| Colon cancer is a common digestive tract disease,and its incidence is increasing year by year.Since lactic acid bacteria(LAB)exopolysaccharides(EPS)was found to inhibit tumor in1982,its inhibitory effect on proliferation of human colon cancer cells has attracted extensive attention.In this study,human colon cancer cell line HT-29 was used as a cell model to study the intrinsic mechanism of the HT-29 cells apoptosis induced by EPS from lactic acid bacteria isolated in our laboratory.The EPS of 14 lactobacillus strains,including Lactobacillus plantarum 3,L.plantarum 4,L.plantarum 9,L.plantarum 12,L.plantarum 13,L.plantarum 14,L.plantarum 24,L.plantarum 27,L.plantarum 31,L.plantarum 32,L.plantarum 37,L.plantarum 44,L.plantarum 49,L.plantarum 50,and the standard strain(LGG)were extracted and the effects of different strains EPS on HT-29 cells proliferation were determined by MTS method.The results showed that the EPS of L.plantarum 12 had the best inhibitory effect on HT-29 cell proliferation,when the concentration of L.plantarum 12 EPS was 500 ?g/mL,the inhibitory rate could reach 32%,therefore,L.plantarum 12 EPS was studied in the subsequent experiments.L.plantarum 12 EPS was separated into two monosaccharides(EPS 12-11 and EPS 12-2)by ion column and gel column,and the effects of EPS 12-1 and EPS 12-2 on the proliferation of HT-29 cells were compared by methylene blue method.The results showed that the inhibition rate of EPS 12-1 on HT-29 was higher than that of EPS 12-2,when the concentration of EPS 12-1 was 500 ?g/mL,the inhibition rate was up to 67%?Then the Tunel apoptosis assay kit was used to detect the effect of the L.plantarum 12 EPS on HT-29 cells apoptosis,the results showed that the apoptosis rate of HT-29 cells was50.3% at the concentration of 500 ?g/mL,which was significantly higher than those at the other three concentrations(p<0.05).After EPS treatment,the expression levels of reactive oxygen species(ROS),apoptosis related proteins(Bax,Bcl-2 and PCNA),cytochrome c(cyt c)and caspase family(caspase 3,caspase 8 and caspase 9)in the treated HT-29 cells were studied,the results showed that the expression of ROS increased with the increase of EPSconcentration,and reached 8514 at the concentration of 500 ?g/mL.The expression of Bax increased with the increase of EPS concentration,while Bcl-2 and PCNA decreased with the increase of EPS concentration,and the ratio of Bax/Bcl-2 increased with the increase of EPS concentration;the expression of cyt c increased with the increase of EPS concentration,and reached 364.99 ng/L at 500 ?g/mL;caspase 3,caspase 8 and caspase 9 were activated at the concentration of 500 ng/mL of L.plantarum 12 EPS,and the activation degree of caspase 3,caspase 8 and caspase 9 was 1.62,2.95 and 1.65 respectively,which indicated that caspase 3,caspase 8 and caspase 9 in HT-29 cells were activated after the treatment by L.plantarum 12 EPS,and played a role in HT-29 cells apoptosis.Based on the results of this study,it was found that L.plantarum 12 EPS could increase the amount of ROS,Bax/Bcl-2 ratio and decrease the expression of PCNA in HT-29 cells,all of these lead to the decrease of mitochondrial membrane potential and increase of cyt c release,when a large amount of cyt c entered mitochondria,caspase 8 and caspase 9 were activated,which lead to the activation of caspase 3,eventually lead to HT-29 cells apoptosis. |
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