Impact Of Intestinal Flora Fermentation Broth On Biological Behaviors Of Colorectal Tumor Cells During Formation Of Aberrant Crypt Foci In Colons

Objective: As the third most commonly seen cancer,colorectal cancer(CRC)witnesses its morbidity growing year by year.Though researches reveal that CRC is caused by a variety of factors,gene and environment,and a major contribution by intestinal flora,the specific pathogenesis is still unknown.Aberrant Crypt Foci(ACF)has been confirmed a pre-cancer mutation,which occurs before adenoma and is currently presumed to follow the order from ACF to adenoma and to colorectal cancer.Previous experiments have proved that intestinal flora and its metabolic ability have changed at the ACF period.Thus,with stool samples collected at the ACF period,we amplified the anaerobe(accounting for over 99% of the intestinal flora),prepared a certain amount of substrate and extracted fermentation broth of the flora.We try to discuss on the impact upon colorectal cancer cells by flora's metabolite and its possible functioning mechanism through observation of the impact of intestinal flora fermentation broth upon the proliferation,apoptosis,invasion and migration and the its impact upon the expression of colorectal cancer cell interleukin-6(IL-6),signal transducer and activator of transcription-3(STAT3)and gene mRNA of phosphatidyl inositol 3-kinase catalytic subunit alpha isoform(PI3KCA).Methods: Sample collection: In preliminary experimental stage,we built models and divided them into two groups at random(Inexperimental group,we had 30 SD mice.We injected carcinogen1,2-dimethylhydrazine(DMH)to the neck and back of the mice twice a week.In control group,we injected the same volume of blank buffer to 12 SD mice),collected stool samples of all mice once every two weeks(3-4 particles,around1g)and killed 3 mice from the experimental group and 1 from the control group at random to collect whole colons of them to calculate the ACF.According to the ACF quantity and same class subset form upon analysis of ACF count variance of the entire colon of two groups of mice at different time point upon modeling at early experimental stage,we used stools of the two groups collected at the 2nd(Week 3),4th(Week 7)and 8th(Week 15)times for subsequent research.We made the bacteria proliferate,gave cell culture medium DMEM/F12 as the base for anaerobic fermentation and extracted the fermentation broth of the flora.According to the source of fermentation broth stool samples and time to collect the stool sample,they were grouped as: Group S2 and D2 for stools collected at the 2nd time,Group S4 and D4 for those collected at the 4th time and Group S8 and D8 for those collected at the 8th time.After interfering colorectal cancer LoVo cell strain with such groups of flora fermentation broth(set a Group NC.When other groups are interfered with a certain concentration and gradient of fermentation broth,Group NC functions as the blank control group with DMEM/F12 as the complete culture medium),we adopted the Counting Kit-8(CCK-8)method to inspect the proliferation and constraining status of each cell group;the flow cytometry,wound healing and Transwell method to inspect the apoptosis rate,migrationand invasion capacity of each cell group;and the Real-time PCR technology to inspect the expression of IL-6,STAT3 and PI3 KCA gene mRNA of each cell group.Results: 1.With same interfering time(24h/48h/72h)and same concentration gradient(20%/10%/5%).Upon 24 h of cell interference at the20% concentration of fermentation broth,proliferation prohibition rates of all the groups were compared as: all S2 V.S.D2,S4 V.S.D4,S8 V.S.D8,S2 V.S.S4 and S2 V.S.S8 showed statistical significances(P<0.01).Proliferation prohibition rates of the experimental groups were all higher than the control groups.Then,comparisons as S4 V.S.S8 and D2 V.S.D4 V.S.D8 showed no statistical significance(P>0.05).Upon 24 h of cell interference at 5% or 10%concentration of fermentation broth for all groups,the results were the same as mentioned above.Upon 48 h of cell interference at 20% concentration of fermentation broth,proliferation prohibition rates of all groups were comparisons as: S2 V.S.D2,S4 V.S.D4,S8 V.S.D8,S2 V.S.S4,S2 V.S.S8 and S4 V.S.S8 showed statistical significances(P<0.05).Proliferation prohibition rates of the experimental groups were all higher than the control groups and the prohibition rates of three experimental groups S2,S4 and S8 showed gradual growths.S2 V.S.S4 V.S.S8 showed no statistical significance(P>0.05).Upon 48 h of cell interference at 5% or 10%concentration of fermentation broth,the results were the same with those from48 h of cell interference at 20% concentration of fermentation broth.Upon 72 h of cell interference at 20% concentration of fermentation broth,the proliferation prohibition rates of all groups were compared as S2 V.S.D2,S4 V.S.D4,S8 V.S.D8,S2 V.S.S4,S2 V.S.S8 and S4 V.S.S8 showed statistical significances(P<0.01).Proliferation prohibition rates of the experimental groups were all higher than the control groups and the prohibition rates of three experimental groups S2,S4 and S8 showed gradual growths.S2 V.S.S4 V.S.S8 showed no statistical significance(P>0.05).Upon 72 h of cell interference at10% concentration of fermentation broth,the