Cancer Associated Fibroblasts Induce The Normal Colon Epithelial Cells And The Effect Of Berberine And Evodiamine To CAFs

Objective1. Cancer associated fibroblasts can induce the cancergenesis and promote the cancer development through autocrine and paracrine, which are very important and play critical role in tumor microenvironment. The study of Berberine and Evodiamine’s effect to CAFs play a significant role in the treatment of colon cacer.2. Induce the normal colon epithelial cells to cancer cells or pro-cancer cells with CAFs can build a useful culture system to study the process of cancer and cancergenesis. The culture system can be used to investigate the induction function of CAFs to epithelial cells and the inhibition effect of Chinese traditional medicine to colon cancer.Methods1. Use the real-time cell analysis system to analyse the effect of Berberine and Evodiamine to the proliferation of cancer associated fibroblasts and the effect of TGF-beta, Berberine and Evodiamine to normal colon epithelial cells.2. The immunofluorescence method is used to analyse the expression level of protein marker after the treatment of Berberine and Evodiamine in CAFs, analyse the effect of Berberine and Evodiamine through the expression level change of α-SMA. CAFs and TGF-beta were used to induce the normal epithelial cells, comparative analysis of the results can help to understand the possible mechanism of CAFs in cancer progress.3. The gene expression level of CAFs were detected by PCR method after the treatment of Berberine and Evodiamine. The gene expression and immunofluorescence results of α-SMA in CAFs were analysed synthetically. The gene express level of α-SMA, FAP and FSP in normal colon epithelial cells after treatment with TGF-beta and coculture with CAFs were detected by PCR method.Results1. The Evodiamine can inhibit the growth of CAFs after analysed in RTCA system, where the cells grow slower than blank group. The Berberine can signigicantly inhibit the proliferation of CAFs, this effect was concentration dependent, which completely inhibit the cells’growth in the highest concentration.There is not enough evidence to prove the promotion effect of TGF-3 to normal epithelial cells, the low concentration can slightly promote cell growth, but high concentration first inhibit cell growth and then convert to promotion effect, which prove the complex function of TGF-β.2. The result of immumofluorescence test show that the Berberine and Evodiamine can inhibit the expression of α-SMA, the inhibition was not correlated to concentration.The expression level of α-SMA was not change in normal epithelial cells which coculture with CAFs. After the treatment of TGF-β, the expression level of α-SMA increased with 5ng/ml treatment of TGF-β, the other concentration could not detect any change.3. The Berberine and Evodiamine can down-regulate the expression level of α-SMA in CAFs, the other marker FAP was decreased after treatment, otherwise the expression level of FSP increased after treatment. After cocultrued with CAFs, the expression level of α-SMA in epithelial was up-regulated. This kind of change was similar as TGF-β treatment especially at the 5ng/ml concentration. The expression level of DNMTs changed after treatment, the epigenetic regulation was included.Conclusion1. Berberine and Evodiamine can inhibit the growth of CAFs and change the expression of protein marker through protein and gene level. Although the Evodiamine can not apparently inhibit the growth of cancer associated cells the protein and gene expression was decreased.2. After the treatment by TGF-β the expression of α-SMA in gene and protein was higher than blank group, although the growth of epithelial cells was not apparently promoted. When normal epithelial cells cocultured with cancer associated cells, the expression level of α-SMA increased in epithelial cells and similar with TGF-β treatment which demonstrated similar promotion effect of CAFs to induce normal epithelial’s pro-cancer change.