重组变构人肿瘤坏死因子相关凋亡诱导配体逆转慢性粒细胞白血病细胞对伊马替尼耐药的机制研究

目的探讨重组变构人肿瘤坏死因子相关凋亡诱导配体(CPT)逆转慢性粒细胞白血病(CML)细胞伊马替尼耐药的相关机制。方法选择2016年至2020年内蒙古医科大学附属医院就诊的5例CML患者,分别采集初诊和伊马替尼耐药状态下的肝素化骨髓血标本,分离单个核细胞。初诊时采集的单个核细胞依次命名为A1~E1,伊马替尼耐药后采集的单个核细胞分别命名为A2~E2。使用人CML野生型K562细胞株(K562-W),采用低浓度伊马替尼小剂量逐步加量的方法,获得伊马替尼耐药的K562细胞(K562-R)。采用20μg/L CPT培养K562-R细胞,设为CPT-K562-R细胞组。CCK-8法检测细胞对伊马替尼的半数抑制浓度(IC50)。采用K562-W、K562-R细胞构建CML移植瘤裸鼠模型,将裸鼠分为K562-W、K562-R、CPT-K562-R移植瘤组,三组均经口灌注伊马替尼,CPT-K562-R组同时皮下注射CPT;比较三组裸鼠移植瘤在伊马替尼治疗前、治疗4周后的肿瘤体积,以及三组裸鼠的存活时间。蛋白质印迹法检测CML患者骨髓单个核细胞、K562细胞株及其移植瘤组织中酪氨酸蛋白激酶受体B4(EphB4)、髓细胞白血病蛋白1(Mcl-1)蛋白水平的变化。结果5例CML患者A2~E2细胞中EphB4蛋白表达水平均较A1~E1细胞增高(均P<0.01)。K562-W、K562-R、CPT-K562-R细胞对伊马替尼的IC50分别为(0.160±0.015)mg/L、(5.450±0.460)mg/L、(0.300±0.035)mg/L,差异有统计学意义(F=390.65,P<0.01)。K562-W组细胞中EphB4、Mcl-1蛋白均呈低水平表达(0.54±0.02和0.70±0.08);K562-R组细胞中EphB4、Mcl-1蛋白表达水平升高(3.04±0.11和2.88±0.04);CPT-K562-R组细胞中EphB4、Mcl-1蛋白表达水平下降(0.57±0.03和0.38±0.04)。伊马替尼治疗前,K562-W、K562-R和CPT-K562-R移植瘤组裸鼠移植瘤体积差异无统计学意义(F=0.39,P=0.68),提示裸鼠移植瘤成瘤均衡;伊马替尼治疗结束后三组移植瘤体积差异有统计学意义(F=26.16,P<0.01)。K562-W、K562-R和CPT-K562-R移植瘤组裸鼠存活时间分别为(18.5±3.3)d、(10.0±2.4)d、(17.5±1.6)d,差异有统计学意义(F=20.45,P<0.01)。K562-W移植瘤组中EphB4、Mcl-1蛋白均呈低水平表达(0.55±0.06和0.67±0.06);K562-R移植瘤组中EphB4、Mcl-1蛋白表达水平升高(1.95±0.08和6.21±0.53);CPT-K562-R移植瘤组中EphB4、Mcl-1蛋白表达水平下降(0.59±0.04和0.37±0.04),且接近K562-W移植瘤组的水平。结论CPT可能通过抑制EphB4、Mcl-1表达,增强CML对伊马替尼的敏感性,这可能是一种伊马替尼治疗靶向通路。

Study on the mechanism of circular permuted tumor necrosis factor-related apoptosis-inducing ligand reversing the resistance to imatinib in chronic myeloid leukemia cells

Objective:To explore the mechanism of circular permuted tumor necrosis factor-related apoptosis-inducing ligand (CPT) reversing the resistance to imatinib in chronic myeloid leukemia (CML) cells.Methods:Five patients with CML in the Affiliated Hospital of Inner Mongolia Medical University from 2016 to 2020 were selected, and heparinized bone marrow blood samples were collected at the first diagnosis and imatinib resistance phase, and mononuclear cells were isolated. The mononuclear cells collected at the first diagnosis were named A1-E1, and the mononuclear cells collected after imatinib resistance were named A2-E2. Human CML wild-type K562 cell line (K562-W) was given gradually increasing small doses of low-concentration imatinib to obtain imatinib-resistant K562 cells (K562-R). K562-R cells were cultured with 20 μg/L CPT and these cells were set as CPT-K562-R group. The CCK-8 method was used to detect the half inhibitory concentration ( IC50) of cells for imatinib. K562-W and K562-R cells were used to establish CML xenografts nude mice models, then the nude mice were divided into K562-W, K562-R and CPT-K562-R xenograft groups. Imatinib was perfused orally in all three groups, and CPT was injected subcutaneously in the CPT-K562-R group at the same time. The tumor volume of the three groups of nude mice before and 4 weeks after treatment with imatinib, and the survival time of the three groups of nude mice were compared. Western blot was used to detect the changes of tyrosine protein kinase receptor B4 (EphB4) and myeloid cell leukemia protein 1 (Mcl-1) protein levels in bone marrow mononuclear cells, K562 cell line and transplanted tumor tissues of CML patients. Results:The expressions of EphB4 protein in A2-E2 cells of 5 patients with CML were higher than those in A1-E1 cells (all P < 0.01). The IC50 of K562-W, K562-R and CPT-K562-R cells for imatinib were (0.160±0.015) mg/L, (5.450±0.460) mg/L, (0.300±0.035) mg/L, and the difference was statistically significant ( F = 390.65, P < 0.01). In cells of K562-W group, EphB4 and Mcl-1 proteins were expressed at low levels (0.54±0.02 and 0.70±0.08); in cells of K562-R group, the expressions of EphB4 and Mcl-1 proteins were enhanced (3.04±0.11 and 2.88±0.04); in cells of CPT-K562-R group, the expressions of EphB4 and Mcl-1 proteins decreased (0.57±0.03 and 0.38±0.04). Before imatinib treatment, there was no statistically significant difference in the tumor volumes of nude mice among the K562-W, K562-R and CPT-K562-R xenograft groups ( F = 0.39, P = 0.68), suggesting the transplanted tumors formed in nude mice were balanced; after imatinib treatment, the difference in the tumor volumes among the three groups were statistically significant ( F = 26.16, P < 0.01). The survival time of nude mice in the K562-W, K562-R and CPT-K562-R xenograft groups was (18.5±3.3) d, (10.0±2.4) d and (17.5±1.6) d, and the difference was statistically significant ( F = 20.45, P < 0.01). In K562-W xenograft group, both EphB4 and Mcl-1 proteins were expressed at low levels (0.55±0.06 and 0.67±0.06); in K562-R xenograft group, the expressions of EphB4 and Mcl-1 proteins were enhanced (1.95±0.08 and 6.21±0.53); the expressions of EphB4 and Mcl-1 in CPT-K562-R xenograft group decreased (0.59±0.04 and 0.37±0.04) and were close to their expressions in K562-W xenograft group. Conclusion:CPT may enhance the sensitivity of CML to imatinib by inhibiting the expressions of EphB4 and Mcl-1, and this may be a targeted pathway for imatinib therapy.