苯并芘上调食管鳞癌环氧化酶-2表达诱导M2型巨噬细胞浸润增加机制

doi:10.3969/j.issn.1006-7795.2023.05.008

摘要/Abstract

摘要： 目的研究有吸烟史的食管鳞癌患者肿瘤微环境中M2型肿瘤相关巨噬细胞浸润增加的潜在机制。方法通过免疫组织化学法以及免疫浸润分析（CIBERSORT）数据库分析检测M2型肿瘤相关巨噬细胞在有/无吸烟史的食管鳞癌患者中表达差异。通过癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库检测有吸烟史的食管鳞癌患者肿瘤组织中参与调控巨噬细胞招募与极化相关的环氧化酶-2（cyclooxygenase-2，COX2）表达变化。使用烟草中主要致癌物苯并芘处理食管鳞癌细胞，通过蛋白印迹（Western blotting）与实时荧光定量聚合酶链反应法（real time quantitative polymerase chain reaction, RT-qPCR）法检测COX2表达变化。通过Transwell法评估苯并芘刺激前后对肿瘤细胞上清对巨噬细胞招募作用的影响。构建肿瘤细胞上清-巨噬细胞共培养体系，使用Western blotting法与RT-qPCR法评估苯并芘刺激前后肿瘤细胞上清对巨噬细胞M2型标志物表达变化。通过观察巨噬细胞形态变化辅助判断其极化情况。结果有吸烟史的食管鳞癌患者M2型巨噬细胞浸润明显升高；在烟草中主要致癌物苯并芘刺激下，食管鳞癌细胞中COX2明显增多；经苯并芘刺激后肿瘤细胞上清对巨噬细胞招募作用明显增强；经苯并芘刺激后肿瘤细胞上清处理巨噬细胞后可诱导其向M2型巨噬细胞极化。结论在烟草中主要致癌物苯并芘的刺激下，食管鳞癌细胞可诱导巨噬细胞进入肿瘤微环境并诱导其向M2型肿瘤相关巨噬细胞极化。

关键词: 食管鳞癌, 吸烟, 苯并芘, 环氧化酶-2, 肿瘤相关巨噬细胞

Abstract: Objective To investigate the potential mechanism of increased infiltration of M2 tumor-associated macrophages in the tumor microenvironment of esophageal squamous carcinoma patients with a history of smoking. Methods Differential expression of M2 tumor-associated macrophages in esophageal squamous carcinoma patients with/without a history of smoking was examined by immunohistochemistry and CIBERSORT database analysis. The expression of cyclooxygenase-2（COX2）involved in the regulation of macrophage recruitment and polarization was detected in tumor tissues of esophageal squamous cell carcinoma（ESCC）patients with a history of smoking by The Cancer Genome Atlas (TCGA)database. Esophageal squamous carcinoma cell lines were treated with benzo(a)pyrene, the major carcinogen in tobacco. Changes in the expression of COX2 of ESCC cell lines were detected by Western blotting and real time quantitative PCR (RT-qPCR). The effect on macrophage recruitment by tumor cell conditional medium was assessed by Transwell assay before and after benzo(a)pyrene stimulation. A co-culture system of tumor cell conditional medium and macrophage was established. Western blotting and RT-qPCR were used to evaluate the expression changes of M2 tumor associated macrophages （TAM）markers. Macrophage morphological changes were observed to assist in determining their polarization. Results The infiltration of M2 macrophages was significantly increased in patients with a history of smoking. COX2 was significantly increased in esophageal squamous carcinoma cells stimulated by benzo(a)pyrene, the major carcinogen in tobacco. The recruitment of macrophages by tumor cell conditional medium was significantly increased after stimulation with benzo(a)pyrene. The treatment of macrophages with tumor cell conditional medium after stimulation with benzo(a)pyrene induced their polarization towards M2 macrophages. Conclusions Stimulation of esophageal squamous carcinoma cell lines with benzo(a)pyrene induces macrophages to enter the tumor microenvironment and induces polarization towards M2 tumor-associated macrophages.

