硼替佐米靶向NF-κB信号通路抑制结外鼻型NK/T细胞淋巴瘤的研究
Bortezomib Inhibits Extranodal Natural Killer/T Cell Lymphoma, Nasal Type by Targeting NF-κB Signaling Pathway
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摘要:目的 研究硼替佐米对结外鼻型NK/T细胞淋巴瘤(ENKTL)的抗肿瘤作用。方法 单独使用不同质量浓度(0、1、2、4、5、6 ng/mL)硼替佐米处理SNK-6细胞24、48、72 h,及不同浓度核因子-κB (NF-κB)信号通路抑制剂BAY11-7082(0、1、2.5、5、10、20 μmol/L)处理SNK-6细胞24 h后,采用CCK8法检测细胞存活率并计算其半数抑制浓度(IC50)。联合使用30 μmol/L Z-VAD-FMK(Pan-caspase抑制剂)+3 ng/mL硼替佐米,以及5、10 μmol/L BAY11-7082+3 ng/mL硼替佐米处理SNK-6细胞24 h,CCK8法检测细胞存活率。不同质量浓度硼替佐米处理SNK-6细胞24 h后,采用Annexin Ⅴ/PI流式细胞术检测细胞凋亡;Western blot检测凋亡相关蛋白Caspase-3、多聚ADP核糖聚合酶(PARP)和Bcl-2的表达,检测NF-κB信号通路相关蛋白P65和P100/52的表达。结果 硼替佐米可呈剂量依赖性的抑制SNK-6细胞增殖(P<0.05),24 h IC50﹝(2.87±0.06) ng/mL﹞低于48 h和72 h(P<0.05)。BAY11-7082亦可抑制SNK-6细胞增殖,24 h IC50= (9.73±0.36) μmol/L。联合用药结果表明,Z-VAD-FMK能减弱硼替佐米对SNK-6细胞增殖的抑制作用(P<0.05),BAY11-7082能增强硼替佐米对SNK-6细胞增殖的抑制作用(P<0.05)。硼替佐米处理SNK-6细胞24 h后,凋亡相关蛋白Caspase-3裂解、PARP激活,以及Bcl-2裂解;NF-κB信号通路相关蛋白P65磷酸化水平降低,P52减少。结论 硼替佐米通过阻断NF-κB信号通路抑制ENKTL细胞增殖,并且经线粒体介导的Caspase途径诱导ENKTL细胞凋亡。Abstract:Objective To investigate the anti-tumor effect of bortezomib on extranodal natural killer/T cell lymphoma, nasal type (ENKTL).Methods SNK-6 cells were treated with different mass concentrations of bortezomib (0, 1, 2, 4, 5, 6 ng/mL) for 24, 48, 72 h, and different concentrations of nuclear factor-kappa B (NF-κB) signaling pathway inhibitor BAY11-7082 (0, 1, 2, 2.5, 5, 10, 20 μmol/L) for 24 h respectively, then the cell viability was measured by CCK8 kit and the half inhibitory concentration (IC50) was calculated. SNK-6 cells were treated with 30μmol/L Z-VAD-FMK (Pan-caspase inhibitor)+3ng/mL bortezomib, and 5, 10 μmol/L BAY11-7082+3 ng/mL bortezomib for 24 h respectively, then the cell viability was measured by CCK8 kit. After treatment of SNK-6 cells with different mass concentrations of bortezomib for 24 h, apoptosis was detected by AnnexinⅤ/PI flow cytometry; the expression of apoptosis-related protein Caspase-3, poly ADP-ribose polymerase (PARP) and Bcl-2 and NF-κB signaling pathway key proteins P65 and P100/P52 were detected by Western blot.Results Bortezomib inhibited the proliferation of SNK-6 cells in a dose-dependent manner (P<0.05), and IC50﹝(2.87±0.06) ng/mL﹞at 24 h was lower than that at 48 h and 72 h (P<0.05). BAY11-7082 also inhibited the proliferation of SNK-6 cells with an IC50= (9.73±0.36) μmol/L at 24 h. The combination treatment indicated that Z-VAD-FMK could attenuate the inhibitory effect of bortezomib on the proliferation of SNK-6 cells (P<0.05), while BAY11-7082 could enhance the inhibitory effect of bortezomib on the proliferation of SNK-6 cells (P<0.05). After treatment of SNK-6 cells with bortezomib for 24 h, apoptosis-related protein Caspase-3 cleavage, PARP activation, and Bcl-2 cleavage; NF-κB signaling pathway-related protein P65 phosphorylation level decreased, and P52 decreased.Conclusion Bortezomib inhibits ENKTL cells proliferation by inhibiting NF-κB signaling pathway and induces apoptosis of ENKTL cells via mitochondria-mediated caspase pathway.
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