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
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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、20μmol/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(IC_(50))。联合使用30μmol/L Z-VAD-FMK(Pan-caspase抑制剂)+3 ng/mL硼替佐米,以及5、10μmol/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AnnexinⅤ/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/P52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 IC_(50)[(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 IC_(50)为(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。
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(IC_(50)) was calculated. SNK-6 cells were treated with 30 μmol/L Z-VAD-FMK(Pan-caspase inhibitor)+3 ng/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 IC_(50)[(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 IC_(50)=(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.
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(IC_(50)) was calculated. SNK-6 cells were treated with 30 μmol/L Z-VAD-FMK(Pan-caspase inhibitor)+3 ng/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 IC_(50)[(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 IC_(50)=(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.
引文
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[14] LEONARD JP,FURMAN RR,COLEMAN M.Proteasome inhibition with bortezomib:a new therapeutic strategy for non-Hodgkin′s lymphoma.Int J Cancer,2006,119(5):971-979.
[15] HIDESHIMA T,IKEDA H,CHAUHAN D,et al.Bortezomib induces canonical nuclear factor-κB activation in multiple myeloma cells.Blood,2009,114(5):1046-1052.
[16] ZOU P,KAWADA J,PESNICAK L,et al.Bortezomib induces apoptosis of epstein-barr virus (EBV)-transformed B cells and prolongs survival of mice inoculated with EBV-transformed B cells.J Virol,2007,81(18):10029-10036.
[17] MORI N,YAMADA Y,IKEDA S,et al.Bay 11-7082 inhibits transcription factor NF-κB and induces apoptosis of HTLV-Ⅰ-infected T-cell lines and primary adult T-cell leukemia cells.Blood,2002,100(5):1828-1834.
[2] SUN J,YANG Q,LU Z,et al.Distribution of lymphoid neoplasms in China analysis of 4638 cases according to the World Health Organization classification.Am J Clin Pathol,2012,138(3):429-434.
[3] SUZUKI R.NK/T cell lymphoma:updates in therapy.Curr Hematol Malig Rep,2018,13(1):7-12.
[4] BONIZZI G,KARIN M.The two NF-κB activation pathways and their role in innateand adaptive immunity.Trends Immunol,2004,25(6):280-288.
[5] MITCHELL S,VARGAS J,HOFFMANN A.Signaling via the NF-κB system.Wiley Interdiscip Rev Syst Biol Med,2016,8(3):227-241.
[6] HUANG Y,DE REYNIéS A,DE LEVAL L,et al.Gene expression profiling identifiesemerging oncogenic pathways operating in extranodal NK/T-cell lymphoma,nasaltype.Blood,2010,115(6):1226-1237.
[7] LIU X,WANG B,MA X,et al.NF-κB Activation through the alternative pathway correlates with chemoresistance and poor survival in extra-nodal NK/T-cell lymphoma,Nasal type.Jpn J Clin Oncol,2009,39(7):418-424.
[8] MUJTABA T,DOU QP.Advances in the understanding of mechanisms and therapeutic use of bortezomib.Discov Med,2011,12(67):471-480.
[9] SORS A,JEAN-LOUIS F,PELLET C,et al.Down-regulating constitutive activation of the NF-κB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis.Blood,2006,107(6):2354-2363.
[10] PHAM LV,TAMAYO AT,YOSHIMURA LC,et al.Inhibition of constitutive NF-κB activation in mantle cell lymphoma B cells leads to induction of cell cycle arrest and apoptosis.J Immunol,2003,171(1):88-95.
[11] SHEN L,AU WY,GUO T,et al.Proteasome inhibitor bortezomib-induced apoptosis in natural killer (NK)-cell leukemia and lymphoma:an in vitro and in vivo preclinical evaluation.Blood,2007,110(1):469-470.
[12] NAGATA H,KONNO A,KIMURA N,et al.Characterization of novel natural killer (NK)-cell and γδ T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus.Blood,2001,97(3):708-713.
[13] BONVINI P,ZORZI E,BASSO G,et al.Bortezomib-mediated 26S proteasome inhibition causes cell-cycle arrest and induces apoptosis in CD-30+ an aplastic large cell lymphoma.Leukemia,2007,21(40):838-842.
[14] LEONARD JP,FURMAN RR,COLEMAN M.Proteasome inhibition with bortezomib:a new therapeutic strategy for non-Hodgkin′s lymphoma.Int J Cancer,2006,119(5):971-979.
[15] HIDESHIMA T,IKEDA H,CHAUHAN D,et al.Bortezomib induces canonical nuclear factor-κB activation in multiple myeloma cells.Blood,2009,114(5):1046-1052.
[16] ZOU P,KAWADA J,PESNICAK L,et al.Bortezomib induces apoptosis of epstein-barr virus (EBV)-transformed B cells and prolongs survival of mice inoculated with EBV-transformed B cells.J Virol,2007,81(18):10029-10036.
[17] MORI N,YAMADA Y,IKEDA S,et al.Bay 11-7082 inhibits transcription factor NF-κB and induces apoptosis of HTLV-Ⅰ-infected T-cell lines and primary adult T-cell leukemia cells.Blood,2002,100(5):1828-1834.