Anti-cancer effect of methanol extract of Piper aduncum L. in human osteosarcoma cell lines

Abstract

Osteosarcoma is the most frequent primary malignant tumor of the skeleton and occurs mainly in children and adolescents. Several chemotherapeutic agents are used to treat osteosarcoma, but these agents have side effects such as congestive heart failure. To minimize these side effects, attempts are being made to develop anti-cancer drugs using plant resource extracts. In this study, the anti-cancer effect of methanol extract of Piper aduncum L. (MEPA) on human osteosarcoma cell lines MG-63 and Saos-2 was investigated. The WST-1 assay was used to confirm the cell viability of the osteosarcoma cell lines MG-63 and Saos-2 and the normal cell line HaCaT. As a result, MEPA showed high cytotoxicity in a dose-dependent manner in the osteosarcoma cell lines. The results of DAPI staining and the Annexin V/PI assay showed MEPA induces apoptosis of MG-63 and Saos-2. To confirm the mechanism of apoptosis, western blot was performed. MEPA induced apoptosis in MG-63 and Saos-2 by up-regulating the expression of caspase and pro-apoptotic proteins while down-regulating the expression of anti-apoptotic proteins. The cell cycle arrest of human osteosarcoma cells was confirmed through cell cycle analysis. The expression levels of cell cycle arrest-related proteins were measured using western blot. These findings suggested that MEPA induces cell cycle arrest of MG-63 and Saos-2 at the G2/M phase. Furthermore, the expression of the PI3K/Akt signaling pathway- and the Wnt/β-catenin signaling pathway-related proteins was investigated through western blot. The expression levels of PI3K 110α and p-Akt were decreased in a dose-dependent manner. In addition, the phosphorylation level of GSK-3β was increased; however, the level of p-β-catenin was decreased in MEPA-treated cells. These results showed that MEPA has anti-cancer activity through apoptosis and cell cycle arrest at the G2/M phase and inhibition of the PI3K/Akt signaling pathway and the Wnt/β-catenin signaling pathway. In conclusion, Piper aduncum L. has positive potential in anti-cancer effects in osteosarcoma.