| Objective: Testing the growth and proliferation inhibition effect of AFC in five human colorectal cancer HCT116、SW620、HCT8、HT29、LOVO cell lines,found that AFC exhibits the most sensitive inhibition in HCT116 cells.Research on the autophagy and apoptosis induced by AFC in HCT-116 cells,and clarify the relationship between autophagy and apoptosis by treatment with autophagy-specifc inhibitors 3-methyladenine(3-MA)or baflomycin A1(BA).Reveal signaling pathways involved in AFC inducing autophagy and apoptosis of human colorectal carcinoma cells.Methods 1.Cell lines and cell culture The human colorectal carcinoma cell line HCT116,SW620,HCT8,HT29 and LOVO were grown in McCoy’s5A,RPMI-1640,DMEM or F12 culture medium containing 10% heat-inactivated fetal bovine serum(FBS),penicillin(100U/mL)and streptomycin(100 mg/mL)at 37°C under an atmosphere of 95% air and 5% CO2.2.Cell viability assay(CCK8 assay)(1)HCT116,SW620,HCT8,HT29 and LOVO cells were treated with AFC which was regulated to final concentrations of 25,50,100,200 and 400μg/ml,and cultured for 48 hours.The growth inhibitory potencies of AFC against various Human colorectal cancer cell lines are detected by CCK8 assay.(2)HCT116 cells were treated with AFC(25,50,100μg/ml)for 24 h,48 h,and 72 h respectively.Cell viability assays was used to estimate whether AFC inhibited HCT116 cell growth in a dose-and time-dependent manner.3.Quantification of apoptosis After treatment of AFC at concentration of 25μg/ml,50μg/ml,100μg/ml for 48 h,the effect of AFC on the apoptosis of HCT116 cells was observed by Hoechst 33258 staining and flow cytometry with AnnexinV/PI staining.To further confirm that apoptosis was induced by AFC,western blotting was performed to detect the cleavage of caspase 3,caspase 9 as well as PARP.4.Determination of autophagy(1)GFP-LC3 transfection: The HCT116 cells were transfected with AdmCherry-GFP-LC3 B for 24 h before receiving AFC 100 μg/ml treatments.After 48-hours treatment,the cells were then fixed with 4% polyoxymethylene and observed under an EVOS? FL Imaging System.(2)Transmission electron microscopy:HCT116 cells were collected by trypsinization,fixed in 2.5% glutara-ldehyde and post-fxed in 1% osmium tetraoxide.Then the cells were progressively dehydrated in ascending grades of ethanol solutions,and embedded in Epon resin.The ultrathin sections were contrasted with uranyl acetate and lead citrate for electron microscopy.(3)After the cells were exposed to 25,50 and 100μg /ml AFC for 12 h,24 h,or 48 h,cell lysates were subjected to western blotting with an anti-LC3 and Beclin-1 antibody.5.The potential crosstalk between AFC-induced apoptosis and autophagy: HCT116 cells were cultured and divide into six groups: In the Control group,cells were cultured in McCoy’s5A medium only;3-MA group(2mM);BA group(1nM);AFC group(100μg/ml);the AFC+BA group(contained 1nM of bafilomycin A1 and 100μg/ml AFC);the AFC+3-MA(contained 2mM of 3-MA and 100μg/ml AFC).The growth inhibitory potencies of different groups are detected by CCK8 assay.By flow cytometry analysis of AnnexinV-FITC/PI double-staining,the percentages of apoptosis in different groups were detected respectively.Western blot analyses were performed for checking the autophagy related protein levels of LC3 and Beclin-1.The expression of cleaved caspase-3 and PARP was also detected by immunoblot analysis.6.Western blot assay was used to determine the activity of PI3 K / Akt/mTOR signalling pathway:(1)HCT116 cells were treated with AFC at concentrations ranging from 25 to 100μg/mL for 48 h.Western blotting analysis was performed to study the effects of AFC on PI3K/Akt/mTOR signaling.(2)HCT116 cells were cultured and divide into four groups: Control group;AFC group(100μg/ml);LY294002 group(LY294002 is a well-characterized inhibitor of PI3 K,10μM.);LY294002+ AFC group(10μM of LY294002 +100μg/ml AFC).Western blot analyses were performed for checking the protein levels of Akt,p-Akt at Ser473,mTOR and p-mTOR at Ser2448.Results 1.The results showed that HCT116 cell was the most sensitive to AFC treatment than the other cell lines with an IC50 value of 120.4±4 μg/ml,and showed lower cytotoxicity on LOVO(IC50: 280.5±12.28 μg/ml),SW620(IC50: 174±10 μg/ml),HCT8(IC50: 212.6±19 μg/ml),and HT-29(IC50: 232.6±27 μg/ml).Compared with control group,treatment with AFC(25μg/mL,50μg/mL,100μg/mL)for 48 h signifcantly inhibited cell viability to 84%±9.39%,79.31%±6.14% and 69.81%±5.35%,respectively,and 25,50 and 100μg/mL AFC treatment for 72 h inhibited cell viability to 81.03±7.2,74.33±7.51 and 61.43±4.32%,respectively.The cell viability of the AFC groups(50 and 100μg/mL)were significantly lower than the control group after treatment for 48 or 72h(P <0.01).However,there was no significant inhibitory effect after treatment for 24 h.2.After treatment of different concentration of AFC,typical morphologyical changes of cell apoptosis such as karyopyknotic pyknic hyperfluorescence bolus,nuclear fragmentation and apoptotic body were observed by Hoechst 33258 staining in a dose-dependent manner,but dispersed adqulis light blue hypofluorescence was observed in the negative control group and solvent control group.The percentage of apoptosis was determined by flow cytometry analysis.After 25,50 and 100 μg/ml AFC treatment,cells exhibited 8.35%,17.68% and 26.95% apoptosis.The apoptosis rate of the AFC groups(50 and 100μg/ml)was different from the control group(P<0.01).Western blotting was performed to detect the cleavage of caspase3,caspase9 as well as PARP.After treatment,AFC clearly cleaved pro-caspase3,pro-caspase9 and PARP to their active forms in a dose-dependent manner.Results showed that the protein expression of Cleaved Caspase-3/Procaspase-3,Cleaved PARP/PARP and Caspase-9/ Actin after treatment of AFC(50 and 100μg/ml)for 48 h were significantly increased,which was different from the control group(P<0.01).3.