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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.

Ying L, Zhu Z, Xu Z, He T, Li E, Guo Z, Liu F, Jiang C, Wang Q - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, the Second Hospital of Dalian Medical University, Dailan, China.

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.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.A549 cells were cultured in the condition as described above and the relative levels of phosphorylated Met, PI3Kp85, AKT and GRP78 expression in the different groups of cells were characterized by Western blot assays and quantified. Data are representative images and expressed as the means ± SD of each protein in individual groups of cells from three separate experiments. *P<0.05; **P<0.01 vs. the controls.
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pone.0129593.g003: Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.A549 cells were cultured in the condition as described above and the relative levels of phosphorylated Met, PI3Kp85, AKT and GRP78 expression in the different groups of cells were characterized by Western blot assays and quantified. Data are representative images and expressed as the means ± SD of each protein in individual groups of cells from three separate experiments. *P<0.05; **P<0.01 vs. the controls.

Mentions: In addition, treatment with an inhibitor for Met attenuated CAF-mediated Met phosphorylation and reduced the levels of PI3Kp85 and AKT phosphorylation and GRP78 expression in A549 cells (Fig 2B). Furthermore, treatment with a PI3K inhibitor reduced the CAF-mediated PI3Kp85 and AKT phosphorylation and GRP78 expression, but did not change the CAF-mediated Met phosphorylation in A549 cells. Moreover, treatment with a GRP78 inhibitor only down-regulated CAF-mediated GRP78 expression, but did not modulate the CAF-mediated Met, PI3Kp85 and AKT phosphorylation in A549 cells. A similar pattern of the CAF-mediated Met/PI3K/AKT phosphorylation and GRP78 expression in A549 cells was observed by Western blot assay (Fig 3). Collectively, these data indicated that HGF in the CAF stimulated the Met/PI3K/AKT activation and up-regulated GRP78 expression in A549 cells.


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.

Ying L, Zhu Z, Xu Z, He T, Li E, Guo Z, Liu F, Jiang C, Wang Q - PLoS ONE (2015)

Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.A549 cells were cultured in the condition as described above and the relative levels of phosphorylated Met, PI3Kp85, AKT and GRP78 expression in the different groups of cells were characterized by Western blot assays and quantified. Data are representative images and expressed as the means ± SD of each protein in individual groups of cells from three separate experiments. *P<0.05; **P<0.01 vs. the controls.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482748&req=5

pone.0129593.g003: Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.A549 cells were cultured in the condition as described above and the relative levels of phosphorylated Met, PI3Kp85, AKT and GRP78 expression in the different groups of cells were characterized by Western blot assays and quantified. Data are representative images and expressed as the means ± SD of each protein in individual groups of cells from three separate experiments. *P<0.05; **P<0.01 vs. the controls.
Mentions: In addition, treatment with an inhibitor for Met attenuated CAF-mediated Met phosphorylation and reduced the levels of PI3Kp85 and AKT phosphorylation and GRP78 expression in A549 cells (Fig 2B). Furthermore, treatment with a PI3K inhibitor reduced the CAF-mediated PI3Kp85 and AKT phosphorylation and GRP78 expression, but did not change the CAF-mediated Met phosphorylation in A549 cells. Moreover, treatment with a GRP78 inhibitor only down-regulated CAF-mediated GRP78 expression, but did not modulate the CAF-mediated Met, PI3Kp85 and AKT phosphorylation in A549 cells. A similar pattern of the CAF-mediated Met/PI3K/AKT phosphorylation and GRP78 expression in A549 cells was observed by Western blot assay (Fig 3). Collectively, these data indicated that HGF in the CAF stimulated the Met/PI3K/AKT activation and up-regulated GRP78 expression in A549 cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, the Second Hospital of Dalian Medical University, Dailan, China.

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.

No MeSH data available.


Related in: MedlinePlus