Honokiol Induces Apoptosis And Autophagy Via The ROS/ERK1/2 Signaling Pathway In Human Osteosarcoma Cells

Purpose:Osteosarcoma is the most common primary malignant tumor of bone.Although the current survival rate of osteosarcoma patients can reach about 70%,there has been no breakthrough in the treatment of osteosarcoma in the past decades.Moreover,the metastasis and recurrence of osteosarcoma greatly affect the prognosis of patients.Therefore,it is an urgent clinical task to find new therapeutic methods for osteosarcoma.Honokiol is a biphenyl compound extracted from the Magnoliaceae plant and has been found to have therapeutic effects on cerebral ischemia,anxiety,and pain,as well as anti-microbial,anti-inflammatory,and anti-tumor properties.In view of the anti-tumor effect of honokiol,this study aimed to explore the therapeutic effect of honokiol on osteosarcoma.Methods:In this study,we examined the inhibitory effect of honokiol on the proliferation of human osteosarcoma cells through cell viability assay and colony formation assay.The effects of honokiol on cell cycle were investigated by cell flow cytometry and cycle-related protein detection.The effects of honokiol on apoptosis were determined by flow cytometry,mitochondrial membrane potential detection,and apoptosis-related protein detection.Using AO staining,MDC staining,electron microscopy and autophagy-related protein detection to explore the effect of honokiol on cell autophagy.We used Z-VAD-FMK and 3-MA to inhibit apoptosis and autophagy,respectively,and observe their interactions.After treatment with honokiol,the expression levels of ROS and ERK1/2 were examined.ROS scavengers and the ERK1/2 pathway inhibitor PD98059 were then used to examine whether honokiol-induced apoptosis and autophagy were altered.A nude mouse xenograft tumor model was established.The nude mice were given an intraperitoneal injection of honokiol and the antitumor effect of honokiol was observed.Results:Honokiol can inhibit the proliferation of osteosarcoma cells,cause G0/G1 cell cycle arrest,promote cell apoptosis and autophagy.Using Z-VAD-FMK and 3-MA to inhibit honokiol-induced apoptosis and autophagy,respectively,we found that apoptosis decreased after inhibion of autophagy and autophagy increased after inhibition of apoptosis,suggesting that honokiol induced cytotoxic autophagy.We observed that honokiol can induce the production of ROS in osteosarcoma cells and activation of ERK1/2 signaling pathway.When ROS scavenger was used,honokiol-induced apoptosis and autophagy were significantly inhibited,and ERK1/2 phosphorylation levels decreased.In addition,pretreatment with PD98059 also inhibited honokiol-induced apoptosis and autophagy.This shows that ROS/ERK1/2 is the main pathway for honokiol to exert anti-tumor effects.Finally,we found that honokiol can inhibit the growth of osteosarcoma in xenograft tumor models in nude mice,suggesting that honokiol also has anti-tumor function in vivo.Conclusion:Our study showed that honokiol can induce cell cycle arrest in G0/G1 phase and activate osteosarcoma cell apoptosis and autophagy through ROS/ERK1/2 pathway.Therefore,honokiol is a potential anti-osteosarcoma drug.