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Introduction
Neuroblastoma is the most common extracranial solid tumor in children; approximately half of patients with neuroblastoma present with metastatic disease at the time of diagnosis (1,2). Metastasis of neuroblastoma can occur by the hematogenous route, seeding bone marrow, liver, and bone. The treatment of patients with disseminated neuroblastoma is one of the greatest challenges for pediatric oncologists, given that the five-year survival rate remains as low as 40-45%, despite advanced treatment options (3). Therefore, it is essential to develop effective strategies to inhibit tumor metastasis.
Cancer metastasis begins with the detachment of metastatic cells from the primary tumor, migration of the cells to distal sites through blood vessels, settlement, and growth at the distal site (4). During this process, metastatic cells go through detachment, migration, invasion, and adhesion (4). Each of these processes involves rate-limiting steps influenced by non-malignant cells of the tumor microenvironment (5). Tumor vascularization is a rate-limiting step for metastasis. Vascular endothelial growth factor (VEGF) is a highly potent molecule that increases vessel permeability, endothelial cell growth, proliferation, migration, and differentiation (6).
Nafamostat mesylate is a synthetic serine protease inhibitor approved for pancreatitis and disseminated intravascular coagulation (DIC) (7). This agent inhibits thrombin, plasmin, kallikrein, trypsin, and C1 esterase in the complement system and factors VIIa, Xa, and XIIa in the coagulation cascade. Moreover, nafamostat mesylate inhibits the metastasis of colon cancer, pancreatic cancer, breast cancer, and squamous cell carcinoma (8-14). However, the effects of nafamostat mesylate on other types of cancer have been less extensively studied.
In the present study, the effects of nafamostat mesylate on neuroblastoma regarding cell proliferation, motility, cell-invasive potential, and growth factor production were investigated. It is suggested that the results of the present study will contribute to developing new aspects of treatment for neuroblastoma.
Materials and methods
Reagents
Nafamostat mesylate was obtained from Nichi-Iko Pharmaceutical Co., Ltd. (product no. 873999), dissolved in distilled water, and stored at -80˚C until use.
Cell culture
The murine neuroblastoma cell line, Neuro-2a (cat. no. ATCC-CCL-131; American Type Culture Collection), was maintained in RPMI-1640 medium (product no. R8758; Sigma-Aldrich: Merck KGaA) and 10% fetal bovine serum (FBS; cat. no. CCP-FBS-BR-500; Cosmo Bio Co., Ltd.). The cells were cultured in a standard T75 cell culture flask (cat. no. 156499; Thermo Fisher...