proliferation prohibition rates of all groups were compared as: S2 V.S.D2,S4 V.S.D4,S8 V.S.D8,S2 V.S.S4,and S2 V.S.S8 showed statistical significances(P<0.01),and proliferation prohibition rates of the experimental groups were all higher than the control groups.S4 V.S.S8 and S2 V.S.S4 V.S.S8 showed no statistical significance(P>0.05).Upon 72 h of cell interference at 5% concentration of fermentation broth of all groups,the results were the same with those upon 72 h of cell interference at 10% concentration of fermentation broth.With three concentration gradients(20%/10%/5%)of the same fermentation broth under different interference time(24h,48 h and 72h),comparisons between any two concentration gradients showed statistical significances(20% V.S.10%(P<0.001),20% V.S.5%(P<0.001),10% V.S.5%(P<0.001).For the same fermentation broth interfered with same time,the higher the interference concentration,the higher the cell proliferation prohibition rate.Comparisons between any two groups under three interference time also showed statistical significances(24h V.S.48h(P<0.001),24 h V.S.72h(P<0.001),48 h V.S.72h(P<0.001)).For interference with the same concentration gradients with the same fermentation broth,the longer the interference time,the higher the cellproliferation prohibition rate.2.For 72 h interference with 20% concentration gradient,apoptosis of such groups: Group NC lower than the other groups(Group S2,D2,S4,D4,S8 and D8)(P<0.05),while the three experimental groups S2 V.S.D2,S4 V.S.D4 and S8 V.S.D8 showed higher apoptosis than the control groups(P<0.01).The three experimental groups S2,S4 and S8 showed gradually increasing apoptosis(P<0.001)and three D groups showed no statistical significances(P>0.05).3.Upon 72 h interference with20% concentration gradient,wound healing found the migration distance of each cell group for 6h,12 h,24h and 48 h that: the 48 h test result turned out the same with that from the Transwell migration experiment.All groups showed shorter distances than Group NC(P<0.001),and experimental groups S2 V.S.D2,S4 V.S.D4 and S8 V.S.D8 showed shorter distances than their corresponding control groups(P<0.01).Comparisons among the three S groups showed statistical significances(P<0.05)and S2,S4 and S8 groups showed an increasingly shorter distance.Comparisons among the three D groups showed no statistical significances(P>0.05).4.Upon 72 h interference with 20%concentration gradient,migration prohibition rates of each cell group worked out through the Transwell migration experiment: S2 V.S.D2,S4 V.S.D4 and S8 V.S.D8.Comparisons among three S groups showed statistical significances(P<0.001),while the experimental groups showed higher migration prohibition rates than the control groups,three S groups(S2,S4 and S8)showed gradually increasing migration prohibition rates,and the comparisons among three D groups(S2,S4 and S8)showed no statistical significances(P>0.05).5.Upon72 h interference with 20% concentration gradient,invasion prohibition rates of each cell group: S2 V.S.D2,S4 V.S.D4 and S8 V.S.D8.Comparisons among three S groups showed statistical significances(P<0.001),while the experimental groups showed higher invasion prohibition rates than the control groups,three S groups(S2,S4 and S8)showed gradually increasing invasion prohibition rates,and the comparisons among three D groups(S2,S4 and S8)showed no statistical significances(P>0.05).6.Upon 72 h interference with20% concentration gradient,the expression of IL-6,STAT3,PI3 KCA gene mRNA of each group: IL-6: NC V.S.S2,NC V.S.S4 and NC V.S.S8 all showed statistical significances(P<0.05).NC V.S.D2,NC V.S.D4,NC V.S.D8,S2 V.S.D2 and S4 V.S.D4 showed no statistical significances(P>0.05).The expression amount of IL-6 gene mRNA of S8 V.S.D8 experiment group was lower(P<0.05).The three S groups showed statistical significances(P<0.01),and the expression of IL-6 gene mRNA of S2,S4 and S8 declines one by one.Comparisons among three D groups showed no statistical significances(P>0.05).STAT3 and PI3KCA: Comparison between Group NC and the others showed no statistical significances(P>0.05).Three S groups and D groups,S2 V.S.D2,S4 V.S.D4 and S8 V.S.D8,all showed no statistical significances(P>0.05).Conclusions: 1.With cell culture medium DMEM/F12(carbonhydrate and amino acid as main components)as the substrate,the intestinal flora undergoes anaerobic fermentation to possibly promote the production of beneficial metabolite SCFAs,thus prohibiting the proliferation of colorectal cancer cells,inducing their apoptosis,and loweringthe invasion and migration capacities of cancer cells.2.With the same base(cell culture medium DMEM/F12)given to the experimental group and corresponding reference groups,fermentation broths of flora of the experimental group and corresponding reference groups showed different impact upon such biological behaviors of colorectal cancer cells as the proliferation,apoptosis,invasion and migration,which may be caused by the different varieties of those groups of intestinal floras and their different metabolic capacities.3.With cell culture medium DMEM/F12 as the base,during anaerobic fermentation process of intestinal flora at the ACF period and at normal status,metabolites of the former may generate more SCFAs,thus regulating the expression of IL-6 gene.