The Study Of Multi-target Tyrosine Kinase Inhibitors To Regulate C-Cbl Gene-mediated Chemoresistance In Acute Myeloid Leukemia Cells

Background and objective:The overall therapeutic effect of acute leukemia is still unsatisfactory.The relapse and refractory were the two main reasons that restricted the improvement of therapies.Currently,intracellular signaling disorder caused by molecular genetic abnormalities was considered to be the main cause of the pathogenesis of acute leukemia.Therefor,the kinase inhibitor drugs,which could target on suppress disease-related signaling pathways,were expected to be the solution for future therapy.Multi-target tyrosine kinase inhibitors,represented by sorafenib,have been widely used in patients with refractory and relapse leukemia,but thetherapeutic effect varies between different patients significantly.The reason behind such is not clear.Our early studies found that the therapeutic effect of patients with high expression of c-kit(CD 117),one of the drug target,was better than those with low or without expression.Apparently,the drug efficacy can be related to the expression of target receptors.In recent years,it has been found there are two systems being involved in tyrosine kinase signaling regulation under physiological state.One of these was activation(positive)while the other one was inactivation(negative).c-CBL protein is the core of the negative regulation system of tyrosine kinase,and the decrease in its expression would directly lead to the increased expression of the tyrosine kinase receptor(e.g.,c-kit)and downstream signal.As such,we are interested in discovering the relationship between c-Cbl gene expression and leukemia cell chemoresistance.Through regulating the expression of c-Cbl gene,can it affect the chemoresistance of leukemia cells by changing the positive and negative balance of tyrosine kinase,which can further improve the therapeutic effect of clinical patients?Therefore,we conducted a study about the functional status of c-Cbl gene and cell chemoresistance and explored the relationship between sorafenib treatment and c-CBL and its related signaling pathway proteins.Methods:1.The basic expression of c-Cbl gene in refractory and relapsed leukemia patients was detected by quantitative PCR.2.Recombinated lentiviruses and adenovirus vector with c-Cbl gene silenced expression and over-expression were constructed by genetic recombination technology respectively.After lentivirus and adenovirus packaging,HL60 and THP1 leukemia cells were infected by viral particles.To confirm the success of viruses packaging,quantitative PCR and immunoblotting were used to detect the changes of mRNA transcription and protein translation of the c-Cbl gene respectively.3.Infected by lentiviral virus and adenovirus,leukemia cells with c-Cbl gene silenced expression and over-expression could be obtained respectively.In order to compare with normal leukemia cells,the differences in self-proliferation,ligand-induced proliferation and drug sensitivity between these cells were detected and measured by cck-8 and trypan blue rejection methods.Cellular cycle and apoptosis induced by sorafenib and cytarabine were measured by flow cytometry.4.The proteins,including T-CBL,p-CBL,c-kit(CD117),PDGFRβ(CD140b),MDR1,T-Akt,p-Akt,T-ERK and p-ERK,of the cells with c-Cbl gene silenced expression,over-expression and normal expression are detected and measured by immunoblotting before and after sorafenib treatment in order to track the changes in expression.Results:1.Low expression of c-Cbl gene can be commonly in patients with relapse and refractory acute leukemia.2.Recombinated lentiviruses were successfully constructed,which could infect HL60 and THP1 cells effectively.Leukemia cells with c-Cbl gene stable silencing could be easily filtered out by puromycin culture.Compared with the control leukemia cells,the cells with c-Cbl gene silencing had been indicated that the proliferation was increased,the proportion in S phase cell was decreased,the sensitivity to SCF-induced proliferation was increased,the chemoresistance to sorafenib was increased and the apoptosis induced by sorafenib and cytarabine was decreased.3.Recombinated adenovirus was successfully constructed,which could also effectively infect leukemia cells.High expression of c-Cbl gene and protein could be detected in both HL60 and THP1 cells after adenovirus infection.Compared with the control leukemia cells,the cells with c-Cbl gene over-expression had been indicated that the proliferation was decreased,the proportion of G2/M phase cell was decreased,the sensitivity to SCF-induced proliferation was decreased,the chemoresistance to sorafenib was decreased and the apoptosis induced by sorafenib and cytarabine was increased.4.Immunoblot analysis showed that the expression of c-Cbl protein in all cells was down-regulated after sorafenib treatment,meanwhile the expression of c-kit and PDGFRβ was being up-regulated,while the expression of MDR1 had no significant changes.When the expression of c-Cbl gene had been silenced,activation of both ERK and AKT signal protein could be detected in HL60 cells.However,in THP1 cells,only AKT signal protein was being activated.When c-Cbl gene was over-expressed,neither ERK nor AKT signal protein was activation in both cells.Treated by sorafenib,ERK signal protein activation could be detected in HL60 cells while AKT signal protein was found to be activated in the THP1 cells.Under the same condition,both ERK and AKT signal protein activation could be detected in HL60 and THP1 cells when the c-Cbl gene’s expression had been silenced or over-expressed.Conclusion:1.The patients with c-Cbl gene low expression might be associated with chemoresistance and disease refractory.2.With the downward regulation of c-Cbl gene expression,the cell proliferation by themselves and induced by ligand was thus increased,and the chemoresistance to sorafenib and cytarabine was also being increased.3.With the upward regulation of c-Cbl gene expression,the cell proliferation by themselves and induced by ligand was then decreased,and the cell chemoresistance to sorafenib and cytarabine was also being decreased.4.The different expression states in c-Cbl gene are related to the chemoresistance of leukemia cells.The use of sorafenib had resulted in the downward regulation of c-Cbl protein and upward regulation of target receptor protein on membrane surface.The forced change in expression of c-Cbl gene can affect the intrinsic signal activation pathway in leukemia cells.For cell with altered c-Cbl gene expression treated by sorafenib,AKT and ERK signal proteins were both involved in intracellular signals activation.5.Based on the function of c-Cbl gene,sorafenib can aggravate the chemoresistance in leukemia cells.Therefore,patients should be carefully selected for clinical use appropriately.Proved by actual clinical results,most patients still have a certain curative effect by sorafenib treatment,and other pathways and mechanisms maybe involved in the development of chemoresistance in leukemia cells.