新型SMO抑制剂SI-4650对人神经胶质瘤U87MG细胞增殖、凋亡和自噬能力的影响
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摘要
目的研究新型精胺氧化酶(spermine oxidase, SMO)小分子抑制剂SI-4650对人胶质瘤U87MG细胞增殖、凋亡和自噬的影响及其分子机制。方法 MTT法检测细胞增殖情况;化学发光法检测SMO和乙酰多胺氧化酶(N~1-acetylpolyamine oxidase, APAO)的酶活性;高效液相色谱法(HPLC)检测细胞内多胺含量;Transwell法分析U87MG细胞的迁移能力;PI单染后结合流式细胞术分析细胞周期;PI/FITC-Annexin V双染后结合流式细胞术、Western blot分析细胞凋亡;激光共聚焦显微镜(LSCM)和Western blot分析细胞自噬。结果 SI-4650可明显抑制U87MG细胞内SMO和APAO的酶活性,干扰多胺代谢,并降低U87MG细胞内总多胺含量。用SI-4650处理能抑制U87MG细胞的增殖和迁移能力,使细胞发生G_0/G_1周期阻滞,并诱导U87MG细胞发生凋亡和自噬性死亡。结论 SI-4650具有杀伤神经胶质瘤U87MG细胞的药理活性,其机制可能与干扰多胺代谢和诱导细胞凋亡和自噬相关。
Aim To investigate the effects of a novel spermine oxidase(SMO) inhibitor SI-4650 on proliferation, apoptosis and autophagy of human glioma U87 MG cells as well as its possible mechanism.Methods MTT assay was used to detect cell proliferation. The chemiluminescence assay was used to determine enzyme activities of SMO and N~1-acetylpolyamine oxidase(APAO). High performance liquid chromatography(HPLC) was performed to detect cellular polyamine content. Transwell assay was used to evaluate the migration ability of U87 MG cells. PI single-staining/flow cytometry(FCM) were used to analyze cell cycle. PI/FITC-Annexin V double-staining/FCM and Western blot were used to analyze apoptosis. Confocal laser scanning microscopy(LSCM) and Western blot were used to analyze autophagy.Results SI-4650 could significantly inhibit the activities of SMO and APAO, interfere with polyamine metabolism and reduce the content of total polyamine in U87 MG cells. Treatment of U87 MG cells with SI-4650 inhibited the proliferation and migration, caused G_0/G_1 cell cycle arrest and induced apoptotic and autophagic cell death.Conclusions SI-4650 has the pharmacological activity of killing glioma U87 MG cells, and its mechanism may be related to the interference of polyamine metabolism and induction of cell apoptosis and autophagy.
Aim To investigate the effects of a novel spermine oxidase(SMO) inhibitor SI-4650 on proliferation, apoptosis and autophagy of human glioma U87 MG cells as well as its possible mechanism.Methods MTT assay was used to detect cell proliferation. The chemiluminescence assay was used to determine enzyme activities of SMO and N~1-acetylpolyamine oxidase(APAO). High performance liquid chromatography(HPLC) was performed to detect cellular polyamine content. Transwell assay was used to evaluate the migration ability of U87 MG cells. PI single-staining/flow cytometry(FCM) were used to analyze cell cycle. PI/FITC-Annexin V double-staining/FCM and Western blot were used to analyze apoptosis. Confocal laser scanning microscopy(LSCM) and Western blot were used to analyze autophagy.Results SI-4650 could significantly inhibit the activities of SMO and APAO, interfere with polyamine metabolism and reduce the content of total polyamine in U87 MG cells. Treatment of U87 MG cells with SI-4650 inhibited the proliferation and migration, caused G_0/G_1 cell cycle arrest and induced apoptotic and autophagic cell death.Conclusions SI-4650 has the pharmacological activity of killing glioma U87 MG cells, and its mechanism may be related to the interference of polyamine metabolism and induction of cell apoptosis and autophagy.
