Yong Xiao, Wang Peiqin, Jiang Tao, Yu Wenchen, Shang Yan, Han Yiping, Zhang Pingping, Li Qiang
Department of Pneumology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Chin Med J (Engl). 2014;127(11):2091-6.
Non-small cell lung cancer (NSCLC) is the most common lung malignancy worldwide. The metastatic potential of NSCLC cells has been shown to be associated with the tumor microenvironment, which consists of tumor cells, stroma, blood vessels, immune infiltrates and the extracellular matrix. Fibroblasts can produce numerous extracellular matrix molecules and growth factors. Gefitinib has been evaluated as a first-line treatment in selected patients, and it has shown favorable efficacy especially in NSCLC, but it is not effective for everyone.
In this study, we examined the antitumor activity of gefitinib on lung fibroblasts co-cultured of lung cancer cells. A series of co-culture experiments that employed cell counting kit-8 (CCK8), transwells, real-time polymerase chain reaction (RT-PCR) and Western blotting with HFL-1 fibroblasts and A549 human lung carcinoma cells were performed to learn more about tumor cell proliferation, migration and invasion; and to determine any change of epithelial mesenchymal transition (EMT)-associated tumor markers vimentin, matrix metallopro-teinase 2 (MMP2) and chemotaxis cytokines receptor 4 (CXCR4) mRNA levels.
A549 cell proliferation in the presence of HFL-1 cells was not significantly increased compared with A549 cells alone, but A549 cell spheroid body formation was increased after co-culture, and treatment with gefitinib increased further. Our study also revealed that fibroblasts attenuated the lung cancer cell inhibition ratio of migration and invasion after gefitinib treatment in vitro. To further study this mechanism, RT-PCR analysis showed that vimentin, MMP2 and CXCR4 mRNA levels were more highly expressed in the lung cancer cells after co-culture, but did not obviously decrease compared with the control cells following gefitinib treatment. This suggests the mechanism by which fibroblasts attenuate gefitinib-induced expression of EMT-associated tumor markers. Finally, our results demonstrated that co-culture with A549 lung cancer cells does not alter the cell cycle distribution of HFL-1 fibroblasts. Furthermore, HFL-1 fibroblasts had no effect on the cell cycle distribution of HFL-1 cells treated with gefitinib.
Gefitinib has lower anti-tumor activity on A549 lung cancer cells when co-cultured with HFL-1 fibroblasts.
非小细胞肺癌（NSCLC）是全球最常见的肺部恶性肿瘤。NSCLC细胞的转移潜能已被证明与肿瘤微环境有关，肿瘤微环境由肿瘤细胞、基质、血管、免疫浸润细胞和细胞外基质组成。成纤维细胞可产生多种细胞外基质分子和生长因子。吉非替尼已被评估用于特定患者的一线治疗，尤其在NSCLC中显示出良好疗效，但并非对所有人都有效。
在本研究中，我们检测了吉非替尼对与肺癌细胞共培养的肺成纤维细胞的抗肿瘤活性。进行了一系列共培养实验，采用细胞计数试剂盒-8（CCK8）、Transwell小室、实时聚合酶链反应（RT-PCR）以及对HFL-1成纤维细胞和A549人肺癌细胞进行蛋白质印迹分析，以进一步了解肿瘤细胞的增殖、迁移和侵袭情况；并确定上皮-间质转化（EMT）相关肿瘤标志物波形蛋白、基质金属蛋白酶2（MMP2）和趋化因子受体4（CXCR4）mRNA水平的任何变化。
与单独的A549细胞相比，在HFL-1细胞存在的情况下，A549细胞的增殖没有显著增加，但共培养后A549细胞球状体形成增加，用吉非替尼处理后进一步增加。我们的研究还表明，在体外，成纤维细胞减弱了吉非替尼处理后肺癌细胞的迁移和侵袭抑制率。为进一步研究此机制，RT-PCR分析显示，共培养后肺癌细胞中波形蛋白、MMP2和CXCR4 mRNA水平表达更高，但与吉非替尼处理后的对照细胞相比没有明显降低。这提示了成纤维细胞减弱吉非替尼诱导的EMT相关肿瘤标志物表达的机制。最后，我们的结果表明，与A549肺癌细胞共培养不会改变HFL-1成纤维细胞的细胞周期分布。此外，HFL-1成纤维细胞对用吉非替尼处理的HFL-1细胞的细胞周期分布没有影响。
与HFL-1成纤维细胞共培养时，吉非替尼对A549肺癌细胞的抗肿瘤活性较低。
