国际口腔医学杂志 ›› 2020, Vol. 47 ›› Issue (4): 406-412.doi: 10.7518/gjkq.2020079

• 论著 • 上一篇    下一篇

吡咯喹啉醌对舌鳞状细胞癌细胞上皮间质转化的抑制作用研究

吴南1,李斌2()   

  1. 1.永州职业技术学院医学院 永州 425000
    2.湖南医药学院口腔医学院 怀化 418000
  • 收稿日期:2019-12-28 修回日期:2020-04-27 出版日期:2020-07-01 发布日期:2020-07-10
  • 通讯作者: 李斌 E-mail:2996967631@qq.com
  • 作者简介:吴南,主治医师,硕士,Email:673686406@qq.com
  • 基金资助:
    湖南省教育厅科学研究项目(18C1132);湖南省自然科学青年基金(2019JJ50423)

Inhibitory effect of pyrroloquinoline quinone on epithelial-mesenchymal transition in tongue squamous cell carcinoma cells

Nan Wu1,Bin Li2()   

  1. 1. School of Medicine, Yongzhou Vocational Technical College, Yongzhou 425000, China
    2. School of Stomatology, Hunan University of Medicine, Huaihua 418000, China
  • Received:2019-12-28 Revised:2020-04-27 Online:2020-07-01 Published:2020-07-10
  • Contact: Bin Li E-mail:2996967631@qq.com
  • Supported by:
    This study was supported by Research Project of Hunan Provincial Department of Education(18C1132);Youth Program of Hunan Natural Science Foundation(2019JJ50423)

摘要:

目的 探讨吡咯喹啉醌(PQQ)对舌鳞状细胞癌细胞CAL27上皮间质转化(EMT)的抑制作用及可能机制。方法 采用MTT法检测PQQ对CAL27细胞增殖作用的影响;Transwell和划痕实验检测PQQ对CAL27细胞侵袭及迁移能力的影响;电子荧光显微镜下检测PQQ对CAL27细胞内产生活性氧(ROS)水平的影响。分别采用PQQ、ROS清除剂N-乙酰基-L-半胱氨酸(NAC)和核因子-κB(NF-κB)激动剂佛波醇酯(PMA)处理细胞,Western blot检测EMT相关蛋白(上皮钙黏蛋白、波形蛋白、锌指转录因子Snail)和NF-κB通路活化水平的变化。结果 PQQ作用CAL27细胞24 h后,随着PQQ浓度的增加,CAL27细胞增殖受抑制,24 h的半数抑制浓度为6.69 μmol·L -1。2 μmol·L -1 PQQ作用CAL27细胞24 h后,细胞的侵袭及迁移能力明显受到抑制(P<0.05)。2 μmol·L -1 PQQ可有效诱导细胞内ROS的产生,上调上皮钙黏蛋白的表达,下调波形蛋白和Snail的表达,抑制NF-κB活化水平(P<0.001)。NAC可以减弱PQQ对EMT相关蛋白的调控作用,降低PQQ对NF-κB通路的抑制作用(P<0.01);PMA同样可以减弱PQQ对EMT相关蛋白的调控作用(P<0.05)。结论 PQQ能够抑制CAL27细胞的EMT进程,ROS和NF-κB信号通路可能在该进程中发挥重要作用。

关键词: 舌鳞状细胞癌, 吡咯喹啉醌, 上皮间质转化, 活性氧, 核因子-κB

Abstract:

Objective To study the effect of pyrroloquinoline quinone (PQQ) on epithelial-mesenchymal transition (EMT) and its underlying mechanisms in human tongue squamous cell carcinoma cells. Methods Human tongue squamous cell carcinoma cell line CAL27 was selected. MTT assay was used to determine the effect of PQQ on cell viability. The invasion and migration abilities of CAL27 cells were checked by Transwell assay and wound healing test. Fluorescence microscopy was performed to measure the effect of PQQ on the generation of reactive oxygen species (ROS) in CAL27 cells. The expression levels of EMT-related proteins (E-cadherin, Vimentin, and zinc-finger transcription factor Snail) and nuclear factor-κB (NF-κB) activator protein in CAL27 cells under the treatment of PQQ, N-acetyl-L-cysteine (NAC), and phorbol-12-myristate-13-acetate (PMA) were determined by Western blot. Results PQQ inhibited the proliferation of CAL27 cells in a dose-dependent manner, and the half maximal inhibitory concentration of PQQ after 24 h was 6.69 μmol·L -1. Transwell assay and wound healing test showed that 2 μmol·L -1 PQQ inhibited the invasion and migration abilities of CAL27 cells (P<0.05). Cellular ROS level was signi-ficantly boosted (P<0.001) after treatment with 2 μmol·L -1 PQQ for 24 h. PQQ (2 μmol·L -1) increased the expression level of E-cadherin and inhibited the expression levels of Vimentin, Snail, and NF-κB (P<0.001). The coincubation of PQQ with NAC or PMA yielded the following experimental results: 1) NAC could reduce the regulatory effect of PQQ on EMT-related proteins and NF-κB activator protein (P<0.01) such that the increased expression level of E-cadherin was inhibited and the inhibited expression levels of Vimentin, Snail, and NF-κB were increased; 2) PMA could also reduce the regulatory effect of PQQ on EMT-related proteins (P<0.05). Conclusion PQQ can inhibit the EMT process in tongue squamous cell carcinoma cells, and ROS and the NF-κB signaling pathway may play an important role in this process.

Key words: human tongue squamous cell carcinoma, pyrroloquinoline quinone, epithelial-mesenchymal transition, reactive oxygen species, nuclear factor-κB

中图分类号: 

  • R730.5

图 1

PQQ对CAL27细胞存活率的影响"

图 2

Transwell侵袭实验结果 倒置相差显微镜 × 200 A:空白对照组;B、C、D:分别为1、2、4 μmol·L-1 PQQ处理组。"

图 3

不同浓度PQQ对CAL27细胞侵袭能力的影响 ???:P<0.001。"

图 4

不同浓度PQQ对CAL27细胞迁移能力的影响 ?:P<0.05;???:P<0.001。"

图 5

细胞划痕实验结果 倒置相差显微镜 × 40 上:0 h;下:培养24 h;从左到右分别为空白对照组和1、2、4 μmol·L-1PQQ处理组。"

图 6

不同浓度PQQ诱导细胞内ROS的产生状况 电子荧光显微镜 × 200 A:空白对照组;B、C、D:分别为1、2、4 μmol·L-1PQQ处理组。"

图 7

PQQ和NAC作用下,CAL27细胞中EMT相关蛋白和NF-κB表达的Western blot电泳图 A:空白对照组;B:PQQ组;C:PQQ+NAC组;D:NAC组。"

图 8

PQQ和NAC作用下,CAL27细胞中EMT相关蛋白和NF-κB表达的统计图 A:E-cadherin;B:Vimentin;C:Snail;D:NF-κB。??:P<0.01;???:P<0.001。"

图 9

PQQ和PMA作用下,CAL27细胞中EMT相关蛋白表达的Wes-tern blot电泳图 A:空白对照组;B:PQQ组;C:PQQ+PMA组;D:PMA组。"

图 10

PQQ和PMA作用下,CAL27细胞中EMT相关蛋白表达的统计图 A:E-cadherin;B:Vimentin;C:Snail。?:P<0.05;??:P<0.01;???:P<0.001。"

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