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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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Activation of the platelet derived growth factor receptor (PDGFR)-Akt signaling axis in MG63 cells co-cultured with platelets. (a, b) Platelets prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were co-cultured with buffer alone (MG63 + Buffer) or platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-RTK array were performed according to the manufacturer's protocol. Representative images of the probed arrays are shown (a). The signal intensity of each spot was determined using an LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of eight reference spots (within red squares) in each membrane were measured and defined as 100%. The relative intensities of duplicate spots are shown (b). (c, d) Analysis of the human phospho-kinase array using lysates prepared from MG63 cells co-cultured with platelets in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were cultured with buffer alone (MG63 + Buffer) or with platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-kinase array were performed according to the manufacturer's protocol. Representative images of reacted membranes are shown (c). The signal intensity of each spot was measured using a LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of six reference spots (red squares) in each membrane were defined as 100%. The relative intensities of duplicate spots are shown (d). (e, f) Platelets prepared with 0.38% sodium citrate were resuspended Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen (e) or HOS/ZsGreen (f) cells were incubated with buffer alone or platelets (2.0 × 107/24-well) for 2 h. Platelets alone, osteosarcoma cells alone (+Buffer) or co-cultures of osteosarcoma cells with platelets (+Platelet) were lysed and immunoblotted using the antibodies to phospho-PDGFRβ, PDGFRβ, phospho-Akt (S473), Akt, or α-tubulin.
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fig02: Activation of the platelet derived growth factor receptor (PDGFR)-Akt signaling axis in MG63 cells co-cultured with platelets. (a, b) Platelets prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were co-cultured with buffer alone (MG63 + Buffer) or platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-RTK array were performed according to the manufacturer's protocol. Representative images of the probed arrays are shown (a). The signal intensity of each spot was determined using an LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of eight reference spots (within red squares) in each membrane were measured and defined as 100%. The relative intensities of duplicate spots are shown (b). (c, d) Analysis of the human phospho-kinase array using lysates prepared from MG63 cells co-cultured with platelets in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were cultured with buffer alone (MG63 + Buffer) or with platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-kinase array were performed according to the manufacturer's protocol. Representative images of reacted membranes are shown (c). The signal intensity of each spot was measured using a LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of six reference spots (red squares) in each membrane were defined as 100%. The relative intensities of duplicate spots are shown (d). (e, f) Platelets prepared with 0.38% sodium citrate were resuspended Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen (e) or HOS/ZsGreen (f) cells were incubated with buffer alone or platelets (2.0 × 107/24-well) for 2 h. Platelets alone, osteosarcoma cells alone (+Buffer) or co-cultures of osteosarcoma cells with platelets (+Platelet) were lysed and immunoblotted using the antibodies to phospho-PDGFRβ, PDGFRβ, phospho-Akt (S473), Akt, or α-tubulin.

Mentions: Platelets contain many growth factors and cytokines, including transforming growth factor-β, vascular endothelial growth factors, and PDGFs,(6,7) which are stored in platelet granules and released on platelet aggregation. Such platelet-derived factors promote the epithelial-mesenchymal transition, tumor vascular angiogenesis, and tumor growth.(8) To identify the mechanism that mediated the effects of platelets and supernatants of osteosarcoma-platelet reactants on osteosarcoma cell proliferation, we used arrays comprising a panel of antibodies specific for cytokines, growth factor receptors, or downstream signaling components. Incubation of the array with cell lysates prepared from reactants of MG63 cells and platelets increased the intensity of the spots corresponding to the positions of antibodies against phosphorylated PDGFRα, phosphorylated PDGFRβ, phosphorylated epidermal growth factor receptor (EGFR), and phosphorylated Akt1/2/3 antibodies (Figs. 2a–d). To exclude the possibility that the addition of platelets altered the respective protein expression levels in MG63 cells, we performed western blot analysis. Consistent with the array data, we confirmed the increase in phospho-PDGFRβ in MG63 cells that were co-cultured with platelets (Fig. 2e). Because PDGFRβ was not detected in the platelet lysate, indicating that co-culture induced PDGFRβ phosphorylation in MG63 cells. An increase in the level of phospho-Akt induced by co-culture was also detected using western blotting (Fig. 2e). Because the elecrophoretic mobility of mouse Akt in platelets appeared higher compared with human Akt in MG63 cells, the intensity of the phospho-Akt signal may represent phosphorylation of human but not mouse Akt. We obtained similar results using another HOS cell line (Fig. 2f). These data suggest that osteosarcoma-platelet interactions activate the PDGFR-Akt signaling pathway.


