Zhong Bei, Li Yu, Liu Xiaodong, Wang Dongsheng
Department of Hyperbaric Oxygen, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
Department of General Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
Oncol Lett. 2018 Jan;15(1):755-764. doi: 10.3892/ol.2017.7380. Epub 2017 Nov 9.
The present study aimed to determine the expression of mast cells, C-C motif chemokine ligand 2 (CCL-2) and C-C motif chemokine receptor 2 (CCR2) in gastric cancer tumor tissue; and the association of mast cells with the proliferation, migration, invasion and apoptosis of gastric cancer cells. In addition, whether the stem cell factor (SCF)/c-Kit pathway was associated with the secretion of CCL-2 by gastric cancer cells was explored. Flow cytometry analysis and immunohistochemistry were used to observe the relative number of mast cells, and reverse transcription-quantitative polymerase chain reaction and western blot analysis were utilized to determine the expression of CCL-2 and CCR2 mRNA and protein. Following the co-culture of the mast cell line HMC-1 and the gastric cancer cell line BGC-823, a Transwell assay was used to validate the effect of mast cells on the migration and invasion of gastric cancer cells. Furthermore, Cell Counting kit-8 and dual acridine orange/ethidium bromide fluorescent staining assays were performed to determine the proliferation and apoptosis of gastric cancer cells, following co-culture with mast cells. The expression of SCF and c-Kit were also determined with a western blot analysis. A specific phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, was used to test the effect of PI3K inhibition on the secretion of CCL-2 in gastric cancer. The results demonstrated that the proportion of infiltrating mast cells, and the mRNA/protein expression of CCL-2 and CCR2, were significantly increased in tumor tissue relative to adjacent tissues. In addition, the migration and invasion of gastric cancer cells were significantly increased when mast cells were used as an attractant. When co-cultured with mast cells, the viability of gastric cancer cells was significantly increased and HO-induced apoptosis was inhibited. In gastric cancer tissue samples, the expression of SCF, c-Kit and phosphorylated (p)-Akt protein were significantly increased compared with normal adjacent tissues. It was hypothesized that SCF/c-Kit signaling pathway was activated by PI3K-Akt, resulting in an increase in the expression of CCL-2 mRNA and protein. Furthermore, it was demonstrated that CCL-2 mRNA and protein expression was significantly inhibited by treatment with the PI3K inhibitor wortmannin. Additionally, wortmannin intervention significantly inhibited gastric cancer cell migration and invasion. Therefore, the results of the present study demonstrated that mast cells may promote gastric cancer cell proliferation, migration and invasion, and inhibit apoptosis. In addition, the activation of the SCF/c-Kit signaling pathway was identified to promote the expression of CCL-2, which is associated with the development and metastasis of gastric cancer.
本研究旨在确定肥大细胞、C-C基序趋化因子配体2（CCL-2）和C-C基序趋化因子受体2（CCR2）在胃癌肿瘤组织中的表达；以及肥大细胞与胃癌细胞增殖、迁移、侵袭和凋亡的关联。此外，还探讨了干细胞因子（SCF）/c-Kit信号通路是否与胃癌细胞分泌CCL-2有关。采用流式细胞术分析和免疫组织化学观察肥大细胞的相对数量，利用逆转录-定量聚合酶链反应和蛋白质印迹分析确定CCL-2和CCR2 mRNA及蛋白的表达。将肥大细胞系HMC-1与胃癌细胞系BGC-823共培养后，采用Transwell实验验证肥大细胞对胃癌细胞迁移和侵袭的影响。此外，在与肥大细胞共培养后，进行细胞计数试剂盒-8实验和双吖啶橙/溴化乙锭荧光染色实验，以确定胃癌细胞的增殖和凋亡情况。还通过蛋白质印迹分析确定SCF和c-Kit的表达。使用特异性磷酸肌醇3-激酶（PI3K）抑制剂渥曼青霉素，检测PI3K抑制对胃癌细胞分泌CCL-2的影响。结果表明，相对于癌旁组织，肿瘤组织中浸润肥大细胞的比例以及CCL-2和CCR2的mRNA/蛋白表达均显著增加。此外，当以肥大细胞作为趋化剂时，胃癌细胞的迁移和侵袭显著增加。与肥大细胞共培养时，胃癌细胞的活力显著增加，且过氧化氢诱导的凋亡受到抑制。在胃癌组织样本中，与正常癌旁组织相比，SCF、c-Kit和磷酸化（p）-Akt蛋白的表达显著增加。据推测，PI3K-Akt激活了SCF/c-Kit信号通路，导致CCL-2 mRNA和蛋白表达增加。此外，证明渥曼青霉素处理可显著抑制CCL-2 mRNA和蛋白表达。此外，渥曼青霉素干预显著抑制胃癌细胞的迁移和侵袭。因此，本研究结果表明，肥大细胞可能促进胃癌细胞增殖、迁移和侵袭，并抑制凋亡。此外，已确定SCF/c-Kit信号通路的激活可促进CCL-2的表达，这与胃癌的发生和转移有关。
