The Effect Of Naringenin Combined With 5-FU On The Invasion And Apoptosis Of Colorectal Cancer And Its Mechanism

Objective: To study the effect of naringenin combined with 5-FU on the proliferation,invasion and apoptosis of colorectal cancer,and to explore the effect and mechanism of naringenin combined with 5-FU alone or in combination on AMPK-mTOR signaling pathway,so as to provide new methods and ideas for the treatment of colorectal cancer.Methods:1.CCK-8 kit was used to detect the effect of different concentrations,different times of naringenin and different concentrations of 5-FU on LoVo cell proliferation,and calculate the IC50.2.To investigate the synergistic effect of naringenin and 5-FU in combination,LoVo cells from human colorectal cancer were divided into 4 groups: normal control group,naringenin group,5-FU group,naringenin and 5-FU Group.The combination index CI will be calculated by Compusyn software with different concentration combinations to select the optimal concentration combinations.3.Transwell chamber was used to detect the effects of naringenin and naringenin combined with 5-FU on the migration and invasion of LoVo cells.4.Inverted microscope was used to observe the changes in cell number and morphology,and flow cytometry was used to determine the apoptosis rate.5.Western blot analysis was used to detect the apoptosis-related protein levels of Bax、Bcl-2、Cleaved caspase-3,as well as the total and phosphorylation levels of AMPK,mTOR and p70S6 k proteins.Results:When used alone,Naringenin or 5-FU could effectively inhibit the proliferation of LoVo cells,among which,naringenin showed time-concentration dependence on LoVo cell proliferation(P <0.05).At 48 h,the IC50 of naringenin was329.30 μmol/l,and the IC50 of 5-FU was 87.61μmol/l.Naringenin and 5-FU could synergistically inhibit the proliferation of colorectal cancer LoVo cells in vitro,and the combination index was lower than 1 at a fixed concentration ratio of 1:4.Compared with the single drug group,naringenin combined with 5-FU enhanced bothcell migration and invasion(P<0.05).In addition,our results showed that naringenin can promote apoptosis activation,up-regulate the apoptosis-related factors of Bax and Cleared-caspase-3,and down-regulate Bcl-2.Naringenin can increase the phosphorylation of AMPK and decrease the phosphorylation of mTOR and p70S6 k,The combination of naringenin and 5-FU makes this trend more obvious.Conclusion:Naringenin combined with 5-FU significantly reduced the proliferation of LoVo cells in colorectal cancer compared with single administration,and showed a synergistic effect;naringenin combined with 5-FU group can significantly inhibit the migration and invasion of cancer cells compared with the single drug group.The mechanism may be related to the activation of AMPK,and the inhibition of phosphorylation of mTOR and downstream p70S6 k.Naringenin combined with 5-FU drug group can significantly promote apoptosis than the single drug group,which may be related to the up-regulation of Bax and Cleaved caspase-3and down-regulation of Bcl-2.