Multi-Omics Study Of Bufadienolides On Lipid Metabolism In Prostate Cancer

Metabolic reprogramming has become one of the hallmarks in cancers.Lipid metabolism participates in various biological processes of cancer cells including proliferation,apoptosis,migration,invasion,and maintenance of membrane homeostasis.Prostate cancer is one of the leading malignant tumors in males.Many studies have shown that the enhanced de novo synthesis of lipids,beta-oxidation of fatty acids and storage of lipid droplets are features of prostate cancer.Therefore,lipid metabolism is closely related to the occurrence and development of prostate cancer.Bufadienolides,the active ingredients of Chansu,show a robust anti-proliferative effect against cancer cells in vitro including prostate tumor cells.In this study,human prostate cancer cell line（PC-3）was adopted as a model to explore the relationship between the lipid metabolism regulation and anti-prostate tumor activity of bufadienolides.Through systematic analyses of lipidomics and transcriptomics,combining with molecular biology experimental verification,the potential biomarkers of bufadienolides against prostate cancer were explained based on lipid metabolism and gene expression levels.CCK-8 method was used to evaluate the in vitro anti-prostate tumor activity of the37 bufadienolides isolated and purified in our laboratory.Combining IC₅₀ concentration and structural characteristics,CS21 and CS34 and CS23 and CS30 were chosen for further analyses based on their similar chemical structures yet different anti-tumor activities.The IC₅₀ concentration of CS21（10 n M）was selected as the dosage of CS21and CS34,while IC₅₀ concentration of CS23（80 n M）was chosen for CS23 and CS30for the controlled comparative study on lipid metabolism.Untargeted lipidomics combined with data dependent acquisition of high-resolution liquid chromatography-mass spectrometry were used to comprehensively explore the lipid metabolism regulation of different bufadienolides in prostate cancer.The results showed that after bufadienolides with high（CS21,CS23）and low activity（CS34,CS30）intervention respectively,the relative concentrations of long-chain phospholipids and glycerides of PC-3 cells altered significantly.Specifically,the bufadienolides with high anti-tumor activity（CS21,CS23）could significantly decrease the levels of long-chain phospholipids and glycerides.The results demonstrated the underlying mechanisms of bufadienolides in promoting the hydrolysis of long-chain lipids or inhibiting the synthesis of long-chain lipids,which might be closely related to their therapeutic effects on prostate cancer.To further explain the altered lipid metabolism by bufadienolides intervention,transcriptomics was used to characterize the regulation of gene expression,and the target genes related to the anti-prostate tumor activity of bufadienolides were screened out.RT-q PCR verification and KEGG gene function annotation were used to systematically reveal the gene expression changes under intervention of different bufadienolides.The results showed that lipid metabolism related genes of PC-3 cells are the most regulated genes under intervention of active bufadienolides,which indicated that lipid metabolism is likely related to the anti-prostate tumor activity of bufadienolides.After the intervention of PC-3 cells with CS21 and CS23,the common key target genes with significant change but not found in CS34 and CS30 were GPX2,ELOVL6,PLD1,PLA2G10,SPTLC3,GAL3ST1,GPX3 and CYP2E1.To explore the relationship between the target genes and lipid metabolism,bioinformatics analysis of TCGA database and bibliography retrieval were used to further verify the clinical significance of the key genes above.The results showed that high expression of ELOVL6 was associated with the reduction of ten-year survival rate in patients with prostate cancer,and it catalyzed the prolongation of fatty acid chain in cancer cells.The high expression of PLD1 promoted the development and progression of prostate cancer,and it functioned in the conversion of phosphatidylcholine into phosphatidylic acid and choline in prostate cancer cells.Western Blot was applied to further analyze the protein levels of ELOVL6 and PLD1 under bufadienolides intervention,and the results indicated that PLD1 was the key protein during lipid metabolism that was under regulation by CS21 and CS23.In summary,this study first explored the relationship between the specific regulation of different structures of bufadienolides on lipid metabolism and their anti-prostate cancer activity through activity evaluation,multi-omics analyses and molecular biology verification.PLD1 is found to be the key protein of bufadienolides in regulating lipid metabolism of prostate cancer,which lays a foundation for further study on the mechanism.This study provides a promising treatment direction for prostate cancer by regulating the expression and activity of PLD1,and also presents a new idea for the treatment of prostate cancer with bufadienolides as PLD1 inhibitors.