Objective: To explore the combined pro-apoptosis effect of HSP90 inhibitor BIIB021 and chloroquine (CQ) in chronic myeloid leukemia (CML) cells bearing T315I mutation and its mechanism.
Methods: The p210-T315I cells were divided into 4 groups by different treatment: control, BIIB021, CQ, and BIIB021 + CQ. After treated with BIIB021 or/and CQ for 24 hours, Annexin V/PI binding assay was used to detect apoptosis rates of CML cells. DAPI staining was used to observe nuclear fragmentation, and Western blot was used to detect the expression of caspase 3, PARP (apoptosis related proteins) and p62, LC3-I/II (autophagy related proteins). P210-T315I cells were inoculated subcutaneously into mice and CML mouse models were established. The mice in treatment groups were injected with BIIB021 and/or CQ while mice in control group were treated with PBS and normal saline. The tumor volume of mice was measured every 4 days, and protein level of cleaved-caspase 3 and LC3-II in tumor tissue were detected by immunohistochemistry.
Results: The results showed that BIIB021 induced apoptosis of CML cells in a dose-dependent manner ( r=0.91). CQ could enhance the apoptosis-inducing effect of BIIB021. Flow cytometry analysis results showed that the apoptosis rate of p210-T315I cells in combination group was higher than that in BIIB021 or CQ only group (P<0.05). DAPI staining showed nuclear fragmentation in combination group could be observed more obviously. Western blot analysis showed that BIIB021 could induce LC3-I to convert to LC3-II and decrease p62 protein levels (P<0.05). Moreover, the combination group had higher expression of LC3-II, p62 (P<0.05), activated PARP and activated caspase 3 than BIIB021 only group (P<0.05). Besides, experiment in vivo showed the mean tumor volume in co-treatment group was lower than that in single drug group (P<0.01). Immunohistochemistry of tumor tissue also showed the protein level of cleaved-caspase 3 and LC3-II in combined group was higher than that in BIIB021 only group.
Conclusion: HSP90 inhibitor BIIB021 induced significant apoptosis of CML cells bearing T315I both in vivo and in vitro. CQ can enhance this effect probably by autophagy inhibition.
题目: 氯喹增强BIIB021对T315I突变的慢性粒细胞白血病细胞促凋亡的研究.
目的: 探讨氯喹（CQ）联合热休克蛋白90抑制剂BIIB021对T315I突变的慢性粒细胞白血病（CML）p210-T315I细胞凋亡的作用及相关机制。.
方法: 将p210-T315I 细胞株按不同处理分为对照、BIIB021、氯喹、BIIB021+氯喹共4组。经过BIIB02和/或CQ处理24 h后，流式细胞术检测细胞凋亡，DAPI染色观察细胞核形态及凋亡情况；Western blot检测凋亡相关蛋白caspase 3、PARP，自噬相关蛋白p62、LC3-I/II的表达情况。皮下注射p210-T315I细胞，构建CML小鼠荷瘤模型，模型建立后治疗组分别给药（BIIB021和/或氯喹），对照组给PBS和生理盐水，每4 d观察记录荷瘤小鼠局部肿瘤体积，免疫组织化学法检测瘤体组织cleaved-caspase 3和LC3-II表达水平。.
结果: BIIB021有促p210-T315I细胞凋亡的作用，随着药物浓度升高，细胞凋亡率越高（r=0.91）。氯喹能增强BIIB021相关的促凋亡作 用，流式细胞术检测结果显示，联合用药组p210-T315I细胞凋亡率明显高于BIIB021、氯喹单用药组（P<0.05）。DAPI染色显示，与药物单用组比较，药物联合应用组细胞中点状浓染更多，细胞核碎裂更明显。凋亡和自噬相关蛋白检测显示，与对照组比较，BIIB021单药组细胞中LC3-I向LC3-II转变增加，p62减少（P<0.05）。当氯喹联合BIIB021时，与BIIB021单用相比，LC3-II和p62的表达量增多（P<0.05），此时PARP和caspase 3的激活带增多（P<0.05）。动物实验显示两种药物联合使用与药物单用相比，小鼠肿瘤体积明显变小（P<0.01）。免疫组织化学显示氯喹+BIIB021联用组肿瘤组织中cleaved-caspase 3、LC3-II表达量高于BIIB021单用组。.
结论: 热休克蛋白90抑制剂BIIB021对T315I突变的CML细胞、小鼠模型有促进凋亡的作用，氯喹能增强这一效应，其机制可能与抑制自噬有关。.
Keywords: BIIB021; HSP90 inhibitor; T315I mutation; chloroquine; chronic myeloid leukemia.