The Inhibitory Effect Of Brucine Combined With 5-fluorouracil On HepG2 Cells And Its Mechanism

Objective:To investigate the inhibitory effect of brucine,5-fluorouracil and two drugs on human HepG2 cells and its mechanism.And to provide a certain experimental basis for the clinical treatment of liver cancer.Methods:1.The effects of different concentrations of brucine,5-fluorouracil and the combination on the proliferation of HepG2 cells were detected by MTT assay.In order to scientifically evaluate the combined effect of the two drugs,we used calcusyn software to calculate the combined index.2.The morphological changes of brucine,5-fluorouracil and the combination on HepG2 cells were observed by AO/EB double fluorescence staining and confocal laser scanning microscopy.Flow cytometry was used to quantify the apoptosis rate of cells.3.The expression of Fas,FasL and FADD genes was detected by real-time PCR in the presence of brucine,5-fluorouracil and combination on HepG2 cells.The expression of Fas and FasL protein was detected by Western blotting.Results:1.MTT results indicated that brucine and 5-fluorouracil both showed a certain inhibitory effect on HepG2 cells alone,with the increase of concentration showing adose-dependent.Combination treatment obviously decreased the viability of HepG2 cells compared to brucine or 5-fluorouracil treatment alone.The difference was statistically significant(P < 0.01).Calcusyn software results show that the combination of the two drugs at a variety of concentrations was less than 1,indicating a synergistic effect between the two drugs.2.Uniformly green live cells with normal morphology were observed in the control group.After treated by 100 ?g/mL brucine,a small fraction of naturally apoptotic cells presented condensed green or depressed orange nuclei,whereas orange/red apoptotic cells with fragmented chromatin and apoptotic bodies occurred in 40 ?g/mL 5-fluorouracil group which showing more apoptotic cells.In contrast,the combination of 100 ?g/mL brucine and 40 ?g/mL 5-fluorouracil obviously enhanced these morphological changes and increased the cell apoptosis in HepG2 cells compared with 5-fluorouracil alone.3.As shown by flow cytometry,the ratios of apoptotic cells,as evidenced by Annexin V/PI positivity in the control and brucine groups were(7.13 ± 0.48)% and(19.69 ±3.25)%,respectively,whereas treatment with 40 ?g/mL of 5-fluorouracil increased this ratio to(27.74 ± 1.76)%.It is noteworthy that the percentage of total apoptotic cells after co-incubation with brucine and 5-fluorouracil was(55.49 ± 3.47)%,significantly higher than that of the brucine or 5-fluorouracil group(P < 0.01),indicating that combination of two agents dramatically promoted apoptosis of HepG2 cells.4.The mRNA expression of Fas,FasL and FADD was examined by real-time PCR method.Compared to the control group,expression levels of Fas,FasL and FADD in burcine group were higher,however,no statistical differences were observed.Compared to the control group,the expression level of Fas,FasL and FADD in 5-fluorouracil group was more higher,and the difference was statistically significant(P < 0.05,P < 0.01).Moreover,HepG2 cells exposed to 5-fluorouracil and brucine for 24 h showed significantly increased mRNA expression levels for Fas,FasL,and FADD,equivalent to 1.56,1.62,and 2.60-fold,respectively,when compared to cells treated with 5-fluorouracil alone.5.Western blotting results showed that,compared to the control group,the expression levels of Fas and FasL protein in burcine group was higher,the difference was statisticallysignificant(P < 0.05,P < 0.01).Compared to the control group,the expression level of Fas and FasL in 5-fluorouracil group was more higher,and the difference was statistically significant(P < 0.01).Moreover,HepG2 cells exposed to 5-fluorouracil and brucine for 24 h showed significantly increased protein expression levels for Fas and FasL,equivalent to1.606,1.600-fold,respectively,when compared to cells treated with 5-fluorouracil alone.Conclusion:In summary,the present study provided evidence of the synergistic anticancer effect of5-fluorouracil and brucine in human hepatocellular carcinoma HepG2 cell line.Combination treatment of 5-fluorouracil and brucine significantly increased the anticancer efficacy,which might be related with apoptosis by activating the Fas/FasL pathway.This finding is encouraging for the development of advantageous treatment regimens for hepatocellular carcinoma.