| Objective:Recent study shows, the Clostridium perfringens enterotoxin (CPE) had cytoxicity on prostate cancer and ovarian caner which hihgly expressed Claudin-4 protein (CL4). However, little was known about the effect of CPE on gastric cancer cells. The objective of this study is to investigate the cytoxicity of CPE on gastric cell lines and the related mechanism.Method:1. Clinical assessment:Cancer tissue (30 cases), adjacent tissue (30 cases) and normal gastric mucosa (30 cases) samples were handled with immunostaining. The intensity of immunoreactivity for CL4 was subjectively scored from 1 to 5.2. Prepatation of CPE:CPE gene was synthesized according to the sequence of Clostridiun perfringens SMI01 CPE gene,and the correct Expression vector plasmid which contains this gene was constructed. This plasmid was transfected to DH5α E.coli, the transformed bacteria was cultured at room temperature and the expression of CPE protein was induced by IPTG. The media of Ni-IDA was used to obtain the purified CPE protein.3. In vitro, human gastric cancer cell line SGC7901 and normal gastric mucose cell line GES-1 was respectively treated by diffirent dose of CPE, then MTT assay was used to dectect the cytoactive of those cells. We used a laser scanning confocal microscope and the method of Western Blot to examine the expression and distribution of CL4. MTT was used to examine the cytoactive of those two group cell lines again after the expression of CL4 was silenced.4. In vivo, SGC7901 cells were injected subcutaneously to form tumor mass in nude mice models.28 mice were divided into 4 groups:control group, low-dose group, middle-dose group and high-dose group.100μL different dose of CPE were injected around the tumor, the control group was injected PBS every day for 10 days. The growth status, body weight, tumor size and side effects of CPE were recorded.Results:1. The positive rate of CL4 was 66.7% in cancer tissue and 46.7% in the adjacent tissue, both were higher than 16.7% in normal mucosa evidently.2. The sequence of The expression vector plasmid Pet28a-CPE was conformed to sequence of CPE gene in gene bank. We found the production of the transformed E.coli had a molecular weight of 35×103 by western blot, this result was corresponding with other reports. The pureness of the production was above 98% and the concentration was about 10mg/mL.3. In vitro, CPE had distinct cytotoxicity for SGC7901 cell, after 24 hours the CPE (0.2mg/L to 10mg/L) added, MTT assay detected the cytoactive of human gastric cancer cells SGC7901was obvious lower than control group, and this cytotoxicity was dose depended. This cytotoxicity was distinctly decreased when the CL4 was silcent. However, CPE has no effect on GES-1. The levels of CL4 in GES-1 was lower than SGC7901 significantly. After treated by CPE, the membrane of SGC7901 cells was disrupted but the GES-1 cells could maitain intact structure of membrane.4. In vivo, we found CPE could inhibit the tumor growth at concentration of 20mg/L, but it caused the side effect such as enterorrhagid and cutaneous necrosis on the injection site. CPE also coused death from the third day after injected if the dose over 30mg/L.Conclusion:Clostridium perfringens enterotoxin CPE expressed cytotoxicity on gastric cancer cell lines SGC7901, and the machanism was related with the expression of CL4 protein. |
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