Melittin inhibits osteosarcoma growth in vitro and in vivo by inactivating the Wnt/β-catenin signaling pathway

Objective: Osteosarcoma is a highly aggressive primary bone tumor that predominantly affects pediatric and adolescent populations. This study aims to explore the therapeutic potential and underlying mechanisms of melittin in treating osteosarcoma both in vitro and in vivo. Materials and methods: Two osteosarcoma cell lines, namely 143B and U-2 OS, were utilized to assess the impact of melittin on cellular proliferation, apoptosis, and cell cycle progression. The effect of melittin on cell viability was evaluated using the CCK-8 assay. Flow cytometry was employed to assess apoptosis and cell cycle progression, while Western blotting analyzed the expression of key proteins, including Cdc-2, c-Myc, and ꞵ-catenin. Specifically, we employed small interfering RNA (siRNA) to selectively knock down the expression of β-catenin in two osteosarcoma cell lines. In vivo studies, tumor growth in nude mice was evaluated through measurements of tumor weight, volume. Additionally, immunohistochemical analysis was performed to assess Ki-67 and active ꞵ-catenin expression in tumor tissues. Results: Melittin induced apoptosis in 143B and U-2 OS osteosarcoma cells in a concentration-dependent manner and caused S-phase cell cycle arrest as the drug concentration increased. Mechanistic investigations revealed that melittin's efficacy was associated with the inactivation of the Wnt/ꞵ-catenin signaling pathway. Specifically, melittin treatment reduced the expression of phosphorylated glycogen synthase kinase-3 beta (p-GSK-3ꞵ) and active ꞵ-catenin. Notably, silencing β-catenin in 143B and U-2 OS osteosarcoma cells significantly enhanced the anti-proliferative and pro-apoptotic effects of melittin. This was evidenced by reduced cell viability and increased apoptosis, thereby further confirming that the anti-tumor efficacy of melittin is dependent on the inhibition of the Wnt/β-catenin signaling pathway. In vivo studies confirmed that melittin significantly inhibited the growth of subcutaneously implanted tumors in nude mice. This inhibition was further supported by Ki-67 analyse and H&E staining, while immunohistochemical analysis revealed an obvious reduction in active ꞵ-catenin expression. Conclusions: Our results offer deeper mechanistic insights into the inhibitory impact of melittin on osteosarcoma progression, at least in part by suppressing the Wnt/β-catenin signaling pathway. We expand upon previous research by providing more comprehensive and robust evidences that underscore the potential of melittin as a viable and safe therapeutic agent for osteosarcoma.