切换至 "中华医学电子期刊资源库"

中华口腔医学研究杂志(电子版) ›› 2012, Vol. 6 ›› Issue (03) : 223 -231. doi: 10.3877/cma.j.issn.1674-1366.2012.03.002

基础研究

转化生长因子β1 诱导人舌鳞状细胞癌细胞上皮-间质转化及其顺铂耐药性研究
刘墨1, 张斌2, 王成2, 刘习强2, 王剑宁2, 黄洪章2,()   
  1. 1.510120 广州,中山大学孙逸仙纪念医院口腔科
    2.510120 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2011-10-23 出版日期:2012-06-01
  • 通信作者: 黄洪章
  • 基金资助:
    国家自然科学基金(81072223)

Study on epithelial-mesenchymal transition induced by TGF-β1 enhances cisplatin-resistance capacity of human tongue squamous cell carci

Mo LIU1, Bin ZHANG1, Cheng WANG1, Xi-qiang LIU1, Jian-ning WANG1, Hong-zhang HUANG1,()   

  1. 1.Department of Stomatology, The Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
  • Received:2011-10-23 Published:2012-06-01
  • Corresponding author: Hong-zhang HUANG
引用本文:

刘墨, 张斌, 王成, 刘习强, 王剑宁, 黄洪章. 转化生长因子β1 诱导人舌鳞状细胞癌细胞上皮-间质转化及其顺铂耐药性研究[J/OL]. 中华口腔医学研究杂志(电子版), 2012, 6(03): 223-231.

Mo LIU, Bin ZHANG, Cheng WANG, Xi-qiang LIU, Jian-ning WANG, Hong-zhang HUANG. Study on epithelial-mesenchymal transition induced by TGF-β1 enhances cisplatin-resistance capacity of human tongue squamous cell carci[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2012, 6(03): 223-231.

目的

利用转化生长因子β1(TGF-β1)诱导人舌鳞状细胞癌(TSCC)细胞CAL27、顺铂耐药细胞CAL27-res 发生上皮-间质转化(EMT),建立TSCC 细胞发生EMT 的实验研究模型。 研究EMT 对TSCC 细胞顺铂耐药性及细胞增殖、凋亡的影响。

方法

10 ng/ml 的TGF-β1 处理CAL27、CAL27-res 细胞8 d,观察细胞形态学变化,Western blot 和细胞免疫荧光验证EMT 相关上皮标记蛋白及间质标记蛋白表达的变化,划痕试验检测细胞迁移能力的变化,MTT 法检测细胞半抑制率(IC50),免疫荧光检测MDR、MRP、TopoⅡβ 的表达,流式细胞仪分析细胞周期及凋亡。

结果

TGF-β1可诱导CAL27、CAL27-res 向间质细胞形态转化,并且下调上皮相关标记蛋白E-cadherin 的表达,上调间质相关标记蛋白Vimentin 的表达,细胞的迁移能力明显增强。 IC50 分别提高2.31 倍(CAL27)和1.72 倍(CAL27-res),MDR 和MRP 表达升高,TopoⅡβ 表达降低。 细胞增殖指数(PI)均明显降低(CAL27:χ2=16.799,P<0.001;CAL27-res:χ2=25.375,P<0.001),细胞凋亡指数(AI)均明显升高(CAL27:χ2=165.0797,P<0.001;CAL27-res:χ2=196.5640,P<0.001)。

结论

TGF-β1 能够成功诱导CAL27、CAL27-res 发生EMT 并且明显增强细胞对顺铂的耐药性,同时抑制细胞增殖,促进凋亡。

Objective

The aim of this study was to establish an Epithelial-mesenchymal Transition model of oral squamous cell carcinoma cell line CAL27 and cisplatin-resistant cell line CAL27-res with TGF-β1 and investigate the effect of Epithelial-mesenchymal Transition on cisplatinresistance capacity, cell growth, and apoptosis.

Methods

Cultured CAL27 and CAL27-res cells were treated with 10 ng/ml TGF-β1 for 8 days. The morphological changes were observed by microscope; the changes of EMT relative marker proteins were assessed by Western blot and immunoflurescence staining.In addition, Pirquet test was used to investigate the change of motility in CAL27 and CAL27-res. A MTT-based method was used to analyze the half maximal inhibitory concentration (IC50) values.Immunofluorescence staining was used to investigate the expression of MDR, MRP and TopoⅡβ. Flow cytometry was used to analyze the growth and apoptosis.

Results

The results showed that TGF-β1 could induce CAL27 and CAL27-res cells morphological alteration from epithelial to mesenchymal and downregulate the expression of epithelial maker protein E-cadherin, upregulate the expression of mesenchymal maker protein Vimentin. Furthermore the motility was significantly enhanced. The IC50 of CAL27 and CAL27-res increased by 2.31 and 1.72 fold accompanied by MDR, MRP up-regulation and Topo Ⅱβ down-regulation. The Proliferation index (PI) of CAL27 and CAL27-res were significantly decreased (CAL27: χ2=16.799, P<0.001; CAL27-res: χ2=25.375, P<0.001). The Apoptosis Index(AI) of CAL27 and CAL27-res increased significantly (CAL27: χ2=165.0797, P<0.001; CAL27-res:χ2=196.5640, P<0.001).

