Anoikis‑resistant human osteosarcoma cells display significant angiogenesis by activating the Src kinase‑mediated MAPK pathway

Abstract

Tumor cells must resist anoikis to metastasize. There is a key role of angiogenesis in the growth and metastasis of tumors. However, the relationship between anoikis resistance and angiogenesis has not been well explored in human osteosarcoma. In the present study, we reported the higher expression of vascular endothelial growth factor‑A (VEGF‑A) in osteosarcoma cells that were resistant to anoikis than in parental osteosarcoma cells, promoting the proliferation, tube formation, and migration of human umbilical vein endothelial cells (HUVECs). Src, JNK (Jun amino‑terminal kinase) and ERK (extracellular signal‑regulated kinase) signaling pathway phosphorylation was activated in anoikis‑resistant cells; Src inhibitor reduced the expression of VEGF‑A and angiogenesis and inhibited JNK and ERK pathway activity. Overexpression of phosphorylated (p)‑Src and VEGF‑A was positively correlated to the metastatic potential in human osteosarcoma tissues, as quantified by immunohistochemistry. In addition, p‑Src expression was directly correlated with VEGF‑A expression and microvessel density in vivo. Our findings revealed that anoikis resistance in osteosarcoma cells increased the expression of VEGF‑A and angiogenesis through the Src/JNK/ERK signaling pathways. Thus, Src may be a potential therapeutic alternative in osteosarcoma angiogenesis and metastasis.

Figure 1.

Characteristics of parental and anoikis-resistant osteosarcoma cells (MTH, MTHar, U2OS and U2OSar). (A) Osteosarcoma cells were round and grew in clusters gradually in suspension culture. Scale bar, 100 µm. (B) The morphology of parental and anoikis-resistant osteosarcoma cells. Scale bar, 25 µm. (C) Flow cytometry demonstrating that anoikis-resistant osteosarcoma cells had significantly decreased apoptosis compared with parental cells under suspension conditions. Results are expressed as the mean ± SD. *P<0.05.

Figure 2.

Anoikis-resistant osteosarcoma cells enhances angiogenesis by increasing VEGF-A expression. (A-C) Cultured medium was collected as CM (MTH, MTHar, U2OS and U2OSar cells) and applied to HUVECs. HUVEC capillary-like cell migr ation, structure formation, and proliferation were examined by (A) wound healing, (B) tubeformation and (C) cell proliferation assays, respectively. Scale bar, 100 µm. (D-F) VEGF-A mRNA and protein expression in parental and anoikis-resistant osteosarcoma cells was detected by (D) RT-qPCR, (E) western blotting and (F) ELISA. Each experiment was performed in triplicate. Results are expressed as the mean ± SD. *P<0.05.

Figure 3.

Src inhibitor reduces VEGF-A expression and inhibits angiogenesis in anoikis-resistant osteosarcoma cells. Anoikis-resistant osteosarcoma cells were pre-treated with Src inhibitor (PP2) and dimethyl sulfoxide (DMSO) for 24 h. (A-C) CM was applied to HUVECs. HUVEC capillary-like cell migration, structure formation, and proliferation were examined by (A) wound healing, (B) tube formation and (C) cell proliferation assays, respectively. Scale bar, 100 µm. (D-F) VEGF-A mRNA and protein expression were detected by (D) RT-qPCR, (E) western blot analysis and (F) ELISA. (G) Western blot detection of Src, JNK, ERK and p38 phosphorylation. Each experiment was performed in triplicate. Results are expressed as the mean ± SD. *P<0.05.

Figure 3.

Src inhibitor reduces VEGF-A expression and inhibits angiogenesis in anoikis-resistant osteosarcoma cells. Anoikis-resistant osteosarcoma cells were pre-treated with Src inhibitor (PP2) and dimethyl sulfoxide (DMSO) for 24 h. (A-C) CM was applied to HUVECs. HUVEC capillary-like cell migration, structure formation, and proliferation were examined by (A) wound healing, (B) tube formation and (C) cell proliferation assays, respectively. Scale bar, 100 µm. (D-F) VEGF-A mRNA and protein expression were detected by (D) RT-qPCR, (E) western blot analysis and (F) ELISA. (G) Western blot detection of Src, JNK, ERK and p38 phosphorylation. Each experiment was performed in triplicate. Results are expressed as the mean ± SD. *P<0.05.

Figure 4.

Representative IHC staining of p-Src, VEGF-A and CD31 in osteosarcoma specimens. Images for p-Src and VEGF-A were obtained under an ×400 magnification and that for CD31 was obtained under an ×200 magnification. Scale bar, 30 µm.

Figure 5.

Anoikis resistance in human osteosarcoma cells promotes angiogenesis in a nude mouse xenograft model. (A) Representative immunohistochemical staining of p-Src, VEGF-A and CD34 inxenograft tumors. Scale bar, 30 µm. (B) p-Src and VEGF-A expression was higher in anoikis-resistant cells, as was MVD. Results are expressed as the mean ± SD. *P<0.05. (C) Proposed model for illustrating the mechanism of angiogenesis:anoikis-resistant human osteosarcoma cell enhanced VEGF-A expression andangiogenesis via the Src/JNK/ERK signaling pathways.