Effects And Mechanism Of Cytochalasin Q Targeting Creatine Kinase B Against Melanoma

Melanoma is a highly heterogeneous and aggressive malignant tumor,which can rapidly multiply and migrate depending on the polymerization of cytoskeletal microfilaments.Targeting microfilaments is considered a promising anti-tumor strategy.Cytochalasins exhibit anti-tumor activity with microfilament depolymerization.However,whether cytochalasin Q(CQ)can effectively inhibit melanoma is still unknown,and its underlying mechanism remains to be clarified.This study aims to explore the anti-melanoma activity and underlying mechanism of CQ,the secondary metabolite of the fungus Xylaria sp.DO1801.Firstly,in vivo B16-allogeneic mouse subcutaneous tumor model was constructed,and the effects of CQ on proliferation,apoptosis,migration and microfilament depolymerization of melanoma cell were determined.The results revealed that CQ exerted a good anti-melanoma effect by inducing microfilament depolymerization and leading to inhibited proliferation,increased apoptosis,and restricted migration in melanoma cells.Then,non-targeted LC-MS metabolomics was designed to analyze the effects of CQ on small molecule metabolites in B16 cells and explore its underlying molecular mechanisms.The results demonstrated that CQ mainly affected the creatine metabolism pathway in melanoma cells.Pretreatment with phosphocreatine restored intracellular ATP levels,microfilament depolymerization,proliferation inhibition,and apoptosis induction.Additionally,detection of intracellular glucose metabolism revealed that CQ inhibited glucose uptake,glycolysis,and oxidative phosphorylation in melanoma cells.These results suggest that CQ suppresses creatine metabolism and glucose metabolism in melanoma cells,thereby inhibiting ATP regeneration and inducing the depolymerization of the cytoskeletal microfilaments.Finally,based on the core role of creatine kinase B(CKB)in creatine metabolism,cell thermal transition assay(CETSA),microthermophoresis(MST)assay,CKB activity detection and molecular docking were employed to study the action of CQ on CKB and elucidate the target and molecular mechanisms of CQ.It was showed that CQ inhibited the enzyme activity of CKB in a substrate-competitive manner.Moreover,melanoma cells with low CKB expression manifested reduced ATP level,perturbed microfilament,inhibited proliferation and induced apoptosis,but showed lower sensitivity to CQ.Tumor-related metabolic pathways and key enzymes,guanididacetate-n-methyltransferase(GAMT)and CKB,in creatine metabolism were then analyzed.The results showed that CQ treatment or CKB silencing down-regulated the PI3K/AKT/FoxO1 signaling pathway in melanoma cells,while creatine and phosphocreatine reversed the down-regulation of PI3K/AKT/FoxO1 signaling pathway by CQ;Meanwhile,CQ treatment and CKB silencing inhibited the expression of GAMT and CKB,which could be restored by creatine,phosphocreatine and PI3 K activation.It is indicated that creatine metabolism forms a feedback loop with PI3K/AKT/FoxO1 pathway and mediates microfilament depolymerization and anti-melanoma effects of CQ.In conclusion,CQ interferes with creatine metabolism through inhibiting CKB and blocks the PI3K/AKT/FoxO1 pathway in a feedback loop,thereby promoting microfilament depolymerization and mediating its cascade of anti-melanoma effects.