Conclusion

EMT induced by TGF-β1 enhanced the cisplatin-resistance capacity in CAL27 and CAL27-res cells, accompanied by growth inhibition and apoptosis accelerated.

图1 CAL27、CAL27-res 细胞经10 mg/ml TGF-β1 处理8 d 后的形态学变化(× 100) CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
图2 Western blot 检测CAL27、CAL27-res 细胞EMT 相关标记蛋白E-cadherin 和Vimentin 的表达变化 CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
图3 免疫荧光检测CAL27、CAL27-res 细胞EMT 相关标记蛋白E-cadherin、Vimentin 的表达变化 E-cadherin,Vimentin 红染,细胞核蓝染。 CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
图4 划痕实验检测CAL27、CAL27-res 细胞迁移能力的变化(×10) CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
图5 根据MTT 结果绘制的CAL27 和CAL27-res 细胞生长抑制曲线 CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
图6 免疫荧光检测MDR、MRP 及TopoⅡβ 的表达变化 MDR、MRP、TopoⅡβ:红染;细胞核:蓝染。 CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
图7 流式细胞仪分析CAL27 和CAL27-res 细胞周期的变化 CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
表1 CAL27 和CAL27-res 细胞周期、增殖指数和凋亡指数的变化(±s,%)
图8 双染法分析CAL27 和CAL27-res 的凋亡变化 CAL27/EMT、CAL27-res/EMT 分别表示经TGF-β1 处理后的CAL27、CAL27-res 细胞
1
Franceschi D, Gupta R, Spiro RH, et al. Improved survival in the treatment of squamous carcinoma of the oral tongue. Am J Surg, 1993,166(4):360-365.
2
Kajiyama H, Shibata K, Terauchi M, et al. Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells. Int J Oncol, 2007,31(2):277-283.
3
Thomson S, Buck E, Petti F, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res, 2005,65(20):9455-9462.
4
Yauch RL, Januario T, Eberhard DA, et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients.Clin Cancer Res, 2005,11(24 Pt 1):8686-8698.
5
Yang AD, Fan F, Camp ER, et al. Chronic oxaliplatin resistance induces epithelial-to-mesenchymal transition in colorectal cancer cell lines. Clin Cancer Res, 2006,12(14 Pt 1):4147-4153.
6
Sabbah M, Emami S, Redeuilh G, et al. Molecular signature and therapeutic perspective of the epithelial-to-mesenchymal transitions in epithelial cancers. Drug Resist Updat, 2008,11(4-5):123-151.
7
Wendt MK, Allington TM, Schiemann WP. Mechanisms of the epithelial-mesenchymal transition by TGF-beta. Future Oncol,2009,5(8):1145-1168.
8
Yang AD, Fan F, Camp ER, et al. Chronic oxaliplatin resistance induces epithelial-to-mesenchymal transition in colorectal cancer cell lines. Clin Cancer Res, 2006,12(14 Pt 1):4147-4153.
9
Kajiyama H, Shibata K, Terauchi M, et al. Chemoresistance to paclitaxel induces epithelial-mesenchymal transition and enhances metastatic potential for epithelial ovarian carcinoma cells. Int J Oncol, 2007,31(2):277-283.
10
Hiscox S, Jiang WG, Obermeier K, et al. Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation. Int J Cancer, 2006,18(2):290-301.
11
Güngör C, Zander H, Effenberger KE, et al. Notch signaling activated by replication stress-induced expression of midkine drives epithelial-mesenchymal transition and chemoresistance in pancreatic cancer. Cancer Res, 2011,71(14):5009-5019.
12
Guttilla IK, Phoenix KN, Hong X, et al. Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs. Breast Cancer Res Treat, 2012,132(1):75-85.
13
Hoshino H, Miyoshi N, Nagai K, et al. Epithelialmesenchymal transition with expression of SNAI1-induced chemoresistance in colorectal cancer. Biochem Biophys Res Commun, 2009,390(3):1061-1065.
14
Su HY, Lai HC, Lin YW, et al. Epigenetic silencing of SFRP5 is related to malignant phenotype and chemoresistance of ovarian cancer through Wnt signaling pathway. Int J Cancer,2010,127(3):555-567.
15
Miettinen PJ, Ebner R, Lopez AR, et al. TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of typeⅠreceptors. J Cell Biol, 1994,127(6 Pt 2):2021-2036.
