Ying Li, Zhu Ziwei, Xu Zhiyun, He Tianrui, Li Encheng, Guo Zhe, Liu Fen, Jiang Chunmeng, Wang Qi
Department of Gastroenterology, the Second Hospital of Dalian Medical University, Dailan, China.
Department of Respiratory, the Second Hospital of Dalian Medical University, Dailan, China.
PLoS One. 2015 Jun 26;10(6):e0129593. doi: 10.1371/journal.pone.0129593. eCollection 2015.
Tumor stroma and growth factors provide a survival environment to tumor cells and can modulate their chemoresistance by dysregulating several signal pathways. In this study, we fabricated a three-dimensional (3D) microfluidic chip using polydimethylsiloxane (PDMS) to investigate the impact of hepatocyte growth factor (HGF) from cancer-associated fibroblasts (CAF) on the Met/PI3K/AKT activation, glucose regulatory protein (GRP78) expression and the paclitaxel-induced A549 cell apoptosis. With a concentration gradient generator, the assembled chip was able to reconstruct a tumor microenvironment in vitro. We found high levels of HGF in the supernatants of CAF and the CAF matrix from the supernatants of activated HFL1 fibroblasts or HGF enhanced the levels of Met, PI3K and AKT phosphorylation and GRP78 expression in A549 cells cultured in a 3D cell chamber, which was abrogated by anti-HGF. Inhibition of Met attenuated the CAF matrix-enhanced PI3K/AKT phosphorylation and GRP78 expression while inhibition of PI3K reduced GRP78 expression, but not Met phosphorylation in A549 cells. Inhibition of GRP78 failed to modulate the CAF matrix-enhanced Met/PI3K/AKT phosphorylation in A549 cells. Furthermore, inhibition of PI3K or GRP78 enhanced spontaneous and paclitaxel-induced A549 cell apoptosis. Moreover, treatment with the CAF matrix inhibited spontaneous and medium or high dose of paclitaxel-induced A549 cell apoptosis. Inhibition of PI3K or GRP78 attenuated the CAF matrix-mediated inhibition on paclitaxel-induced A549 cell apoptosis. Our data indicated that HGF in the CAF matrix activated the Met/PI3K/AKT and up-regulated GRP78 expression, promoting chemoresistance to paclitaxel-mediated apoptosis in A549 cells. Our findings suggest that the microfluidic system may represent an ideal platform for signaling research and drug screening.
肿瘤基质和生长因子为肿瘤细胞提供了生存环境，并可通过失调多种信号通路来调节其化疗耐药性。在本研究中，我们使用聚二甲基硅氧烷（PDMS）制作了一个三维（3D）微流控芯片，以研究来自癌症相关成纤维细胞（CAF）的肝细胞生长因子（HGF）对Met/PI3K/AKT激活、葡萄糖调节蛋白（GRP78）表达以及紫杉醇诱导的A549细胞凋亡的影响。借助浓度梯度发生器，组装好的芯片能够在体外重建肿瘤微环境。我们发现，CAF的上清液以及来自活化的HFL1成纤维细胞上清液中的CAF基质中存在高水平的HGF，且HGF增强了在3D细胞室中培养的A549细胞中Met、PI3K和AKT的磷酸化水平以及GRP78的表达，而抗HGF可消除这种作用。抑制Met可减弱CAF基质增强的PI3K/AKT磷酸化和GRP78表达，而抑制PI3K可降低GRP78表达，但对A549细胞中的Met磷酸化无影响。抑制GRP78未能调节CAF基质增强的A549细胞中Met/PI3K/AKT磷酸化。此外，抑制PI3K或GRP78可增强自发的和紫杉醇诱导的A549细胞凋亡。而且，用CAF基质处理可抑制自发的以及中、高剂量紫杉醇诱导的A549细胞凋亡。抑制PI3K或GRP78可减弱CAF基质介导的对紫杉醇诱导的A549细胞凋亡的抑制作用。我们的数据表明，CAF基质中的HGF激活了Met/PI3K/AKT并上调了GRP78表达，从而促进了A549细胞对紫杉醇介导的凋亡的化疗耐药性。我们的研究结果表明，微流控系统可能是信号研究和药物筛选的理想平台。
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