修回日期: 2014-01-13
接受日期: 2014-01-18
在线出版日期: 2014-03-18
目的: 探讨衣霉素(tunicamycin, TM)诱导的内质网应激, 对胃癌细胞侵袭力的影响及机制.
方法: 以浓度为3 μmol/L的TM处理胃癌SGC7901细胞24 h, Transwell侵袭实验检测细胞侵袭能力, 并应用Western blot检测pPERK和GSK-3β蛋白Ser9位点的表达.
结果: Transwell侵袭实验显示, TM处理胃癌SGC7901细胞24 h, 细胞的侵袭性明显减弱. Western blot结果显示, 相对于DMSO对照组, TM处理组pPERK蛋白的表达明显高于DMSO对照组, 而Ser9-GSK-3β蛋白的表达明显降低, 差异均有统计学意义(P<0.01).
结论: TM诱导的内质网应激, 通过激活GSK-3β, 降低胃癌细胞的侵袭力.
核心提示: 衣霉素(tunicamycin)诱导的内质网应激, 可通过降低糖原合成激酶-3β(glycogen synthase kinase-3β, GSK-3β)的Ser9位点磷酸化, 激活GSK-3β, 从而明显降低胃癌细胞的侵袭力.
引文著录: 付政祺, 邹丰, 王绪明, 李艳, 刘丽江. 衣霉素诱导的内质网应激对胃癌细胞侵袭力的影响及机制. 世界华人消化杂志 2014; 22(8): 1101-1105
Revised: January 13, 2014
Accepted: January 18, 2014
Published online: March 18, 2014
AIM: To investigate the involvement of tunicamycin (TM)-induced endoplasmic reticulum stress in the invasion of gastric cancer cells and the underlying mechanisms.
METHODS: SGC7901 cells were treated with TM at a concentration of 3 μmol/L for 24 h. After treatment, the invasion of gastric cancer cells was evaluated by Transwell chamber assay. The phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (pPERK) and glycogen synthase kinase-3β (GSK-3β) at Ser9 was examined by Western blot.
RESULTS: TM treatment induced endoplasmic reticulum stress, which was demonstrated by increased pPERK. Endoplasmic reticulum stress decreased the invasion ability of gastric cancer cells and the phosphorylation of GSK-3β at Ser9.
CONCLUSION: Endoplasmic reticulum stress induced by TM may decrease the invasion of gastric cancer cells by activation of GSK-3β.
- Citation: Fu ZQ, Zou F, Wang XM, Li Y, Liu LJ. Involvement of tunicamycin-induced endoplasmic reticulum stress in invasion of gastric cancer cells. Shijie Huaren Xiaohua Zazhi 2014; 22(8): 1101-1105
- URL: https://www.wjgnet.com/1009-3079/full/v22/i8/1101.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v22.i8.1101
胃癌是我国最常见的恶性肿瘤之一, 易发生侵袭转移是其死亡率高的常见原因[1-4]. 近年研究发现, 内质网应激在肿瘤的发生发展中发挥重要作用. 在胃癌裸鼠移植瘤模型中, 敲除内质网陪伴分子葡萄糖调节蛋白78(glucose-regulated protein, GRP78)可抑制体外肿瘤细胞的侵袭生长与转移[5]. 内质网应激诱导剂衣霉素(tunicamycin, TM)可通过降低KL-6 mucin(Krebs von den Lungen-6)的表达, 降低人肝内胆管癌RBE细胞的侵袭力[6]. 但内质网应激在胃癌细胞侵袭中的作用及机制尚不完全清楚.
