| [Background&Objective]Following detached from the primary tumor, aggressive malignant cells intravasation in blood or lymphatic vessels generate circulating tumor cells (CTC). The first description of the presence of CTCs in the peripheral blood of cancer patients, so far, was a more than100years history. The CTC has always been looked as the source of tumor recurrence and metastasis. Most cells suffered apoptosis after they lose contact with their extra cellular matrix or their neighbouring cells. Encounted extremely terrible external condition, how to survive and acquired the anoikis resistance ability were still uncovered. It was reported that CXCL8could promote the proliferation, invation, migration and angiogenesis in the recent studies. It also shown tha CXCL8could active multi-survival pathways to inhibit the apoptotic signal. So, in our study we will investigate the role of CXCL8in the anoikis resistance of colorectal cancer, further explore the molecular detail during the process. We would also detect the expression of the CXCL8and associated protein in the surgical resection colorectal cancer (CRC) specimen, analyze its corralation with clinical features, trying to find and block the key targets promoting cell survival signal mediated by CXCL8, in order to raise a new treatment modalities to improve the clinical efficacy and reduce the recurrence and metastasis of CRC, as well as provide a new indicator of metastasis and recurrence for colorectal cancer patient.[Methods]1. Studies on the anoikis resistance induced by CXCL8in vitro.(1) Construct the aniokis resistance model:colorectal cancer cells were seeded in a Poly-HEMA coated plates to inhibit the cells attachment and simulate the state of CTC in the blood.(2) Studies on the correlation of CXCL8and colorectal cancer anoikis sensitivity: Immunofluorescence assay, RT-PCR and Western blot were used to test the expression level of vary colorectal cancer cells. Application of FITC-Annexin V/PI double staining to identify the apoptotic cells and test the apoptotic rate of detached and attached cells by flow cytometry at different time points, analyze the correlation of CXCL8and anoikis sensitivity.(3) The influence of CXCL8on the anoikis of CXCL8:stimulate the colorectal cancerlls, SW480Caco2with different concentration of CXCL8. Apoptotic cell ratios were examined by flow cytometry and the Annexin V-FITC apoptotic detection kit. MTS assay was used test the proliferation rate of colorectal cancer cells on the condition of attachment and detachment by the stimulation of CXCL8. Immunoblotting was employed to analyze the expression of the apoptosis associated protein Bcl2and caspase-3. Lovo, HT29and HCT116were treated by the optimal concentration of CXCL8for48hours, the change of cell apoptosis was detected again to confirm the effect of CXCL8.2. Explored the molecular detail of anoikis resistance induced by CXCL8.(1) Western blot were also utilized to determine the changes of T-AKT, P-AKT, T-ERK, P-ERK and TOPK in the colorectal cancer cell treated by CXCL8.(2) Inhibition test. Western blot analysis of P-AKT/AKT, P-ERK1/2/ERK1/2, and apoptotic markers in SW480cells which were pretreated the cells with AKT inhibitor MK2206and ERK1/2inhibitor U0126before stimulated with CXCL8.(3) RNA interference experiments. Cells were transfected with siRNA-targeting AKT, ERK1/2or control siRNA before the stimulation of CXCL8,48hours later, detect the anoikis rate by flow cytometry and the Annexin V-FITC/PI stain. The associated signal proteins were analyzed by Western blot.(1) Detection the expression of CXCL8and TOPK by immunohistochemical analysis in CRC tissues and normal mucosa.(2) Correlation analysis of the expression of CXCL8and TOPK with clinicopathological characteristics in CRC.(3) The clinical outcomes of co-existence of CXCL8and TOPK were investigated.[Results]1. CXCL8can protect the CRC cells from anoikis in vitro. (1) The expressions of CXCL8and anoikis rates were negative correlation in CRC cell lines, but there was no assotiation for attached cells.(2) Exogenous CXCL8could promote colorectal cancer cells resist anoikis.(3) PI3K/AKT and ERK1/2pathways have participated in the anoikis resistance of CRC cell lines induced by CXCL8.(4) TOPK was the crosstalk between PI3K/AKT and ERK1/2during the anoikis resistance of CRC cell lines.2. Significance of expressions of CXCL8and TOPK in colorectal cancer tissues.(1) A significantly higher CXCL8and TOPK levels in cancer tissues compared to paired normal tissues (χ2=53.281,P=0.000;χ2=372.000,P=0.000).(2) Significant positive correlations were observed between CXCL8and TOPK expressions (r=0.254,P=0.000).(3) The expression of CXCL8was associated with cell differentiation, tumor invation depth, lymph node metastasis, liver metestasis. However, the TOPK level was associated with the location of tumor, cell differentiation, invade depth, lymph node metastasis, liver metestasis and CEA (P<0.05).(4) The Kaplan-Meier analysis and the log-rank test shows that patients with higher CXCL8and TOPK expressions had a shorter overall survival (OR) time and disease-free survival (DFS)(OR:log rank=11.302, P=0.001; log rank=10.620, P=0.001;PFS:log rank=9.6,P=0.02;log rank=10.502,P=0.001), compared with the low one. The patients who showed a high espression of both CXCL8and TOPK had a shorter OR and DFS compare with CXCL8or CCL20expression alone.(5) Multivariate Cox regression analysis revealed that tumor invade depth, lymph node metastasis, liver metestasis, CXCL8and TOPK expression were independent predictors of overall survival. Tumor invade depth, CEA, lymph node metastasis, liver metastasis, CXCL8and TOPK expression could direct influence the DFS of colorectal cancer patients. Furthermore, co-expression of CXCL8and TOPK was an indepndent prognostic factor to the overall survive and disease-free survive (OR:log rank=4.210, P=0.04, log rank=4.386, P=0.036:DFS:log rank=10.024, P=0.001; log rank=4.662, P=0.032).[Conclution]Anoikis resistance of CRC induced by CXCL8depends on the activation of PI3K/AKT and ERK1/2. TOPK serves as the crosstalk between PI3K/AKT and ERK1/2. CXCL8plays an important role in the process of metastasis and recurrence of CRC. Co-existence of CXCL8and TOPK overexpression would potentially assist in TNM staging systems to predict the prognosis of these patients who would benefit from target the CXCL8associated pathway. |
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