Chemopreventive Effects And Mechanisms Of Silibinin On Colitis-associated Cancer

Background and ObjectiveInflammatory bowel disease(IBD)is a chronic and recurrent disease of unknown etiology which affects the intestinal mucosa.Several studies have confirmed that IBD patients are at a higher risk of developing to colitis-associated cancer(CAC)than average population.CAC has also become the leading cause of death in patients with IBD,therefore effective strategies for IBD prevention are required.As a plant extract,silibinin(SB)has anti-inflammatory,anti-oxidant and anti-cancer effects,while its effects and mechanism on colitis have not been clarified.We evaluated the chemopreventive effects of silibinin on a CAC mice model,and its impact on IL-6/STAT3 signaling pathways.Intestinal tumor cells(IMCE and HCT-116 cell lines)were also treated by silibinin.Moreover,it provides the reliable experimental basis for exploring a new method to prevent and control CAC.Methods1.The female C57BL/6 mice(6-week-old)were fed in the environment for 1week and then were put into the experiment at the age of 7 weeks.The mice were divided into 3 groups randomly: control group,AOM/DSS group and silibinin treatment group(AOM/DSS/SB).The control group was fed normally.The AOM/DSS group and the AOM/DSS/SB group were given one intraperitoneal injection of AOM(10mg/kg)on day 0.At the same time,2%DSS was added to sterile water for drinking daily for 5 days,followed by normal sterile water for 16 days.Every 21 days was a cycle,and there were totally three cycles.Silibinin(750mg/kg/d)was continuously administered daily in the intervention group for 10 weeks.Weight,stool frequency and character changes of mice were recorded every week.Mice were sacrificed at the end of the experiment at week 10.The colon length,number and diameter of the tumors were documented.Tissue H&E staining was used for histological injury score.Immunohistochemical staining was performed for cell proliferation,TUNEL for apoptosis assessment,and tight junction protein ZO-1 was evaluated by immunofluorescent staining for colonic mucosa barrier.The production of inflammatory cytokines was determined by Realtime-PCR.The inhibitory effect ofsilibinin on IL-6/STAT3 pathway activation was evaluated by Western blot and Immunohistochemical staining.2.Effects of silibinin on cell lines(IMCE and HCT 116)with different concentration gradients were determined by MTT,Realtime-PCR and Western blot.Results1.It was observed that silibinin significantly reduced the amount and size of tumor in AOM/DSS mice,compared with that of AOM/DSS group(6.9±3.1 vs13.1±5.9),and especially the difference of tumor diameter(≥4mm)between the two groups was significant(t=4.12,P < 0.001).The Ki-67 positive cells of the mice of the silibinin treated group were significantly lower than that of the AOM/DSS group.Silibinin treated group showed more apoptotic cells in tumors compared with AOM/DSS group(TUNEL).Intestinal histological injury scores and tumor scores of silibinin treated group was significantly lower than that of AOM/DSS group.The m RNA expression levels of inflammatory cytokines in the intestinal tissues of silibinin treated group were significantly lower than those of AOM/DSS group.The value of phosphorylated STAT3 expression in intestinal tissues of silibinin treated group was significantly lower than that of AOM/DSS group,while there was no significant difference of the total STAT3 protein expression between the two groups.2.MTT assay showed that cell viability of IMCE and HCT-116 cell lines were significantly inhibited.Relative expressions of IL-6,IL-1β and TNF-α in IMCE and HCT-116 cells were significantly suppressed by silibinin,STAT3 phosphorylation were blocked in tumors of silibinin group compared with that of AOM/DSS group.ConclusionsOur study demonstrated that silibinin could inhibit the proliferation of colon cancer cell line,inhibit colitis associated cancer mouse model of intestinal mucosal inflammation,restrain the growth of the intestinal tumors,and suppress colitis-associated tumorigenesis in mice via inhibiting IL-6/STAT3 signaling.These results showed silibinin may be a CAC potential preventive medication.