Vitamin D3 Inhabits The Growth Of Colon Cancer Cells Which Are Dependent Runt Related Transcription Factor 3

Colon cancer is the one of most common human malignant tumor throughout the world. In present, its incidence is rapidly increasing because of changes in lifestyle and diet, and it is the fourth malignant tumor in our country. Epidemiological survey and several human clinical trials suggest that vitamin D has a protective and therapic effect on colon carcinogenesis, which has also been confirmed by abundant experimental studies. In order to improve the clinical effective application, it is very important to clearly understand the mechanism of the anti-cancer action of vitamin D. Vitamin D3 inhibits cell growth by increasing expression of either transforming growth factor-β(TGF-β) or its receptors, which potently inhibits proliferation of many cell types and is involved in cell cycle control and apoptosis. In some models, Vitamin D can restore sensitivity to TGF-βby inducing the expression of the TGF-βreceptor. However, it has been verified that Runx3 is a target protein in TGF-β-mediated tumor suppressor pathway.Runx3, one of Runt-related genes, is a novel tumor suppressor. The studies showed gene methylation and silencing of Runx3 in several malignancies of stomach, liver, pancreas, lung, prostate, etc. Furthermore, Runx3 expression was decreased in human colon cancer, and restoration of Runx3 expression in colon cancer cells attenuated the growth and abrogated the metastasis of colon cancer in an animal model. In light of common signal pathway, whether there is any relationship between vitamin D3 anti-cancer and the expression of Runx3. In addition, vitamin D3 can up-regulate Runx1 and Runx3, but down-regulate Runx2. It has reported that vitamin D3 can increase the expression of Runx3 in HL-60 cells which was induced by retinoic aid. So in the present work, we explored the relative mechanism involved in colon cancer cells.【Objectives】(1)To investigate whether vitamin D3 anti-cancer is assocciated with Runx3 expression in colon cancer cell lines;(2)To examine the possible mechanisms underlying the induction Runx3 effect by vitamin D3 in colon cancer cells.【Methods】(1) The expression level of Runx3 in colon cancer cell lines were determined by western blot.(2) Runx3 mRNA and protein expression treatment with vitamin D3 were determined by RT-PCR and western blot respectively.(3) The Runx3-specific siRNA vector was designed and constructed by DNA recombinant technology.(4) The Runx3-siRNA was transfected into SW480 cells using lipofectamine 2000 reagent. Empty vector was used as control. The transfected cells were screened with G418 for 1 month, single cell colonies were obtained by limited dilution method. Semi-quantitative RT-PCR and Western blot were used to detect the Runx3 expression in the transfected cells for confirmation of transfection.(5) The effect of vitamin D3 on the growth of transfected cells and control cells were detected by MTT and flow cytometry.(6) Western blot was used to detect cyclin A, cyclin D1, cyclin E, cdk2, cdk4, PCNA, Rb and p27 protein expression.【Results】1. Runx3 expression in colon cancer cellsWestern blot examination found that Runx3 expression was undetectable in Lovo Cell line; SW620 and HT-29 cell lines has a low level of Runx3 expression; Runx3 was expressed at a high level in SW480 cell line.2. Runx3 is up-regulation by vitamin D3 in time and dose-dependent mannerWe used RT–PCR and western blot to detect Runx3 mRNA and protein level respectively. Time course (from 6 hours to 72 hours) experiments showed vitamin D3 stimulation of Runx3 mRNA expression in SW480 cells. Runx3 gene expression was up–regulated after 6 hours of treatment with vitamin D3. It was enhanced at 72h. VitaminD3 induced Runx3 mRNA production in SW480 cells in a dose-dependent manner. SW480 cells were treated with vitamin D3(10^-10,10^-9,10^-8,10^-7 mol /L respectively) for 48 hours. Vitamin D3 caused increase of Runx3 mRNA and the expression of Runx3 was up-regulated obviously on the concentration of 10-8 mol/L. Western blot analysis was performed to further detect protein level. It was clear that the results of western blot was coincidence with RT-PCR in a time and dose-dependent manner.3. Deletion of Runx3 weaken the effective of vitamin D3 on SW480 cellsAs described previously, Runx3 is an upregulated gene by vitamin D3 in SW480 cells. Vitamin D3 treatment increased the expression of Runx3 in a time and dose-dependent expression. Next, we tested to determine whether or not deletion of Runx3 expression affect vitamin D3 induced SW480 proliferation. We designed and constructed Runx3- siRNA to downregulate the expression of Runx3 in colon cancer cells. After cell transfection and antibiotic screening for 1 month, the expression of Runx3 in stably transfected cells was determined by RT-PCR and western blot analysis. Runx3-siRNA could downregulate the expression of SW480 effectively compared with those transfeted with empty vectors and parent cells. The growth curves showed that SW480-si growth slowly than SW480 and SW480-vector after tre -atment with vitamin D3, namely ,the growth curves for Runx3-si cells were significantly lower than those for control cells (P <0.05 on days 4–7), whereas there was no difference between SW480 and SW480-vector.4. Runx3-siRNA Lead to the Cell Cycle Arrest of Colon Cancer Cells Treatment with Vitamin D3We studied the effects of vitamin D3 on the cell cycle after deletion of Runx3 by flow cytometry analysis to further investigate the mechanism .The results of the cell cycle showed that 31.3% of SW480-si cells were in S-phase compared to 12.6% of SW480 cells and 11.2% SW480-vector at 48 hours after vitamin D3, whereas 37% of SW480-si cells were in S-phase compared to 23.5% of SW480 cells when no drug (P < 0.05). To further investigate the mechanism of vitamin D3 induce Runx3 expression lead to cell cycle arrest in colon cancer cells, we examined cell cycle protein by western blot analysis. Our results indicated that inhibition the expression of Runx3 can lead to an increase cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and PCNA proteins, correlated with a decrease in p27 and Rb proteins. Conversely,vitamin D3 downregulate cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and PCNA proteins, but with upregulation p27 and Rb proteins. Compare with control group, it was not obvious that the change of cell cycle proteins in SW480-si cells treatment by vitamin D3.【Conclusion】We provide evidence which Runx3 expression was up-regulated by vitamin D3 in colon cancer cell lines. Simultaneously, our study presented evidence for Runx3-si led to upregulation cyclin A, cyclin D1, cyclin E, cdk2, cdk4 and PCNA, but to downregulation Rb and p27 expressions, which affect cell cycle development. Conversely, vitamin D3 inhabited the development of cell cycle. These data may be helpful for elucidation the mechanism of vitamin D3 anti-proliferation in colon cancer.