曲格列酮诱导胃癌MKN45细胞株凋亡

摘要

研究不同浓度曲格列酮对胃癌MKN45细胞过氧化物酶体增殖激活受体γ(PPARγ)激活水平变化及细胞凋亡的作用.

方法: 胃癌MKN45细胞培养后分为4组, 分别为生理盐水对照组和1、5、10 μmol/L曲格列酮实验组. 用EMSA测定不同浓度曲格列酮对胃癌MKN45细胞PPARγ激活水平的变化, 用流式细胞术检测caspase-3蛋白的表达、细胞周期的变化及凋亡.

结果: 随曲格列酮浓度的增加, PPARγ活性明显升高, 以对照组活性为100, 1、5、10 μmol/L曲格列酮组的PPARγ平均活性分别为155.8、218.7和307.6(P<0.01). 流式细胞术证实MKN45细胞经1, 5, 10 μmol/L曲格列酮的作用后, 随着曲格列酮剂量的增加, Caspase-3蛋白的表达均值分别为4.51, 10.95, 20.49, 33.56(P<0.01), G0/G1期细胞逐渐增加, S期细胞下降, 凋亡细胞增多.

结论: 曲格列酮通过对胃癌MKN45细胞PPARγ的激活可引发胃癌MKN45细胞株细胞周期抑制及凋亡, 可为胃癌的治疗提供新的靶点.

关键词: 曲格列酮; 过氧化物酶体增殖激活受体γ; 胃癌; 凋亡; EMSA; 流式细胞术

Troglitazone induces gastric cancer cell line MKN45 apoptosis

Chun-Fang Jiang, Qing Chen, Jie Zhou, Hai Zheng, Juan Chen

Chun-Fang Jiang, Qing Chen, Hai Zheng, Department of General Surgery, Union Hospital, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China

Jie Zhou, Juan Chen, Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China

Supported by: the Scientific Research Fund from the Health Department of Hubei Province, No. 2003JX1B006.

Correspondence to: Chun-Fang Jiang, Department of General Surgery, Union Hospital, Huazhong University of Science and Technology, 1277 Jiefang Road, Wuhan 430022, Hubei Province, China. jiangcfun@126.com

Received: March 4, 2007
Revised: October 4, 2007
Accepted: October 28, 2007
Published online: October 18, 2007

Abstract

AIM: To observe the effects of different concentrations of troglitazone on the activity of peroxisome proliferators-activated receptor γ (PPARγ), and apoptosis of the MKN45 gastric cell line.

METHODS: Cultured MKN45 gastric cells were randomly divided into four groups: control, 1, 5 and 10 μmol/L troglitazone. The change in activity of PPARγ was detected by electrophoretic mobility shift assay (EMSA). Flow cytometry was used to detect changes in the expression of caspase-3, cell cycle and apoptosis.

RESULTS: TPPARγ activity of cultured MKN45 gastric cells increased significantly with the concentration of troglitazone. Data were quantified for the control group as 100, the mean activity of 1 μmol/L troglitazone was 155.8, 5 μmol/L was 218.7, and 10 μmol/L was 307.6 (P < 0.01). Flow cytometry confirmed that the mean expression of caspase-3 protein was increased to 4.51, 10.95, 20.49, 33.56 with increasing concentration of troglitazone. The cells in G0/G1 stage increased steadily and those in S stage decreased at the same time (P < 0.01). The rate of apoptosis also increased.

CONCLUSION: Troglitazone could activate PPARγ and cell-cycle arrest and apoptosis of MKN45 gastric cells. PPARγ may become a new target in the treatment of gastric cancer.

Key Words: Troglitazone; Peroxisome proliferation activated receptor γ; Gastric cancer; Apoptosis; Electrophoretic mobility shift assay; Flow cytometry

0 引言

胃癌是我国最常见的消化系肿瘤, 其发病机制尚未阐明. 胃癌在早期即可发生转移, 对放疗敏感性差, 生存率低. 研究证实, 过氧化物酶体增殖激活受体γ(peroxisome proliferation activated receptor γ, PPARγ)与多种癌症的发生、发展密切相关[1-4]. PPAR属于激素核受体超家族, 能被某些降脂药物、苯氧乙酸脂和脂肪酸等物质激活[5-8]. PPAR基因在不同物种中存在几种亚型, 包括较为清楚的α, β, γ 3种亚型和δ亚型[9]. 最新研究初步证实, PPARγ的激活可促进大肠癌、乳腺癌等肿瘤细胞系的分化, 抑制肿瘤细胞系的增殖, 并可导致肿瘤细胞系凋亡[10-11]. 我们研究曲格列酮(troglitazone)对胃癌MKN45细胞PPARγ激活水平变化及细胞凋亡的作用, 为临床胃癌的辅助治疗提供新的靶点.