引文
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[12] 刘芳,周小江,陈乃宏. Cdk5介导的凋亡与自噬交互作用对脑缺血的调控[J]. 中国药理学通报, 2018, 34(4): 463-6.
[12] Liu F, Zhou X J, Chen N H. Regulation on intercross effect between Cdk5 mediated apoptosis and autophagy after cerebral ischemia[J]. Chin Pharmacol Bull, 2018, 34(4): 463-6.
[13] Choi A M, Ryter S W, Levine B. Autophagy in human health and disease[J]. N Engl J Med, 2013, 368(19):1845-6.
[14] Chen H Y, Huang T C, Shieh T M, et al. Isoliquiritigenin induces autophagy and inhibits ovarian cancer cell growth[J]. Int J Mol Sci, 2017, 18(10): 2025.
[15] Li C J, Liao W T, Wu M Y, et al. New insights into the role of autophagy in tumor immune microenvironment[J]. Int J Mol Sci, 2017, 18(7): 1566.
[2] Bachrach U. Naturally occurring polyamines: interaction with macromolecules[J]. Curr Protein Pept Sci, 2005, 6(6):559-66.
[3] Miller-Fleming L, Olin-Sandoval V, Campbell K, et al. Remaining mysteries of molecular biology: the role of polyamines in the cell[J]. J Mol Biol, 2015, 427(21): 3389-406.
[4] Battaglia V, DeStefano Shields C, Murray-Stewart T, et al. Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention[J]. Amino Acids, 2014, 46(3):511-9.
[5] Murray-Stewart T R, Woster P M, Casero R A. Targeting polyamine metabolism for cancer therapy and prevention[J]. Biochem J, 2016, 473(19): 2937-53.
[6] Cervelli M, Salvi D, Polticelli F, et al. Structure-function relationships in the evolutionary framework of spermine oxidase[J]. J Mol Evol, 2013, 76(6):365-70.
[7] Cervelli M, Amendola R, Polticelli F, et al. Spermine oxidase: ten years after[J]. Amino Acids, 2012, 42(2-3): 441-50.
[8] Goodwin A C, Murray-Stewart T R, Casero R A. A simple assay for mammalian spermine oxidase: A polyamine catabolic enzyme implicated in drug response and disease[J]. Methods Mol Biol, 2011, 720:173-81.
[9] Dai F, Yu W, Song J, et al. Extracellular polyamines-induced proliferation and migration of cancer cells by ODC, SSAT, and Akt1-mediated pathway[J]. Anticancer Drugs, 2017, 28(4): 457-64.
[10] Abdulhussein A A, Wallace H M. Polyamines and membrane transporters[J]. Amino Acids, 2014, 46(3): 655-60.
[11] 唐琪,布文奂,王丹丹,等. 自噬与凋亡的相互作用及其对肿瘤发展过程影响的研究进展[J]. 吉林大学学报(医学版), 2015, 41(6):1303-6.
[11] Tang Q, Bu W H, Wang D D, et al. Advance research on interaction between autophagy and apoptosis and its influence in development of tumors[J]. J Jilin Univ(Med Sci Ed), 2015, 41(6): 1303-6.
[12] 刘芳,周小江,陈乃宏. Cdk5介导的凋亡与自噬交互作用对脑缺血的调控[J]. 中国药理学通报, 2018, 34(4): 463-6.
[12] Liu F, Zhou X J, Chen N H. Regulation on intercross effect between Cdk5 mediated apoptosis and autophagy after cerebral ischemia[J]. Chin Pharmacol Bull, 2018, 34(4): 463-6.
[13] Choi A M, Ryter S W, Levine B. Autophagy in human health and disease[J]. N Engl J Med, 2013, 368(19):1845-6.
[14] Chen H Y, Huang T C, Shieh T M, et al. Isoliquiritigenin induces autophagy and inhibits ovarian cancer cell growth[J]. Int J Mol Sci, 2017, 18(10): 2025.
[15] Li C J, Liao W T, Wu M Y, et al. New insights into the role of autophagy in tumor immune microenvironment[J]. Int J Mol Sci, 2017, 18(7): 1566.