The Proliferative Inhibition Of Chronic Myeloid Leukemia Gleevec Resistant Cell Line K562/G01 By The Berbamine And Its Mechanism Study

Leukemia is a clonal hematopoietic stem cell disorder that jeopardizes human's health.Chronic myeloid leukemia(CML)is a clonal hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance to apoptosis.The dysregulated activity of the bcr/abl oncoprotein tyrosine kinase,which is encoded by the bcr/abl fusion gene generated from the chromosomal translocation of t(9;22)and present in approximately 95%of CML patients,has been shown to be responsible for these malignant phenotypes.Numerous studies have demonstrated that being phosphorylated that p210bcr/abl oncoprotein can promote cell proliferation and survival,and block apoptosis of tumor cells.The blockade of Bcr/Abl kinase activity represents a rational strategy to treat leukemias caused by this oncogenic fusion protein.Gleevec, which is a selective inhibitor of the bcr/abl tyrosine kinase target to bcr/abl,is effective as a single-agent therapy for CML.Nearly all patients with CML achieve a partial or complete hematological remission.In 2001,Gleevec has been a recommend therapy by FDA.However,at the same time,resistance has been reported,particularly in patients with advanced-stage disease.Due to continued therapy,resistance can seriously cut down patients' life span.So,it is necessary to find some other compounds that inhibit bcr/abl oncoprotein.Here we describe the activity of a natural small molecular compound,berbamine,as a new Calmodulin antagonist that can selectively induce cell death of mang human cancer cells including melanoma cells, carcinoma of prostate cells,uterine cervix cancer cells and leukemia cells,et al.Previously, Our study team show berbamine can inhibit the growth of CML K562 cell lines and CML cells from patients.Meaningfully,BBM also can inhibit the growth of Gleevec resistant cell lines,but little is known about its exact mechanisms.Based on previous work,in this study,we observed the mRNA levels of BCR/ABL,TCF7,c-JUN, c-MYC genes and protein expression levels of p210bcr/abl protein,phosphorylated p210bcr/abl protein and NF-κB(P65),IκB in K562/G01 cells treated with BBM by means of various technologies,including cell culture systems,MTT assay,FCM, RT-PCR,western blots etc.Materials and methodsOur cell lines is human Ph+ CML leukemia K562,K562/G01 cells,which express endogenous P210 bcr/abl protein,were cultured in RPMI 1640 supplemented with 10%new bovine serum and treated with berbamine as indicated time and dose. We use MTT assays as Proliferation detection showed cell viability treated with berbamine;Flow cytometry(FCM)and Annexin V-FLUOS/PI staining kit were used to evaluate apoptosis of leukemic cells;p210bcr/abl protein and Phosphorylation of p210bcr/abl protein in leukemic cells were assessed by Western blots with c-abl antibody and p65,IκB antibody.The transcriptional levels of BCR/ABL,TCF7,c-MYC,c-JUN in leukemic cells were determined by RT-PCR with primers to BCR/ABL,TCF7,c-MYC,c-JUN,respectively.Results(1)Gleevec resistance detection of K562/G01 cells:The inhibition rates of K562 cells and K562/G01 cells treated with 1μg/ml Gleevec at 48h are 31.82%and 91.87%. K562/G01 cell lines have high resistant index,IC₅₀value is 0.19μg/ml,3.25μg/ml respectively,K562/G01 cells Gleevec resistant index is 17.22 times than that of K562 cells.(2)Proliferative inhibition of K562,K562/G01 cells treated with BBM:The inhibition rates of K562 cells treated with 8μg/ml BBM are 53.52%and 58.43%.The inhibition rates of K562/G01 cells treated with 8μg/ml BBM are 36.34%and 57.60%.After treatment with berbamine at different concentrations for 24h,the value of K562 cells,K562/G01 cells IC₅₀was.5.02μg/mlå’Œ9.07μg/ml;48h: 3.28μg/ml and 4.43μg/ml,respectively.(3)Flow cytometry(FCM)and Annexin V-FLUOS/PI staining detection:After treatment with berbamine at 4μg/ml,8μg/ml,16μg/ml for 24h,the percentages of apoptosis leukemic cells increased from 13.90%to 53.21%,70.30%,79.58%, respectively.(4)The change of BCR/ABL gene in mRNA levels and p210,p-p210 in protein expression in K562/G01 cells by BBM:Western Blots results showed that K562 cells,K562/G01 cells treated with 8μg/ml berbamine,10μg/ml berbamine(0,6h, 12h,24h)inhibited BCR/ABL gene in mRNA levels,also down-regulated phosphorylation of p210bcr/abl protein and total p210bcr/abl protein in leukemia cells remarkedly.The ratio of BCR/ABLmRNA andβ-actin after K562/G01 cultured with BBM 6h is 0.036136 while 0.491887 before BBM treated.The ratio of p210 protein andβ-actin at 6h is 0.093922 treated with 8μg/ml BBM in K562 cells while 0.557711 before BBM treated;the ratio of P-p210 protein andβ-actin at 6h is 0 while 1.291786 before BBM treated.(5)The change of NF-κB(p65)and IκB protein in K562/G01 cells by BBM:0,6h, 12h,24h K562/G01 cells treated with 10μg/ml BBM,the ratio of nuclear p65 protein and Lamin B at 6h is 0.005912 while 0.382442 before BBM treated;The ratio of Iκ3 andβ-actin at 6h is 0.163192 while 0.003516 before BBM treated,but, the total of p65 has no abviously changed.(6)After K562/G01 resistant cells treated with berbamine at 10μg/ml,Wnt pathway major gene TCF7,c-myc was down-regulated remarkably,and c-jun gene were up-regulated obviously.To the specific,the ratio of TCF7 andβ-actin from 0.247446 before BBM treated cut down to 0.032716 at 12h;the ratio of c-MYC andβ-actin from 0.733604 before BBM treated down to 0.211018 at 12h;the ratio of c-JUN andβ-actin is 0.001173 at Oh up to 0.138436 after treat with BBM at 24h.Conclusion(1)BBM could inhibit proliferative and induce apoptosis of Gleevec resistant cell line K562/G01 cells remarkably.(2)BBM is an inhibitor of phosphorylate-p210 protein,it can down-regulate p210 protein,so do BCR/ABL genes in mRNA levels,especially for P-p210.(3)BBM can inhibit translocation to nucleus of NF-κB(p65),up-regulate IκB expression;it can also inhibit TCF7,c-MYC genes,up-regulate c-JUN genes in mRNA levels.We can conclude that BBM induce apoptosis of K562/G01 cells via NF-κB signaling pathway and Wnt pathway.On a view of effective time,at 6h the expression of NF-κB(p65)showed a noticeable decrease while the downregulation of TCF7 gene at 12h.Consequently,we presume that the proliferation inhibitory and induced apoptosis of BBM predominant mainly via NF-κB(p65) signaling pathway.