Experimental Study On Chemoprevention Of Familial Adenomatous Polyposis By Berberine

Objective FAP is associated with mutation of Apc,and the current treatments mainly include whole colorectum resection and chemoprevention by COX-2 inhibitors.Surgery cannot prevent upper gastrointestinal adenomas,and may lead to the increased economic load and poor life quality.The chemopreventive effect of celecoxib is limited,and it also brings potential cardiovascular and gastrointestinal side effects,which restricts its long term clinical use.Recent studies and our previous data have shown that Berberine may have antineoplastic effects,and can also inhibit the expression of COX-2.Our current study was aimed to investigate the inhibitory effects on polyps growth by Berberine and associated mechanisms in FAP models.Methods Part 1 Four-week-old Min mice were randomized into two groups(Berberine prevention group and Control group 1,n=8),eight-week-old Min mice were randomized into three groups(Berberine treatment group,celecoxib group,and Control group 2),four-week-old wild-type C57BL/6J mice were selected as Control group 3.Drugs were given in drinking water.After 12 weeks,all animals were killed.The number and size of polyps were scored under a dissecting microscope.HE staining,PCNA and Ki-67 staining,and TUNEL assay were used to evaluate pathological analysis,cell proliferation and apoptosis.Gene microarray was used to assess the gene profiles in Berberine prevention group and control group 1.In addition,immunohistochemistry,Western blot,Realtime-Q-PCR and ELISA were used to detect Wnt and EGFR pathways,COX-2 expression,and PGE2 level.Part 2 IMCE cells were treated with Berberine in the presence or absence of EGF and TNFa.Cell proliferation and apoptosis were identified.Western blot was performed to detect the total and phosphorylation of EGFR and protein kinase B(Akt).Results Part 1 1.Berberine was well tolerated,and all mice grew slowly without significant difference.2.The total number of intestinal polyps in Berberine prevention(10.38±1.85)and treatment group(14.83±2.93)decreased 66%and 57%compared to the control groups.Berberine can reduce 75%of the colon polyps(0.25±0.46 vs 1.00±0.82,P<0.001).In Berberine prevention group,all sizes of polyps were significantly reduced,while<lmm and 1mm-2mm size polyps were decreased in Berberine treatment group.In Berberine prevention group,the numbers of polyps were significantly reduced in middle(P=0.002)and distal(P=0.001)portions of small intestine,while the numbers were decreased in proximal(P=0.001)and middle(P=0.018)portions in Berberine treatment group.Importantly,polyps in Berberine treatment group were significantly fewer than those in celecoxib group(P=0.027).3.The percent of PCNA and Ki-67 positive cells in Berberine prevention group decreased by 32%and 55%,while apoptotic cells increased to 2.14 fold(P<0.001).4.Gene microarray indicated the different gene expression profiles between Berberine and control group 1.5.Berberine could block cytoplasm and nuclear translocation of P-catenin,and reduce the expression of cyclin D1 and c-myc.6.Moreover,phosphorylation of EGFR,ERK,and Akt were inhibited by Berberine.7.Berberine could down-regulate the expression of COX-2,and also decrease the level of PGE2.Part 2 1.Proliferating activity of IMCE cells was increased after adding EGF and the proportion of cell proliferation was(10.6411.41)%.The proportion was significantly decreased in EGF and Berberine group(1.81±0.85)%compared to EGF group(P<0.01),while the least was the Berberine group(0.49±0.42)%.2.The proportions of cell apoptosis were(8.47±2.52)%and(9.39±2.13)%in Berberine group and TNFa group respectively,which were significantly increased compared to normal control(0.27±0.30)%,P<0.01.3.The p-EGFR was significantly increased after adding EGF,and was decreased in EGF and Berberine group at a concentration-dependent manner.4.Moreover,the p-Akt was increased after adding TNFa,and was decreased in TNFa and Berberine group compared to TNFa group.Conclusions Long term use of Berberine is safe,and can significantly prevent intestinal polyposis in FAP models,which seems to be better than celecoxib.Moreover,Berberine can reduce proliferation,induce apoptosis,cause gene expressions change,modulate the Wnt and EGFR pathways,and down-regulate the expression of COX-2,suggesting its chemopreventive potential against FAP and colorectal cancer.