1 材料和方法

1.1 材料

PPARγ寡聚核苷酸探针(Invitrogen CO.), Caspase-3 antibody(Santa Cruz), ³²P-ATP(中科院高能物理所), T₄-多聚核苷酸激酶(Promega), 二硫苏糖醇(DTT)和苯甲基磺酰氟(PMSF)(Sigma); NP-40(Fluka), Sephadex G-50(Pharmacia), X光底片(Kodak), HEPES, RPMI 1640培养基(中健公司), 显影及定影试剂(乐凯), 胃癌MKN45细胞株(同济医学院生化教研室惠赠).

1.2 方法

胃癌MKN45细胞株常规RPMI 1640培养基加100 mL/L胎牛血清培养, 70%传代, 大量扩增, 随机分4组: 即生理盐水对照组和1, 5, 10 μmol/L曲格列酮实验组, 分别给药. 经过处理24 h的细胞株经胰酶消化, 洗涤(TBS), 离心(1000 r/min×5 min), 收集处理后的细胞, TBS 10 mL重悬并计数, 至少达5×10⁸ /L, 再次离心后弃上清, 加入TBS 1 mL重悬沉淀并转移至1.5 mL EP管中, 置4℃使之温度平衡. 4℃, 3000 r/min离心15 s, 小心弃上清; 用冰冷的Buffer A 400 μL小心重悬沉淀, 置冰浴15 min使细胞肿胀; 加入NP-40使其终浓度达1 g/L, 剧烈振荡裂解细胞; 4℃, 3000 r/min离心4 min, 小心弃净上清. 用冰冷的Buffer C 50 μL小心重悬沉淀, 4℃剧烈振摇15 min; 4℃, 12 000 r/min离心10 min; 上清经考马斯亮兰G-250法测定蛋白质浓度后, 各取核抽提物8 μg, 与³²P-ATP标记的PPARγ寡聚核苷酸探针1 μg结合, 4℃下行EMSA, 2 h后凝胶取出, 置干胶仪干胶, -70℃放射自显影[12]. 另各组细胞培养48 h后收集, 40 g/L多聚甲醛室温下固定30 min, 再用PBS洗3次, 每次5 min, 加入抗caspase-3的一抗, 置室温60 min, PBS洗3遍, 加入FITC标记的二抗, 室温避光反应30 min. PBS洗3次后, 移入测试管, 上机检测细胞平均荧光强度. 阴性对照不加FITC标记的二抗, 代之以同型IgG. 避光30 min后用流式细胞仪检测. 每管计数10 000个细胞. 离心收集细胞, 调整浓度为1×10⁹ /L, -20℃的700 mL/L乙醇固定1 h; -20℃冰箱保存待染色. 0.01 mol/L PBS(pH7.2)洗涤2次, 离心弃上清, 加入PBS 1 mL溶解细胞沉淀; 加入RNase至终浓度100 mg/L, 37℃ 孵育30 min; 然后加入PI染液至终浓度5 mg/L混匀, 4℃避光染色30 min. 取经PI新近染色的胃癌细胞悬液1 mL(细胞数10 000个左右), 在FCM下检测. PI及annexin V抗体双染胃癌细胞, 流式细胞术检测凋亡[13].

统计学处理 影像使用Scion Image软件量化, 利用SPSS10.0软件包进行统计学处理, 数据采用各组的mean±SD表示, 各组间比较采用t检验.

2 结果

2.1 PPARγ活性和caspase-3蛋白表达

随曲格列酮浓度的增加, PPARγ活性明显升高, 以生理盐水对照组活性均数为100, 1, 5, 10 μmol/L曲格列酮组PPARγ活性均数分别为155.8, 218.7和307.6(P<0.01, 图1). 流式细胞术证实MKN45细胞经1, 5, 10 μmol/L曲格列酮的作用后, 随着曲格列酮剂量的增加, caspase-3蛋白的表达均值逐渐增加, 分别为4.51, 10.95, 20.49和33.56, 各组间存在显著性差异(P<0.01, 图2).

图1 曲格列酮作用细胞核提取物凝胶滞留电泳. 1: 对照(生理盐水)组; 2-4: 1, 5, 10 μmol/L曲格列酮组

图2 不同浓度曲格列酮作用MKN45细胞的caspase-3蛋白的表达. A: 0 μmol/L; B:1 μmol/L; C: 5 μmol/L; D: 10 μmol/L

2.2 细胞周期和凋亡

MKN45细胞经0, 1, 5和10 μmol/L的troglitazone作用后, 随着剂量的增加, G0/G1期细胞逐渐增加, S期细胞下降, 与对照组比较, 差异有显著性(P<0.01, 表1). MKN45细胞经1, 5, 10 μmol/L troglitazone作用后, 随着剂量的增加, 凋亡细胞增加. 凋亡率分别为1.07%, 11.63%, 12.96%和23.29%, 差异有显著性(P<0.01).

