Abstract
Endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling is important for osteosarcoma (OS) progression. Monoclonal antibodies (mAbs) targeting ganglioside GD2 reportedly inhibit tumor cell viability independent of the immune system. A recent study suggests that ganglioside GD2 may play an important role in OS progression. In the present study, we for the first time explored the effects of anti-GD2 mAb alone or in combination with ETAR antagonist on OS cell invasiveness and viability. Human OS cell lines Saos-2, MG-63 and SJSA-1 were treated with control IgG (PK136 mAb, 50 µg/mL), anti-GD2 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), or 14G2a (50 µg/mL)+BQ123 (5 µM). Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) were used as a positive control. Our results showed that BQ123, ETAR-shRNA and 14G2a mAb individually decreased cell invasion and viability, matrix metalloproteinase-2 (MMP-2) expression and activity, PI3k activity, and phosphorylation at serine 473 (ser473) of Akt in OS cells. 14G2a mAb in combination with BQ123 or ETAR-shRNA showed significantly stronger inhibitory effects compared with each individual treatment. In all three cell lines tested, 14G2a mAb in combination with BQ123 showed the strongest inhibitory effects. In conclusion, we provide the first in vitro evidence that anti-ganglioside GD2 14G2a mAb effectively inhibits cell invasiveness, MMP-2 expression and activity, and cell viability in human OS cells. ETAR antagonist BQ123 significantly enhances the inhibitory effects of 14G2a mAb, likely mainly through inhibiting the PI3K/Akt pathway. This study adds novel insights into OS treatment, which will serve as a solid basis for future in vivo studies on the effects of combined treatment of OS with anti-ganglioside GD2 mAbs and ETAR antagonists.
Highlights
Osteosarcoma (OS) is the most frequent primary bone malignancy and the eighth most common type of cancer among children, comprising 2.4% of all malignancies in pediatric patients and approximately 35% of all bone cancers [1]
matrix metalloproteinase-2 (MMP-2) protein levels were determined by Western blot analyses in in Saos-2 (A), MG-63 (B) and SJSA-1 (C) cells treated with control IgG (50 mg/mL, lane 2), selective endothelin A receptor (ETAR) antagonist BQ123 (5 mM, lane 3), stably-transduced ETAR-shRNA, 14G2a Monoclonal antibodies (mAbs) (50 mg/mL, lane 5), 14G2a+BQ123, 14G2a+ETAR-shRNA, and selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 mM, lane 8)
In addition to blocking ET-1/ETAR signaling with BQ123, we knocked down ETAR in the cell lines to verify the role of ETAR in OS invasiveness and viability
Summary
Osteosarcoma (OS) is the most frequent primary bone malignancy and the eighth most common type of cancer among children, comprising 2.4% of all malignancies in pediatric patients and approximately 35% of all bone cancers [1]. OS is a devastating disease, characterized by high local aggressiveness and a tendency to metastasize to the lungs and distant bones. The cure rate of OS is approximately 65% for patients with localized diseases. When presenting with metastases at the time of diagnosis, the survival rate is 25% [2,3]. Despite modern multidisciplinary treatments including chemotherapy and surgery, the 5-year survival rate of osteosarcoma patients remains 60%–70% [1]. There is an urgent need to develop novel approaches to treat OS patients, to identify and confirm potential therapeutic targets involved in OS progression
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