Plumbagin Regulates JNK And AkT/mTOR Signaling Pathway By Inducing Reactive Oxygen Species Production To Increase The Chemosensitivity Of Tongue Squamous Cell Carcinoma To Cisplatin

Background:Tongue squamous cell carcinoma(TSCC)is the most common malignant tumor of epithelial origin in oral cancers.Patients with advanced TSCC often have a poor prognosis due to chemoresistance.Cisplatin is the best chemotherapy drug for patients with advanced tongue squamous cell carcinoma,but it often results in poor efficacy due to chemotherapy resistance.In recent years,with the in-depth research on the drug resistance mechanism of tumor chemotherapy,the level of intracellular reactive oxygen species(ROS)is considered to be a key factor affecting the cytotoxicity of chemotherapy drugs.The relationship between the level of ROS in tongue squamous cell carcinoma cells and its resistance to cisplatin has not been reported yet.Recently,the combination therapy of phytochemicals and traditional chemotherapeutics are widely used to overcome the multidrug resistance of cancer,which have achieved remarkable results.Plumbagin is a chemical substance of naphthoquinone extracted from the root of plumbagin.Our previous studies have found that plumbagin play a key role to inhibit the growth of TSCC,and it can induce the production of intracellular ROS.However,whether plumbagin exert its cytotoxicity by regulating the intracellular ROS level and thereby regulate the chemoresistance of TSCC to cisplatin and its mechanism are still unclear.Objective:The main aim of the present study is:to investigate the relationship between the level of intracellular ROS and cisplatin resistance in TSCC cells.Next,to explore whether plumbagin exerts its cytotoxicity by inducing the production of intracellular ROS and affects the chemosensitivity and mechanism of TSCC to cisplatin.Methods:Human tongue squamous cell carcinoma CAL27 and cisplatin-resistant CAL27/CDDP cells were used in this research.CCK-8 cell viability detection kit was used to detect the cell viability.CM-H₂DCFDA reactive oxygen detection probe and Mito SOX Red mitochondrial ROS detection probe were used to detect the changes in the levels of ROS.Clone formation experiment was used to test the growth of TSCC cells.DAPI nuclear staining kit and flow cytometry assay were used to detect the apoptosis.Cyto-ID autophagy detection kit,Lyso Tracker autophagy-lysosome detection kit and transmission electron microscopy were used to detect the autophagy.Western Blotting used to detect the expression levels of apoptosis,autophagy and signaling pathway-related proteins.BALB/c nude mice xenograft models were used to study the growth inhibitory effect and side effects on the body of the combination of plumbagin and cisplatin in vivo.Results:The present study found that:1.Down-regulated the level of intracellular ROS in TSCC cells can reduce the cytotoxicity of cisplatin.2.Plumbagin inhibits the activity and proliferation of TSCC cells by inducing the production of intracellular ROS.Plumbagin induces the production of ROS,activates JNK,and inhibits the AKT/m TOR signaling pathway to induce apoptosis and autophagy in TSCC cells;.3.The combination of plumbagin and cisplatin exert a synergistic anticancer effect by generating ROS,activating JNK,and inhibiting the AKT/m TOR pathway in TSCC cells.4.The nude mouse xenograft tumor model has verified the synergistic anti-tumor effect of plumbagin and cisplatin without obvious side effects.Conclusions:In summary,our study shows that the down-regulation of intracellular ROS is one of the main mechanisms of cisplatin resistance in TSCC.Plumbagin can exert its cytotoxic effect by up-regulating the level of intracellular ROS.The combination of plumbagin and cisplatin exert a synergistic anticancer effect by upregulating ROS,activating JNK,and inhibiting the AKT/m TOR pathway in TSCC cells.The combination of plumbagin and cisplatin exert a synergistic anticancer effect in nude mouse xenograft tumor model without obvious side effects.