Licochalcone A And B From Glycyrrhiza Uralensis:Extraction,Anti-cancerous Potential And The Molecular Mechanism

Glycyrrhiza uralensis,as one of the medicinal and food resources,has many activities such as anti-cancer,anti-bacterial,anti-virus,anti-inflammatory and immune regulation.In this study,Glycyrrhiza uralensis was used to study the extraction of flavonoids.At the same time,Licochalcone A and Licochalcone B were separated by HSCCC and silica gel column chromatography to study their anti-cancer effects on human hepatocellular carcinoma HepG2 cells in vitro.Finally,transcriptome sequencing was used to study anti-cancer mechanism of Licochalcone A and Licochalcone B in the RNA and miRNA level.The optimum conditions of ultrasonic-assisted extraction of flavonoids from licorice were as followed:extraction time of 2.1h,Ultrasonic power 112.42W,liquid-solid ratio of 30.15:1 and 64.38%ethanol.The flavonoids yield obtained under these conditions was 6.56 mg/g.Licochalcone A was isolated by high-speed countercurrent chromatography and Licochalcone B was isolated by silica gel column chromatography.The anticancer activity of Licochalcone A was further studied in HepG2 cells.The results showed that Licochalcone A significantly inhibited the proliferation of HepG2cells.The IC₅₀0 was 65.96?M after treated for 24 h.Licochalcone A blocked cell cycle at G2/M phase,induced cell apoptosis and increased ROS level.The Licochalcone A treatment altered the expression of cell cycle related genes.The mRNA level of Survivin,Cyclin B1 and CDK1 decreased,while the mRNA level of Weel,P21,Cyclin D1 and JNK1 increased.In addition,both death receptor pathway and mitochondrial pathway are involved in cell apoptosis.The expressions of DR3,DR5,Caspases 3,Caspases 8,Caspases 10,Fas,Bad,Bax,Bcl-2,Bak and PUMA participated in above pathways were improved,while the expressions of PKC?,p70S6K and Akt were decreased.Therefore,Licochalcone A induced cell apoptosis by regulating the expression of related genes in death receptor signaling pathway and mitochondrial signaling pathway.The anticancer activity of Licochalcone B was further studied in HepG2 cells.Licochalcone B showed significantly inhibition on the proliferation of HepG2 cells.The IC₅₀0 of Licochalcone B on HepG2 cells for 24h was 110.15?M.Licochalcone B blocked cell cycle at G2/M phase,induced cell apoptosis and increased ROS level in cells.Licochalcone B affected the expression of cell cycle related genes.The mRNA expression of cell cycle genes such as CDK1,Cyclin B1,CHK2,CDC14B,and CDC7were down regulated,while ZBTB17,CDC20,PKMYT1,GADD45A,GADD45B,SFN,CDKNIC,p21 and p53 were significantly up regulated.The protein expression of Cyclin B1,p21,p53,CDK1 and p-CHK2 was consistent with the mRNA expression.The mRNA expression of receptor-mediated pathway genes?TNF,TNF-R1,Caspases 8,Fas,FasL,FOS,JUN and protein expression of TNF-R1,Fas,Caspases 8?was up regulated.For mitochondrial pathway,the mRNA expression of Bid,Bak,PUMA,DIABLO,ENdoG,Caspase 9 and Caspase 3and protein expression of Bak,Caspase 3and Caspases 9 were increased except the mRNA and protein expression of Bcl-xl.The inhibition of Caspase 8 and Caspase 9 proteins in these pathways abolished the Licochalcone B induced apoptosis.The present work suggested that Licochalcone B could promote the apoptosis of HepG2 cells by regulating the expression of related genes for death receptor signaling pathways and mitochondrial signaling pathway.Based on the IC₅₀0 value obtained previously,HepG2 was treated with Licochalcone A and Licochalcone B at 70?M and 120?M.The differential expression profiles of their mRNA levels were determined by transcriptome.Compared with the untreated cells,there were 3414 up-regulated genes and 2647 down-regulated genes after Licochalcone A treatment.Meanwhile,there were 2928 up-regulated genes and 2601down-regulated genes after Licochalcone B treatment,.Then,KEGG analysis was used to analyze the important metabolic pathways enriched by differentially expressed genes.The differentially expressed genes treated with Licochalcone A were mainly enriched in the MAPK signaling pathway,the FoxO signaling pathway and the p53 signaling pathway.The differentially expressed genes treated with Licochalcone B were mainly concentrated in PI3K-Akt signaling pathway,MAPK signaling pathway and Wnt signaling pathway.These results suggest that Licochalcone A and Licochalcone B can regulate various complex physiological processes in hepatocellular carcinoma cell HepG2,and reveal the anti-cancer mechanism of Licochalcone A and Licochalcone B on hepatocellular carcinoma at the mRNA level.Based on the IC₅₀0 value obtained previously,HepG2 was treated with Licochalcone A and Licochalcone B at 70?M and 120?M to investigate the miRNA expression profile.Licochalcone A induced 54 miRNAs up-regulated and 48 miRNAs down-regulated when compare with the untreated cells.Relatively,Licochalcone B increased 50 miRNAs expression and decreased 42 miRNAs expression.KEGG analysis was used to analyze important metabolic pathways enriched by target genes of differentially expressed microRNAs.The target genes treated with Licochalcone A were mainly concentrated in the MAPK signaling pathway,PI3K-Akt signaling pathway and Ras signaling pathway.The target genes treated with Licochalcone B were mainly concentrated in the PI3K-Akt signaling pathway,MAPK signaling pathway and Rap1signaling pathway.These results suggest that Licochalcone A and Licochalcone B can regulate various complex physiological processes in HepG2 at the level of miRNA.