16
Yu C, Liu Y, Huang D, et al. TGF-β1 mediates epithelial to mesenchymal transition via the TGF-β/Smad pathway in squamous cell carcinoma of the head and neck. Oncol Rep,2011,25(6):1581-1587.
17
Pirozzi G, Tirino V, Camerlingo R, et al. Epithelial to mesenchymal transition by TGF-β1 induction increases stemness characteristics in primary non small cell lung cancer cell line.PLoS One, 2011,6(6):e21548.
18
Wendt MK, Smith JA, Schiemann WP. Transforming growth factor-β-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression.Oncogene, 2010,29(49):6485-6498.
19
Richter P, Umbreit C, Franz M, et al. EGF/TGFβ1 costimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332. J Oral Pathol Med, 2011,40(1):46-54.
20
Azarova AM,Lyu YL,Lin CP,et al.Roles of DNA topoisomeraseⅡisozymes in chemotherapy and secondary malignancies. Proc Natl Acad Sci U S A, 2007,104(26):11014-11019.
21
Kim SH, Bark H, Choi CH. Mercury induces multidrug resistance-associated protein gene through p38 mitogen-activated protein kinase. Toxicol Lett, 2005,155(1):143-150.
22
Guo X, Ma N, Wang J, et al. Increased p38-MAPK is responsible for chemotherapy resistance in human gastric cancer cells. BMC Cancer, 2008,8:375.
23
Meyerzu Schwabedissen HE, Grube M, Dreisbach A, et al.Epidermal growth factor-mediated activation of the map kinase cascade results in altered expression and function of ABCG2(BCRP). Drug Metab Dispos, 2006,34(4):524-533.
24
Yoon H, Min JK, Lee JW, et al. Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2. Biochem Biophys Res Commun, 2011,405(3):333-337.
25
Nickl-Jockschat T, Arslan F, Doerfelt A, et al. An imbalance between Smad and MAPK pathways is responsible for TGF-beta tumor promoting effects in high-grade gliomas. Int J Oncol,2007,30(2):499-507.
26
Song J. EMT or apoptosis: a decision for TGF-β. Cell Res,2007,17(4):289-290.
[1] 王雪菲, 海琳悦, 李立方, 肖春花. Luminal A型乳腺癌的内分泌治疗与化疗[J/OL]. 中华乳腺病杂志(电子版), 2023, 17(05): 294-300.
[2] 林昌盛, 战军, 肖雪. 上皮性卵巢癌患者诊疗中基因检测及分子靶向药物治疗[J/OL]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 505-510.
[3] 涂家金, 廖武强, 刘金晶, 涂志鹏, 毛远桂. 严重烧伤患者鲍曼不动杆菌血流感染的危险因素及预后分析[J/OL]. 中华损伤与修复杂志(电子版), 2023, 18(06): 491-497.
[4] 袁柳凤, 徐文绮, 朱小宇, 王慧珠, 伦文辉. 283株淋球菌对七种常见抗菌药物的耐药性分析[J/OL]. 中华实验和临床感染病杂志(电子版), 2024, 18(02): 108-112.
[5] 韦涌涛, 王松霞, 苏爱美, 王东平. 耐碳青霉烯类铜绿假单胞菌耐药性及联合药敏试验研究[J/OL]. 中华实验和临床感染病杂志(电子版), 2024, 18(01): 43-48.
[6] 张海金, 王增国, 蔡慧君, 赵炳彤. 2020至2022年西安市儿童医院新生儿细菌感染分布及耐药监测分析[J/OL]. 中华实验和临床感染病杂志(电子版), 2023, 17(04): 222-229.
[7] 尚峰进, 陈陆尧, 刘亚星, 张浩然, 连长红. 肿瘤相关中性粒细胞在胃癌发生发展和治疗中的研究进展[J/OL]. 中华普通外科学文献(电子版), 2024, 18(01): 58-61.
[8] 陈浩, 王萌. 胃印戒细胞癌的临床病理特征及治疗选择的研究进展[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 108-111.
[9] 巨春蓉, 门同义, 薛武军. 实体器官移植后难治性/耐药性巨细胞病毒感染诊疗进展[J/OL]. 中华移植杂志(电子版), 2024, 18(02): 86-92.
[10] 胡思平, 熊性宇, 徐航, 杨璐. 衰老相关分泌表型因子在前列腺癌发生发展中的作用机制[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 425-434.
[11] 赛甫丁·艾比布拉, 买买提·依斯热依力, 李义亮, 王永康, 王志, 克力木·阿不都热依木. 不同材质补片修补对腹壁疝大鼠腹横筋膜组织转化生长因子-β1及Collagen合成代谢的作用[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(02): 161-167.
[12] 邢嘉翌, 龚佳晟, 祝佳佳, 陆群. 肺癌化疗患者继发肺部感染的病原菌耐药性及炎症因子变化分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 714-718.
[13] 杨慧, 郭丽娟, 冯晓丹, 李静, 黄成谋, 蔡兴锐, 覃英娇, 王远礼. 非小细胞肺癌铂类药物耐药mi RNA表达特征及预测分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 719-724.
[14] 王蕊, 林先萍, 李盼盼. 铜绿假单胞菌感染肺炎菌血症危险因素及耐药性分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(03): 478-480.
[15] 蔡小芳, 高慧, 葛军, 邢慧芸, 庄小燕, 李小丁. 多重耐药性肺结核治疗依从性预测分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(01): 51-56.
阅读次数
全文


摘要