Study On Anti-tumor Activity Of Cembranoid Type Diterpenes Of Tobacco

The cembranoid type diterpenes in tobacco has a good antimicrobial,antitumor and neuroprotective activity as previous studies.Hepatocellular carcinoma has been the world's second largest malignant tumor in recent years,with the incidence gradually increased.The inhibitory effect of cembranoid type diterpenes on hepatoma cell lines HepG2 and SMMC-7721 was rarely reported.In order to explore the antitumor activity of cembranoid type diterpenes in tobacco and make full use of tobacco resources,?-2,7,11-cyprotermine-4,6-diol(?-CBD)was used to investigate the antitumor activity of hepatoma Hep G2 and SMMC-7721 cells in vitro in this study.1.HepG2,SMMC-7721 and HL-7702 cells were treated with different concentrations of ?-CBD for 24 h,48 h and 72 h after MTT assay.The results showed that ?-CBD had inhibitory effect on the three kinds of cells in a time-dependent and concentration-dependent manner.But the inhibitory effect on HL-7702 was weaker than that of HepG2 and SMMC-7721 cells.According to the result,HepG2 and SMMC-7721 cells were treated with 20 mg / L ?-CBD at 24 h,48 h and 72 h and the clone formation ability of was measured.The colony formation rates of HepG2 were 80.92%,35.42%,6.10% and SMMC-7721 were 76.63%,56.70% and 29.13%.The results showed that ?-CBD could reduce the cell clone formation rate and inhibit the proliferation of hepatocellular carcinoma cells.2.The morphological changes of HepG2 and SMMC-7721 cells treated with ?-CBD were observed under light microscope after Giemsa staining,which find the experimental group of cells showing apoptotic morphology: shrink,round,the volume shrink,shed from the wall and suspended in the culture medium.Through the AO / EB fluorescence double staining test,the control group showed uniform green fluorescence,while the experimental group of cells appeared in varying degrees of orange,orange-red fluorescence.Indicating that ?-CBD changes the plasma membrane permeability and likely to have the role of induction of apoptosis.3.The expression of transcripts in HepG2 cell treated with 20 mg / L ?-CBD for 48 h showed that there were 3068 genes with significant differences,among which 1289 were up-regulated and 1779 were down-regulated genes.The differential gene GO enrichment analysis showed that the down-regulated genes included cell attachment,cell migration,mitosis,cell cycle,DNA strand extension in DNA replication,growth factor binding,which inhibit cell survival and proliferation.Upregulated genes include protein localization,transition metal ions,cell death,programmed cell death,apoptosis progression,apoptotic pathway,ubiquitin transferase activity,endoplasmic reticulum signaling pathway,protein transport,which could promote apoptosis.Differential gene KEGG pathway enrichment analysis showed that the up-regulation pathway include NF-kB signaling pathway,HIF-1 signaling pathway,apoptosis,cancer pathway,autophagy pathway,and down-regulation pathway include adhesion,citric acid cycle,conduction pathway,AMPK signaling pathway,TGE-? signaling pathway,P13K-Akt signaling pathway,cell cycle.Apoptosis of cells may be mediated by p53-PUMA,PI3K-Akt,IL-1-NFkB-IAP pathway.4.Treatment of HepG2 at 20 mg / L ?-CBD for 24 h,48 h and 72 h after flow cytometry.The number of S phase of HepG2 was 10.4%,16.9%,22.0% and 29.8%.The number of S phase cells increased significantly,which indicated that ?-CBD inhibit the proliferation of hepatocarcinoma cells.The transcriptome gene expression of the cell cycle arrest was verified.The apoptotic rate of HepG2 cells was increased with the increase of ?-CBD time,which showed that ?-CBD could induce HepG2 cell apoptosis.Six differentially expressed genes were selected for qRT-PCR,and the expression trend of the gene was consistent with the DGE analysis,which indicated that the DGE analysis was reliable.In conclusion,?-CBD could decrease the viability of hepatocarcinoma cells,inhibit cell proliferation,change plasma membrane permeability,apoptotic morphology,cell cycle S phase arrest,and induce apoptosis through p53-PUMA,PI3K-Akt,IL-1-NFkB-IAP pathway.