二氯化钴诱导唾液腺腺样囊性癌ACC-M细胞自噬的实验研究

摘要/Abstract

摘要： 目的：研究二氯化钴(CoCl2)对唾液腺腺样囊性癌ACC-M细胞增殖和自噬的影响。方法：应用MTT法对ACC-M细胞进行细胞活力测验，以检测CoCl2对细胞增殖的抑制作用；透射电镜观察自噬体形成；双免疫荧光标记、实时定量PCR（RT-PCR）及Western 蛋白免疫印迹检测自噬相关基因HIF-1α/BNIP3通路相关基因、自噬相关蛋白微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3, LC3）及Beclin 1的表达。采用SPSS15.0软件包对数据进行双尾 t检验。结果：CoCl2可降低ACC-M细胞的活性，同时诱导自噬体的形成；透射电镜下，实验组细胞内可见自噬体样的双层膜结构；RT-PCR及Western 蛋白免疫印迹检测显示，CoCl2可使HIF-1α/BNIP3信号通路和自噬相关蛋白的表达显著提高。结论：CoCl2可模拟低氧环境，从而诱导ACC-M细胞产生自噬，HIF-1α/BNIP3信号通路在这一过程中扮演着重要角色。

关键词: 二氯化钴, 自噬, 腺样囊性癌, 低氧, HIF-1α

Abstract: PURPOSE : Adenoid cystic carcinoma (ACC) is one of the common types of salivary gland malignancies in the head and neck. Our previous study confirmed that autophagy is closely related to tumorigenesis of ACC. This study investigated the expression of Cobalt chloride (CoCl2)-induced autophagy and its role in tumor invasion. METHODS : Autophagosome formation and upregulation of autophgy-related microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 were detected in ACC-M cells in response to CoCl2. HIF-1α/BNIP3 signaling pathway was investigated in hypoxia-induced autophagy in ACC. Statistical comparisons between groups were performed using two-tailed Student’s t test with SPSS 15.0 software package. RESULTS : CoCl2 reduced cell viability of ACC-M cells, and induced formation of autophagosomes. RT-PCR and Western blot showed that the expression of HIF-1α/BNIP3 signal pathway of autophagy related protein was significantly increased. CONCLUSIONS : The results revealed that CoCl2 mimetic hypoxia could induce autophagy in ACC, and HIF-1α/BNIP3 pathway plays an important role in activation of hypoxia-induced autophagy in ACC.

Key words: Cobalt chloride, Autophagy, Adenoid cystic carcinoma, Hypoxia, HIF-1α

中图分类号:

R739.8

尹长伟，吴海威，黄圣运，马猛，陈占伟，张东升,. 二氯化钴诱导唾液腺腺样囊性癌ACC-M细胞自噬的实验研究[J]. 中国口腔颌面外科杂志, 2015, 13(6): 491-496.

YIN Chang-wei, WU Hai-wei, HUANG Sheng-yun, Ma Meng, CHEN Zhan-wei, ZHANG Dong-sheng,.. Cobalt chloride induced autophagy in salivary adenoid cystic carcinoma[J]. China J Oral Maxillofac Surg, 2015, 13(6): 491-496.

