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Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

Takagi S, Takemoto A, Takami M, Oh-Hara T, Fujita N - Cancer Sci. (2014)

Bottom Line: The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt.Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt.Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

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Co-culture with platelets promotes platelet aggregation and the proliferation of osteosarcoma cells. (a) Platelets (4.0 × 107) prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 2% PPP and 250 μM CaCl2, followed by incubation with PBS (red lines) or osteosarcoma MG63 (left panel, black line) and HOS (right panel, black line) cells (1.0 × 108 cells). The transmission of light by the samples was measured using an aggregometer to determine the aggregation rate. (b) MG63 and HOS cells stably transfected with ZsGreen gene, MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel), respectively, were co-cultured with the indicated number of washed mouse platelets in medium containing 0.5% FBS. TENSV buffer was added to each well at the times indicated, and the fluorescence of ZsGreen was used to determine the number of viable osteosarcoma cells. The error bars indicate the mean ± standard deviation (SD) of triplicate experiments. (c) Washed mouse platelets (2.0 × 107 platelets) were resuspended in modified Tyrode's buffer containing 1% PPP and 200 μM CaCl2 followed by incubation with PBS or osteosarcoma cells (2.5 × 104 cells) for 30 min. After centrifugation twice at 10 000 g for 10 min, the supernatant of osteosarcoma-platelet reactants or the PBS-platelet reactant was added to the cultures of MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel) cells. After 24 h, TENSV buffer was added to each well and the fluorescence of ZsGreen was used to determine the relative number of viable cells. The error bars indicate the mean ± SD of triplicate experiments. **P < 0.01 by the Student t-test.
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fig01: Co-culture with platelets promotes platelet aggregation and the proliferation of osteosarcoma cells. (a) Platelets (4.0 × 107) prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 2% PPP and 250 μM CaCl2, followed by incubation with PBS (red lines) or osteosarcoma MG63 (left panel, black line) and HOS (right panel, black line) cells (1.0 × 108 cells). The transmission of light by the samples was measured using an aggregometer to determine the aggregation rate. (b) MG63 and HOS cells stably transfected with ZsGreen gene, MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel), respectively, were co-cultured with the indicated number of washed mouse platelets in medium containing 0.5% FBS. TENSV buffer was added to each well at the times indicated, and the fluorescence of ZsGreen was used to determine the number of viable osteosarcoma cells. The error bars indicate the mean ± standard deviation (SD) of triplicate experiments. (c) Washed mouse platelets (2.0 × 107 platelets) were resuspended in modified Tyrode's buffer containing 1% PPP and 200 μM CaCl2 followed by incubation with PBS or osteosarcoma cells (2.5 × 104 cells) for 30 min. After centrifugation twice at 10 000 g for 10 min, the supernatant of osteosarcoma-platelet reactants or the PBS-platelet reactant was added to the cultures of MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel) cells. After 24 h, TENSV buffer was added to each well and the fluorescence of ZsGreen was used to determine the relative number of viable cells. The error bars indicate the mean ± SD of triplicate experiments. **P < 0.01 by the Student t-test.

Mentions: Osteosarcomas form pulmonary metastasis by inducing platelet aggregation.(16,17) To assess the role of osteosarcoma-platelet interactions in determining the malignant phenotype of osteosarcomas, we first measured the abilities of the human osteosarcoma cell lines MG63 and HOS to induce platelet aggregation. We found that each osteosarcoma cell line induced platelet aggregation to an extent that is consistent with published studies (Fig. 1a). We next examined the influence of platelets on the growth of the osteosarcoma cell lines. Because of the high concentration of adenosine triphosphate (ATP) in platelets, we were unable to determine the growth of osteosarcoma cells using proliferation assays that measure ATP. Therefore, we generated stable transfectants of MG63 and HOS cells that expressed ZsGreen (MG63/ZsGreen and HOS/ZsGreen, respectively) and measured ZsGreen fluorescence to determine the number of viable cells. Although the growth rates of MG63/ZsGreen and HOS/ZsGreen cells were slow in the presence of 0.5% FBS, the growth rate of each cell line was significantly enhanced in proportion to the number of washed platelets added to the cultured cells (Fig. 1b). We found that the addition of the supernatant of an osteosarcoma-platelet reactant, but not that of the PBS-platelet reactant, significantly enhanced the growth of MG63/ZsGreen and HOS/ZsGreen cells (Fig. 1c). These results indicate that the proliferation of osteosarcoma cells is increased in the presence of platelets as well as by supernatants of osteosarcoma-platelet reactant.


Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

Takagi S, Takemoto A, Takami M, Oh-Hara T, Fujita N - Cancer Sci. (2014)

Co-culture with platelets promotes platelet aggregation and the proliferation of osteosarcoma cells. (a) Platelets (4.0 × 107) prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 2% PPP and 250 μM CaCl2, followed by incubation with PBS (red lines) or osteosarcoma MG63 (left panel, black line) and HOS (right panel, black line) cells (1.0 × 108 cells). The transmission of light by the samples was measured using an aggregometer to determine the aggregation rate. (b) MG63 and HOS cells stably transfected with ZsGreen gene, MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel), respectively, were co-cultured with the indicated number of washed mouse platelets in medium containing 0.5% FBS. TENSV buffer was added to each well at the times indicated, and the fluorescence of ZsGreen was used to determine the number of viable osteosarcoma cells. The error bars indicate the mean ± standard deviation (SD) of triplicate experiments. (c) Washed mouse platelets (2.0 × 107 platelets) were resuspended in modified Tyrode's buffer containing 1% PPP and 200 μM CaCl2 followed by incubation with PBS or osteosarcoma cells (2.5 × 104 cells) for 30 min. After centrifugation twice at 10 000 g for 10 min, the supernatant of osteosarcoma-platelet reactants or the PBS-platelet reactant was added to the cultures of MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel) cells. After 24 h, TENSV buffer was added to each well and the fluorescence of ZsGreen was used to determine the relative number of viable cells. The error bars indicate the mean ± SD of triplicate experiments. **P < 0.01 by the Student t-test.
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Related In: Results  -  Collection

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fig01: Co-culture with platelets promotes platelet aggregation and the proliferation of osteosarcoma cells. (a) Platelets (4.0 × 107) prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 2% PPP and 250 μM CaCl2, followed by incubation with PBS (red lines) or osteosarcoma MG63 (left panel, black line) and HOS (right panel, black line) cells (1.0 × 108 cells). The transmission of light by the samples was measured using an aggregometer to determine the aggregation rate. (b) MG63 and HOS cells stably transfected with ZsGreen gene, MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel), respectively, were co-cultured with the indicated number of washed mouse platelets in medium containing 0.5% FBS. TENSV buffer was added to each well at the times indicated, and the fluorescence of ZsGreen was used to determine the number of viable osteosarcoma cells. The error bars indicate the mean ± standard deviation (SD) of triplicate experiments. (c) Washed mouse platelets (2.0 × 107 platelets) were resuspended in modified Tyrode's buffer containing 1% PPP and 200 μM CaCl2 followed by incubation with PBS or osteosarcoma cells (2.5 × 104 cells) for 30 min. After centrifugation twice at 10 000 g for 10 min, the supernatant of osteosarcoma-platelet reactants or the PBS-platelet reactant was added to the cultures of MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel) cells. After 24 h, TENSV buffer was added to each well and the fluorescence of ZsGreen was used to determine the relative number of viable cells. The error bars indicate the mean ± SD of triplicate experiments. **P < 0.01 by the Student t-test.
Mentions: Osteosarcomas form pulmonary metastasis by inducing platelet aggregation.(16,17) To assess the role of osteosarcoma-platelet interactions in determining the malignant phenotype of osteosarcomas, we first measured the abilities of the human osteosarcoma cell lines MG63 and HOS to induce platelet aggregation. We found that each osteosarcoma cell line induced platelet aggregation to an extent that is consistent with published studies (Fig. 1a). We next examined the influence of platelets on the growth of the osteosarcoma cell lines. Because of the high concentration of adenosine triphosphate (ATP) in platelets, we were unable to determine the growth of osteosarcoma cells using proliferation assays that measure ATP. Therefore, we generated stable transfectants of MG63 and HOS cells that expressed ZsGreen (MG63/ZsGreen and HOS/ZsGreen, respectively) and measured ZsGreen fluorescence to determine the number of viable cells. Although the growth rates of MG63/ZsGreen and HOS/ZsGreen cells were slow in the presence of 0.5% FBS, the growth rate of each cell line was significantly enhanced in proportion to the number of washed platelets added to the cultured cells (Fig. 1b). We found that the addition of the supernatant of an osteosarcoma-platelet reactant, but not that of the PBS-platelet reactant, significantly enhanced the growth of MG63/ZsGreen and HOS/ZsGreen cells (Fig. 1c). These results indicate that the proliferation of osteosarcoma cells is increased in the presence of platelets as well as by supernatants of osteosarcoma-platelet reactant.

Bottom Line: The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt.Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt.Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus