Effects And Mechanism Of Baicalin On An Orthotopic Transplantation Mouse Model Of Mismatch Repair Gene Deficient Colorectal Cancer

Part I:Establishment of orthotiopic model of human colon cancer marked by green fluorescent protein and its biological characteristicsObjective To establish a stable orthotiopic model with high green fluorescent protein (GFP) expressing in nude mice.Methods Human HCT-116colon cancer cells transfected with GFP were used to build a subcutaneous tumor model in nude mice. Fifteen BALB/C nude mice were selected to undergo orthotiopic transplantation of colon when the GFP-labeled tumor grew to lOmm×lOmm as observed by in vivo fluorescent microscopy. The growth and metastasis of orthotopically implanted colon cancer cells were observed with fluorescent imaging system at different time points.Results GFP-labeled colon cancer models were successfully established in all the15nude mice, and there was no surgery-related complications or death. Tumors marked by GFP were observed under fluoroscope in week3. The size of the tumors progressively increased with time. The volumes of the orthotopically transplanted tumors obtained from global measurement using fluorescent imaging system was larger than those measured by peripheral vernier calipers at postoperative week3,4,5,6,7, while no statistically significant difference was observed (t=-1.280,-1.115,0.718,-0.199,-0.386, P>0.05); There was a significant difference in the interation of measure method and different time points (F=29.546, P(?)0.05). Eight nude mice survived at the end of the experiment, and tumor metastasis was observed in6mice.Conclusion It is technically feasible to construct GFP-labeled colon cancer orthotiopic transplantation model with high survival rate. The mice model could be used for real-time, in vivo, no-invasive and dynamic observation and analysis of the growth and metastasis of tumor cells. PartⅡ:Effects of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancerObjective To study the anticancer effects of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer.Methods Sixty orthotopic transplantation mice model of human colon cancer cell line HCT-116expressing GFP were established, which were divided randomly into negative controlled group (5%NaHCO3) and50,100,200mg/kg Baicalin groups. The nude mice were treated with intragastric infusion twice a day. Nude mice growth state, average weigh, inhibition rate of transplanted tumor, tumor metastasis and survival state were observed.Results At14,21and28days after treatment with different dose of Baicalin, tumor growth velocity was significantly slower in the treatment groups, and tumor volume was significantly smaller than the controlled group (there were832±637,2012±1566and2494±1557mm3respectively in14,21and28days)(F=4.433, P<0.05). At the end point of study, survival state of100mg/kg group(13/15)was superior to controlled group (8/15) and200mg/kg group (8/15)(x2=4.665and3.980, P<0.05). However, there were no significant differences in tumor metastasis and tumor surface vessel density.Conclusions Baicalin has statistically significant effects in inhibiting tumor growth in an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer, and100mg/kg may be an ideal treatment dose. PartⅢ:Mechanism of inhibitation of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancerObjective To study the mechanism of Baicalin on an orthotopic transplantation mice model of mismatch repair gene deficient colorectal cancer.Methods Sixty orthotopic transplantation mice model of human colon cancer cell line HCT116expressing GFP were established, which were divided randomly into four equal-sized groups, including a negative control group (G1) and three treatment groups that received50mg/kg (G2),100mg/kg (G3), or200mg/kg (G4) of baicalin. The cell cycle, apoptosis, microsatellite, and relative gene were detected.Results The proportion of tumor cells in G2/M phase in experimental groups was higher than that in control group signigicantly (G2vs G1, p=0.000; G3vs G1, p=0.003; G4vs G1,p=0.004). The apoptotic rates were significantly increase in experimental groups when compared with the control group (G2vs Gl,p=0.000; G3vs G1,p=0.004; G4vs G1,p=0.028). There were higer significant differences in PCNA, P53, hMLH1, hMSH2, TGF-β1, and IGF-IIR genes, but no difference in MSI, Bcl-2, and Bax genes.Conclusion The mechanism of the inhibitory effects of Baicalin is related to inhibit the expression of PCNA. P53、hMLH1、hMSH2、TGF-β1and IGF-ⅡR, induce apoptosis of tumor cells, and cell cycle progression is blocked at G2/M phase.