Nf-¦Êb In Colorectal Cancer Metastasis Related Gene Regulation And Significance

Colorectal cancer remains one of the most common cancer types and its incidence has gradually increased in recent years in China. The clinical outcome for colorectal cancer patients remains poor, and the important reason for death is invasion and metastasis of the cancer. The role of NF-κB in malignancies has been explored in relation to regulation of apoptosis, tumor progression, and responses to chemotherapy and ionizing radiation. But its role specifically in the process of metastasis, especially in the colorectal cancer, has not been examined in detail.Tumor metastasis is a complex series process, including three key steps, tumor cell deviation,transfer and development. Among these,the most important step is tumor invasion and new vessels formation. Blockage any of these steps,the tumor metastasis could be inhibited. In these years, nuclear transcription factor kappa B, (NF-κB) was proved to play critical role in the control of tumor metastasis.The progression of colorectal cancer relies mostly on angiogenesis. The tumor grows very slowly without blood vessels but it grows very fast with new vessels formation for enough oxygen and nutrition are supplied. As a factor of angiogenesis , NF-κB partakes of the invasion and metastasis of tumor. Moderate expression of cell adhesion molecules is vital to life, but they turn out to be an important factor in tumor invasion and metastasis in a state of disorder. The functional disorder or absence of E-cadherin , as one of the cell adhesion molecules, is one of the key factors in tumor invasion and metastasis.It has been reported recently that many genes concerning with cell adhension and angiogenesis are regulated by NF-κB, which plays an important role in invasion and metastasis of tumor by regulating the expression of these genes.It has been reported that NF-κB modulates the expression of E-cadherin gene. And the similar results, which NF-κB modulates the expression of IL-8 gene, has been also reported . But there is little reports about the relationship among the IL-8, E-cadherin and the activation of NF-κB in colorectal cancer.We speculate that two important genes, IL-8 and E-cadherin, which partake of the invasion and metastasis of tumor, are modulated by NF-κB.We undertook the present study to determine whether NF-κB is constitutively activated in colorectal carcinoma tissues, at the same time, to evaluate thecorrelation between NF-kB activity and clinicopathological features, and to examine relativity between IL-8 and E-cadherin genes expression and NF-k B activation in colorectal carcinoma.Stable transfection of a mutant IkBo lacking Ser32 and Ser36 that was insensitive to IKK-mediated phosphorylation and degradation resulted in inhibition of NF- k B activity. After stable transfection of colorectal cancer cell line with pCDNA-MIkBa plasmid, the regulation of gene expression of NF-k B downstream was studied. And the tumor cell invasion activity was studied at the same time with the Boyden chamber. In the paper, the study was separated two parts.Part One. The correlation between NF- k B activity and the expression of IL-8, E-cadherin in colorectal carcinoma.Aim: The purpose of this study was to determine whether NF-k. B is constitutively activated in 46 cases of human colorectal carcinoma tissue and, if so, to determine whether there is corelation between NF-k B activity and the IL-8 and E-cadherin gene expression, to determine the relationship between gene expression and clinicopathological features of colorectal carcinoma.Methods: NF-k B activation was determined by immunohistochemical analysis of formalin-fixed, paraffin-embedded specimens, Dig-EMSA from fresh tissue specimens and fluorescence quantitative-PCR for mRNA.Rusults: Nuclear translocation of RelA was significantly higher in tumor cells in comparison to normal epithelial cells (P<0. 05);NF-k B activation was correlated with tumor invasion related clinicopathological features such as lymphatic invasion, the invasion of vessels, pathological stage and metastasis. (P<0. 05)The IL-8 mRNA was highly expressed in colorectal cancer compared with normal (P<0. 05);IL-8 mRNA expression was correlated with clinicopathological features such as invasion of vessels, lymphatic invasion, pathological stage, tumor differentiation and metastasis. (P<0. 05)The E-cadherin mRNA was highly expressed in normal compared with colorectal cancer (P<0. 05);E-cadherin mRNA expression was correlated with clinicopathological features such as the invasion of vessels, lymph nodes , tumor size, tumor stage and metastasis (P<0. 05).There was a significant correlation between NF- k B activation (nuclear translocation of RelA) and expression of IL-8 gene and E-cadherin gene (P<0. 05),Conclusion: collectively, these findings suggest that NF-kB is constitutively activated in human colorectal carcinoma tissues;NF-kB activity is correlated with gene expression of the IL-8 and E-cadherin, and NF-kB with invasion-related genes expression is correlated with clinicopathological features of colorectal carcinoma. Part Two. The relationship between the tumor-related metastasis gene expression and NF-kB activity in colorectal cell lineAim: To study the effects of restrained NF-kB activation on the tumor-related metastasis gene expression and the invasion activity of colorectal cell line.Methods: The plasmid transfection, fluorescence quantitative PCR for mRNA, and immunohistochemical analysis were used in this study.Results: Stable transfection of colorectal cancer cell line DLD-1, with recombinant pCDNA 3. 1 (-)-MIkBo resulted in the IL-8 gene expression of colorectal cancer cell line decreased greatly, but E-cadherin increased markedly and the invasion activity was inhibited significantly.Conclusion: Stable transfection of a mutant I KBa lacking Ser32 and Ser36 into colorectal cancer cell line could inhibit the NF-kB activation greatly. And through inhibition of the activity, the gene expressions related to colorectal cancer cell metastasis was regulated greatly, and also the invasion activity of colorectal cancer cell was decreased markedly. These suggest that NF-k B might play a critical role in the regulation of tumor metastasis through controlling the related gene expression.Conclusion:a. NF- k B is constitutively activated in human colorectal carcinoma tissues and plays a central and specific role in the regulation of tumor metastasis, and it suggests that NF-kB activity is related to tumor progression due to its transcriptional regulation of invasion-related factors such as IL-8 and E-cadherin gene expression.b. NF-kB and invasion-related gene expression was correlated with clinicopathological features of colorectal cancer.c. Stable transfection of a mutant I k B a lacking Ser32 and Ser36 into colorectal cancer cell line could inhibit the NF-kB activation greatly.d. Through inhibition of the activity, the gene expression related to colorectal cancer cell metastasis was regulated greatly.e. Through inhibition of the activity, the invasion activity of colorectal cancer cell was decreased markedly.f. NF- k B might play a critical role in the regulation of colorectal tumor metastasis and invasion, and the inhibition of the activity of NF- k B might be as a therapeutic method.