Exploring The Anti-metastatic Mechanism Of Sparganium Stoloniferum Component Kaempferol Based On Glycolysis Rate-limiting Enzyme

Background and PurposeMelanoma is a kind of skin cancer with high malignant degree and high metastasis.the incidence of melanoma in China is increasing year by year,while metastasis has greatly reduced the survival rate of patients.Therefore,how to inhibit metastasis is the most important problem in tumor treatment.Aerobic glycolysis,also known as "Warburg effect",is not only a common metabolic feature of tumors,but also the result of poor adaptation to the living environment of tumors.Warburg effect provides tumor cells with the advantage of growth and proliferation,and provides sufficient energy and macromolecular synthetic substrate.The acidic microenvironment is conducive to neovascularization and cellular pseudopodia formation,as well as tumor immune escape and chemotherapy resistance mediated by acidic microenvironment,which are conducive to tumor metastasis.The three key kinases HK,PFK and PK in the glycolysis pathway regulate the three irreversible reaction steps respectively.Interfering with the activity or expression of one of the kinases can significantly inhibit the glycolysis of tumors.At present,the abnormal expression of three kinases has been detected in a variety of tumors,so metabolic kinase is a promising target for tumor therapy.Kaempferol,which comes from the rhizome of Sparganium,is one of the active components of Rhizoma sparganii Flavonoids,and has many effects,such as anti-oxidation,anti-tumor,anti-inflammation and so on.Its anti-tumor effect has been studied,but the specific mechanism is mainly focused on the promotion of apoptosis,cell cycle arrest and so on.There is still a lack of exploration and report on its effect on Warburg effect and its in-depth mechanism.Therefore,the main purpose of this study is to explore the effect of kaempferol on melanoma glycolysis metabolism,observe its effect on melanoma metastasis,and take its effect on metabolic kinase as a foothold to conduct a more in-depth study of the mechanism.Research Contents and ResultsThe main results were as follows:(1)After screening the concentration of drugs that did not inhibit the proliferation of tumor cells by MTT,the effects of kaempferol on melanoma migration and invasion were investigated in vitro by scratches test,Transwell migration test and 3D invasion test.The results showed that kaempferol could effectively inhibit the migration and invasion of A375 and B16F10 cells.ECAR and OCR were detected by Seahorse XFe96 Extracellular Flux Analyzer.Glucose uptake,lactic acid,pyruvate and ATP production were detected by glycolysis related kit to investigate the effect of kaempferol on glycolysis of melanoma cells.Kinase activity assay kit and Western blot were preformed to detect the changes of kinase activity and protein level.The results showed that kaempferol significantly inhibited the glycolysis of A375 and B16F10,and significantly inhibited the activity of HK,but had no effect on protein level.(2)To study the mechanism of kaempferol inhibiting HK activity.The mitochondrial localization of HK and HK2-VDAC1 co-localization were detected by immunofluorescence,and the mitochondrial proteins were extracted to detect the protein levels of HK2 and VDAC1 in the mitochondria.The protein levels of upstream factors AKT,p-AKT,GSK-3β,p-GSK-3β and PKA regulating HK2-VDAC1 binding in mitochondria were detected to explore the specific mechanism.The results showed that kaempferol inhibited the binding of HK2-VDAC1 to mitochondria,which may be achieved by acting on AKT/p-GSK-3β signal pathway.(4)Finally,the anti-metastasis effect of kaempferol in vivo was verified by constructing the caudal vein-lung metastasis model of A3 75 and B16F10,and the effect of kaempferol on glycolysis and metabolic kinase was detected.The results showed that 100mg/kg kaempferol could significantly inhibit the lung metastasis of melanoma and the glycolysis of metastasis.Correspondingly,kaempferol could inhibit the activity of HK in metastasis in vivo,and the immunofluorescence results showed that kaempferol inhibited the co-localization of HK2-VDAC1 in pulmonary metastases.Conclusion and SignificanceAccording to the experimental results,we draw the following conclusions:kaempferol can resist melanoma metastasis,which is related to the inhibition of glycolysis of melanoma,and its specific mechanism may be achieved by inhibiting the binding of HK2-VDAC1 in mitochondria by acting on AKT/p-GSK-3β signal pathway.Based on the theory of Warburg effect,this paper explores the new mechanism of anti-tumor metastasis of kaempferol from the perspective of glycolysis metabolism,and provides a basis for the development of clinical anti-tumor metastasis drugs.