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)

Activation of the platelet derived growth factor receptor (PDGFR)-Akt signaling axis in MG63 cells co-cultured with platelets. (a, b) Platelets prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were co-cultured with buffer alone (MG63 + Buffer) or platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-RTK array were performed according to the manufacturer's protocol. Representative images of the probed arrays are shown (a). The signal intensity of each spot was determined using an LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of eight reference spots (within red squares) in each membrane were measured and defined as 100%. The relative intensities of duplicate spots are shown (b). (c, d) Analysis of the human phospho-kinase array using lysates prepared from MG63 cells co-cultured with platelets in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were cultured with buffer alone (MG63 + Buffer) or with platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-kinase array were performed according to the manufacturer's protocol. Representative images of reacted membranes are shown (c). The signal intensity of each spot was measured using a LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of six reference spots (red squares) in each membrane were defined as 100%. The relative intensities of duplicate spots are shown (d). (e, f) Platelets prepared with 0.38% sodium citrate were resuspended Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen (e) or HOS/ZsGreen (f) cells were incubated with buffer alone or platelets (2.0 × 107/24-well) for 2 h. Platelets alone, osteosarcoma cells alone (+Buffer) or co-cultures of osteosarcoma cells with platelets (+Platelet) were lysed and immunoblotted using the antibodies to phospho-PDGFRβ, PDGFRβ, phospho-Akt (S473), Akt, or α-tubulin.
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fig02: Activation of the platelet derived growth factor receptor (PDGFR)-Akt signaling axis in MG63 cells co-cultured with platelets. (a, b) Platelets prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were co-cultured with buffer alone (MG63 + Buffer) or platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-RTK array were performed according to the manufacturer's protocol. Representative images of the probed arrays are shown (a). The signal intensity of each spot was determined using an LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of eight reference spots (within red squares) in each membrane were measured and defined as 100%. The relative intensities of duplicate spots are shown (b). (c, d) Analysis of the human phospho-kinase array using lysates prepared from MG63 cells co-cultured with platelets in modified Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen cells were cultured with buffer alone (MG63 + Buffer) or with platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-kinase array were performed according to the manufacturer's protocol. Representative images of reacted membranes are shown (c). The signal intensity of each spot was measured using a LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of six reference spots (red squares) in each membrane were defined as 100%. The relative intensities of duplicate spots are shown (d). (e, f) Platelets prepared with 0.38% sodium citrate were resuspended Tyrode's buffer containing 200 μM CaCl2. MG63/ZsGreen (e) or HOS/ZsGreen (f) cells were incubated with buffer alone or platelets (2.0 × 107/24-well) for 2 h. Platelets alone, osteosarcoma cells alone (+Buffer) or co-cultures of osteosarcoma cells with platelets (+Platelet) were lysed and immunoblotted using the antibodies to phospho-PDGFRβ, PDGFRβ, phospho-Akt (S473), Akt, or α-tubulin.
Mentions: Platelets contain many growth factors and cytokines, including transforming growth factor-β, vascular endothelial growth factors, and PDGFs,(6,7) which are stored in platelet granules and released on platelet aggregation. Such platelet-derived factors promote the epithelial-mesenchymal transition, tumor vascular angiogenesis, and tumor growth.(8) To identify the mechanism that mediated the effects of platelets and supernatants of osteosarcoma-platelet reactants on osteosarcoma cell proliferation, we used arrays comprising a panel of antibodies specific for cytokines, growth factor receptors, or downstream signaling components. Incubation of the array with cell lysates prepared from reactants of MG63 cells and platelets increased the intensity of the spots corresponding to the positions of antibodies against phosphorylated PDGFRα, phosphorylated PDGFRβ, phosphorylated epidermal growth factor receptor (EGFR), and phosphorylated Akt1/2/3 antibodies (Figs. 2a–d). To exclude the possibility that the addition of platelets altered the respective protein expression levels in MG63 cells, we performed western blot analysis. Consistent with the array data, we confirmed the increase in phospho-PDGFRβ in MG63 cells that were co-cultured with platelets (Fig. 2e). Because PDGFRβ was not detected in the platelet lysate, indicating that co-culture induced PDGFRβ phosphorylation in MG63 cells. An increase in the level of phospho-Akt induced by co-culture was also detected using western blotting (Fig. 2e). Because the elecrophoretic mobility of mouse Akt in platelets appeared higher compared with human Akt in MG63 cells, the intensity of the phospho-Akt signal may represent phosphorylation of human but not mouse Akt. We obtained similar results using another HOS cell line (Fig. 2f). These data suggest that osteosarcoma-platelet interactions activate the PDGFR-Akt signaling pathway.

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