Study On Effects Of Clofarabine Combined With Bortezomib In K562Cell Line

Objective: Leukemia is a kind of malignant myeloproliferative diseaseobserved in hematopoietic stem cells, including acute leukemia and chronicleukemia. Chronic myelogenous leuke-mia (CML) occupies15%~20%ofleukemia occurred in adults, which characterized in slow development ofdisease course. Recently, thanks to the fast development in molecular biologyand cytogenetics, we have got more in-depth understanding on thepathogenesis of CML and lots of new treatments were developed.Clofarabine is the second generation of purine nucleoside derivatives andFDA has approved its application in children refractory or relapse ALL. It isthe first novel medicine that FDA has approved its application in children withleukemia during the past10years. Part of the children obtained continuousalleviation and had the opportunity to receive bone marrow transplantation.This medicine also brings hope to refractory or relapse AML children. Clinicalstudies indicated that this medicine had exhibited broad-spectrum anti-tumoractivities and had obtained satisfactory effects in children and adults with ALL,AML, MDS and CML.Bortezomib is a kind of proteasome inhibitor. Previous study hasdemonstrated that bortezomib inhibited the proliferation and enhanced theapoptosis in K562cell lines, and could increase the expression of SHIP genein K562cells too.Lots of researches suggest that bortezomib showed certaininhibition in various malignant tumor cell lines. Synergistic or sensitizingeffect, not single additive effect, was observed by combining with many kindsof drugs[32].SHIP belongs to inositol5’-phosphatase family and its main functionsinclude inhibiting PI3K/Akt pathway and inducing programmed cell death.Previous study of this project had demonstrated the lack of SHIP gene expression in leukemia cells K562. SHIP gene inhibits the proliferation ofK562cells and mediates the apoptosis of K562cells.Recently, reports on inhibition of Clofarabine on K562cells are rare. Inorder to get a better understanding of the exact mechanism of Clofarabine’sinhibition on proliferation as well as promotion on apoptosis in K562cells,Clofarabine was used alone or combining with bortezomib in K562cells andthe inhibition in proliferation and promotion in apoptosis were observed, totest the changes in expression level of SHIP in K562cells treated byClofarabine alone or bortezomib and Clofarabine,which provides theoreticalbasis for the treatment of chronic myeloid leukemia.Method:1The influence of Clofarabine used alone or combination with bortezomib indifferent concentrations on the proliferation of K562cells were detected byCCK8;2The influence of Clofarabine used alone or in combination with bortezomibon apoptosis of K562cells were detected by flow cytometric analysis;3The changes in expression of SHIP mRNA were determined by RQ-PCRafter Clofarabine were given alone or in combination with bortezomib.4Statistical analysis was performed.Result:1Inhibition of Clofarabine in proliferation of K562cellsK562cells were treated with Clofarabine of different concentrations.24hlater, the results were analyzed, which indicated that, within certainconcentration range, the inhibition on the proliferation of K562cells showeddoes-dependent manner. When clofarabine were used in K562cell lines, theinhibition rate gradually increased as the gradual increase of the concentration.2Pro-apoptotic effects of clofarabine used alone in K562cellsIn the group clofarabine was used alone, the proliferation of K562cellswere inhibited and the apoptosis rate increased as the concentration increased.3Inhibition of Clofarabine combined with bortezomib on proliferation andPro-apoptotic effects of K562cells Clofarabine and bortezomib(10nmol/L)were combined and used,theresults indicated that the inhibition gradually increased. Compared with theconditions when clofarabine or bortezomib were used alone, within sameintervene time, the inhibition rate increased obviously as the concentrationincreased (p<0.05). Whether synergistic or antagonistic effects existed whenthe two drugs were used in combination was evaluated with Jin’s formula andthe results suggested synergistic effect. When Clofarabine and bortezomibwere combined,the apoptosis rate increased as the concentration increased,and the differences were statistically significance when compared with thesingle drug group (P﹤0.05). The results indicated that when clofarabine wasused in combination with bortezomib in K562cells, the effects wereenforced. At the same time point, the apoptosis rate observed in thecombined group is higher than that in the single group, which indicatedsynergistic effect in promotion of apoptosis.4Changes in expression of SHIPin K562cellsWhen clofarabine were used alone and in combination with bortezomib,the expression level of SHIP increased as the concentration of drugs usedincreased. The amount of gene expression in the combined group is higherthan that in the single group and the difference was statistically significant(P<0.05).The differences of average expression level between the drug-treatedgroup and non-treated group is statistically significant (P﹤0.05) anddose-effect relationship was observed between expression level variation inK562cells and the concentration of drugs used.Conclusion:Clofarabine and bortezomib has a dose-and time-dependentantiproliferation and proapoptotic effect on K562cells. With prolongation oftime and increase of concentration of bortezomib and clofarabine, the effecthas gradually increased. Synergistic or sensitizing effect, was observed bycombining with clofarabine.Besides, clofarabine could up-regulate theexpression of SHIP gene in K562cells and bortezomib could enhance thisactivity. Clofarabine and bortezomib could inhibit the proliferation and induce the apoptosis of K562cells, and SHIP gene may play a certain role.