糖原合成激酶-3β(glycogen synthase kinase-3β, GSK-3β)是Wnt信号转导通路的重要分子. 研究发现GSK-3β参与肿瘤的发生、侵袭与转移[7]. 在人体胃癌组织中GSK-3β的阳性率为46%(129/281), 与淋巴结浸润、转移和生存率负相关[8]. 抑制GSK-3β表达可促进胃癌细胞SGC7901的迁移能力[9,10]. 为了探讨GSK-3β是否受内质网应激调控, 是否参与介导胃癌细胞的侵袭, 本研究拟给予内质网应激特异性诱导剂TM处理胃癌细胞系SGC7901, 通过检测内质网跨膜蛋白pPERK蛋白表达的变化及其与胃癌细胞侵袭力改变的关系, 明确内质网应激对胃癌细胞侵袭力的影响. 并通过检测GSK-3β蛋白Ser9位点表达的改变情况, 探讨GSK-3β途径在内质网应激调控胃癌细胞侵袭中的作用.
人胃癌细胞系SGC7901由华中科技大学同济医学院免疫学系惠赠, 为本实验室保存. TM购自Alexis, 溶于DMSO以3 mmol/L浓度-20 ℃保存. pPERK(Thr 981)和β-actin(C4)抗体购自Santa Cruz公司; Transwell侵袭试剂盒购自Millipore公司; Bicinchoninic acid(BCA)蛋白测定试剂盒购自Pierce公司.
1.2.1 细胞培养及药物处理: SGC7901细胞接种在含10%小牛血清的RMPI 1640培养基中, 置于37 ℃, 5%CO2培养箱中培养, 每2-3 d换1次液. 细胞增长至约70%-80%左右融合时胰酶消化传代. TM处理细胞的浓度为3 μmol/L, 时间为24 h[11].
1.2.2 Transwell侵袭实验: 向侵袭小室(Transwell chamber)中加入制备好的密度为0.5×109/L-1.0×109/L的无血清SGC7901细胞悬液, 将小室置于含10%胎牛血清培养基的24孔板内培养24 h. 用棉签轻轻刮除小室内未侵袭的细胞后, 将小室底部置于试剂盒中的染色液里染色20 min, 漂洗数次、风干. 在显微镜下观察, 细胞计数.
1.2.3 Western blot技术检测: SGC7901细胞收集后, BCA法测蛋白, 进行SDS-PAGE电泳, 电泳后湿转至PVDF膜, 含5%脱脂奶粉的TBS-T(含0.05%Tween-20的TBS)37 ℃封闭60 min, 加1:1000稀释的抗pPERK(Thr 981)和β-actin抗体4 ℃孵育过夜. TBS-T漂洗5 min×3次, 加入1:4000稀释的辣根过氧化物酶标记的羊抗兔和羊抗鼠IgG 37 ℃孵育45 min. TBS-T漂洗5 min×3次, ECL化学发光试剂检测[12].
统计学处理 全部数据经SPSS13.0统计学软件处理, 数据均采用mean±SD表示, 相关因素分析采用Spearman等级相关分析. P<0.05为差异有统计学意义.
Western blot结果显示, TM处理SGC7901细胞24 h后, pPERK蛋白的表达明显高于DMSO对照组(图1), 提示TM处理诱导内质网应激.
与对照组(DMSO处理组)相比, TM处理组的SGC7901细胞的侵袭性明显减弱(图2).
胃癌细胞SGC7901经TM处理24 h后, Ser9-GSK-3β蛋白表达下降(图3), 提示内质网应激通过降低GSK-3β的Ser9位点磷酸化, 激活GSK-3β, 影响细胞侵袭能力.
内质网是细胞内蛋白质合成、折叠及转运的重要场所, 并利用其严格的质控系统, 保证其输出蛋白具有正常结构功能. 当多种生理或病理因素, 如缺氧、钙超载等, 引起细胞内未折叠/错误折叠蛋白质增多, 内环境失衡时, 将导致内质网应激. 此时, 一系列级联反应即未折叠蛋白反应(unfolded protein response, UPR)被激活, 通过限制未折叠/错误折叠蛋白的合成, 加强对蛋白的折叠能力及加速未折叠/错误折叠蛋白的降解对细胞发挥保护作用. 但当这种紊乱持续发展, 超过了自身调节, 将导致细胞的凋亡和死亡[13,14]. 近年研究发现, 内质网应激可能在肿瘤的发生、侵袭、转移中发挥重要作用. 内质网陪伴分子GRP78和GRP94在胃癌组织中高表达, 且与肿瘤大小、侵袭深度和淋巴结转移等相关[15-17]. 内质网应激诱导剂TM可通过降低KL-6 mucin的表达, 降低RBE细胞的侵袭力[6]. 本研究发现TM诱导的内质网应激可降低胃癌细胞的侵袭力.
