李舜,杨智松,韩国征,李红艳,齐浩明
LI Shun,YANG Zhisong,HAN Guozheng,LI Hongyan,QI Haoming

• 1:大庆龙南医院
• 2:石河子大学第一附属医院中医二科
• 3:渭南市中心医院心血管内科

摘要(Abstract)：

目的:探讨阿立塞替(MLN8237)对人非小细胞肺癌放射敏感性的影响及作用机制。方法:用siPORT Neo FX转染试剂将阴性siRNA、si Aurora Kinase A分别转染NCI-H1975细胞阴性对照组(Si control)及靶向Aurora激酶A组(si Aurora Kinase A),未转染si PORT Neo FX的NCI-H1975细胞作为单纯对照组(Control);CCK8实验检测抑制Aurora激酶A的抑制剂(MLN82370)对NCI-H1975细胞生长的影响,细胞凋亡实验检测抑制Aurora激酶A及加入MLN8237对NCI-H1975细胞凋亡的影响,克隆形成实验检测了MLN8237对NCI-H1975细胞放射敏感性的影响影响,qRT-PCR实验评估了NCI-H1975细胞中p16及p21的表达,荧光素酶分析实验测定细胞周期相关转录因子(E2F)和应激活化蛋白激酶(JNK)的活性。结果:NCI-H1975细胞的转染效率达89. 91%,抑制Aurora激酶A及加入MLN8237可明显抑制NCI-H1975细胞的生长,差异有统计学意义(P <0. 01);细胞凋亡实验结果显示,抑制Aurora激酶A及加入MLN8237可明显提高NCI-H1975细胞的凋亡率;克隆形成实验显示,MLN8237能够显著降低照射后NCI-H1975细胞的克隆形成能力对NCI-H1975细胞有放射增敏效果,且随剂量增大其增敏效果越显著; qRT-PCR结果显示,加入MLN8237(Aurora激酶A抑制剂)照射后,p16及p21的表达明显上调;荧光素酶分析实验结果显示,抑制Aurora激酶A显著降低了E2F的活性,而JNK的活性显著提高,差异均有统计学意义(P <0. 01)。结论:阿立塞替能通过靶向Aurora激酶A,而抑制NCI-H1975细胞增殖及诱导其凋亡,从而提高NCI-H1975细胞的放射敏感性。
Objective: To investigate the effect of alisertib on the radiosensitivity of human non-small cell lung cancer by targeting Aurora kinase A. Methods: NCI-H1975 cells were transfected with si PORT NeoFX transfection reagent and divided into Si control group,Si Aurora Kinase A group; NciH1975 cells that were not transfected with si PORT NeoFX were used as the control group. The effects of inhibitors that inhibit Aurora kinase A( MLN82370) on NCI-H1975 cells growth were detected by CCK8 assay; the effects of Aurora kinase A inhibition and the addition of MLN8237 on the apoptosis of NCI-H1975 cells were detected by apoptosis assay; the effects of MLN8237 on radiosensitivity of NCIH1975 cells were examined by cloning formation experiment; the expression of p16 and p21 in NCIH1975 cells was evaluated by qRT-PCR; the activities of E2 F and JNK were determined by luciferase assay. Results: The transfection efficiency of NCI-H1975 cells was 89. 91%; CCK8 results showed the growth of NCI-H1975 cells were significantly inhibited by Aurora kinase A or adding the MLN8237,the difference was statistically significant( P < 0. 01). The results of apoptosis assay showed the apoptosis of NCI-H1975 cells were significantly increased by inhibiting of Aurora kinase A or adding MLN8237; The cloning formation experiments showed the cloning forming ability of NCIH1975 cells were significantly reduced with MLN8237 after irradiation,the radiosensitivity of NCIH1975 cells were significantly increased with MLN8237; The qRT-PCR results showed the expression of p16 and p21 was significantly up-regulated after IR with MLN8237( Aurora kinase A inhibitor);The results of Luciferase analysis showed that the activity of E2 F was significantly decreased while the activity of JNK was significantly increased by inhibition of Aurora kinase A( P < 0. 01). Conclusion:Alisertib can inhibit the proliferation and induce apoptosis of NCI-H1975 cells by targeting Aurora kinase A,thereby increasing the radiosensitivity of NCI-H1975 cells.

关键词(KeyWords)： 癌,非小细胞肺;转染;辐射耐受性;细胞凋亡;细胞增殖;阿立塞替
carcinoma,non-small-cell lung(NSCLC);transfection;radiation tolerance;apoptosis;cell proliferation;alisertib

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31560270)

作者(Author): 李舜,杨智松,韩国征,李红艳,齐浩明
LI Shun,YANG Zhisong,HAN Guozheng,LI Hongyan,QI Haoming

