Emodin Inhibits Mice Colon Cancer Development By Affecting Regulator T Cell Trafficking

ObjectiveColon cancer is a malignant tumor, and the incidence rate increased year by year in recent years, which become a great threat to the human health. The current treatment methods are not applicable to all patients with colon cancer, especially the older patients. To improve patients’quality of life is becoming the new target for cancer therapy. Traditional Chinese medicine has a distinct characteristics and advantages of application in the clinical therapy of tumor, the effector of " strengthening the body resistance to eliminate pathogenic factors " was significantly better than that of chemotherapy treatment in improving quality of life in patients.Most of tumor studies have focused on the pathogenesis mechanism of cancer, especially tumor immune escape mechanism. Although it is complicated, the tumor immune escape mechanism is related to regulatory T cells (regulatory T cells, Tregs).Regulatory T cells are recognized with the immunosuppressive effect in immunity, and CD4+D25+Tregs are the most well-understood Tregs. Tregs migrate in tissues before immunosuppression. The accumulation of Tregs in different areas of the body is caused by directional trafficking. Tregs are often enriched in response to a network of chemotactic stimuli involving chemokine and chemokine receptors, resulting in increased ratios of Tregs among conventional effector populations, and leading to changes in immune suppression and homeostasis. The enrichment of Tregs at the tumor microenvironment is due in part to selective recruitment mediated by chemokine, and heightened expression of these chemokine is correlated with elevated Treg levels in many cancer types. By far, the most described axis in selective Treg recruitment to the tumor is through the CCR4and the CCR4-associated chemokine CCL22and CCL17.NF-κB is an important transcription factors, regulating the expression of cytokine, chemokine and other inflammatory factors. Abnormal activation of NF-κB has been found in different development stages of a variety of tumors, and the activation can contribute to regulation of chemokine, which aid to recruit the immune cells in the tumor microenvironment.Rhubarb is a traditional Chinese medicine with widely clinical application, and the effective component is emodin. The immune pharmacological action of emodin is involved in anti-infective, anti-inflammatory and antitumor. It is interesting these diseases that emodin can treat have existed Tregs imbalance of local lesion. And we also found that the ratio of Tregs can affected by emodin.In this study, we observed the inhibitory effects of emodin on CT26cells in vitro and in vivo, and its relationship with CD4+CD25+Tregs migration, to focus on the mechanism of emodin on alterations CD4-CD25+Tregs trafficking manner.Methods and Results1. Inhibition of emodin on CT26cell line in vitroWe observed the effects of different concentration of emodin on CT26cell.①Monolayer wound healing experiments,②Annexin V-FITC/PI kit for apoptosis assay,③Foxp3expression by flow cytometry,④Statistical analysis.Results show Emodin has significantly inhibited the migration of CT26cell, promoted CT26cell apoptosis, down-regulated Foxp3expression in CT26cell.2. Observing the pharmacological action of emodin in vivo2.1The inhibition of emodin on colon cancer①To establish tumor model of colon cancer, and then study antitumor effect of emodin in vivo.②Animal and drugs:animal were divided into four groups, the negative control group, tumor-bearing model group, emodin treatment group and FTY720treatment group, in turn given0.5%sodium carboxymethyl cellulose solution, emodin (100mg/kg), FTY720(3mg/kg) and0.5%sodium carboxymethyl cellulose solution once every day, continuous14d.③To calculate tumor inhibition rate.The results show a success rate of more than98%on establishment tumor model. The tumor weight of tumor-bearing group was (1.01±0.14) g, that of emodin group was (0.26±0.08) g, and that of FTY720group was (0.34±0.11) g, the inhibition rate of emodin group was74%, FTY720inhibition rate of66%, more than30%, suggesting emodin can inhibit the tumor growth in a mouse model (p<0.05).2.2The observation of CD4+CD25+Tregs trafficking in cancer model①Detection of the percent of CD4+CD25+Tregs by flow cytometry,②Detection of CCR4expression in Tregs,③Detection of chemokine CCL17and CCL22expression in tumor by confocal fluorescence microscopy.Results show that CD4+CD25+Tregs have enriched in tumor and emodin and FTY720can inhibit the accumulation. Emodin and FTY720can significantly reduce CCR4+Tregs ratio in tumor. Emodin and FTY720have significantly decreased the expression of CCL17in tumor tissue.2.3Effects of emodin on Tregs function①The ratio CD8+CD3+T cell by flow cytometry,②Detection of IFN-γ, TNF-β1, IL-10by ELISA,③IFN-γ secretion of T cells by flow cytometry,④IL-10secretion of CD4+CD25+Tregs by flow cytometry.The results showed that:①Emodin and FTY720have significantly reduced the percent of CD8+CD3+T cells in the peripheral blood and lymph nodes, but increased the proportion in tumor;②The levels of IFN-γ, TNF-β land IL-10in tumor-bearing group were significantly higher than that of the negative control group, emodin and FTY720can reduce the levels of all cytokines in the serum;③Emodin and FTY720also can inhibit CD4+CD25+Tregs on secretion of IL-10.3. Investigation the effect mechanism of CD4+CD25+Tregs migrationFirst, to establish CT26cell and spleen lymphocytes co-culture system to observe the intervention effect of emodin, to explore the mechanisms involved. ①Foxp3expression by flow cytometry,②The ratio of CD4+CD25+Tregs by flow cytometry and the expression of CCR4on CD4+CD25+Tregs surface,③RT-PCR for CCR4mRNA expression in CD4+CD25+Tregs,④activation of NF-κ B in CT26cell.The results showed:①Emodin has down-regulated Foxp3expressing of CT26cell in co-culture system,②Emodin has significantly reduced CD4+CD25+Treg in co-culture cell system, and down-regulated CCR4expression of CD4+CD25+Tregs,③Emodin has reduced the expression of CCR4mRNA in CD4+CD25+Treg cells,④Emodin has significantly inhibited the activation of NF-κBin CT26cell.DiscussionEmodin inhibited CT26cell growth in vivo and in vitro. CD4+CD25+Tregs ratio in peripheral lymphoid organs has enriched by induced colon cancer CT26cells, and emodin and FTY720can reduce the accumulation in tumor through down-regulation of CCL17/CCR4expression. Emodin inhibit CD4+CD25+Tregs function has not only induced the level IFN-gamma, TNF-β and IL-10in serum, but also increased the ratio of CD3+CD8+T cells in tumor. The activation of NF-κB in CT26cells was inhibited because emodin has reduced the expression of chemokine CCL17. Then the CD4+CD25+Tregs trafficking manner was altered by CCL17/CCR4axis.This study explored knowledge about emodin:①To reveal the CD4+CD25+Tregs trafficking associated with the CCL17/CCR4axis in CT26tumor mouse,②Emodin affects regulatory T-cell trafficking and inhibits colon cancer cell growth.③To provide new experimental evidence for anticancer effect of emodin, and provide a new way of thinking on study of traditional Chinese medicine which can regulate immunity system.