Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform.

Li Ying,Qi Wang,Encheng Li,Chunmeng Jiang,Zhiyun Xu,Tianrui He,Zhe Guo,Fen Liu,Ziwei Zhu,Eric Asselin

doi:10.1371/journal.pone.0129593

Abstract

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.

Highlights

The development of chemoresistance is a major obstacle for the successful treatment of nonsmall cell lung cancer (NSCLC)[1]
We examined the impact of Cancerassociated fibroblasts (CAF) or Hepatocyte growth factor (HGF) on the Met/phosphoinositide 3-kinase (PI3K)/AKT phosphorylation, Glucose-regulated protein 78 (GRP78) expression and paclitaxel-induced apoptosis in human non-small cell lung cancer A549 cells cultured in the 3D matrix
HGF can be produced by stromal fibroblasts in a tumor microenvironment and is responsible for activation of the HGF/c-Met signaling during the advancement of lung cancer [33]

Summary

Introduction

The development of chemoresistance is a major obstacle for the successful treatment of nonsmall cell lung cancer (NSCLC)[1]. CAF can produce growth factors that provide pro-survival signaling to the adjacent tumor cells in a paracrine manner [8, 9]. Hepatocyte growth factor (HGF) is one of the growth factors produced by CAF in tumor microenvironment and can bind its receptor of Met and activate the downstream phosphoinositide 3-kinase (PI3K)/AKT, which regulate chemoresistance and cell apoptosis [8]. This suggests that the HGF/Met and downstream PI3K/AKT signaling may be potential therapeutic targets against chemoresistant cancers. While dozens of inhibitors targeting the CAF-mediated pathways are currently under clinical studies, the efficacy of these inhibitors is clinically disappointing due to their poor specificity and severe adverse effects and functional overlap of many kinases [10]

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Results

Conclusion