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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。
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关键词:
- 结外鼻型NK/T细胞淋巴瘤（ENKTL） /
- 硼替佐米 /
- 凋亡 /
- NF-κB信号通路 /
- BAY11-7082
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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图 3 线粒体介导的Caspase途径凋亡相关蛋白表达（A）与硼替佐米联合Z-VAD-FMK对SNK-6细胞增殖（B）的影响
Figure 3. Effect of mitochondria-mediated Caspase pathway apoptosis-related protein（A） and bortezomib combined with Z-VAD-FMK on proliferation of SNK-6 cells（B）
A：Western blot；B：CCK8 kit﹝ a: Saline （control group）; b: 30 μmol/L Z-VAD-FMK; c: 3 ng/mL bortezomib ; d: 30 μmol/L Z-VAD-FMK+3 ng/mL bortezomib﹞
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图 4 NF-κB信号通路蛋白表达（A）与BAY11-7082单独处理（B）及联合硼替佐米处理（C）对SNK-6细胞增殖的影响
Figure 4. Effect of NF-κB signaling pathway protein（A） and single BAY11-7082（B） and BAY11-7082 combined with Bortezomib（C） on proliferation of SNK-6 cells
A：Western blot; B: 0，1，2.5，5，10，20 μmol/L BAY11-7082 treatment of SNK-6 cells 24 h respectively，CCK8 kit detect cell viability（n=3，*P<0.01，**P<0.001，vs. former concentration）; C: Saline （control group，a），5 μmol/L BAY11-7082 （b），10 μmol/L BAY11-7082 （c），3 ng/mL bortezomib （d），5 μmol/L BAY11-7082+3 ng/mL bortezomib （e），10 μmol/L BAY11-7082+3 ng/mL bortezomib （f） treatment of SNK-6 cells 24 h respectively，CCK8 kit detect cell viability （*P<0.01）
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