鼻咽癌肿瘤干细胞高表达SOD2可对抗顺铂的杀伤作用  

Nasopharyngeal carcinoma stem cells develop resistant against Cisplatin through upregulating SOD

作　　者：林碧华[1] 陈婧[2] 郭春连[3] 余海波[4] 张鑫 周克元[1,5] 

机构地区：[1]广东医学院生物化学与分子生物学教研室,523808 [2]东莞市人民医院 [3]广东医学院药剂学教研室 [4]广东医学院招投标中心 [5]广东省医学分子诊断重点实验室

出　　处：《天津医药》2015年第6期577-581,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81272434);湛江市科技攻关计划(2013B01091);广东医学院青年基金(Q2012005);国家级大学生创新创业训练计划项目(201310571004);广东省大学生创新创业训练计划项目(1057113030);广东医学院大学生创新实验项目立项(ZZDM012;ZZDM013)

摘　　要：目的探讨鼻咽癌(NPC)肿瘤干细胞对抗顺铂诱导氧化应激损伤的机制。方法利用CCK-8法测定顺铂对NPC细胞CNE-2与NPC肿瘤干细胞CNE-2S的半数抑制浓度。观察不同浓度(0.1、0.5、1.0μmol·L-1)顺铂作用后,细胞内活性氧(ROS)、总谷胱甘肽(GSH)的含量及总超氧化物歧化酶(SOD)的活性改变。实时定量RT-PCR测定1μmol·L-1顺铂作用于2组细胞48 h后,谷胱甘肽合成酶(GSS)、γ-谷氨酰半胱氨酸合成酶催化亚基(GCLC)、γ-谷氨酰半胱氨酸合成酶调节亚基(GCLM)、SOD1和SOD2 m RNA的表达情况,免疫印迹法检测SOD2蛋白的表达情况。利用小干扰RNA技术沉默SOD2,并与1μmol·L-1顺铂共处理2组细胞,用台盼蓝染色观察细胞的存活情况。结果顺铂对CNE-2S的半数抑制浓度显著高于CNE-2(μmol·L-1:9.8±1.1 vs 2.4±0.6,P<0.05)。在不同浓度的顺铂处理后,2组细胞内ROS水平升高,但CNE-2S在处理前后的ROS水平均低于CNE-2(P<0.05)。1μmol·L-1顺铂刺激后,CNE-2S与CNE-2细胞中GSH含量均上升,但2组细胞间无明显差异(P>0.05);2组细胞中SOD活性均上升,且CNE-2S显著高于CNE-2(P<0.05)。顺铂处理前后,2组细胞的GSS、GCLC、GCLM和SOD1的m RNA水平无明显差异,但CNE-2S中SOD2的m RNA及蛋白表达水平高于CNE-2(P<0.05)。沉默SOD2与顺铂共处理2组细胞,能有效抑制其存活率。结论 NPC肿瘤干细胞CNE-2S高表达SOD2后,抗氧化应激能力增强,从而导致对顺铂的耐药。Objective To investigate the way that nasopharyngeal carcinoma （NPC） and NPC stem cells develops resistance to cisplatin through anti-reactive oxygen species mechanism. Methods Using CCK-8 cell counting kit, we measured the half inhibitory concentration of cisplatin against NPC cells“CNE-2”and NPC stem cells“CNE-2S”, and compared their resistant index. We examined the differences in the reactive oxygen species （ROS） levels, total glutathi-one （GSH） levels, and total superoxide dismutase （SOD） levels between CNE-2 and CNE-2S at different concentrations of cisplatin administration （0.1,0.5 and 1.0μmol·L-1）. Using q-PCR, we determined the mRNA expression level of GSS, GCLC, GCLM, SOD1 and SOD2 after 48 hours administration of cisplatin at 1 μmol · L-1. Protein expression level of SOD2 was also tested using Western Blot after 48 hours administration of cisplatin at 1μmol · L-1. Upon silencing the SOD2 in NPC cell through siRNA, Trypan blue was used to analyze cell survival after cisplatin was administrated at 1μmol · L-1. Results The inhibition concentration of cisplatin against CNE-2 was higher than that against CNE-2S （μmol · L-1：9.8 ± 1.1 vs 2.4 ± 0.6,P〈0.05）. ROS levels in CNE-2 and CNE-2S both rise with cisplatin administration, but ROS levels of CNE-2 before and after cisplatin treatment were both higher than those in CNE-2S （P〈0.05）. The total gluta-thione levels in CNE-2 and CNE-2S were both increased after 1μmol·L-1 cisplatin treatment but there is no significant dif-ference in levels of glutathione between these two cell lines. After treated with cisplatin, SOD level were increased in both CNE-2S and CNE-2, but it is higher in CNE-2S than that in CNE-2 （P〈0.05）. The mRNA levels of GSS, GCLC, GCLM, and SOD1 were not different significantly between in CNE-2 and in CNE-2S with or without cisplatin treatment. However, SOD2 in CNE-2S were higher than that in CNE-2 on both mRNA and protein levels （P〈0.05）. Silenced SOD2 disrupted the resistance o

关 键 词：鼻咽癌 肿瘤干细胞 顺铂 耐药 氧化应激 谷胱甘肽 超氧化物歧化酶 RNA干扰 

分 类 号：R739.6[医药卫生—肿瘤]