Combined Use Of Vitamin D3 Enhances Chemopreventive Effects Of Metformin On Mouse Colitis-associated Colon Neoplasia

Objective:To study the synergistic effects and underlying mechanism of vitamin D3 that enhances chemopreventive effects of metfoemin on mouse colitis-associated colon neoplasia.Method:Colitis-associated colon enoplasia was established using ICR male mice via intraperitoneal administration(20 mg/kg body weight)of 1,2-dimethylhydrazine(DMH)and 1-week oral exposure(2%in drinking water)of a carcinogen,dextran sodium sulfate(DSS).A total of 276 ICR male mice,5-week old,were divided into 7 groups:1)normal controls;2)DMH controls;3)DSS controls;4)DMH + DSS controls;5)vitamin D3(3 dosage groups);6)metformin(3 dosage groups);and 7)vitamin D3 + metformin(3 dosage combinations).Different treatments were carried out from the 4th day after intraperitoneal administration of DMH,and lasted for 137 days.All mice were sacrificed at 20th week,with large intestine and serum samples collected.Tumors were then counted and measured.Aberrant crypt foci(ACF)were observed under low power microscope after methylene blue staining.Concentrations of insulin,insulin like growth factor 1(IGF-1),insulin like growth factor binding protein 1(IGFBP-1)and insulin like growth factor binding protein 3(IGFBP-3)in serum was determined using ELISA Kit.Expression levels of Phospho-IGF-I Receptor ?(Tyr1135/1136)/Insulin Receptor ?(Tyr1150/1151),Phospho-AMPKa(Thr172),Phospho-Akt(Ser473),Phospho-mTOR(Ser2448),Phospho-p70 S6 Kinase(Thr389),Phospho-S6 Ribosomal Protein(Ser235/236)and Cyclin D1 were determined using Western Blotting.Results:Compared with DMH+DSS group,combined use of VD3(200 IU/kg/day)and metformin(240 mg/kg/day)significantly reduced the number and volume of colon tumors,and also the number of ACF.Serum concentrations of insulin,IGF-1 and IGFBP-3 in the DMH+DSS group significantly decreased compared with normal control group,whereas concentrations of IGFBP-1 increased.Combined use of VD3(200 IU/kg/day)and metformin(240 mg/kg/day)also significantly increased serum concentration of IGFBP-3 compared with DMH+DSS group and VD3(200 IU/kg/day)group respectively;however,levels of insulin,IGF-1 and IGFBP-1 in serum were not affected.Compared with normal control group,expression levels of p-mTOR,p-P70S6K,p-S6P,and cyclin D1 in DMH+DSS group significantly increased,whereas the expression level of p-AMPK significantly decreased,yet with no significant difference for that of p-AKT.Compared with DMH+DSS group and VD3(200 IU/kg/day)group respectively,combined use of VD3(200 IU/kg/day)and metformin(240 mg/kg/day)increased the expression level of p-AMPK and decreased that of p-AKT,p-mTOR,p-P70S6K,p-S6P and cyclinDl significantly.Compared with metformin group,combined use of VD3(200 IU/kg/day)and metformin(240 mg/kg/day)increased the expression level of p-AMPK and decreased that of cyclin D1 significantly.Conclusion:Inhibitory effects of vitamin D3(VD3)or metformin alone on mouse colitis-associated colon neoplasia induced by 1,2-dimethylhydrazine(DMH)and dextran sodium sulfate(DSS)were not obvious and unstable,though that on colon aberrant crypt foci(ACF)were significant yet not dose dependent.In contrast,combined use of Vitamin D3(VD3)and metformin significantly inhibited aberrant crypt foci(ACF)formation and colitis-associated colon neoplasia.The underlying anti-tumor mechanism may be related to synergic effects of Vitamin D3(VD3)on metformin that increased serum concentration of IGFBP-3,activated expression of p-AMPK,as well as inhibited expressions of p-mTOR,p-P70S6K,p-S6P,and Cyclin D1.