DOI: 10.19367/j.cnki.2096-8388.2020.05.006

参考文献(References)：

• [1]TORRE L A,FREDDIE B,SIEGEL R L,et al. Global cancer statistics,2012[J]. Ca A Cancer Journal for Clinicians,2015,65(2):87-108.
• [2]TAN W L,JAIN A,TAKANO A,et al. Novel therapeutic targets on the horizon for lung cancer[J]. Lancet Oncology,2016,17(8):e347-e362.
• [3] ENGSTROM P F,JUAN PABLO A,BENSON A B,et al. NCCN Clinical practice guidelines in oncology:colon cancer[J]. J Natl Compr Canc Netw,2009,4(8):350-391.
• [4]TAN D S W,YOM S S,MING S T,et al. The international association for the study of lung cancer consensus statement on optimizing management of egfr mutation-positive non-small cell lung cancer:status in 2016[J]. Journal of Thoracic Oncology,2016,11(7):946-963.
• [5]EL-SHEIKH A,FAN R,BIRKS D,et al. Inhibition of aurora kinase a enhances chemosensitivity of medulloblastoma cell lines[J]. Pediatric Blood&Cancer,2010,55(1):35-41.
• [6]ZHAO L,ZHANG Y,ZHOU J,et al. Advances in research on drugs targeting non-small cell lung cancer[J].Journal of China Pharmaceutical University,2014,45(2):136-144.
• [7]NOY A,DE VOS S,THIEBLEMONT C,et al. Targeting Bruton tyrosine kinase with ibrutinib in relapsed/refractory marginal zone lymphoma[J]. Blood,2017,129(16):2224-32.
• [8]BARTON V N,FOREMAN N K,DONSON A M,et al.Aurora kinase A as a rational target for therapy in glioblastoma[J]. Journal of Neurosurgery Pediatrics,2010,6(1):98.
• [9]WANG R,WANG J H,CHU X Y,et al. Expression of STK15 mRNA in hepatocellular carcinoma and its prognostic significance[J]. Clinical Biochemistry,2009,42(7):641-647.
• [10]YOSHIFUMI B,KATSUHIKO N,KAORI S,et al. Aurora-A expression is independently associated with chromosomal instability in colorectal cancer[J]. Neoplasia,2009,11(5):418-425.
• [11]OGAWA E,TAKENAKA K,KATAKURA H,et al.Perimembrane aurora-A expression is a significant prognostic factor in correlation with proliferative activity in non-small-cell lung cancer(NSCLC)[J]. Annals of Surgical Oncology,2008,15(2):547-554.
• [12]MILAM M R,GU J,YANG H,et al. STK15 F31I polymorphism is associated with increased uterine cancer risk:A pilot study[J]. Gynecologic Oncology,2007,107(1):71-74.
• [13]SHANG-BIN Y,XIAO-BO Z,HONG-XIA Z,et al. Amplification and overexpression of Aurora-A in esophageal squamous cell carcinoma[J]. Oncology Reports,2007,17(5):1083-1088.
• [14]ASHTON S,SONG Y H,NOLAN J,et al. Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo[J]. Science Translational Medicine,2016,8(325):325ra17-ra17.
• [15]LEE S,CIMICA V,RAMACHANDRA N,et al. Aurora A is a repressed effector target of the chromatin remodeling protein INI1/h SNF5 required for rhabdoid tumor cell survival[J]. Cancer Research,2011,71(9):3225-3235.
• [16]MIGNOGNA C,STAROPOLI N,BOTTA C,et al. Aurora Kinase A expression predicts platinum-resistance and adverse outcome in high-grade serous ovarian carcinoma patients[J]. Journal of Ovarian Research,2016,9(1):31.
• [17]LYKKESFELDT A E,IVERSEN B R,JENSEN M B,et al. Aurora kinase A as a possible marker for endocrine resistance in early estrogen receptor positive breast cancer[J]. Acta Oncologica,2017,57(1):1-7.
• [18]TEKATLI H,DUIJM M,OOMEN-DE H E,et al. Normal tissue complication probability modeling of pulmonary toxicity after stereotactic and hypofractionated radiation therapy for central lung tumors[J]. Int J Radiat Oncol Biol Phys,2018,100(3):738-747.
• [19]GYULAVRI P,SZOKOL B,SZABADKAI I,et al.Discovery and optimization of novel benzothiophene-3-carboxamides as highly potent inhibitors of Aurora kinases A and B[J]. Bioorganic&Medicinal Chemistry Letters,2018,28(19):3265-3270.
• [20]HARRINGTON E A,BEBBINGTON D,MOORE J,et al. VX-680,a potent and selective small-molecule inhibitor of the Aurora kinases,suppresses tumor growth in vivo[J]. Nat Med,2004,10(3):262-267.
• [21]ALRIFAI D,PETTENGELL R. MLN8237(alisertib)and its role in peripheral T-cell lymphoma[J]. Expert opinion on investigational drugs,2014,23(12):1731-1736.
• [22]KILINC K,DEMIR S,TURAN I,et al. Rosa canina extract has antiproliferative and proapoptotic effects on human lung and prostate cancer cells[J]. Nutrition and cancer,2019,1-10.
• [23]VENKATARAMAN S,ALIMOVA I,TELLO T,et al.Targeting aurora kinase a enhances radiation sensitivity of atypical teratoid rhabdoid tumor cells[J]. J Neurooncol,2012,107(3):517-526.

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