promoter activity in K562 cells was also observed.It is a foundation for further exploration of autophagy death by Beclin-1 overexpression in CML cells and the down-regulation mechanism of Bcr-Ab1.In the second part,investigate the effect of Beclin-1 overexpression on cell growth,autophagy and related genes in K562 cells.CD34+CD38-cells isolated from bone marrow of CML patients with acute phase are also investigated.To observe the regulatory effect of Beclin-1 overexpression on Bcr-Abl protein.To study the effect of autophagy regulators and siRNA of ATG7/UVRAG gene on the down-regulation of Bcr-Abl protein.To discusse the effect of Beclin-1 overexpression on the co-localization of Bcr-Abl and p62/SQSMT1.In addition,Beclin-1 was overexpressed in 32D-p210-T315I cell line which is Bcr-Abl T315I mutation,and the changes of cell growth and autophagy-related genes were observed,as well as the regulation of Bcr-Abl.Part ?:Study on the interaction between autophagy gene Beclin-1 and Bcr-Abl fusion gene protein in chronic myelogenous leukemia Objective:Analyze and study the interaction between Beclin-1 and Bcr-Abl protein.Methods:Bioinformatics was used to analyze whether there was direct or indirect interaction between the two genes.Beclin-1 and Bcr-Abl interactions in vitro and in vivo were studied by Gst-Pull down assay,immunoprecipitation and Western blot.Furthermore,the effect of Beclin-1 overexpression on Bcr-Abl promoter activity in K562 cells was tested by double luciferase activity assay.Results:1.According to the predicted results of bioinformatics,Beclin-1 and bcl-2 have direct interactions.Beclin-1 may interact with Bcr-Abl through bcl-2 under the condition of medium confidence.2.Gst-Pull down experiment found that C-terminal of Beclin-1 450-300 and 450-250 amino acid deficiency affected the binding of Beclin-1 to GST.It is suggested that Beclin-1 protein and Bcr-Abl binding sites may be located at C-terminal 450-250 amino acid.3.The results of co-immunoprecipitation showed that Beclin-1 C-terminal 150 amino acid deletion protein could not precipitate Bcr-Ab1 protein,while C-terminal 100 amino acid deletion protein could precipitate Bcr-Abl protein.It was further confirmed that the site of Beclin-1 protein interaction with Bcr-Abl was at the C-terminal 100-150 amino acid residues.4.Double luciferase activity assay showed that it was significantly decreased in beclin-1-k562 co-transfected cells,indicating that Beclin-1 overexpression significantly inhibited the activity of Bcr-Abl promoter.Conclusion:Although bioinformatics analysis using STRING database showed that there was no direct interaction between Beclin-1 and Bcr-Abl.In this study,Beclin-1 may confirm to have a direct interaction with Bcr-Abl by Gst-Pull down and immunoprecipitation experiment.The site of C-terminal 100-150 amino acid may be the binding area.Double-luciferase biopsy showed that Beclin-1 overexpression inhibited the transcriptional activity of Bcr-Abl in K562 cells.The results of this study and previous studies in literature suggested that Beclin-1,a key autophagy gene,was closely related to CML,and targeted autophagy pathway may be a new strategy to treatment of CML.Part ?:Beclin-1 overexpression induced autophagy death of CML cells and its molecular mechanismObjective:To analyze and study the molecular mechanism of autophagy in CML cells with Beclin-1 overexpression.Methods:1.Western blot was used to confirm that the down-regulation of Bcr-Abl may be related to autophagy pathway and not related to Caspase signaling pathway.2.Autophagy inhibitor 3-MA and proteasome inhibitor epoxomicin Epo were used to analysis the effect on autophagy after Beclin-1 overexpression in K562 cells by electron microscopy and Western blot.3.The effect of autophagy regulators and ATG7/UVRAG gene siRNA on the down-regulation of Bcr-Ab1 was studied.4.To investigate the effect of Beclin-1 overexpression on co-localization of Bcr-Abl and p62/SQSMT1.5.Combined with 3-MA or Epo respectively to inhibit the growth