Blockage of Store-operated Ca Entry Antagonizes Epstein-Barr Virus-promoted Angiogenesis by Inhibiting Ca Signaling-regulated VEGF Production in Nasopharyngeal Carcinoma

Background: Epstein-Barr virus (EBV) actively contributes to the pathological process of nasopharyngeal carcinoma (NPC) by enabling NPC cells to acquire various capacities required for their malignant biological actions. Our earlier works demonstrated that EBV-encoded latent membrane protein 1 (LMP1) enhanced vascular endothelial growth factor (VEGF)-mediated angiogenesis by boosting store-operated Ca entry (SOCE) upon extracellular epidermal growth factor (EGF) stimulation. However, the antagonistic effects of SOCE blockage on EBV-promoted angiogenesis must be appropriately evaluated in vivo, and the global effect of EBV infection on the EGF-elicited cytosolic Ca signaling, which regulates VEGF-mediated angiogenesis remains to be further clarified.

Materials And Methods: Two EBV-infected NPC cell lines, CNE2-EBV and HK1-EBV, along with their parental cell lines were employed in the present study. Dynamic cytosolic Ca changes were measured in individual fluorescent Ca indicator-loaded cells. Amounts of VEGF production were determined by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs)-formed tube networks were quantitatively evaluated as an in vitro angiogenesis assay. A mouse model concurrently bearing EBV-positive/negative xenografts was utilized to evaluate the tumor growth and angiogenesis in vivo.

Results: EBV infection reliably promoted transplanted tumor growth while enhancing angiogenesis. Introduction of EBV into EBV-negative NPC cells increased the EGF-stimulated VEGF production while amplifying the EGF-evoked Ca responses. Inhibition of the EBV-boosted Ca signaling using 2-aminoethyl diphenylborinate (2-APB), a specific SOCE inhibitor, effectively antagonized the EBV-promoted VEGF production and endothelial tube formation in vitro. Pharmacological blockage of SOCE exhibited anti-angiogenic effect in the EBV-positive xenografts.

Conclusion: SOCE can serve as a candidate pharmacological target for treating NPC, as blockage of the Ca signaling via SOCE is a feasible strategy to suppress the EBV-driven malignant profiles in NPC cells.