Mucronulatol Induces Apoptosis in Oral Squamous Cell Carcinoma by c-Jun N-terminal kinase Signaling Pathway

Abstract Background: Oral squamous cell carcinoma (OSCC) is the most common type of cancer affecting the oral cavity. Mucronulatol is a methoxyisoflavan and its anti-tumor role remains nearly unknown. Aims and Objectives: This study aimed to determine if there is a potential link between mucronulatol and OSCC tumorigenesis and to further investigate the possible regulatory mechanisms involved. Materials and Methods: Human OSCC cell lines SAS and SCC9 were treated with mucronulatol at various concentrations and time points. Cell viability, cell-cycle distribution, mitochondrial membrane potential, apoptosis-related protein expression, and signaling pathway activation were assessed using flow cytometry and western blot analysis. Pharmacological inhibition of JNK and siRNA-mediated knockdown of XIAP were employed to clarify the mechanistic pathways involved. Results: Mucronulatol dose and time dependently reduced cell viability, increased cell cycle arrest at G2/M phase, thereby inhibiting cell cycle proteins-cyclin A, D3, cyclin-dependent kinase 2 (CDK2), CDK4, and CDK6 in SAS and SCC9 cells. Meanwhile, mucronulatol upregulated cleaved caspase 9, caspase 3 and poly (ADP-ribose) polymerase which were then accompanied by increased mitochondrial membrane depolarization in oral cancer cells. Oral cancer cells pretreated with the c-Jun N-terminal kinase (JNK) inhibitor decreased caspase 3/7 and caspase 9 expressions were rescued after mucronulatol treatment. We also investigated the effect of mucronulatol on protein kinase B, extracellular signal-regulated kinase, p38, and JNK pathways. Our results, from western blot analysis, suggest that mucronulatol decreases phosphorylation of JNK at treatment concentrations of 100 μM in both the cell lines. JNK inhibitor decreased cleaved caspase 3/7 and caspase 9 expressions were elicit by mucronulatol treatment. Mucronulatol specifically decreased X-linked inhibitor of apoptosis (XIAP) expression to execute its pro-apoptotic activities. Si-XIAP decreased cleaved caspase 3/7 and caspase 9 expressions were reversed after mucronulatol treatment. Conclusions: From these results, it may be inferred that mucronulatol is a good chemotherapeutic candidate for oral cancer, as it activates JNK in response to XIAP inhibition, exhibiting anti-tumor efficacy.