The Study Of Expression Of Thymic Stromal Lymphopoietin In Colorectal Cancer And Its Regulation On Growth Of Colorectal Cancer Cell

Colorectal cancer is a common gastrointestinal malignant tumor that threatens human health. Till now, regular treatment methods such as operation accompanied by radiotherapy and chemotherapy can not reach a satisfactory result. Therefore, early discovery, early diagnosis and early therapy that is made individually are the optimal methods to improve recovery rate. Thymic stromal lymphopoietin(TSLP) was previously considered to be associated with allergic diseases like asthma and inflammatory bowel disease. The pathogenesis was due to the Th2 type immuno-reaction elicited by TSLP. Chronic inflammation is believed to be related to occurrence of tumor. It has been reported that TSLP could induce chronic inflammation through Th2 cells resulting in malignant tumor such as breast and pancreatic cancer. However, it has also been reported that TSLP could induce dermatitis while inhibit dermatoma. Furthermore, there has been no report about the direct effect of TSLP on tumor cells. Therefore, the role of TSLP and its mechanism in a variety of tumors may have diversity to be determined. The occurrence of colorectal tumor is a chronic progress during which colon epithelial cells make malignant transformation. Intestinal epithelial cells can secrete TSLP under normal condition, which performs the role of maintaining the stability of lumen microenvironment. But it has been no report about the role,of TSLP in occurrence of colorectal cancer.Aim:To determine the expression of TSLP in human colorectal cancer, the direct effect of TSLP on cancer cells and the regulation mechanism of tumor growth, we separated the topic into four parts:(1) investigation of expression of TSLP in human colorectal tumor and tumor adjacent tissues, and the correlation between TSLP expression and tumor stage;(2) investigation of expression of TSLPR on colorectal cancer cells including sw1116,sw480 and DLD-1;(3)investigation of regulatory effect of TSLP-TSLPR axis on the proliferation, apoptosis and cell cycle of colorectal cancer cells;(4) investigation of the downstream signaling pathway and apoptotic pathway in colorectal cancer cells initiated by TSLP-TSLPR activation;(5) investigation of regulatory effect of exogenous TSLP on a nude mice xenograft model of human colorectal cancerMethod:(1) specimens of forty cases of colorectal cancer patients after operation were collected and were classified in terms of Duke’s criteria. The expression of TSLP on mRNA and protein level in tumor and tumor adjacent tissue was detected by realtime-PCR, ELISA and immunohistochemistry method. The expression level was evaluated in correlation with Duke’s criteria; (2) The expression of TSLPR on colorectal cancer cell lines was detected by RT-PCR, immunocytochemistry and flow cytometry;(3) After the colorectal cell lines were treated by diverse concentration of TSLP for 48h, the apoptotic rate was measured by Annexin-V-PI.cell cycle by PI, proliferation rate by CCK-8 kit and caspase3, PARP by western-blot; (4) After the colorectal cell lines were treated by diverse concentration of TSLP for 48h, molecules such as P-JNK and P-P38 in MAPK pathway, STAT3, P53 and p21 as transcription factors, caspase8 and caspase9 in endogenous and exogenous apoptotic pathway and mitochondrial apoptotic molecules such as bid, bim, bad, bax and bak were detected by western-blot. When caspase8 inhibitor was added with TSLP, the apoptotic rate and caspase3 expression were again investigated. JC-1 was used to detect the alteration of mitochondrial membrane potential at early apoptosis; (5) Female nude mice were classified into control group and TSLP-intervention group. After the nude mice xenograft model of human colorectal cancer was created by implanting sw1116 cells into the blank of mice, PBS and 5ug/kg TSLP were administered around tumor every other day respectively. When the mice were sacrificed and tumors were resected at end, the tumor volume and weight were measured, the necrotic area and infiltration of leukocytes were evaluated by HE, the apoptotic rate by TUNEL method and expression of cleaved-caspase3 by western-blot.Result:(1) In human colorectal cancer, TSLP could be expressed by tumor cells or stromal cells while in tumor adjacent tissues, TSLP could be expressed by colon epithelial cells. The expression on mRNA and protein level in the former was less than that in the latter (p<0.05). The expression level of TSLP in tumor, which is highest in Duke’s A and lowest in Duke’s D, was negatively correlated with Duke’s criteria (p<0.05). (2) There was active TSLP receptor(TSLPR) expression on colorectal cancer cell lines such as sw1116, sw480 and DLD-1.(3) After the colorectal cell lines were treated by diverse concentration of TSLP for 48h, firstly, the total apoptotic rate and the expression level of cleaved-caspase3 and cleaved-PARP was obviously elevated. This pro-apoptotic effect was correlated with TSLP in a dose-dependent manner(p<0.05);secondly, the proliferation rate of sw1116 and sw480 other than DLD-1 was significantly decreased, which was also correlated with TSLP in a dose-dependent manner(p<0.05);lastly, the proportion of G1 phase and S phase in cell cycle did not change obviously, whereas that of G2/M phase was decreased according to TSLP in a dose-dependent manner(p<0.05). This inhibitory effect was most significant at dose of 200ng/ml. (4) After the colorectal cancer cell lines were treated by diverse concentration of TSLP for 48h, theexpressionlevelofphosphorylated-JNK, phosphorylated-P38, P53,p21 , cleaved-caspase8 and cleaved-caspase9 was elevated in a dose-dependent manner whereas that of phosphorylated-STAT3 was decreased. When caspase8 inhibitor was added with TSLP, the increase of apoptotic rate and expression of cleaved-caspase3 was effectively inhibited. The expression level of mitochondrial apoptotic molecules such as bid, bim, bad, bax and bak was positively correlated with TSLP in a dose-dependent manner, whereas the mitochondrial membrane potential was decreased according to TSLP adversely and the proportion of JC-1 monomer was increased. (5) After the nude mice xenograft model of human colorectal cancer was treated with exogenous TSLP, the volume and weight of tumors in TSLP-intervention group was less than that in control group. However, the necrotic area, extent of leukocyte infiltration, apoptotic cell number and expression level of cleaved-caspase3 were highly increased in TSLP-intervention group compared with control group(p<0.05).Conclusion:The expression of TSLP in human colorectal cancer might perform as a role of inhibiting tumor growth. And the expression level was correlated with tumor severity, indicating that TSLP might serve as a valuable marker to evaluate the prognosis of colorectal tumor. Active TSLPR was expressed on surface of colorectal cancer cell lines, therefore, exogenous TSLP could bind to it followed by activation of MAPK signaling pathway and extrinsic apoptotic pathway, resulting in the promotion of apoptosis. This pro-apoptotic effect was dose-dependent and mitochondria-dependent apoptotic pathway also contributed to this effect. TSLP could efficiently inhibit the growth of nude mice xenograft tumor of human colorectal cancer, implying that TSLP possessed clinical application prospect and feasibility to some extent.