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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

Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip.A549 cells were cultured in triplicate in the maintenance medium alone, mixed with the CAF matrix in the presence or absence of anti-HGF or containing 40 ng/ml of human HGF in the 3D chambers for 48h. The cells were stained with the indicated FITC-conjugated antibodies, and examined under a fluorescent microscope. Furthermore, the cells were cultured in the mixture of maintenance medium and CAF matrix in the presence or absence of an inhibitor for c-Met, PI3K or GRP78 for 48h and stained as described above. Data are representative images (magnification x 200) from three separate experiments. (A)The CAF matrix or HGF enhances the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. (B)The effect of an inhibitor of c-Met, PI3K or GRP78 on the CAF-enhanced c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.
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pone.0129593.g002: Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip.A549 cells were cultured in triplicate in the maintenance medium alone, mixed with the CAF matrix in the presence or absence of anti-HGF or containing 40 ng/ml of human HGF in the 3D chambers for 48h. The cells were stained with the indicated FITC-conjugated antibodies, and examined under a fluorescent microscope. Furthermore, the cells were cultured in the mixture of maintenance medium and CAF matrix in the presence or absence of an inhibitor for c-Met, PI3K or GRP78 for 48h and stained as described above. Data are representative images (magnification x 200) from three separate experiments. (A)The CAF matrix or HGF enhances the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. (B)The effect of an inhibitor of c-Met, PI3K or GRP78 on the CAF-enhanced c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.

Mentions: To investigate the potential mechanisms underlying the effect of tumor stroma on cancer cells, we examined the Met, PI3K and AKT phosphorylation in A549 cells cultured in the 3D chambers by immunofluorescence assay. As expected, A549 cells cultured in CAF matrix displayed higher levels of anti-phosphorylated Met, PI3K and AKT and anti-GRP78 staining compared to those cultured without CAF matrix (Fig 2A). Previous studies have shown that tumor stromal cells, such as fibroblasts, can secrete HGF that can promote the proliferation of cancer cells [28]. We questioned whether HGF in the CAF promoted the Met, PI3K and AKT activation and up-regulated GRP78 expression in A549 cells. To address this question, we first determined the concentrations of HGF in the CAF collected from activated HLF1 cells that had been cultured for varying periods. We found that the levels of HGF in the supernatants of cultured HLF1 cells gradually increased with time and peaked (400 ng/2×105 cells) at 72 h post culture. However, there was no detectable level of HGF in the supernatants of cultured A549 cells (data not shown). Next, we tested whether HGF mediated the Met/PI3K/Akt activation and up-regulated GRP78 expression in A549 cells. We found that addition of 40 ng HGF to maintenance medium also increased anti-phosphorylated Met, PI3K and AKT as well as anti-GRP78 staining in A549 cells. More importantly, addition of human HGF neutralizing antibody (20 μg/ml) into the mixture of maintenance medium and CAF containing almost prevented anti-phosphorylated Met, PI3K and AKT as well as anti-GRP78 staining 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)

Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip.A549 cells were cultured in triplicate in the maintenance medium alone, mixed with the CAF matrix in the presence or absence of anti-HGF or containing 40 ng/ml of human HGF in the 3D chambers for 48h. The cells were stained with the indicated FITC-conjugated antibodies, and examined under a fluorescent microscope. Furthermore, the cells were cultured in the mixture of maintenance medium and CAF matrix in the presence or absence of an inhibitor for c-Met, PI3K or GRP78 for 48h and stained as described above. Data are representative images (magnification x 200) from three separate experiments. (A)The CAF matrix or HGF enhances the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. (B)The effect of an inhibitor of c-Met, PI3K or GRP78 on the CAF-enhanced c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482748&req=5

pone.0129593.g002: Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip.A549 cells were cultured in triplicate in the maintenance medium alone, mixed with the CAF matrix in the presence or absence of anti-HGF or containing 40 ng/ml of human HGF in the 3D chambers for 48h. The cells were stained with the indicated FITC-conjugated antibodies, and examined under a fluorescent microscope. Furthermore, the cells were cultured in the mixture of maintenance medium and CAF matrix in the presence or absence of an inhibitor for c-Met, PI3K or GRP78 for 48h and stained as described above. Data are representative images (magnification x 200) from three separate experiments. (A)The CAF matrix or HGF enhances the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. (B)The effect of an inhibitor of c-Met, PI3K or GRP78 on the CAF-enhanced c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.
Mentions: To investigate the potential mechanisms underlying the effect of tumor stroma on cancer cells, we examined the Met, PI3K and AKT phosphorylation in A549 cells cultured in the 3D chambers by immunofluorescence assay. As expected, A549 cells cultured in CAF matrix displayed higher levels of anti-phosphorylated Met, PI3K and AKT and anti-GRP78 staining compared to those cultured without CAF matrix (Fig 2A). Previous studies have shown that tumor stromal cells, such as fibroblasts, can secrete HGF that can promote the proliferation of cancer cells [28]. We questioned whether HGF in the CAF promoted the Met, PI3K and AKT activation and up-regulated GRP78 expression in A549 cells. To address this question, we first determined the concentrations of HGF in the CAF collected from activated HLF1 cells that had been cultured for varying periods. We found that the levels of HGF in the supernatants of cultured HLF1 cells gradually increased with time and peaked (400 ng/2×105 cells) at 72 h post culture. However, there was no detectable level of HGF in the supernatants of cultured A549 cells (data not shown). Next, we tested whether HGF mediated the Met/PI3K/Akt activation and up-regulated GRP78 expression in A549 cells. We found that addition of 40 ng HGF to maintenance medium also increased anti-phosphorylated Met, PI3K and AKT as well as anti-GRP78 staining in A549 cells. More importantly, addition of human HGF neutralizing antibody (20 μg/ml) into the mixture of maintenance medium and CAF containing almost prevented anti-phosphorylated Met, PI3K and AKT as well as anti-GRP78 staining 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