| BackgroundColon cancer is ranked second and third place respectively of the most common tumor in both men and women worldwide, and shows a rapid tumor progression. Due to the high invasiveness of colon cancer,20%-50% of patients with colon cancer will be dead in five years after diagnosis due to extensive spread. Recurrence and metastasis of colon cancer is a major factor for death, and more than 50% of patients can not get radical tumor resection. Although progress has been made in the early and rapid diagnosis and treatments of colon cancer, the 5-year survival rate is still very low, especially for those patients with distant metastasis. Molecular pathogenesis of colon cancer remains largely unclear, therefore, to further understand the molecular mechanisms of colon cancer, will be a great help to improve the treatment.Interactions between tumor and stroma, can promote tumor progression, which can be mediated by a variety of factors, including chemokines. Chemokines are a class of low-molecular-weight chemokine protein in general, secreted from the cells to the extracellular stroma. They were originally named because they can attract leukocyte to sites of inflammation. They play an important role in inflammation, lymphoid homing, tissue development and other physiological and pathological processes. Interleukin-8 (IL-8), also known as CXCL8, is a cytokine that is the most characteristic and effective in the class of human CXC chemokines. It has been well studied for the role in inflammation, infection and other disease states. There are two kinds of IL-8 receptors, CXCR1 (IL-8RA) and CXCR2 (IL-8RB), which are widely distributed in a variety of normal and abnormal cell membrane. IL-8 has high binding affinity with CXCR1 and CXCR2, which could further activate the downstream biological effects of cell-mediated pathway. In tumors, IL-8 could promote angiogenesis by stimulating endothelial cells, induce chemotaxis of neutrophils to the tumor site, promote the proliferation and migration of tumor cells, and inhibit apoptosis of tumor cells, which are important for the involvement in tumor progression. Numerous studies have demonstrated the key role of IL-8 in the angiogenesis, growth, invasion and metastasis of colon, stomach and pancreatic tumor. In addition, IL-8 has a prognostic value in various kinds of malignancies, including liver cancer, nasopharyngeal cancer, stomach cancer, lung cancer and prostate cancer.Invasion and metastasis belong to the major characteristics of tumor cells, which involves a variety of cell biological activities, such as interactions between the invasive cells and the extracellular matrix. Integrins are a class of membrane proteins that mediate the interaction between tumor cells and extracellular matrix. Integrins are a large family of heterodimeric transmembrane receptors, consisting of a a subunit and α β subunit, and they play a key role in tumor migration and metastasis, thereby contributing to tumor progression. Integrin αvβ6 is the only heterodimer that 06 subunit could only form and αvβ6 is usually only expressed in epithelial cells. Integrin αvβ6 is hardly expressed in normal tissues, but it becomes highly expressed in embryonic tissue formation, tissue repair and pathological conditions, especially in the process of tumor formation. Our previous studies have demonstrated that integrin αvβ6 plays an important role in tumor proliferation, apoptosis, and directional migration, invasion and metastasis, matrix metalloproteinase (MMP) secretion and chemo-resistance.Studies have shown that IL-8 is closely related to the invasion and metastasis of colon cancer cells. IL-8 is almost undetectable in normal healthy tissues, but in pathological conditions IL-8 may be induced rapidly by pro-inflammatory cytokines such as TNF-a or IL-1β, where integrin αvβ6 can be similarly upregulated. The specific mechanism of IL-8 in tumor invasion and its relationship with the integrin avP6 is still not very clear. Whether there is cross talk between IL-8 and integrin αvβ6 or not needs to be further understood.In the above context, based on previous studies, the present study was to collect clinical colon cancer specimens and following up the patients, so as to investigate the roles of IL-8 and integrin αvβ6 in the invasion and metastasis of colon cancer at the clinical and cell levels, and the underlying molecular mechanisms of biological effects through in vitro study, immunohistochemistry, ELISA, plasmid transfection, qRT-PCR and RNA interference, so as to develop new understanding and potentially useful therapeutic targets for colon cancer.PartⅠ Expression of IL-8 and αvβ6 in colon cancer tissues and different colon cancer cell linesObjective To investigate the expression of IL-8 and αvβ6 in colon cancer tissues together with different colon cancer cell lines, and to study the correlation between the expression of IL-8 and αvβ6.Methods30 specimens from 2013 to 2014 were collected from Qilu Hospital of Shandong University, and all these patients were diagnosed and received radical surgery.During radical operations, colon cancer tissues and matched adjacent normal tissues were got in time. The mRNA and protein expression levels of IL-8 were detected through qRT-PCR and Western Blot. In addition, another 139 cases from 2007 to 2010 were randomly selected from Qilu Hospital of Shandong University and all these patients were diagnosed and received radical surgery. The expression of IL-8 and avP6 were detected by immunohistochemical analysis. The correlation of IL-8 and avP6 with different clinicopathological factors were further analyzed, and the relationship between IL-8 and avP6 was studied by Mann-Whitney