Key words: esophageal squamous cell carcinoma, smoking, benzo(a)pyrene, cyclooxygenase-2, tumor associated macrophages

中图分类号:

R735.1

肖泽儒, 闫锐, 梁紫葳, 葛洋, 安广宇. 苯并芘上调食管鳞癌环氧化酶-2表达诱导M2型巨噬细胞浸润增加机制[J]. 首都医科大学学报, doi: 10.3969/j.issn.1006-7795.2023.05.008.

Xiao Zeru, Yan Rui, Liang Ziwei, Ge Yang, An Guangyu. Major tobacco carcinogen benzo(a)pyrene induces infiltration of M2 type macrophages by upregulating cyclooxygenase-2 expression in esophageal squamous carcinoma[J]. Journal of Capital Medical University, doi: 10.3969/j.issn.1006-7795.2023.05.008.

参考文献

［1］Abnet C C, Arnold M, Wei W Q. Epidemiology of esophageal squamous cell carcinoma［J］. Gastroenterology, 2018, 154(2): 360-373.
［2］Sun D, Li H, Cao M, et al. Cancer burden in China: trends, risk factors and prevention［J］. Cancer Biol Med，2020, 17(4): 879-895.
［3］Simba H, Kuivaniemi H, Lutje V, et al. Systematic review of genetic factors in the etiology of esophageal squamous cell carcinoma in African populations［J］. Front Genet, 2019, 10: 642.
［4］Li W, Hu J, Adebali O, et al. Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo［a］pyrene［J］. PNAS, 2017, 114(26): 6752-6757.
［5］Hakami R, Mohtadinia J, Etemadi A, et al. Dietary intake of benzo(a)pyrene and risk of esophageal cancer in north of Iran［J］. Nutr Cancer, 2008, 60(2): 216-221.
［6］Mantovani A, Allavena P, Marchesi F, et al. Macrophages as tools and targets in cancer therapy［J］.Nat Rev Drug Discov, 2022, 21(11): 799-820.
［7］Christofides A, Strauss L, Yeo A, et al. The complex role of tumor-infiltrating macrophages［J］.Nat Immunol, 2022, 23(8): 1148-1156.
［8］Cheng C, Wang P, Yang Y, et al. Smoking-induced M2-TAMs, via circEML4 in EVs, promote the progression of NSCLC through ALKBH5-regulated m6A modification of SOCS2 in NSCLC cells［J］. Adv Sci (Weinh), 2023: e2300953.
［9］Castro C, Peleteiro B, Lunet N. Modifiable factors and esophageal cancer: a systematic review of published meta-analyses［J］. J Gastroenterol, 2018, 53(1): 37-51.
［10］Qiu F, Liang C L, Liu H, et al. Impacts of cigarette smoking on immune responsiveness: up and down or upside down?［J］. Oncotarget, 2017, 8(1): 268-284.
［11］Wang G, Pan C, Cao K, et al. Impacts of cigarette smoking on the tumor immune microenvironment in esophageal squamous cell carcinoma［J］. J Cancer, 2022, 13(2): 413-425.
［12］Madeen E, Siddens L K, Uesugi S, et al. Toxicokinetics of benzo［a］pyrene in humans: extensive metabolism as determined by UPLC-accelerator mass spectrometry following oral micro-dosing［J］. Toxicol Appl Pharmacol, 2019, 364: 97-105.
［13］Wei Y, Zhao L, He W, et al. Benzo［a］pyrene promotes gastric cancer cell proliferation and metastasis likely through the aryl hydrocarbon receptor and ERK-dependent induction of MMP9 and c-myc［J］. Int J Oncol, 2016, 49(5): 2055-2063.
［14］Salem M L, El-ashmawy N E, Abd El-fattah E E, et al. Immunosuppressive role of benzo［a］pyrene in induction of lung cancer in mice［J］. Chem Biol Interact, 2021, 333: 109330.
［15］Wang H, Liu B, Chen H, et al. Dynamic changes of DNA methylation induced by benzo(a)pyrene in cancer［J］. Genes Environ, 2023, 45(1): 21.
［16］Mantovani A, Allavena P, Sica A, et al. Cancer-related inflammation［J］. Nature, 2008, 454(7203): 436-444.
［17］Liu Q, Tong D, Liu G, et al. Metformin inhibits prostate cancer progression by targeting tumor-associated inflammatory infiltration［J］. Clin Cancer Res, 2018, 24(22): 5622-5634.
［18］Mcneil J J, Nelson M R, Woods R L, et al. Effect of aspirin on all-cause mortality in the healthy elderly［J］. N Engl J Med, 2018, 379(16): 1519-1528.
［19］Meyerhardt J A, Shi Q, Fuchs C S, et al. Effect of celecoxib vs placebo added to standard adjuvant therapy on disease-free survival among patients with stage Ⅲ colon cancer: the CALGB/SWOG 80702 (alliance) randomized clinical trial［J］. JAMA, 2021, 325(13): 1277-1286.

