Induction Of Apoptosis In Leukemia Cell Line K562 By Bortezomib In Combination With Idarubicin

backgroundLeukemia is a group of heterogeneous malignancy of hematopoietic system in which the hematopoietic stem cells and progenitor cells have a malignant change,and the cells blocked in different stages of haematogenesis lose the ability of further differentiation and maturation.Although the effectivity of treatment has been increased in recent years,there are still a considerable numbers of refractory cases of leukemia due to relapse or drug resistance,therefore it is particularly important for us to seek new methods of treatment in the current.Bortezomib(bortezomib,BOR)is a novel anti-tumor targeted therapy drug,mainly used for the treatment of relapsed or refractory multiple myeloma,and has shown good results.In vitro studies,BOR had shown a good effect on a variety of malignant tumor cells,but there is few report in leukemia,and none study report on BOR in combination with Idarubicin (idarubicin,IDA).The effect of BOR in combination with IDA on leukemia cell line K562 was revealed in this study,and this will provide a theoretical basis for the treatment of leukemia.Material and MethodsFor specified time and concentration,the K562 cells was treated with Bortezomib alone or in combination with Idarubicin,detected the viability of K562 cells by MTT,analysed the results and obtained the inhibition rate of growth of K562 cell treated with Bortezomib alone or in combination with Idarubicin for 24 hours;Stained the cells of different groups by Hoechst33342,and observed the morphology change of nucleus;Extracted cell genomic DNA,by gel electrophoresis of DNA,got the characteristics "ladder" DNA bands of apoptotic cells;Detected the expression trends of survivin and bcr / abl gene By RT-PCR;Detected the expression trends of survivin and bcr / abl-p210 protein by Western Blot.Results(1)The inhibition rate of growth of K562 cells treated with 100nmol / L Bortezomib alone for 24 hours was 40.11%,the inhibition rate of growth of K562 cells treated with 200nmol / L Idarubicin alone for 24 hours was 39.72%,and the combination of the two is 76.58%.For the combination,the coefficient of drugs in interaction(CDI)＜1,it indicated that the two had synergistic effect.(2)For the negative control group,the nucleus stained by Hoechst33342 emitted uniform dispersion of blue fluorescent,and the group of Bortezomib alone or in combination with Idarubicin,cell apoptosis was seen,the nucleus performed lectin, pyknosis,formation of dense,bright blue fluorescent particles;Perspective units the combition group had more numbers of apoptotic cells than both single dose treatment groups.(3)50-150nmol / L Bortezomib treated K562 cells for 12 hours,DNA Ladder can be seen obviously by DNA gel electrophoresis,for the 50 nmol / L Bortezomib treatment group,it can also be found the normal genomic DNA band and apoptosis DNA Ladder coexistence.In the same condition,the time extended to 24 hours,each group still can be found apoptosis DNA Ladder,but in the group of 50 nmol / L,normal genomic DNA band disappeared,this showed that with the time extended,the effect of Bortezomib enhanced.100 nmol / L Bortezomib alone or in combination with 200nmol / L Idarubicin treated K562 cell for 24 hours,DNA gel electrophoresis also showed significant DNA Ladder.(4)Compared with control group,the expression of survivin and bcr / abl mRNA of the combination group dereased significantly.Quantitative Analysed by the software, for the expression of survivin mRNA,BOR+IDA group:0.736±0.024,control group:0.895±0.023,P＜0.01.The other two groups' survivin mRNA expression is near to the control group:BOR group:0.881±0.019,IDA group:0.889±0.021,control group:0.895±0.023,P＞0.05.For the expression of bcr/abl mRNA,BOR+IDA group:0.440±0.020,control group:0.870±0.027,P＜0.01.The other two groups' bcr/abl mRNA expression is near to the control group:BOR group:0.858±0.015,IDA group:0.844±0.029,control group:0.870±0.027,P＞0.05.(5)Compared with control group,the expression of survivin and bcr / abl-p210 protein of the combination group dereased significantly.Quantitative Analysed by the software,for the expression of survivin protein,BOR+IDA group:0.371±0.025,control group:0.871±0.032,P＜0.01.The other two groups' survivin protein expression is near to the control group:BOR group:0.862±0.017,IDA group:0.859±0.022,control group:0.871±0.032,P＞0.05.For the expression of bcr/abl fusion protein p210,BOR+IDA group:0.340±0.027,control group:0.865±0.021,P＜0.01.The other two groups' bcr/abl fusion protein expression is near to the control group:BOR group:0.838±0.019,IDA group:0.814±0.029,control group:0.865±0.021,P＞0.05.Conclusion(1)Bortezomib in combination with Idarubicin can inhibit the proliferation of leukemia cell line K562 by induction of apoptosis,and the inhibition effect of combination is much more enhanced than the two drugs used alone.(2)Bortezomib in combination with Idarubicin can inhibit the survivin and bcr / abl gene's expression of K562 cell,it may be one of the mechanisms that mediate the apoptosis of K562 cells.This study indicates that Bortezomib in combination with Idarubicin can be used as a new method in clinical treatment of leukemia.