Anti-hepatocarcinoma Effect And Mechanism Of EFLDO From Euphorbia Lunulate Bge

Natural products have become an important source of new anti-tumor drugs because of their novel skeleton and unique mechanism of action.Based on the privious work of our research group,the abietane diterpenoid ent-3a-formylabieta-8?14?,13?15?-dien-16,12?-olide(EFLDO,C₂₁H₂₈O₄)was isolated from Euphorbia lunulate Bge.Its antitumor activity and mechanism was investigated in vitro and in vivo.The specific research content of this subject is as follows:1.Twelve tumor cell lines from 9 different organ sources were used to evaluate the anti-tumor activity of EFLDO in vitro.2.Human hepatoma HepG2 cells were used as the research object,and the biological activities of EFLDO on HepG2 cells were comprehensively studied in the field of proliferation,cell cycle and apoptosis,and the mechanism was explored.3.To investigate whether EFLDO and human tumor necrosis factor-related apoptosis-inducing ligand TRAIL have a synergistic effect to inhibit the tumor cell activity,and elucidate the mechanism of TRAIL combined with EFLDO-induced apoptosis.4.To investigate the effect of EFLDO on HepG2 cells in invasion and migration,its mechanism was investigated based on biomolecular studies.Through the above experiments,the following research results are obtained:1.EFLDO had different degrees of inhibition on HCT-116,MCF-7,NCI-H460,K562,HeLa,MGC-803,EJ and CNE-2Z.The IC₅₀ values after 72 h of administration were 27.31?M,19.74?M,46.72?M,83.36?M,10.30?M,24.09?M,26.48?M,and 30.23?M.For the hepatoma cells HepG2,Bel7402,QGY7701 and Bel7402/Fu,the IC₅₀ after 72 hours of EFLDO administration were 7.72?M,28.10?M,21.67?M and 32.83?M,respectively.At the same time,in the human normal cell line HUVEC and L02,the IC₅₀ was 40.32?M and65.46?M after 72 hours of EFLDO treatment.2.On HepG2 cells,6.25?M,12.5?M,25?M and 50?M EFLDO showed significant proliferation inhibition after 25 h,48 h and 72 h,in a time and dose-dependent manner.Flow cytometry Annexin V-PI double staining showed that,after 12 h,24 h,36 h and 48 h 62.5?M EFLDO treatment,the apoptosis rates of HepG2 cells were increased as a time and dose-dependent manner.Significant dose-dependent trends.The results of Hoechst 33342 staining showed that the apoptosis of HepG2 cells was observed at 25?M,50?M and 75?M EFLDO for 0,24,48 and 72 hours,withdense granular fluorescence.The JC-1 experiment indicated that EFLDO could damage the mitochondrial membrane potential and induce apoptosis of HepG2 cells.After administration of 6.25?M,12.5?M,25?M,and 50?M EFLDO for 12 h,24 h,36 h,and 48 h,the activated caspase 3,8,and 9 proteins in HepG2 were increased,Bcl-2 and Survivin protein was down-regulated and Bax was up-regulated,all in a time and dose-dependent manner.The results of flow cytometry PI single staining assay showed that EFLDO could induce G2/M phase arrest in HepG2 cells,and this effect was time and dose-dependent.Western Blot assay showed that EFLDO could down-regulate the cycle-related proteins Cyclin B and CDK1 in a time and dose-dependent manner,and significantly down-regulate p-AKT,AKT,p-ERK and ERK protein expression.In the HepG2 xenograft model,EFLDO could dose-dependently inhibit xenograft growth.The tumor inhibition rates of 10 mg/kg,20 mg/kg,and 40 mg/kg EFLDO were24.9%,42.6%,and 57.8%,respectively.Immunohistochemistry results showed that EFLDO could inhibit Ki67 protein expression in a dose-dependent manner.3.Compared with EFLDO group and TRAIL group,3?M EFLDO combined with 25 ng/mL TRAIL significantly inhibited the proliferation of HepG2 cells.Flow cytometry Annexin-V FITC/PI double staining showed that 3?M EFLDO combined with 25 ng/mL TRAIL significantly increased apoptosis in HepG2 cells.Western Blot assay showed that 3?M EFLDO combined with 25 ng/mL TRAIL significantly increased the protein expression of caspase 3 and caspase 8.Meanwhile,Bax and BAD proteins were significantly up-regulated.Bcl-2 protein was significantly down-regulated.The mRNA and protein expression levels of DR4 and DR5 were significantly increased compared with the control group.In p53-siRNA HepG2 cells,after TRAIL combined with EFLDO,caspase 3 activity was up-regulated and HepG2 cell apoptosis was increased.4.Administration of 1?M,2?M,and 4?M EFLDO for 48 h significantly reduced the distance between scratches in cell scratches.After administration of 2?M,4?M and 8?M EFLDO for 24 h,the number of transmembrane cells of HepG2 in Transwell chambers coated with and without Matrigel gel was significantly reduced.After administration of 2?M,4?M and 8?M EFLDO for 24 h,MMP2 and MMP9 in HepG2 cells were significantly down-regulated at protein and gene levels.In this study,the anti-tumor activity of EFLDO was evaluated in an in vitro model.It found EFLDO was effective in inhibiting the growth of hepatoma cells.It was confirmed that EFLDO could inhibit HepG2 cells by inhibiting proliferation,regulating cycle disorders,and inducing apoptosis.It was elucidated that EFLDO could significantly attenuate the migration and invasion of HepG2 cells by regulating MMP2 and MMP9 and sensitize TRAIL to induce apoptosis based on the p53 signaling pathway in HepG2 cells.The results of this subject can help to develop EFLDO combined with TRAIL as a new strategy to treat epatocarcinoma,and also provide a scientific basis for the clinical application of EFLDO to the treatment of cancer.