索拉非尼联合柔红霉素对白血病K562细胞的抑制作用

Combination of sorafenib and daunorubicin has antileukemic activity on K562 cells

目的:探讨小分子Raf激酶抑制剂索拉非尼(sorafenib)联合柔红霉素(DNR)对白血病细胞K562及U937的抑制作用及可能的分子机制。方法:MTT法测定索拉非尼和柔红霉素单独作用于K562和U937细胞的抑制率及DNR IC10联合不同浓度索拉非尼作用于K562及U937的联合抑制率;流式细胞AnnexinⅤ/PI法测定单药及联合用药后K562细胞的凋亡率以及Hoechst33258染色法观察单药及联合作用后细胞凋亡形态的改变;West-ern blotting法测定索拉非尼、DNR及U0126对K562及U937p-ERK1/2的影响;根据金氏方程证明2种药物联合抑制率及凋亡是否有协同作用。结果:MTT法测定索拉非尼联合DNR对K562及U937均有协同抑制作用(q>1.15,P<0.01);流式细胞AnnexinⅤ/PI和Hoechst33258染色法,均证明索拉非尼联合柔红霉素能联合诱导K562细胞凋亡(q>1.15,P<0.05),两者有明显的一致性;K562细胞的基础pERK1/2蛋白水平明显高于U937细胞(P<0.01),索拉非尼和U0126都能够显著抑制K562细胞p-ERK1/2水平;U0126联合DNR存在协同抑制K562细胞的作用。结论:索拉非尼联合DNR作用于白血病细胞K562、U937存在协同抑制及凋亡诱导作用;DNR对U937的抑制作用明显高于K562;索拉非尼对K562的敏感性高于U937细胞;索拉非尼可能通过下调p-ERK1/2水平增加柔红霉素抗白血病细胞的效应。

AIM ： To investigate the synergetic inhibitory effect of sorafenib and daunorubicin （DNR） on K562 and U937 cells. METHODS： The inhibitory rate of sorafenib or daunorubicin alone, and the combined inhibitory rate of sorafenib and IC10 daunorubicin were measured by MTT assay. Apoptotic rate of single drug or combination was assessed by flow cytometry （Annexin V/PI staining） and Hoechst 33258 staining assay, p -ERK1/2 level was detected by Western blotting after the cells were treated with sorafenib, daunorubicin and U0126 or combinations. Synergistic or antagonistic effect of proliferation and apoptosis on K562 and U937 was estimated according to the Jin＇s Method. RESULTS ： Combination of sorafenib and DNR showed synergistic growth inhibition （q 〉 1.15, P 〈0. 01 ） and synergistic promotion of apoptosis （q 〉 1.15, P 〈 0. 05） in K562 and U937 cells. The level of p- ERK1/2 in K562 cells was obviously higher than that in U937 cells （P 〈0. 01 ）. p- ERK1/2 expression was completely inhibited in sorafenib or U0126 treated K562 ceils for 24 h. Combination of U0126 with DNR inhibited the proliferation of K562 cells synergistically. CONCLUSION： Combination of sorafenib with DNR showed synergistic cell growth inhibition and promotion of apoptosis in K562 and U937 cells. U937 cells were more sensitive to DNR than K562 cells while K562 cells were more sensitive to sorafenib. Sorafenib enhances the anti - leukemic activity of DNR in K562 and U937 cells via down - regulation of p - ERK1/2 expression.