Effect And Mechanism Of Evodiamine On Tumor Growth And Aerobic Glycolysis In Wilms Tumor

Background:Wilms tumor（WT）is the most prevalent renal malignancy tumor in infants and children.Existing treatments for WT can cause some serious complications,such as heart failure,renal failure and cognitive impairment secondary to chemoradiotherapy,affecting cancer survivors’normal life and long-term treatment effect,so we expect to find a drug with good anti-tumor effect,little toxicity and less side effects for the treatment of WT.Aerobic glycolysis is a characteristic metabolic mode of tumors,tumors metabolize glucose into lactate and ATP in the presence of O₂.Hyperactive glycolysis can quickly provide energy for tumor cells,and participate in the synthesis process of essential nutrients such as amino acids and lipids,in order to better meet the needs of rapid proliferation of cancer cells.Evodiamine（EVO）is the main effective ingredient of the traditional Chinese medicine Euodia rutaecarpa（Juss.）Benth.,which can relieve pain and stop vomiting and diarrhea,at the same time,studies have proved that EVO has good anti-tumor effect,mainly by inhibiting malignant behaviors such as tumor proliferation,invasion and metastasis.However,it is still unknown whether EVO can inhibit the development of WT and regulate its aerobic glycolysis.Moreover,studies have shown that the AKT pathway is related to abnormal energy metabolism,and AKT may be a key target in the regulation of aerobic glycolysis.Objective:WT cells were treated with different concentrations of EVO,the therapeutic effect of EVO on WT was clarified by observing the influence of EVO on human WT cell growth and aerobic glycolysis in vitro,the role of AKT pathway in aerobic glycolysis was explored,so as to clarify the mechanism of EVO regulating WT aerobic glycolysis,and provide new ideas for the treatment of WT.Method:（1）Changes in viability of human WT cells treated with different concentrations of EVO at 24 h,36 h and 48 h were examined by CCK8 assay,and appropriate drug concentrations and drug treatment time were selected for subsequent experiments.（2）The effects of EVO on tumor proliferation,invasion,metastasis and apoptosis were evaluated by clone formation assay,Transwell assay,Annexin V-FITC/PI staining and Western blot assay.（3）Glucose consumption and lactate production assays were used to analyze the effects of EVO on glucose consumption and lactate production levels in WT cells.（4）The effects of EVO on the expression of AKT pathway molecules（AKT and p-AKT）and aerobic glycolysis-related molecules（GLUT1,PFK1,HK2,PKM2,LDHA）was assessed by q RT-PCR and Western blot.Changes in glucose consumption,lactate production and the expression of aerobic glycolysis-related molecules in WT cells were measured by combining EVO and AKT agonists/inhibitors.Result:（1）EVO intervention significantly inhibited the cell viability,colony formation ability,invasion,migration and promoted tumor apoptosis in WT cells in both a dose-dependent and time-dependent manner（P<0.05）.EVO decreased the expression of proliferation-related molecule Ki67 and enhanced the expression of apoptosis-related molecules such as Caspase-3 and Caspase-9（P<0.05）.（2）EVO intervention reduced glucose consumption and lactate production in WT cells,indicating that EVO could inhibit the aerobic glycolysis of WT.（3）Expression of glycolytic protein PFK1 was confirmed to be significantly down-regulated（P<0.05）,while the expression of protein GLUT1,HK2,PKM2 and LDHA showed little or no significant change（P>0.5）.The above data illustrated that the decrease of aerobic glycolysis in WT cells was mainly due to reduced expression of PFK1 protein induced by EVO.（4）EVO downregulated the expression of AKT pathway molecule p-AKT（P<0.05）,while the expression change of molecule AKT was not statistically different（P>0.05）,suggesting that EVO could induce changes of the AKT pathway in WT cells.Rescue experiments suggested that the combination of EVO and AKT agonist reversed the inhibitory effect on aerobic glycolysis induced by EVO alone,and the inhibitory effect of EVO was enhanced when EVO was used in combination with AKT inhibitor in WT cells.Conclusion:EVO can exert anti-tumor effects by inhibiting the proliferation,invasion,migration,aerobic glycolysis,and inducing cell apoptosis of WT cells,and this study also suggests that EVO may downregulate the expression of the key glycolytic enzyme PFK1 through the AKT pathway,thereby inhibiting the aerobic glycolysis process of WT.Therefore,EVO may be an effective anticancer agent and glucose metabolism regulator.