(1)After transfected with Ad-mCherry-GFP-LC3 B recombinant adenovirus,the GFP-LC3 puncta which gathered on the membrane of autophagy body were frequently seen in the cancer cells treated with AFC for 48 h,and displayed in the form of yellow spots and red spots.However,the control cells had a bright yellow fluorescence in cell cytoplasm.(2)In addition,by using transmission electron microscopy,a large number of autophagy body and autophagy-lysosome in HCT116 cells were monitored after treated for 48 h by AFC(100μg/ml).(3)When the cells were treated with AFC at the indicated concentration(25,50 and 100μg/ml),the expression levels of LC3-II and Beclin-1 were significantly enhanced in a dose-and time-dependent manner.Results showed that the protein expression of LC3-II/ LC3-I in the AFC groups(50 and 100μg/ml)were significantly higher than that in the control group after treatment for 12,24 or 48h(P<0.05);The protein expression of Beclin-1/Actin in the AFC groups(50 and 100μg/ml)were significantly enhanced after treatment for 12,24 or 48 h,which were different from the control group(P<0.01).4.The cytotoxicity of AFC(100μg/ml)combined with 3-MA(2 mM)or BA(1nM)for 48 h in HCT116 cells was measured by CCK8 assay.The BA group,3-MA group,AFC group,AFC+3-MA group and AFC+BA group inhibited cell viability to 91.5% ±7.09%,89.7%±9.01%,74.3%±5.13%,40.5%±2.51% and 54.2% ±4.04%,respectively.Compared with the control group,the BA group and 3-MA group had no significant difference.Compared with the control group,the cell viability of the AFC group was significantly lower(P<0.001).Co-treatment with AFC and 3-MA or BA significantly decreased the cell viability compared with the cells treated with AFC alone(P<0.001).The apoptosis rate was determined by flow cytometry analysis of AnnexinV-FITC/PI doublestaining.The BA group,3-MA group,AFC group,AFC+3-MA group and AFC+BA group increased the percentage of apoptosis to 6.36%±0.31%,6.65%±0.40%,7.21% ±0.69%,27.64% ±1.74%,45.02% ±1.85% and 40.8% ±1.79%,respectively.Compared with the control group,the BA group and 3-MA group had no significant difference.Compared with the control group,the apoptosis rate of the AFC group was significantly higher(P<0.001).Compared with the AFC group,the percentage of apoptosis in the AFC+3-MA group and AFC+BA group were significantly enhanced(P<0.001).Immunoblot assays revealed that the addition of 3-MA was shown to decrease LC3-II levels in AFC-treated cells,while baflomycin A1 had their LC3-II further accumulated during AFC treatment.In addition,the increased of Beclin-1 protein expression by AFC were considerably decreased by pre-treatment with 3-MA or BA.Western blotting was performed to detect the cleavage of caspase3 as well as PARP.Results showed that treating HCT116 cells with AFC increased the activation of cleaved caspase3 and PARP,which were augmented when AFC was combined with 3-MA or BA.Compared with the control group,the expression of Cleaved Caspase-3/Procaspase-3 and Cleaved PARP/PARP in the BA group and 3-MA group had no substantial changes.Compared with the control group,the expression in the AFC group were significantly higher(P<0.01).Compared with the AFC group,the expression in the AFC+3-MA group and AFC+BA group were significantly enhanced(P<0.001).5.Immunoblot assay revealed that compared with the control group,AFC inhibited PI3 K phosphorylation,and dramaticly decreased the levels of phospho-Akt,leading to the down-regulation of downstream phosphomTOR in a dose-dependent manner.The expression of PI3 K,Akt and m-TOR in each groups had no substantial changes.The ratio of p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100μg/ml)was significantly lower than that in the control group(P<0.01).Additionally,after a 48 h treatment with LY294002,AFC,and AFC+LY294002,HCT116 cells were harvested and lysed for an immunoblot assay.Compared with the control group,the expression of p-Akt and p-mTOR were successively decreased,while the expression of Akt and m-TOR in each groups had no substantial changes.The expression of p-Akt/Akt and p-mTOR/m TOR in the AFC group was significantly lower than that in the control group(P<0.001).Compared with the AFC group,the expression of p-Akt/Akt and p-mTOR/mTOR in the AFC+LY294002 group was significantly decreased(P<0.001,and P<0.05).Conclusions 1.AFC exhibits the most sensitive inhibition in HCT116 cells concentration-and time-dependently.2.AFC induced HCT116 cells apoptosis in a dose-dependent manner which may be associated with the intrinsic mitochondrial pathway.3.AFC induced autophagy in HCT116 cells concentration-and timedependently.4.Pre-treatment with 3-MA or BA blocked autophagy induction by AFC,mean while 3-MA and BA increased AFC-induced apoptosis,suggesting that AFC-induced autophagy protects cells from apoptosis.5.With or without pretreatment with LY294002,AFC inhibited the PI3K/Akt/mTOR signalling pathway in a dose-dependent manner,indicated that AFC induced apoptosis and autophagy might be due to its inhibition of the PI3K/Akt/mTOR signalling pathway. |
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