顺铂与足叶乙甙对白血病细胞K562的协同杀伤作用及其机制  

Synergetic Cytotoxicity of Cisplatin and Etoposide to Leukemia Cell Line K562 and Its Mechanism

作　　者：马卫东[1] 阴梅云[1] 蒋常文[1] 徐世荣[1] 翟丽东[1] 郑力芬[1] 王彦玲[1] 闫蕴力[1] 

机构地区：[1]河北医科大学基础医学研究所细胞生物室,河北石家庄050017

出　　处：《癌症》2005年第8期958-964,共7页Chinese Journal of Cancer

摘　　要：背景与目的:足叶乙甙(etoposide,VP鄄16)为白血病化疗最常用的化疗药之一,其临床应用日益受到天然与继发耐药的影响。对一些实体瘤的研究发现顺铂(cisplatin,DDP)与VP鄄16联合应用具有协同效应。本研究通过DDP与VP鄄16联合作用杀伤白血病细胞K562,探讨其增强VP鄄16疗效的作用机制。方法:采用VP鄄16(0,5μg/ml)与不同浓度DDP(0,0.3,3,15,30μg/ml)联合对K562细胞进行处理。应用噻唑蓝(MTT)法检测用药后细胞的存活相对数量,计算VP鄄16和DDP应用对K562细胞的抑制作用;AO/EB双荧光法定量分析细胞凋亡率。半定量RT鄄PCR法和Westernblot检测拓扑异构酶(topoisomerase,TOPO)Ⅱα、Ⅱβ、Sp1、Sp3基因的mRNA和蛋白水平。结果:VP鄄16与DDP合用具有明显的协同效应。两药合用后,细胞凋亡率明显增加。单独应用DDP可以明显上调TOPOⅡ和Sp1表达(呈浓度依赖,TOPOⅡα、Ⅱβ、Sp1在30μg/mlDDP时比正常对照分别上调36%,25%和75%),并使Sp3的表达降低49%;单用5μg/mlVP鄄16时TOPOⅡ则下调(TOPOⅡα下降72%),TOPOⅡβ和Sp1的表达未受影响,Sp3的表达上调14%。5μg/mlVP鄄16与DDP联合应用具有协同效应,使TOPOⅡ和Sp1表达水平升高。TOPOⅡα、Ⅱβ、Sp1在5μg/mlVP鄄16与30μg/mlDDP联合应用时比单用VP鄄16分别上升83%,11%,58%,但低于单独应用DDP的表达水平;而Sp3的表达水平与单独应用DDP比较下调61.3%。Western印迹检测结果与RT鄄PCR结果相符。结论:DDP在化疗中与VP鄄16发挥协同作用,通过上调拓扑异构酶Ⅱ的表达水平,为VP鄄16提供更多的靶酶,使VP鄄16杀伤K562细胞效果明显提高。BACKGROUND ＆ OBJECTIVE. Etoposide （VP-16） is one of the most common chemotherapy drugs, but its usage is limited by drug resistance. Some researches on solid tumors show that cisplatin （DDP） have synergetic effect with VP-16. This study was to evaluate synergetic cytotoxicity of VP-16 and DDP to leukemia cell line K562, and explore the mechanism.METHODS： K562 cells were treated with VP-16 （0 or 5 μg/ml） and DDP （0,0.3, 3, 15, or 30 μg/ml） in different combination patterns. Inhibitory effect of VP-16 and DDP on survival of K562 cells was measured by M-FI assay. Cell apoptosis was evaluated by AO/EB double fluorescent labeling. The expression of topoisomerase （TOPO） II a and II 13, and transcription factor Sp1 and Sp3 were measured by semi-quantitive reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. RESULTS： MTT assay showed significant synergetic cytotoxicity of VP-16 and DDP. VP-16 in combination with DDP obviously enhanced cell apoptosis. RT-PCR showed that DDP significantly up-regulated the expression of TOPO Ⅱ and Sp1 in concentration-dependent manners （TOPO Ⅱα, Ⅱβ, and Spl were up-regulated by 36%, 25%, and 75% of control, respectively, when treated with 30 μg/ml of DDP）, and down-regulated Sp3 by 49% of control; VP-16 （5μg/ml） down-regulated TOPO Ⅱ α by 71.9%, and up-regulated Sp3 by 14%;VP-16 （5 μg/ml） in combination with DDP （30 μg/ml） up-regulated TOPO Ⅱα by 83%, Ⅱβ by 11%, and Sp1 by 58% when compared with using VP-16 alone （but the levels were lower than using DDP alone）, and down-regulated Sp3 by 61.3% when compared with using DDP alone. Western blot showed similar results to RT-PCR. CONCLUSION： Through up-regulating TOPO Ⅱ ,DDP could enhance the chemotherapeutic effect of VP-16 on K562 cells by provide more target enzyme to act on.

关 键 词：足叶乙甙/药理学 顺铂/药理学 拓扑异构酶Ⅱ 转录因子SP1 转录因子Sp3 白血病/药物疗法 K562细胞 

分 类 号：R733.7[医药卫生—肿瘤]