Scutellarein Inhibits Osteosarcoma Growth by Targeting the TLR4/TRAF6/NF-κB Pathway

Abstract

Yingxu Shi,1 Yu Tang,2 Zhiwei Sun,3 Ping Sui,4 Yiming Shao,4 Zhonghao Wang,4 Jian Zhang,1 Ming Gao1 1Department of Trauma Orthopedics, Affiliated Hospital of Jining Medical University, Jining, Shandong, 272007, People’s Republic of China; 2Pharmaceutical Department, Affiliated Hospital of Jining Medical University, Jining, Shandong, 272007, People’s Republic of China; 3Department of Surgery, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA; 4Department of Clinical Medicine, Jining Medical University, Jining, Shandong, 272067, People’s Republic of ChinaCorrespondence: Ming Gao, Department of Trauma Orthopedics, Affiliated Hospital of Jining Medical University, No. 129 hehua Road, Jining, Shandong, 272007, People’s Republic of China, Tel + 86-0537-0298202, Email [email protected]: Osteosarcoma (OS) is the most common malignant tumor associated with poor patient outcomes and a limited availability of therapeutic agents. Scutellarein (SCU) is a monomeric flavone bioactive compound with potent anti-cancer activity. However, the effects and mechanisms of SCU on the growth of OS remain unknown.Methods: The Cell Counting Kit-8, colony formation assay and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays were used to analyze cell proliferation ability in vitro. TLR4/TRAF6/NF-κB signaling transduction was investigated by RNA sequencing analysis, quantitative real-time polymerase chain reaction, Western blotting, NF-κB luciferase reporter assay, immunofluorescent staining, and immunoprecipitation. Molecular docking and cellular thermal shift assay were employed to confirm the binding interaction between SCU and TLR4. The effects of SCU and TLR4 overexpression on OS growth were analyzed using a xenograft tumor model and immunohistochemical staining.Results: SCU was found to significantly inhibit OS cell proliferation, and RNA sequencing analysis suggested that the NF-κB pathway is closely associated with this process. Further studies revealed that SCU inhibited the canonical NF-κB pathway through its binding with TLR4, which disrupted the interaction of TLR4 and TRAF6. Moreover, SCU also repressed NF-κB signal transduction by inhibiting TLR4 expression. Furthermore, SCU was revealed to suppress OS cell proliferation by targeting TLR4 in vitro and in vivo.Conclusion: SCU exhibited a dual impact by inhibiting TLR4 expression and disrupting TLR4–TRAF6 interaction, resulting in NF-κB inactivation, thereby blocking OS growth. Keywords: scutellarein, osteosarcoma, TLR4, NF-κB pathway, molecular modeling

Keywords

• scutellarein
• osteosarcoma
• tlr4
• nf-κb pathway
• molecular modeling