[1]	李雪, 邢洁, 张倩, 李鹏, 吕富靖, 张澍田. 窄带成像技术联合放大内镜JES分型对早期食管癌的诊断价值[J]. 首都医科大学学报, 2022, 43(2): 210-215.
[2]	范晓娜, 姚健楠, 葛洋, 安广宇, 李鹰. DCLK1敲除对食管鳞癌细胞迁移和侵袭能力的影响[J]. 首都医科大学学报, 2019, 40(3): 417-423.
[3]	朱圣韬, 孙秀静, 李鹏, 郭庆东, 朱圣泉, 张澍田. PHF8基因对食管鳞状细胞癌裸鼠移植瘤生长的影响[J]. 首都医科大学学报, 2016, 37(1): 12-16.
[4]	孙秀静, 原标, 朱圣韬, 李鹏, 徐有青. 沉默PHF8重组慢病毒的制备及其对食管鳞癌细胞增生的影响[J]. 首都医科大学学报, 2014, 35(1): 122-128.
[5]	王文莉;段华;张颖;郭银树;成九梅. 子宫腺肌病PGP 9.5、NGF的表达及其与痛经的关系[J]. 首都医科大学学报, 2012, 33(1): 11-15.
[6]	张莉;张澍田;于中麟;李鹏;宗晔;许彩民;华方圆;韩兵社. RNA干扰阻断COX-2基因表达对食管鳞状上皮癌EC109细胞株增生和凋亡的影响[J]. 首都医科大学学报, 2008, 29(3): 295-299.
[7]	林瑾;张澍田. 阿司匹林对食管鳞癌细胞株致凋亡的作用及对烟草提取物作用的影响[J]. 首都医科大学学报, 2006, 27(4): 431-434.
[8]	李鹏;张澍田;于中麟;刘萱;张莉;周巧直;林瑾;胡要君;许彩民. 尼美舒利对食管鳞癌细胞的增生抑制和诱导凋亡作用[J]. 首都医科大学学报, 2006, 27(4): 438-440.
[9]	周巧直;张澍田. 烟草提取物与环氧化酶-2对食管鳞癌细胞株增生作用的研究[J]. 首都医科大学学报, 2006, 27(4): 450-453.
[10]	李海燕;王小平;陈瑞芬;刘国贞;宋爱利. 人食管癌组织中热休克蛋白70和糖调节蛋白94的表达[J]. 首都医科大学学报, 2004, 25(2): 183-185.
[11]	曹秋梅;龚宝丽;刘宾. 内镜下碘染色对早期食管癌的诊断价值[J]. 首都医科大学学报, 2003, 24(2): 153-154.
[12]	战烨;曾辉;张勇;王植平;赵相印. 食道鳞癌凋亡相关基因表达及原位凋亡的同步检测[J]. 首都医科大学学报, 1999, 20(2): 95-98.
[13]	高志;王天佑;蔡执敏. 青年食管癌患者临床特点的探讨[J]. 首都医科大学学报, 1997, 18(3): 253-255.
[14]	王青釭, 孟莹, 朱圣韬, 施海韵, 张澍田. 启动子甲基化调节在低表达环氧合酶-2的食管鳞癌移植模型中的作用[J]. 首都医科大学学报, 2014, 35(6): 798-804.