Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy.
Haematologica. 2012 Nov;97(11):1722-30. doi: 10.3324/haematol.2012.062083. Epub 2012 Jun 11.
Both the multi-kinase inhibitor sorafenib and the small molecule inhibitor of the MDM2/p53 interaction, nutlin-3, used alone, have shown promising anti-leukemic activity in acute myeloid leukemia cells. Thus, in this study we investigated the effect of the combination of sorafenib plus nutlin-3 in acute myeloid leukemia.
Primary acute myeloid leukemia blasts (n=13) and FLT3(wild-type)/p53(wild-type) (OCI-AML3), FLT3(mutated)/p53(wild-type) (MOLM), FLT3(mutated)/p53(mutated) (MV4-11), FLT3(wild-type)/p53(deleted) (HL60) or FLT3(wild-type)/p53(mutated) (NB4) acute myeloid cell lines were exposed to sorafenib, used alone or in association with nutlin-3 at a 1:1 ratio, in a range of clinically achievable concentrations (1-10 μM). Induction of apoptosis and autophagy was evaluated by transmission electron microscopy and by specific flow cytometry analyses. The levels of Mcl-1, p53 and Bak proteins were analyzed by western blotting. Knock-down of Bax and Bak gene expression was performed in transfection experiments with specific short interfering RNA.
The sorafenib+nutlin-3 drug combination exhibits synergistic cytotoxicity in primary acute myeloid leukemia blasts and in acute myeloid leukemia cell lines with maximal cytotoxicity in FLT3(mutated) MV4-11 and MOLM, followed by the FLT3(wild-type) OCI-AML3, HL60 and NB4 cell lines. The cytotoxic activity of sorafenib+nutlin-3 was characterized by an increase of both apoptosis and autophagy. Moreover, Bax and Bak showed prominent roles in mediating the decrease of cell viability in response to the drug combination in p53(wild-type) OCI-AML3 and p53(deleted) HL-60 cells, respectively, as demonstrated in transfection experiments performed with specific short interfering RNA.
Our data demonstrate that acute myeloid leukemia cells show a variable but overall good susceptibility to the innovative therapeutic combination of sorafenib+nutlin-3, which differentially involves the pro-apoptotic Bcl-2 family members Bax and Bak in p53(wild-type) and p53(deleted) cells.
多激酶抑制剂索拉非尼和 MDM2/p53 相互作用的小分子抑制剂 nutlin-3 单独使用时，在急性髓系白血病细胞中均显示出有前景的抗白血病活性。因此，在这项研究中，我们研究了索拉非尼加 nutlin-3 联合治疗急性髓系白血病的效果。
对 13 例急性髓系白血病原始细胞和 FLT3（野生型）/p53（野生型）（OCI-AML3）、FLT3（突变型）/p53（野生型）（MOLM）、FLT3（突变型）/p53（突变型）（MV4-11）、FLT3（野生型）/p53（缺失）（HL60）或 FLT3（野生型）/p53（突变型）（NB4）急性髓细胞系进行检测，这些细胞暴露于索拉非尼中，单独或按 1:1 比例与 nutlin-3 联合使用，范围为临床可达到的浓度（1-10 μM）。通过透射电子显微镜和特定的流式细胞术分析评估细胞凋亡和自噬的诱导。通过蛋白质印迹分析 Mcl-1、p53 和 Bak 蛋白的水平。通过特定的短干扰 RNA 转染实验进行 Bax 和 Bak 基因表达的敲低。
索拉非尼+nutlin-3 药物联合在急性髓系白血病原始细胞和急性髓系白血病细胞系中表现出协同细胞毒性，在 FLT3（突变型）MV4-11 和 MOLM 中表现出最大的细胞毒性，其次是 FLT3（野生型）OCI-AML3、HL60 和 NB4 细胞系。索拉非尼+nutlin-3 的细胞毒性活性表现为细胞凋亡和自噬的增加。此外，Bax 和 Bak 在 p53（野生型）OCI-AML3 和 p53（缺失）HL-60 细胞中分别作为介导细胞活力对药物联合反应下降的重要作用，这在特定的短发夹 RNA 转染实验中得到了证明。
我们的数据表明，急性髓系白血病细胞对索拉非尼+nutlin-3 的创新治疗组合表现出不同但总体较好的敏感性，该组合在 p53（野生型）和 p53（缺失）细胞中差异地涉及促凋亡 Bcl-2 家族成员 Bax 和 Bak。
