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背景与目的:香烟烟雾诱导的炎性反应可显著促进肺肿瘤的生长,本研究通过体内外实验明确香烟烟雾激活肺癌细胞Wnt/β-catenin信号途径的机制。方法:利用尾静脉注射鼠Lewis肺癌细胞方法建立小鼠肺癌模型,然后采用香烟烟雾对其进行刺激,诱发炎性反应。免疫组化方法检测小鼠和人肺肿瘤组织中CD68、肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)和无磷酸化β-catenin的表达。采用Transwell插入式细胞培养皿共培养人巨噬细胞和人肺癌细胞株A549细胞。采用蛋白质印迹法(Western blot)检测A549细胞中无磷酸化β-catenin的表达及磷酸化的GSK-3β和Akt蛋白的表达。结果:免疫组化结果显示,香烟烟雾刺激肺肿瘤负荷小鼠后,其肿瘤组织中巨噬细胞CD68+、TNF-α和无磷酸化β-catenin的表达均明显高于对照组。肺癌患者肿瘤组织中,CD68和TNF-α的阳性表达均明显高于正常组织。肺腺癌和鳞癌的无磷酸化β-catenin的染色阳性率分别为68.9%和62.2%,其阳性率与患者吸烟与否及肿瘤分期有关,差异有统计学意义(P均<0.05),并且CD68+的细胞数在无磷酸化β-catenin染色阳性的标本中显著升高(P<0.01)。Western blot检测结果显示,不同浓度香烟抽提物(cigarette smoke extract,CSE)刺激共培养细胞后,A549细胞中无磷酸化β-catenin的表达随着CSE浓度的增加而显著上升。在加入TNF-α中和抗体后,A549细胞中无磷酸化β-catenin的表达被抑制。用TNF-α处理A549细胞后,磷酸化的GSK-3β和Akt蛋白在2 h后表达增加,4 h后无磷酸化β-catenin蛋白表达也开始上调。结论:香烟烟雾诱导肺肿瘤组织中巨噬细胞释放炎性反应因子TNF-α,进而通过Akt/GSK-3β途径激活肿瘤细胞中Wnt/β-catenin信号通路。
BACKGROUND & OBJECTIVE: The inflammatory response induced by cigarette smoke can significantly promote the growth of lung cancer. In this study, the mechanism of cigarette smoke-activated Wnt / β-catenin signaling in lung cancer cells was studied in vitro and in vivo. Methods: The mouse lung cancer model was established by injecting murine Lewis lung cancer cells through the tail vein, and then the lung cancer model was induced by cigarette smoke to induce inflammatory reaction. Immunohistochemistry was used to detect the expression of CD68, tumor necrosis factor alpha (TNF-α) and phosphorylated β-catenin in mouse and human lung tumors. Human macrophages and human lung cancer cell line A549 cells were co-cultured with Transwell® plug-in cell culture dishes. The expression of phosphorylated β-catenin and phosphorylated GSK-3β and Akt protein in A549 cells were detected by Western blot. Results: The results of immunohistochemistry showed that the expressions of CD68 +, TNF- α and phosphorylated β-catenin in macrophages of tumor-bearing mice after cigarette smoke-stimulated lung tumor-bearing mice were significantly higher than those of control group. The positive expression of CD68 and TNF-α in lung cancer patients was significantly higher than that in normal tissues. The positive rates of non-phosphorylated β-catenin staining in lung adenocarcinoma and squamous cell carcinoma were 68.9% and 62.2%, respectively. The positive rates were related to smoking status and tumor stage (P <0.05) And the number of CD68 + cells was significantly increased in non-phosphorylated β-catenin staining (P <0.01). Western blot results showed that the expression of phosphorylated β-catenin in A549 cells was significantly increased with the increase of CSE concentration after different concentrations of cigarette smoke extract (CSE) stimulated cocultured cells. After addition of TNF-α neutralizing antibody, the expression of phosphorylated β-catenin was inhibited in A549 cells. Phosphorylated GSK-3β and Akt protein increased after 2 h treatment with TNF-α, and the expression of phosphorylated β-catenin began to rise after 4 h. CONCLUSION: The cigarette smoke induces the release of the inflammatory response factor TNF-α by macrophages in lung cancer tissues, and then activates the Wnt / β-catenin signaling pathway in tumor cells through the Akt / GSK-3β pathway.