| Objective:In our country, bladder cancer is one of the most commonly tumor in urinary system. Urinary bladder cancer is characterized by easy recurrence, strong invasiveness and easy to get acquired drug-resistance. So the available protocols are far form satisfaction. Providing new drug target become an important problem to be solved. In our earlier research, we found that EMA and CD44v6might be the markers of bladder cancer stem cells. The cells of CD44v6as a biological tumor marker may be the reasons of recurrence and metastasis. Overexpression of CD44v6is associated with occurrence, development and metastasis of a variety of human malignant tumors. Therefore, on the basis of two-tie grading system, we detect the expression of EMA and CD44v6by using immunocytochemistry(IHC). This study is to investigate CD44v6expression in bladder cancer cell line T24, evaluate the effects of CD44v6blockage via monoclonal antibody method on biological behavior of bladder cancer cell line T24in vitro, and explore the related molecular mechanism.Methods:1. In the current study, the expression of CD44v6and EMA were examined in40cases of bladder transitional cell carcinoma,5normal bladder mucosas by means of immunohistoehemistry(IHC). Aim to evaluate the relationship between the protein expression of the two and the clinical pathological parameter.2. The expressions of CD44v6protein in bladder transitional epithelial cells and bladder cancer T24cells were detected by immunocytochemistry.3. MTT assay was used to detect the growth inhibitory rate of T24cells treated with different concentrations of anti-CD44v6monoclonal antibodies for24,48and72h, respectively. After T24cells were treated with anti-CD44v6monoclonal antibodies, the apoptosis was dectected by flow cytometer, the expressions of apoptosis-related markers bcl-2and Bax were determined by immunohistochemistry.4. The change of migration ability of T24cells was detected by Transwell migration test.Results:1. The positive expression rate of EMA and CD44v6in bladder carcinoma was92.5%and57.5%, respectively. The frequency of EMA-positive cells was not related with the tumor grades and stage. With the invasion of bladder carcinoma, the expression of CD44v6decreased obviously (P<0.01). Mostly EMA negative cells and CD44v6positive cells were expressed in the outer root sheat in normal bladder mucosas and bladder transitional cell carcinoma, while EMA positive cells and CD44v6negative cells were expressed in the position of relative mature.2. CD44v6mostly expressed on membrane and was expressed in T24cells, but rarely expressed in bladder transitional epithelial cells.3. MTT assay demonstrated that anti-CD44v6monoclonal antibodies had inhibitory effect on the proliferation of T24cells (P<0.05). The apoptosis rate of T24cells was increased induced by anti-CD44v6monoclonal antibodies (P<0.01). The expression level of Bax protein was up-regulated, while the expression level of Bcl-2protein was down-regulated in T24cells (P<0.01). Using light microscopy, the cells treated with anti-CD44v6monoclonal antibodies showed the change of cellular morphology.4. The migration of T24cells was inhibited after treatment with anti-CD44v6monoclonal antibodies (P<0.05).Conclusions:1. The results of IHC showed that EMA and CD44v6might be the markers of bladder cancer stem cell.2. Anti-CD44v6monoclonal antibodies can inhibit the abilities of proliferation and also induce apoptosis. The mechanism may be related with the expression regulation of bcl-2and Bax genes.3. CD44v6protein is closely related with tumor invasion and metastasis. Anti-CD44v6monoclonal antibodies can inhibit the abilities of migration of T24cells. |
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