GSK-3β是一种丝氨酸/苏氨酸激酶, 活性受其Ser9位点(抑制性)和Tyr216位点(活性)的磷酸化水平调节[18]. 研究发现, GSK-3β参与细胞内多种信号转导通路, 在胃癌细胞的增殖与凋亡中发挥重要作用. 人体胃癌组织中GSK-3β的阳性率为46%(129/281), 且在早期胃癌组织中阳性率较高, 与淋巴结浸润、转移和生存率负相关[8]. 抑制GSK-3β表达, 可促进胃癌细胞SGC7901的迁移能力[9,10]. 蛋白激酶RNA样内质网激酶[protein kinase RNA (PKR)-like endoplasmic reticulum kinase, PERK]是Ⅰ型内质网跨膜蛋白. 应激状态下, PERK被激活, 与GRP78解聚并发生自身磷酸化, 通过减少蛋白质合成, 以减轻内质网内新生蛋白质折叠需求的压力[19]. 研究发现, 内质网应激可激活GSK-3β[20,21]. 在阿尔茨海默病患者大脑海马神经元中pPERK阳性神经元的GSK-3β表达丰富[22]. 肾脏缺血后处理(ischemic postconditioning)及联合曲美他嗪治疗可降低内质网应激蛋白pPERK等的表达, 并通过降低GSK-3β活性, 改善肾功能提高大鼠生存率[23]. 提示, 内质网应激时, PERK等通路可通过GSK-3β途径, 影响细胞存活. 本研究发现TM诱导的内质网跨膜蛋白PERK的磷酸化, 可通过降低GSK-3β蛋白Ser9位点的磷酸化(非活性位点), 激活GSK-3β, 从而减弱胃癌细胞的侵袭力. 但内质网应激调控GSK-3β影响细胞侵袭的机制有待进一步研究.
总之, 本研究结果显示: TM诱导的内质网应激, 可通过降低GSK-3β的Ser9位点磷酸化, 激活GSK-3β, 从而明显降低胃癌细胞的侵袭力.
胃癌是我国最常见的恶性肿瘤之一, 易发生侵袭转移是其死亡率高的常见原因. 研究发现, 内质网应激在肿瘤的发生发展中发挥重要作用, 但其在胃癌细胞侵袭中的作用及机制尚不完全清楚. 内质网陪伴分子在胃癌组织中高表达, 且与肿瘤大小、侵袭深度和淋巴结转移等相关. 内质网应激可激活GSK-3β, 其在人体胃癌组织中的表达, 与淋巴结浸润、转移和生存率负相关. 抑制GSK-3β表达, 可促进胃癌细胞SGC7901的迁移能力.
余日胜, 主任医师, 浙江大学医学院附属第二医院放射科
本研究发现衣霉素(tunicamycin, TM)诱导内质网跨膜蛋白PERK的磷酸化, 可通过降低GSK-3β蛋白Ser9位点的磷酸化(非活性位点), 激活GSK-3β, 从而减弱胃癌细胞的侵袭力. 但内质网应激调控GSK-3β影响细胞侵袭的机制有待进一步研究.
内质网应激在胃癌细胞侵袭中的作用及机制尚不完全清楚. 本研究发现, TM诱导的内质网应激, 可通过降低GSK-3β的Ser9位点磷酸化, 激活GSK-3β, 从而明显降低胃癌细胞的侵袭力.
探讨内质网应激在胃癌细胞侵袭中的作用及机制, 此成果将对胃癌发生、发展的理论研究以及胃癌治疗新靶点的选择方面, 产生重要影响.
本文具有一定指导意义.
编辑: 田滢 电编: 鲁亚静
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