表1 曲格列酮对MKN45细胞周期的影响(%).

Troglitazone (μmol/L)	G0/G1	G2/M	S
0	67.1	16.7	16.2
1	73.8	16.5	9.7b
5	84.4b	8.3b	7.3b
10	88.2b	5.5b	6.4b

^bP<0.01 vs 0 μmol/L Troglitazone.

3 讨论

PPAR基因在不同物种中存在α, β, γ和δ 4种亚型, 同其他核受体超家族成员一样, PPAR有6个调节功能区: 即A, B, C, D, E, F[9,14]. 过氧化物酶体增殖激活受体γ是配体活化的核受体, 配体与之结合后, 活化PPARγ并与PPAR反应元件(peroxisome proliferator-activated receptor response element, PPRE)结合, 调节基因转录. PPAR通过PPAR-RXR(retinoid-X receptor)二聚体与一个相应元件结合而调控基因的转录, 该元件是由间隔一个核苷酸的PuGGTCA的重复序列组成, 该受体因这些物质也能够促使过氧化物酶体(peroxisome)体积和含量增加而得名. 核激素受体至少有两个二聚作用界面: 一个在他的DNA结合域, 另一个在他的配体结合域. DNA结合域在没有DNA存在时是单体, 有DNA时才发生聚合. 配体结合域二聚作用的功能看来是稳定受体-DNA复合物. PPAR能够调控许多参与细胞内外代谢的目的基因, 尤其是β-氧化过程中的一些重要酶类, 另外PPAR也能够参与脂肪细胞的分化[15-17]. 最新研究初步证实, PPARγ的激活可促进包括大肠癌、乳腺癌等肿瘤细胞系的分化, 抑制肿瘤细胞系的增殖, 并可导致肿瘤细胞系凋亡[18-22].

曲格列酮是PPARγ的配体, 其格列酮(glitazones)部分能与PPAR受体相结合. PPAR受体通过打开和关闭特定的基因来调控基因表达[23-25]. Glitazones含有一个噻唑烷二酮(thiazolidinedione)环的化学集团, 能够和PPAR紧密结合并激活PPAR[26-30]. 我们证实, 曲格列酮浓度的增加可明显升高PPARγ活性, 以对照组活性均值为100, 1 μmol/L曲格列酮组PPARγ活性均值为155.8, 5 μmol/L曲格列酮组PPARγ活性均值为218.7, 10 μmol/L曲格列酮组PPARγ活性均值为307.6(P<0.01). 流式细胞术证实MKN45细胞经1, 5, 10 μmol/L曲格列酮的作用时, 随着曲格列酮剂量的增加, G₀/G₁期细胞逐渐增加, S期细胞下降, Caspase-3蛋白的表达均值分别为4.51, 10.95, 20.49和33.56(P<0.01). 另外, MKN45细胞经1, 5, 10 μmol/L曲格列酮的作用后, 随着曲格列酮剂量的增加, 凋亡细胞明显增多, 凋亡率分别为1.07%, 11.63%, 12.96%和23.29%(P<0.01).

本实验显示, PPARγ激活能够导致肿瘤细胞周期抑制, 并诱导肿瘤细胞凋亡. PPARγ激活的体外试验结果证实其具有抑瘤功能, 给进一步的研究提供了基础和方向. PPARγ激活调控参与许多细胞内外代谢的目的基因, 最终导致肿瘤细胞周期抑制; 诱导肿瘤细胞凋亡的准确途径是进一步的研究目标, 最终可能为临床防治胃癌提供新的药物及靶点.

评论

背景资料

胃癌是我国最常见的消化系肿瘤, 其发病机制尚未完全阐明, 早期预防及治疗极其重要. 最新研究证实, 过氧化物酶体增殖激活受体PPARγ与多种癌症的发生、发展密切相关.

研发前沿

过氧化物酶体增殖激活受体具有6个调节功能区, 即A、B、C、D、E、F, 其中C和E是DNA和配体结合区. 过氧化物酶体增殖激活受体γ是配体活化的核受体, 配体与之结合后, 活化PPARγ并与PPAR反应元件结合, 调节基因转录. 最新研究初步证实, PPAR-γ的激活可促进结肠癌、乳腺癌等肿瘤细胞系的分化, 抑制肿瘤细胞系的增殖,并可导致肿瘤细胞系凋亡.

同行评价

本文研究了曲格列酮对胃癌细胞MKN45的凋亡诱导作用, 目前此类研究较少, 内容新颖, 行文流畅, 结构层次较清晰, 有一定的参考价值.

备注

编辑: 何燕 电编:郭海丽

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