Background and Purpose:Currently,lung cancer has the highest mortality rate in the world,and cisplatin-based therapy is still the standard treatment regimen for lung cancer patients.However,relapse frequently occurs due to resistance to cisplatin,so cisplatin resistance remains a major obstacle to successfully treating cancer patients with anticancer drugs.Glucose metabolism has generated much interest over the past few decades due to its role in cancer.Many studies show that glucose plays a critical role in cell survival and the development of drug resistance.The pentose phosphate pathway(PPP),branching from glycolysis at the first committed step,is the primary source of NADPH.NADPH is required and consumed during fatty acid synthesis and reactive oxygen species clearance.Thus,PPP plays a crucial role in helping cancer cells meet their anabolic needs and combat oxidative stress.Sesquiterpene lactone compound xanthatin,one of the main components of Xanthium compositae,has antitumor effects on various cancers.Previous studies have shown that xanthatin has antitumor effects on various tumor cells and can inhibit the glycolysis pathway in the energy metabolism of tumor cells,thus promoting oxidative phosphorylation.Therefore,this study aimed to investigate whether xanthatin can regulate the pentose phosphate pathway of cisplatin-resistant lung cancer cells by affecting GLUT1 expression,thereby inducing apoptosis of cisplatin-resistant cells.Objective:As an active sesquiterpene lactone compound,xanthatin is isolated from the Xanthium strumarium L.,our previous studies have shown that xanthatin has antitumor effects on various tumor cells and can promote oxidative phosphorylation by inhibiting glycolysis in energy metabolism of tumor cells.This study aimed to investigate whether xanthatin can regulate the pentose phosphate pathway in cisplatin-resistant lung cancer cells by affecting GLUT 1 expression,thereby inducing apoptosis of resistant cells.Methods:1.In vitro cell experiment:Different concentrations of xanthatin treated with A549/DDP and H460/DDP cells,then cell proliferation,migration,and apoptosis were examined by CCK-8,CFDA SE,Transwell,flow cytometry,and Western blot,respectively.Subsequently,intracellular NADPH/NADP+ ratio and ROS levels were assessed in A549/DDP cells after overexpressing the GLUT1.2.In vivo animal experiment:Nude mouse transplanted tumor model of cisplatin-resistant human lung cancer A549/DDP cells were established and randomly divided into a control group,a cisplatin group(2mg/kg/d),xanthatin group(40mg/kg/d).All tumors were harvested to detect the tumor volume and tumor weight,followed by being processed into frozen sections for HE staining,tunnel assay and Ki67 staining.Results:1.Effects of xanthatin on DDP-resistant lung cancer cells viabilityIn the CCK-8 experiment,xanthatin inhibited A549/DDP and H460/DDP cell viability compared with the control group.The results showed that colony formation decreased significantly with the increase of xanthatin concentration.Moreover,the assessment of CFSE by flow cytometry showed much lower cell proliferation in the xanthatin treatment groups than in the control.These data revealed that xanthatin could reduce the cell viability and proliferation capacity of resistance cells.2.Xanthatin potently inhibits migration and increases apoptosis of DDP-resistant lung cancer cellsThe migrations of A549/DDP and H460/DDP cells were measured by transwell assay in the study.Cell migration was remarkably suppressed by xanthatin compared with the control.The results of JC-1 showed that the green fluorescence increased and the mitochondrial membrane potential decreased after treatment with different concentrations of xanthatin.The western blot analysis results demonstrated that xanthatin could markedly increase the protein levels of the cleaved-caspase3 and cleaved-PARP,and decrease the protein expression levels of β-catenin,slug as compared with the control group.These results strongly suggested that xanthatin could suppress migration and induce apoptosis in A549/DDP and H460/DDP cells.3.Xanthatin inhibits DDP-resistance cells survival via inhibiting NADPH/ROS metabolismTo investigate the potential mechanisms,we explored the effect of xanthatin on the intracellular redox status of A549/DDP and H460/DDP cell lines.Notably,these DDPresistance cells exhibited an increased ROS level and decreased intracellular NADPH/NADP+and GSH/GSSG ratios.We used antioxidant GSH(2 mM)and NAC(8 mM)for the rescue experiment.As expected,GSH and NAC significantly reduced xanthatin-induced ROS and apoptosis.Meanwhile,xanthatin considerably decreased cell proliferation through CFDA SE and CCK-8 assays,while supplementation of the NAC or GSH markedly reversed this inhibition.These data suggest that xanthatin inhibits DDPresistance cell growth by promoting ROS-dependent apoptosis via inhibiting PPP and reducing NADPH and GSH production.4.Xanthatin causes PPP suppression and ROS accumulation by inhibiting GLUT1.Western blotting showed that the protein level of GLUT1 was relatively higher in A549/DDP and H460/DDP than in A549 and H460 cells.Furthermore,GLUT1 protein expression was decreased in A549/DDP and H460/DDP cells compared with blank control after treatment with xanthatin.As expected,immunofluorescence analysis showed that the expression of membrane-associated GLUT1 was markedly decreased.We transfected A549/DDP cells with pcDNA-GLUT1 or pcDNA-NC.overexpression of GLUT 1 significantly upregulated the expression of GLUT1 at both mRNA and protein levels.Interestingly,overexpression of GLUT1 significantly increased cell viability of xanthatin-treated conditions.Flow cytometry analysis indicated that GLUT1 overexpression immensely reduced the production of ROS,whereas xanthatin significantly elevated the ROS levels.Thus,overexpression of GLUT1 significantly increased cellular NADPH/NADP+and GSH/GSSG ratios,and the reintroduction of xanthatin rescued the effects of overexpression of GLUT1.The data further verified that xanthatin induces A549/DDP cell apoptosis,which may increase ROS by inhibiting GLUT 1 expression.5.The efficacy of xanthatin in A549/DDP tumor xenograft mouse modelThe effects of xanthatin on DDP resistance cells in vivo were assessed by establishing a xenograft model in mice.Xanthatin-treated group(40mg/kg/d)could effectively inhibit the growth of transplanted tumors in nude mice and produce obvious anti-tumor effects.Further,the mRNA and protein levels of GLUT1 were reduced considerably in xanthatintreated tumors compared with vehicle and cisplatin-treated(2mg/kg/d)tumors.These results indicate that xanthatin could decrease GLUT1 expression while effectively inhibiting tumor cell proliferation and promoting tumor cell apoptosis in vivo.Conclusion:This study shows that xanthatin is a promising antitumor compound that induces apoptosis of cisplatin-resistant lung cancer cells by regulating GLUT 1-mediated ROS accumulation.These findings may provide a possible strategy for treating cisplatinresistant lung cancer. |