| Part One:The inhibitory effect of 1,25-Dihydroxyvitamin D3 on colon cancer cells proliferation and its mechanismObjective:To explore the role of 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) on human colon cancer cells proliferation and its mechanism.Method:The expression levels of nuclear Vitamin D receptor (nVDR) in human colon cancer cell lines SW620, SW480, Caco-2, DLD-1, HT-29 and HCT-116 were detected by Western blot. SW480 and DLD-1 cells were selected as experimental candidates with high expression of nVDR, and further confirmed by immunocytochemistry staining. SW480 and DLD-1 cells were treated with 1,25(OH)2D3 at different concentrations (1,10,50 and 100nmol/L) for 24, 48 and 72h respectively. Cell proliferation was quantified by MTT assay. SW480 cells were treated with either 1,25(OH)2D3 (100nmol/L) or vehicle(control) for 48h, and then several parameters were measured:(1)the mRNA and protein expression levels of β-catenin, nVDR and Forkhead box M1 (FOXM1) by qRT-PCR and Western blot; (2)the transcriptional activity of β-catenin by Dual-Luc if erase(?) Reporter Assay System; (3)the nuclear protein expression level of β-catenin by Western blot and nuclear/cytosol fractionation kit. SW480 cells were subjected to transfecting siRNA to knock down FOXM1 and nVDR, followed by 1,25(OH)2D3 treatment and measurement of the transcriptional activity of β-catenin by Dual-Luciferase(?) Reporter Assay System.Results:(1)SW480 and DLD-1 cells highly expressed nVDR; (2) The proliferation of SW480 and DLD-1 cells was significantly inhibited by 1,25(OH)2D3 in a dose and time dependent manner (p<0.05); (3) 1,25(OH)2D3 significantly repressed the transcriptional activity of β-catenin in SW480 cells via promoting the nuclear export of β-catenin (p<0.05), while didn’t affect its mRNA and protein levels; (4) 1,25(OH)2D3 promoted the mRNA synthesis and protein expression of nVDR and FOXM1; (5) The inhibitory effect of the transcriptional activity of β-catenin by 1,25 (OH)2D3 in SW480 cells could be reversed when FOXM1 and nVDR were knocked down.Conclusion:1,25(OH)2D3 significantly inhibits the proliferation of SW480 and DLD-1 human colon cancer cells. 1,25(OH)2D3 represses the transcriptional activity of β-catenin via increasing FOXM1 expression and promoting the nuclear export of β-catenin.Part Two:The Protective Role of Vitamin D3 on Azoxymethane and Dextran Sulfate Sodium Induced Colitis-associated Colorectal Cancer in MiceObjective:To explore the preventive effect of vitamin D3 (VitD3) on azoxymethane (AOM) and dextran sulfate sodium (DSS) induced colitis-associated colorectal cancer (CAC) in mice and its mechanism.Method:C57BL/6 mice were divided into 6 groups. Group 1 served as naive control, which was given normal saline by intraperitoneal injection and drinking water by gastric lavage. Group 2 to 6 were given AOM (12.5mg/kg) by intraperitoneal injection, and one week later 2.5% DSS in drinking water was fed for 5 days to establish CAC model. Group 3 to 6 served as intervention groups that received VitD3 at different doses by gastric lavage (3 times per week), while group 2 served as model control given the same amount of drinking water. Group 3 to 5 (known as low dose group, medium dose group and high dose group) began to receive 15,30,60IU/g/w VitD3 (3 times per week) respectively two weeks before AOM injection, and group 6 (Post-DSS group, P-D group) began to receive 60IU/g/w VitD35 days after DSS intake. Mice were sacrificed by the end of the 14th week. Macroscopic evaluation of tumor number (TN) and tumor load (TL) as well as pathologic evaluation was carried out to assess CAC severity. The mRNA expression level of β-catenin was determined by qRT-PCR. The Ki-67 positive ratio and protein expression level of β-catenin and forkhead box M1 (FOXM1) were determined by immunohistochemistry. TNF-α levels in both serum and colon tissue were determined by ELISA.Results:(1) In terms of the tumor number (TN) and tumor load (TL), the medium dose group (n=12, TN=5.75±2.60, TL=12.88±5.10mm), the high dose group (n=11, TN=4.00±2.41, TL=10.55±7.20mm) and the P-D group (n=12, TN=4.17±3.21, TL=10.79±6.72mm) were significantly lower than the model control group (n=10, TN=10.9±5.13, TL=25.88±15.47mm) (p<0.05). However, there were no significant differences between the low dose group (n=11, TN=7.27±3.77, TL=15.08±10.32mm) and the model control group, as well as between the high dose group and the P-D group (p>0.05). (2) In term of Ki-67 positive ratio, the medium dose group (47.00±10.40%), the high dose group (42.68±9.75%) and the P-D group (46.63±9.68%) were significantly lower than the model control group (72.38±9.32%) (p<0.05). (3) In term of β-catenin mRNA level, all VitD3 intervention groups were significantly lower than the model control group(p<0.05). The amount of nuclear β-catenin decreased as the given dose of VitD3 increased. In term of β-catenin protein level, the high dose group was significantly lower than the model control group (p<0.05). (4) In term of FOXM1 protein level, the high dose group was significantly lower than the model control group (p<0.05). (5) In term of TNF-α levels in serum and colon tissue, there were no significant differences between VitD3 intervention groups and the model control group (p>0.05), although the level of TNF-α decreased as the given dose of VitD3 increased.Conclusion:VitD3 supplement suppresses AOM/DSS induced CAC in mice. VitD3 inhibits the transformation process from colitis to colorectal cancer possibly via reducing the expression of β-catenin, FOXM1 and TNF-α level in both serum and colon tissue. |
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