of K562 cells with Beclin-1 transfection were detected by MTT assay.6.After overexpression of Beclin-1 in primary cells of T315I mutation positive CML patients and 32D-p210-T315I cells,MTT,colony culture and Western blot were used to determine whether overexpression of Beclin-1 induced autophagy of drug-resistant cells and its effect on growth and colony formation,as well as the regulation of Bcr-Abl protein.7.The effect of Beclin-1 overexpression on CML stem cells was determined by colony culture and Western blot by using CD34+CD38-cells isolated from bone marrow of CML patients with acute phase.Results:1.Bcr-Abl was inhibited in K562 cells with Beclin-1 overexpression and it did not activate the pathways of caspase-3 and PAPR.The down-regulation of Bcr-Abl was unrelated to the activation of Caspase signaling pathway.2.The results of electron microscopy and Western blot showed that the autophagy inhibitor 3-MA could reverse autophagy which was induced by Beclin-1.Epo significantly induced autophagy by Beclin-1 overexpression.However,neither 3-MA nor Epo had significant effect on Bcr-Abl down-regulation.On the other hand,Western blot showed that Beclin-1 overexpression in K562 cells would lead to up-regulation of autophagy related genes ATG7 and UVRAG.3.The results showed that both interference by ATG7 and UVRAG could significantly reverse Beclin-1 overexpression induced autophagy.In the same time,the interference of the two genes can partially reverse the down-regulation of Bcr-Abl.4.Cathesin inhibitor CA074 was applied to observe the effect of CA074 on co-localization of Bcr-Ab1 and p62/SQSTM,and the results showed that CA074 significantly inhibited co-localization of Bcr-Ab1 and p62/SQSTM.5.It was showed that Beclin-1 overexpression inhibited CML growth by MTT method.Epo combined with Beclin-1 induced autophagy and significantly inhibited the growth of CML cells.6.After overexpression of Beclin-1 in 32D-p210-T315I cell line,MTT showed that overexpression of Beclin-1 inhibited cell growth.The overexpression of beclin-1 in primary CML cells with T315I mutation was detected by colony culture,and colony formation decreased.The expression of autophagy-related gene LC3II was increased and the expression of p-Bcr-Abl protein was decreased by Western blot.7.The effect of Beclin-1 overexpression on CFU-GM colony was detected by colony culture method.The results showed that Beclin-1 overexpression of CD34+CD38-significantly reduced the number of CFU-GM colony.We used Western blot to analyze the effect of Beclin-1 overexpression on Bcr-Abl proteins and signaling pathway in CD34+CD38-cells.The results showed that Beclin-1 overexpression up-regulated ATG7 and UVRAG and significantly inhibited Bcr-Abl protein expression.LC3-II significantly increased and P62 expression decreased suggested that autophagy was induced in CML stem cells.Conclusion:The study showed that Beclin-1 overexpression in K562 resulted in up-regulation of ATG7 and autophagy,which significantly inhibited the growth of cells.Beclin-1 overexpression can inhibit the growth of 32D-p210-T315I and induce autophagy.At the same time,it also plays a role in the degradation of Bcr-Abl protein,suggesting that overexpression of Beclin-1 in T315I mutant cells may overcome drug resistance by inducing autophagic death.Beclin-1 overexpression inhibited the CFU-GM colony formation of CD34+CD38-cells in vitro,suggested that induction of autophagy death may be a new method to treatment of CML and the clearance of Bcr-Abl+CML stem cells.Beclin-1 overexpression induced downregulation of Bcr-Abl and partially reversed by interference with ATG and UVRAG.On the other hand,the application of protease Cathesin B can interfere with the co-localization of Bcr-Abl and p62/SQSMT1.It was suggested that Beclin-1 overexpression resulted in downregulation of Bcr-Abl,which was related to autophagy dependent degradation of Bcr-Abl protein.The evidence in CML leukemia stem cells is not enough,and further research is needed in the future.