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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 enhancement of Akt phosphorylation in ganglioside GD3+ N1 melanoma cells by costimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I). Phosphorylation levels of Akt after stimulation with HGF, adhesion stimulation with CL-I, or both HGF and CL-I were compared between GD3+ cells and GD3− cells. 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) Akt and phosphorylated Akt (Ser473 and Thr308) 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 an anti-Akt or an anti-phospho-Akt antibodies. The bands were detected with ECL. The bands were scanned and the relative intensities of phosphorylated bands were presented after correction with intensities of total Akt. (B) Akt and phosphorylated Akt (Ser473 and Thr308) 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 were measured as in (A). (C) Akt and phosphorylated Akt (Ser473 and Thr308) 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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fig03: Synergistic enhancement of Akt phosphorylation in ganglioside GD3+ N1 melanoma cells by costimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I). Phosphorylation levels of Akt after stimulation with HGF, adhesion stimulation with CL-I, or both HGF and CL-I were compared between GD3+ cells and GD3− cells. 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) Akt and phosphorylated Akt (Ser473 and Thr308) 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 an anti-Akt or an anti-phospho-Akt antibodies. The bands were detected with ECL. The bands were scanned and the relative intensities of phosphorylated bands were presented after correction with intensities of total Akt. (B) Akt and phosphorylated Akt (Ser473 and Thr308) 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 were measured as in (A). (C) Akt and phosphorylated Akt (Ser473 and Thr308) 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 analyzed phosphorylation of Akt (p-Ser473, p-Thr308) by either HGF stimulation or adhesion to CL-I, or both of them at the same time in GD3+ cells and GD3− cells. The cells were harvested after starving for 14–16 h in serum-free DMEM and rotated for 1 h at 37°C to reduce basal phosphorylation levels of Akt. After HGF stimulation, phosphorylation of Ser473 in Akt was slightly higher in GD3+ cells, but phosphorylation of Thr308 in Akt was not different between GD3+ and GD3− cells (Fig.3A). Phosphorylation of Ser473 and Thr308 of Akt was higher in GD3+ cells than in GD3− cells during adhesion to CL-I, corresponding with our previous report,22 while the phosphorylation levels were low (Fig.3B). Then, we analyzed phosphorylation of Akt during costimulation of GD3+ and GD3− cells with HGF and adhesion to CL-I. Consequently, phosphorylation levels of Ser473 and Thr308 of Akt were definitely higher in GD3+ cells than in GD3− cells (Fig.3C). These results suggested that phosphorylation of Akt 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 enhancement of Akt phosphorylation in ganglioside GD3+ N1 melanoma cells by costimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I). Phosphorylation levels of Akt after stimulation with HGF, adhesion stimulation with CL-I, or both HGF and CL-I were compared between GD3+ cells and GD3− cells. 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) Akt and phosphorylated Akt (Ser473 and Thr308) 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 an anti-Akt or an anti-phospho-Akt antibodies. The bands were detected with ECL. The bands were scanned and the relative intensities of phosphorylated bands were presented after correction with intensities of total Akt. (B) Akt and phosphorylated Akt (Ser473 and Thr308) 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 were measured as in (A). (C) Akt and phosphorylated Akt (Ser473 and Thr308) 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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getmorefigures.php?uid=PMC4317880&req=5

fig03: Synergistic enhancement of Akt phosphorylation in ganglioside GD3+ N1 melanoma cells by costimulation with hepatocyte growth factor (HGF) and adhesion to collagen type I (CL-I). Phosphorylation levels of Akt after stimulation with HGF, adhesion stimulation with CL-I, or both HGF and CL-I were compared between GD3+ cells and GD3− cells. 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) Akt and phosphorylated Akt (Ser473 and Thr308) 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 an anti-Akt or an anti-phospho-Akt antibodies. The bands were detected with ECL. The bands were scanned and the relative intensities of phosphorylated bands were presented after correction with intensities of total Akt. (B) Akt and phosphorylated Akt (Ser473 and Thr308) 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 were measured as in (A). (C) Akt and phosphorylated Akt (Ser473 and Thr308) 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 analyzed phosphorylation of Akt (p-Ser473, p-Thr308) by either HGF stimulation or adhesion to CL-I, or both of them at the same time in GD3+ cells and GD3− cells. The cells were harvested after starving for 14–16 h in serum-free DMEM and rotated for 1 h at 37°C to reduce basal phosphorylation levels of Akt. After HGF stimulation, phosphorylation of Ser473 in Akt was slightly higher in GD3+ cells, but phosphorylation of Thr308 in Akt was not different between GD3+ and GD3− cells (Fig.3A). Phosphorylation of Ser473 and Thr308 of Akt was higher in GD3+ cells than in GD3− cells during adhesion to CL-I, corresponding with our previous report,22 while the phosphorylation levels were low (Fig.3B). Then, we analyzed phosphorylation of Akt during costimulation of GD3+ and GD3− cells with HGF and adhesion to CL-I. Consequently, phosphorylation levels of Ser473 and Thr308 of Akt were definitely higher in GD3+ cells than in GD3− cells (Fig.3C). These results suggested that phosphorylation of Akt 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