test. Furthermore, the expression of IL-8 and avP6 were detected among different colon cancer cells with different metastatic potential, including HT-29, WiDr and Caco-2.ResultsBy detecting the expression of mRNA and protein levels of IL-8 in the 30 colon cancer tissues and matched adjacent normal tissues by qRT-PCR and Western Blot, we found the mRNA and protein levels of IL-8 in colon cancer were significantly higher than those in adjacent normal tissues. The immunohistochemical analysis of the 139 patients with primary colon cancer showed that integrin avP6 was detected in 58 cases with the positive rate of 41.7%, and the immunostaining αvβ6 was mainly located in the cell membrane and cytoplasm of tumor cells, while IL-8 was detected in 83 cases with the positive rate of 59.7%, and the immunostaining of IL-8 was mainly located in the cytoplasm of tumor cells and stromal cells. The statistical analysis of clinical follow-up data showed that the expression of IL-8 was related to pathological differentiation (p= 0.017), clinical stage M (p= 0.004) and TNM stage (p= 0.007), while the expression of integrin αvβ6 was related to clinical N stage (p= 0.041), M stage (p= 0.012) and TNM stage (p= 0.005). Mann-Whitney test showed that the expression of IL-8 was correlated with the expression of αvβ6. In vitro study demonstrated that the protein levels of IL-8 and integrin β6 were higher in HT-29 and WiDr cell lines, but lower in Caco-2 cell line. In addition, we detected the extracellular extract of these three cell lines using ELISA and we found that the content of IL-8 in HT-29 and WiDr cell lines was significantly higher than that in Caco-2.Conclusion1. The expression levels of IL-8 in colon cancer tissues were significantly higher than in adjacent normal tissues, and the expression of IL-8 was correlated with the expression of integrin avP6.2. Both IL-8 and integrin αvβ6 were highly expressed in colon cancer cells with high metastatic potential while they were lowly expressed in cells with low metastatic potential, which indirectly support the hypothesis that the expression of IL-8 was correlated with the expression of integrin αvβ6.3. The expression of IL-8 was related to pathological differentiation, clinical stage M and TNM stage, while the expression of integrin αvβ6 was related to clinical N stage, M stage and TNM stage. IL-8 and integrin αvβ6 are related to the invasion and metastasis of colon cancer.Part II Effect of IL-8 on the expression of integrin αvβ6ObjectiveTo investigate the effect of IL-8 on the expression of integrin αvβ6 and to study the potential mechanism.MethodsqRT-PCR and Western blot were used to respectively detect the effect of exogenous or endogenous IL-8 on integrin αvβ6 expression; The effect of IL-8 on ERK 1/2 phosphorylation and activation of Ets-1 was studied through Western blot, besides MEK pathway specific inhibitor U0126 or Ets-1 specific siRNA were used to explore their roles in the regulation of integrin αvβ6 induced by IL-8.ResultsIL-8 could induce the expression of mRNA and protein levels of integrin β6 subunit in a concentration-dependent manner in HT-29 and WiDr cell lines. After IL-8 was down-regulated by shRNA interference technology, the expression of (36 subunit decreased, and CXCR1 and CXCR2 antibody significantly inhibited the process. IL-8 could induce the phosphorylation of ERK 1/2 in a time-dependent manner, and MEK pathway specific inhibitor U0126 could significantly inhibit IL-8-mediated ERK 1/2 phosphorylation and upregulation of β6 subunit. IL-8 could induced t-Ets-1 expression level slightly and phospho-Ets-1 level significantly, which could be inhibited by MEK pathway specific inhibitor U0126. Ets-1 specific si-Ets-1 could significantly inhibit IL-8-induced upregulation of integrin αvβ6.Conclusion1. IL-8 could activate ERK 1/2 signaling pathway to further activate the transcriptional activity of Ets-1, thereby contributing to upregulation of integrin αvβ6.2. Both CXCR1 and CXCR2 play a part in the regulation of integrin αvβ6 by IL-8.PartⅢ Role of integrin αvβ6 in IL-8-mediated migration and invasion of colon cancer cellsObjectiveTo explore the role of integrin αvβ6 in IL-8 induced migration and invasion of colon cancer cells.MethodsWound healing assay, migration and invasion assay were used to detect the role of IL-8 in the invasion and migration of colon cancer cells. Through si-RNA and function blocking technique, transwell migration assay was used to detect the role of integrin αvβ6 in IL-8-mediated migration and invasion of colon cancer cells. MEK pathway specific inhibitor U0126 or PD98059, and Ets-1 specific siRNA were used respectively to detect the effect of ERK 1/2 and Ets-1 on IL-8-mediated tumor invasion and migration.ResultsIL-8 could induce the invasion and migration of HT-29 and WiDr cell lines in a concentration dependent manner, and the induction of IL-8 achieved maximum with the concentration of 10 ng/ml. CXCR1 and CXCR2 antibody could significantly inhibite IL-8-induced colon cancer migration. Integrin αvβ6-specific siRNA or function-blocking antibody 10D5 could significantly inhibit IL-8-induced migration of colon cancer cells. MEK pathway specific inhibitors and Ets-1 siRNA could also significantly inhibit IL-8-induced migration of colon cancer cells.Conclusion1. IL-8 could activate ERK 1/2 signaling pathway to further activate the transcriptional activity of Ets-1, so as to upregulate the expression of integrin αvβ6, thereby contributing to promoting invasion and migration of colon cancer cells.2. Both CXCR1 and CXCR2 play a part in IL-8-mediated invasion and migration of colon cancer cells. |
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