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Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas.

Furukawa K, Kambe M, Miyata M, Ohkawa Y, Tajima O, Furukawa K - Cancer Sci. (2013)

Bottom Line: In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I).When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3- cells or GD3+ cells treated with either one of the stimulants.Cell growth measured by 5-ethynyl-2' deoxyuridine uptake also showed synergistic effects in GD3+ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Japan; Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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Synergistic increase of phosphorylation levels of Erks by simultaneous stimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I) in melanoma cells. Phosphorylation levels of Erks after growth factor stimulation with HGF, adhesion stimulation with CL-I, or both in GD3+ cells and GD3− cells were examined. The cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h, then used for each experiment. (A) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after adding HGF (10 ng/mL) in GD3+ and GD3− cell suspensions (1.0 × 106 cells/ 5 mL). After HGF stimulation, cells were lysed and the lysates were used for immunoblotting with anti-Erks or anti-phospho-Erks antibodies. The bands were detected with ECL and scanned. The relative intensities of phosphorylated bands are presented after correction with intensities of total Erks. (B) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after CL-I adhesion stimulation in GD3+ and GD3− cells (0.4 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). (C) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after simultaneous stimulation with HGF (10 ng/mL) and CL-I adhesion in GD3+ and GD3− cells (1.0 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). β-actin bands indicate equal loading of samples.
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fig04: Synergistic increase of phosphorylation levels of Erks by simultaneous stimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I) in melanoma cells. Phosphorylation levels of Erks after growth factor stimulation with HGF, adhesion stimulation with CL-I, or both in GD3+ cells and GD3− cells were examined. The cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h, then used for each experiment. (A) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after adding HGF (10 ng/mL) in GD3+ and GD3− cell suspensions (1.0 × 106 cells/ 5 mL). After HGF stimulation, cells were lysed and the lysates were used for immunoblotting with anti-Erks or anti-phospho-Erks antibodies. The bands were detected with ECL and scanned. The relative intensities of phosphorylated bands are presented after correction with intensities of total Erks. (B) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after CL-I adhesion stimulation in GD3+ and GD3− cells (0.4 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). (C) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after simultaneous stimulation with HGF (10 ng/mL) and CL-I adhesion in GD3+ and GD3− cells (1.0 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). β-actin bands indicate equal loading of samples.

Mentions: We then analyzed phosphorylation of Erks by either HGF stimulation or adhesion to CL-I, or both of them at the same time, in GD3+ and GD3− cells. After starving for 14–16 h in serum-free DMEM, the cells were harvested and rotated for 1 h at 37°C to reduce basal phosphorylation levels of Erks. Phosphorylation levels of Erks by HGF stimulation were slightly higher in GD3+ cells than in GD3− cells (Fig.4A). As for adhesion stimulation to CL-I, phosphorylation levels of Erks were not different between GD3+ cells and GD3− cells (Fig.4B). We then costimulated GD3+ and GD3− cells with HGF and adhesion, and analyzed phosphorylation levels of Erks. In this case, phosphorylation levels of Erks were remarkably increased in GD3+ cells compared with GD3− cells (Fig.4C). Among four bands in total Erks detected after the stimulation, new bands above original Erk1/2, representing phosphorylated forms of Erk1/2, were more prominent in GD3+ cells than in GD3− cells after costimulation. These results suggested that phosphorylation of Erks was synergistically enhanced by costimulation with HGF and adhesion to CL-I in GD3+ cells.


Ganglioside GD3 induces convergence and synergism of adhesion and hepatocyte growth factor/Met signals in melanomas.

Furukawa K, Kambe M, Miyata M, Ohkawa Y, Tajima O, Furukawa K - Cancer Sci. (2013)

Synergistic increase of phosphorylation levels of Erks by simultaneous stimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I) in melanoma cells. Phosphorylation levels of Erks after growth factor stimulation with HGF, adhesion stimulation with CL-I, or both in GD3+ cells and GD3− cells were examined. The cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h, then used for each experiment. (A) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after adding HGF (10 ng/mL) in GD3+ and GD3− cell suspensions (1.0 × 106 cells/ 5 mL). After HGF stimulation, cells were lysed and the lysates were used for immunoblotting with anti-Erks or anti-phospho-Erks antibodies. The bands were detected with ECL and scanned. The relative intensities of phosphorylated bands are presented after correction with intensities of total Erks. (B) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after CL-I adhesion stimulation in GD3+ and GD3− cells (0.4 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). (C) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after simultaneous stimulation with HGF (10 ng/mL) and CL-I adhesion in GD3+ and GD3− cells (1.0 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). β-actin bands indicate equal loading of samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig04: Synergistic increase of phosphorylation levels of Erks by simultaneous stimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I) in melanoma cells. Phosphorylation levels of Erks after growth factor stimulation with HGF, adhesion stimulation with CL-I, or both in GD3+ cells and GD3− cells were examined. The cells were harvested and rotated for 1 h at 37°C after serum starvation for 14–16 h, then used for each experiment. (A) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after adding HGF (10 ng/mL) in GD3+ and GD3− cell suspensions (1.0 × 106 cells/ 5 mL). After HGF stimulation, cells were lysed and the lysates were used for immunoblotting with anti-Erks or anti-phospho-Erks antibodies. The bands were detected with ECL and scanned. The relative intensities of phosphorylated bands are presented after correction with intensities of total Erks. (B) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after CL-I adhesion stimulation in GD3+ and GD3− cells (0.4 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). (C) Total Erk1/2 and phosphorylated Erk1/2 were analyzed at the time points indicated after simultaneous stimulation with HGF (10 ng/mL) and CL-I adhesion in GD3+ and GD3− cells (1.0 × 106 cells/6-cm dish). The relative intensities of phosphorylated bands are presented as in (A). β-actin bands indicate equal loading of samples.
Mentions: We then analyzed phosphorylation of Erks by either HGF stimulation or adhesion to CL-I, or both of them at the same time, in GD3+ and GD3− cells. After starving for 14–16 h in serum-free DMEM, the cells were harvested and rotated for 1 h at 37°C to reduce basal phosphorylation levels of Erks. Phosphorylation levels of Erks by HGF stimulation were slightly higher in GD3+ cells than in GD3− cells (Fig.4A). As for adhesion stimulation to CL-I, phosphorylation levels of Erks were not different between GD3+ cells and GD3− cells (Fig.4B). We then costimulated GD3+ and GD3− cells with HGF and adhesion, and analyzed phosphorylation levels of Erks. In this case, phosphorylation levels of Erks were remarkably increased in GD3+ cells compared with GD3− cells (Fig.4C). Among four bands in total Erks detected after the stimulation, new bands above original Erk1/2, representing phosphorylated forms of Erk1/2, were more prominent in GD3+ cells than in GD3− cells after costimulation. These results suggested that phosphorylation of Erks was synergistically enhanced by costimulation with HGF and adhesion to CL-I in GD3+ cells.

Bottom Line: In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I).When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3- cells or GD3+ cells treated with either one of the stimulants.Cell growth measured by 5-ethynyl-2' deoxyuridine uptake also showed synergistic effects in GD3+ cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Japan; Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Show MeSH
Related in: MedlinePlus