参考文献

[1] Adelstein DJ, Koyfman SA, El-Naggar AK, et al. Biology and management of salivary gland cancers[J]. Semin Radiat Oncol, 2012, 22(3): 245-253.
[2] Dodd RL, Slevin NJ. Salivary gland adenoid cystic carcinoma: a review of chemotherapy and molecular therapies[J]. Oral Oncol, 2006, 42(8): 759-769.
[3] Bradley PJ. Adenoid cystic carcinoma of the head and neck: a review [J]. Curr Opin Otolaryngol Head Neck Surg, 2004, 12(2):127-132.
[4] Janssen HL, Haustermans KM, Balm AJ, et al. Hypoxia in head and neck cancer: how much, how important? [J].Head Neck, 2005, 27(7): 622-638.
[5] Rouschop KM, Wouters BG. Regulation of autophagy through multiple independent hypoxic signaling pathways [J]. Curr Mol Med, 2009, 9(4): 417-424.
[6] 刘晓东, 邓廉夫, 王君, 等. 低氧诱导因子-1α 对成骨细胞功能的调控作用[J]. 上海交通大学学报（医学版）, 2007, 27(3): 298-302.
[7] Toustrup K, S?rensen BS, Alsner J, et al. Hypoxia gene expression signatures as prognostic and predictive markers in head and neck radiotherapy [J]. Semin Radiat Oncol, 2012, 22(2): 119-127.
[8] Adams JM, Difazio LT, Rolandelli RH, et al. HIF-1: a key mediator in hypoxia (Review) [J]. Acta Physiol Hung, 2009, 96(1): 19-28.
[9] Semenza GL. HIF-1 and mechanisms of hypoxia sensing [J]. Curr Opin Cell Biol, 2001, 13(2): 167-171.
[10] Brennan PA, Mackenzie N, Quintero M. Hypoxia-inducible factor 1α in oral cancer [J]. J Oral Pathol Med, 2005, 34(7): 385-389.
[11] Costa AF, Tasso MG, Mariano FV, et al. Levels and patterns of expression of hypoxia-inducible factor-1α, vascular endothelial growth factor, glucose transporter-1 and CD105 in adenoid cystic carcinomas with high-grade transformation [J]. Histopathology, 2012, 60(5): 816-825.
[12] Zhou C, Liu J, Tang Y, et al. Coexpression of hypoxia-inducible factor-2α, TWIST2, and SIP1 may correlate with invasion and metastasis of salivary adenoid cystic carcinoma [J]. J Oral Pathol Med, 2012, 41(5): 424-431.
[13] Chen N, Karantza V. Autophagy as a therapeutic target in cancer [J]. Cancer Biol Ther, 2011, 11(2): 157-168.
[14] 吴媛媛,刘海意,乔福元.二氯化钴化学缺氧对细胞滋养层细胞凋亡及浸润的影响 [J]. 生殖与避孕,2009, 29(6): 354 -356.
[15] Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting [J]. Autophagy, 2007, 3(6) : 542-545.
[16] Jiang L, Huang S, Li W, et al. Expression of autophagy and ER stress-related proteins in primary salivary adenoid cystic carcinoma [J]. Pathol Res Pract, 2012, 208(11): 635-641.
[17] 权晶晶, 凌均棨. 二氯化钴在体外细胞缺氧研究中的应用 [J]. 国际口腔医学杂志, 2009, 36(4): 455-458.
[18] Mazure NM, Pouyss??ur J. Hypoxia-induced autophagy: cell death or cell survival?[J]. Curr Opin Cell Biol,2010,22(2):177-180.
[19] Bellot G, Garcia-Medina R, Gounon P, et al. Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains [J]. Mol Cell Biol, 2009, 29(10): 2570-2581.
[20] Azad MB, Gibson SB. Role of BNIP3 in proliferation and hypoxia-induced autophagy: implications for personalized cancer therapies [J]. Ann N Y Acad Sci, 2010, 1210: 8-16.
[21] 梁军, 黄志权, 游云华, 等. HIF-1α和VEGF的表达与腺样囊性癌临床病理的相关性研究[J]. 中国口腔颌面外科杂志, 2010, 8(6): 548-554.
[22] 李德超, 董洪宇, 于丽. 腮腺腺样囊性癌中HIF-1α和MMP-7的表达及意义[J]. 口腔医学研究, 2010, 26(2): 256-258.
[23] Janssen HL, Haustermans KM, Balm AJ, et al. Hypoxia in head and neck cancer: how much, how important?[J]. Head Neck, 2005, 27(7): 622-638.
[24] Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia [J]. Proc Natl Acad Sci USA, 1993, 90(9): 4304-4308.
[25] Song ZC, Zhou W, Shu R, et al. Hypoxia induces apoptosis and autophagic cell death in human periodontal ligament cells through HIF-1α pathway [J]. Cell Prolif, 2012, 45(3): 239-248.
[26] Naves T, Jawhari S, Jauberteau MO, et al. Autophagy takes place in mutated p53 neuroblastoma cells in response to hypoxia mimetic CoCl2[J]. Biochem Pharmacol, 2013, 85(8): 1153-1161.
[27] Chinnadurai G, Vijayalingam S, Gibson SB. BNIP3 subfamily BH3-only proteins: mitochondrial stress sensors in normal and pathological functions[J]. Oncogene, 2008, 27(Suppl 1): S114-127.
[28] Zhao X, He Y, Chen H. Autophagic tumor stroma: mechanisms and roles in tumor growth and progression [J]. Int J Cancer, 2013, 132(1): 1-8.
[29] Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing [J]. EMBO J, 2000, 19(21): 5720-5728.
[30] Maiuri MC, Le Toumelin G, Criollo A, et al. Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1 [J]. EMBO J, 2007, 26(10): 2527-2539.
[31] Maiuri MC, Criollo A, Tasdemir E, et al. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-XL [J]. Autophagy, 2007, 3(4): 374-376.