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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

The design and validation of a 3D culture microfluidic chip.(a)The schematic design of the microfluidic chip with CGG and downstream cell chambers (the upper panel) and the fabricated chip with pumping machine (the lower panel). (b)The diffused Rh-123 in the 3D chamber within 30 min and >95% cells were viable (green). Magnification ×100. (c) The morphological features of A549 cells in the 3D chamber without or with CAF matrix. The white arrows indicate apoptotic cells. (d)The α-SMA immunofluorescence assay of HFL1 cells. HFL1 cells induced by A549 medium showed a positive α-SMA staining (right) compared to the untreated HFL1 (left). Magnification ×400. (e) Immunohistochemistry assay for lung cancer tissues. The expression of α-SMA protein in the lung cancer tissues is higher than that in adjacent tissues. Magnification ×200.
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pone.0129593.g001: The design and validation of a 3D culture microfluidic chip.(a)The schematic design of the microfluidic chip with CGG and downstream cell chambers (the upper panel) and the fabricated chip with pumping machine (the lower panel). (b)The diffused Rh-123 in the 3D chamber within 30 min and >95% cells were viable (green). Magnification ×100. (c) The morphological features of A549 cells in the 3D chamber without or with CAF matrix. The white arrows indicate apoptotic cells. (d)The α-SMA immunofluorescence assay of HFL1 cells. HFL1 cells induced by A549 medium showed a positive α-SMA staining (right) compared to the untreated HFL1 (left). Magnification ×400. (e) Immunohistochemistry assay for lung cancer tissues. The expression of α-SMA protein in the lung cancer tissues is higher than that in adjacent tissues. Magnification ×200.

Mentions: The schematic design of microfluidic device with a two-layer structure is shown in Fig 1A. The lower layer consisted of a combination of a linear concentration gradient generator (CGG) and four downstream parallel cell culture units with two oval-shape modules. The CGG had two inlets (a diameter of 1.5 mm) for medium and drug solution perfusion and corresponding cascade microchannels (10 mm × 200 μm × 100 μm). The CGG utilized diffusive mixing to generate a mixture of the two inlets at the mixing microchannels. The concentration interval from the channel 1 to channel 4 generated by CGG in theory is (drug concentrationmax—drug concentrationmix)/3, which had been demonstrated in our previous study [25]. The dimensions of each chamber used for cell culture were 800 μm (length) × 400 μm (width) × 100 μm (height). The inlet and outlet diameters of cell chamber were 0.6 mm. Accordingly, the mixture of cell-basement membrane extracts (BME) was seeded in the cell culture chamber, where cells were cultured in 3D. The excess mixture was effused from a cell outlet. The upper PDMS layer possessed two inlets (a diameter of 1.5 mm) and multiplexed perfusion channels (200 μm in width and 100 μm in height). Hence, soluble factors, fibroblast-secreted growth factors and drugs flowed to the cell chambers on the lower layer. The two layers were combined through the exactly matched holes inside the channels of upper and lower layers by using a stereomicroscope with the reference marks.


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)

The design and validation of a 3D culture microfluidic chip.(a)The schematic design of the microfluidic chip with CGG and downstream cell chambers (the upper panel) and the fabricated chip with pumping machine (the lower panel). (b)The diffused Rh-123 in the 3D chamber within 30 min and >95% cells were viable (green). Magnification ×100. (c) The morphological features of A549 cells in the 3D chamber without or with CAF matrix. The white arrows indicate apoptotic cells. (d)The α-SMA immunofluorescence assay of HFL1 cells. HFL1 cells induced by A549 medium showed a positive α-SMA staining (right) compared to the untreated HFL1 (left). Magnification ×400. (e) Immunohistochemistry assay for lung cancer tissues. The expression of α-SMA protein in the lung cancer tissues is higher than that in adjacent tissues. Magnification ×200.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129593.g001: The design and validation of a 3D culture microfluidic chip.(a)The schematic design of the microfluidic chip with CGG and downstream cell chambers (the upper panel) and the fabricated chip with pumping machine (the lower panel). (b)The diffused Rh-123 in the 3D chamber within 30 min and >95% cells were viable (green). Magnification ×100. (c) The morphological features of A549 cells in the 3D chamber without or with CAF matrix. The white arrows indicate apoptotic cells. (d)The α-SMA immunofluorescence assay of HFL1 cells. HFL1 cells induced by A549 medium showed a positive α-SMA staining (right) compared to the untreated HFL1 (left). Magnification ×400. (e) Immunohistochemistry assay for lung cancer tissues. The expression of α-SMA protein in the lung cancer tissues is higher than that in adjacent tissues. Magnification ×200.
Mentions: The schematic design of microfluidic device with a two-layer structure is shown in Fig 1A. The lower layer consisted of a combination of a linear concentration gradient generator (CGG) and four downstream parallel cell culture units with two oval-shape modules. The CGG had two inlets (a diameter of 1.5 mm) for medium and drug solution perfusion and corresponding cascade microchannels (10 mm × 200 μm × 100 μm). The CGG utilized diffusive mixing to generate a mixture of the two inlets at the mixing microchannels. The concentration interval from the channel 1 to channel 4 generated by CGG in theory is (drug concentrationmax—drug concentrationmix)/3, which had been demonstrated in our previous study [25]. The dimensions of each chamber used for cell culture were 800 μm (length) × 400 μm (width) × 100 μm (height). The inlet and outlet diameters of cell chamber were 0.6 mm. Accordingly, the mixture of cell-basement membrane extracts (BME) was seeded in the cell culture chamber, where cells were cultured in 3D. The excess mixture was effused from a cell outlet. The upper PDMS layer possessed two inlets (a diameter of 1.5 mm) and multiplexed perfusion channels (200 μm in width and 100 μm in height). Hence, soluble factors, fibroblast-secreted growth factors and drugs flowed to the cell chambers on the lower layer. The two layers were combined through the exactly matched holes inside the channels of upper and lower layers by using a stereomicroscope with the reference marks.

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