Anti-tumor Activity Of Main Secondary Metabolites From Xylaria Sp.DO1801

Objective:The existence of tumor has a seriously threaten to human health and the quality of life.The purpose of this study was to confirm the anti-tumor activity of the primary secondary metabolites(SM)of Xylaria sp.DO1801 by evaluating the anti-tumor effect of SM in vivo and in vitro.The whole study provided a basic foundation for the clinical application of cytochalasins.Methods:(1)Firstly,the structure of the major secondary metabolites extracted from Xylaria sp.DO1801 was identified by Nuclear Magnetic Resonance(NMR);(2)To clarify the anti-tumor activity of major SM,we examined the effect of SM on the proliferation of different types of cancer cells,and we constructed tumor-bearing mice models by subcutaneously injecting with B16 or LCC tumor cells in the flank to evaluate the influence of SM on the tumor growth;(3)The proliferation of normal pancreatic cells HPDE6-C7 and human embryonic kidney epithelial cells HKC,the ratio of CD3~+CD4~+and CD3~+CD8~+cells in spleen of normal mice were utilized to evaluate the toxicity of the four kinds of cytochalasins;(4)The effect of cytochalasins combined with paclitaxel in multidrug resistant breast cancer cells MCF-7-MDR was demonstrated by calculating the reversal multiple,rhodamine B staining and High Performance Liquid Chromatography(HPLC);(5)qRT-PCR was used to detect the key genes in the possible mechanism of drug resistance in MCF-7-MDR cells,and we found the possible mechanism of change the sensitivity of drug-resistant cells to paclitaxel;(6)The metabolic changes of B16 cells treated with cytochalasins were observed by metabolomics;(7)The ability of oxidative phosphorylation and glycolysis inB16 cells treated with CQ or ECC was detected by SeahorseXF energy analyzer;(8)Glucose and lipid related genes,glucose uptake and lactate dehydrogenase(LDH)activity in B16 cells were detected to further confirm the effects of SM on energy metabolism.Results:(1)The major SM were extracted from Xylaria sp.DO1801 and identified by NMR as 19,20-Epoxy-cytochalasin Q(ECQ);Cytochalasin Q(CQ);21-O-Deacetyl-19,20-epoxycytochalasin Q(DEC);19,20-epoxycytochalasin C(ECC);(2)The SM of Xylaria sp.DO1801 inhibited various cancer cells proliferation.However,the effects of SM on different types of cancer cells are different.However,the four cytochalasins didn't significantly inhibited the proliferation of normal cells,and the proportion of CD3~+CD4~+and CD3~+CD8~+cells had no significant change;(3)In melanoma tumor-bearing mice,the growth of tumor volume in CQ and ECC groups was significantly slower than that in the model group,and the growth trend was similar to that in cisplatin group;(4)The reversal multiples of CQ or ECC combined with paclitaxel were 3.035 and 2.726,respectively.When the dosage of CQ or ECC was increased to 5?M,the reversal multiples were 3.985 and 10.285,respectively;(5)After CQ or ECC treatment,the expression of Abcc1,Casp3,Bax and P53significantly decreased,while the mRNA expression of Bcl-2 increased.There was no significant change in the expression of Top2a and Prkca genes;(6)Compared with untreated cells,CQ and ECC treatment increased intracellular drug concentration,and ECC treatment with 40?M significantly increased intracellular rhodamine B's content;(7)Treatment with CQ or ECC,the content of taurine and bile acid decreased and the corresponding metabolic pathways were suppressed;(8)The results of seahorseXF energy analysis showed that the ability of oxidative phosphorylation and glycolysis were suppressed after treatment with CQ or ECC,accompanying by the decrease of glucose intake and intracellular lactate dehydrogenase's activity.Besides,the expression of genes related to glucose and lipid metabolism decreased.Conclusion:(1)The main SM of Xylaria sp.DO1801,as four cytochalasins,showed anti-tumor activity in vivo and in vitro;(2)CQ or ECC combined with paclitaxel reversed the drug resistance of MCF-7-MDR cells partly,and the possible mechanism was to affect ABC transporters,inhibiting the drug-efflux of tumor cells;(3)This study demonstrated that CQ or ECC influenced the metabolic mode of B16 cells accompanied by the significant changes of metabolites and metabolism pathways;(4)In vitro experiments showed that the activity of oxidative phosphorylation and glycolysis,glucose uptake and intracellular lactate dehydrogenase activity decreased after CQ or ECC treatment.The energy metabolism was influenced by CQ and ECC in B16 cells.Innovative points:From the perspective of metabolism in cancer cell,the mechanism of cytochalasins exerting anti-tumor activity will provide a basis for the clinical application of cytochalasin for the first time.