| Objective:This study choose the leukemia cell line(K562R)which is resistant to Imatinib as research object,to explore the proliferation and apoptosis effect of Sorafenib on K562 R cells,Futher more,to discuss the expression of the RAF/MEK/ERK signal pathway in this process,so that,to provide experimental basis for clinical treatment to chronic myeloid leukemia patients with Imatinib drug-resistant.Methods:We cultured K562 R cells in the system: 1640 basic medium,10% fetal bovine serum,and 1% penicillin streptomycin,with 37 degrees Celsius incubator containing 5%CO2,we passage once every 2-3 days,we use the cells grew well in the next experiment.K562 R cells are divided into different groups according to drug concentration,every group was cultured for 48 hours.Next we measure the inhibitory status caused by Sorafenib,Imatinib,Sorafenib combined with Imatinib via CCK-8.And calculate the IC50 value of Imatinib and Sorafenib,find out the most synergistic concentration of the two drugs.Then According to these concentration results,four groups are reclassified,including control group,sorafenib treatment group,imatinib treatment group,Suolafeini and imatinib treatment group.Annexin V/ 7-AAD double labeling are picked to detect the changes of K562 R cell apoptosis rate measured by flow cytometry.The protein changes,A Raf 、B Raf、C Raf、MEK1+MEK2、p-MEK1+MEK2、ERK1+ERK2、p-ERK1+ERK2,contain in RAF/MEK/ERK signal way are tested by Western blot.Results:1.The results of the CCK-8shows that the proliferation of K562 R cells were significantly inhibited by sorafenib of 5 μmol/ L-100 μmol/ L and imatinib of 10 μmol/L-100 μmol/L(P<0.05),in a dose-dependent manner.sorafenib’s IC 50 for K562 R cells is32.67 μmoll,with a 95 % confidence range of 28.59-37.34 μmol/L and imatinib’s IC 50 is 16.91 μmol/L,with a 95 % confidence range of 9.73-29.40μmol/L.Sorafenib and imatinib had synergistic effects on inhibiting proliferation of K562 R cells.The optimal synergistic concentration is sorafenib 10 μmol/L +imatinib 10 μmol/L.2.Sorafenib or imatinib can promote apoptosis of K562 R cells alone,and the apoptosis rate of K562 R cells,caused by sorafenib and imatinib is significantly increased compared with that of individual drug use(P<0.05).3.The Western blot assay shows that the mean concentrations of A RAF,B RAF,C RAF,MEK1 + MEK2,p-MEK1 +MEK2 decreased with sorafenib group and imatinib group respectively in comparison with the control group,the reduction in sorafenib plus imatinib combined group is more significant than the other groups.But,both of sorafenib and imatinib have no significant effect on the protein expression of ERK1 + ERK2,imatinib can reduce the expression of P-ERK1+ERK2,However sorafenib is able to increase the expression of P-ERK1 + ERK2.Conclusions:1.Sorafenib and imatinib are able to inhibit the proliferation of K562 R,a drug-resistant cell line of leukaemia,in a dose-dependent manner and promote its apoptosis;2.The effect of sorafenib and imatinib on the proliferation inhibition and the apoptosis promotion of K562 R cells is more obvious than that of using imatinib alone,namely sorafenib can improve the sensitivity of imatinib,used for K562 R cells;3.Sorafenib and imatinib can decrease the average protein concentration expression of A RAF,B RAF,C RAF,MEK1+MEK2,P-MEK1+MEK2 of the RAF/MEK/ERK signaling pathway in K562 R cells.and the two drug combination reduced that protein expression is more obvious,but both of them has little effect on the average protein concentration expression of ERK1+ERK2 in the pathway,at the same time,sorafenib enlarges the expression amount of P-ERK1+ERK2.The mechanism may associated with sorafenib’s negative feedbacks on this pathway,thus activating P-ERK1+ERK2reflectively. |
