The Study On The Relationship Between Cytokines And FasL-expressing Human Colon Cancer Cells' Liver Metastasis And Invasion

Colorectal cancer is one of the most common malignancies, and the prognosis of patients with liver metastasis still remains dismal. Approximately half of the patients undergoing apparently curative resection will die within 5 years because of recurrent disease, mostly with liver metastases. Liver was the only site affected in 50 % of these patients. In fact, the liver is the commonest site for the development of metastatic colorectal cancer. However, about 5-10% of the patients with colorectal hepatic metastases undergo resection with curative intention, resulting in a five-year survival of 20⁴0%¹. Most liver metastases were unresectable because of the number, size, position of tumors as well as general conditions unsuitable for liver resection.Liver metastasis is an especially serious problem as the main cause of death in the later stages. A number of events including invasion of cancer cells into the blood vessels at the primary site, engagement with the endothelial cells in the liver, escape of cancer cells into the liver parenchyma and subsequent colonization, are essential prerequisites for the development of metastatic liver tumor. Escape from immune detection and cell invasion are well-known to be essential elements for tumormetastasis. The outcome of cancer metastasis depends on multiple interactions between the malignant cell and the host environment. Such interactions can influence primary cancer growth and metastasis by altering the balance between tumor cell proliferation and death.Apoptosis or programmed cell death plays an important role in normal cell regulation and is regulated by a variety of intracellular and extracellular signals. The Fas/Fas ligand (FasL) system plays an important role in the transduction of apoptotic signal into the cells. Within several hours after the engagement of target cells Fas by effector cells FasL, target cells undergo cell death by way of the Fas-mediated apoptosis. In recent years, numerous studies have demonstrated that Fas is expressed on the surface of cells of various types, whereas FasL expression is restricted to a small number of cell types, such as lymphocytes, cells of the immune-privileged organs and many types of malignant tumor cells. Meanwhile, evidence has pointed to an abnormal increase in apoptosis among activated Fas-positive lymphocytes, mainly in the periphery of the FasL-expressing tumors. On the other hand, to a great extent, the occurrence of tumor is due to the fact that the converted cells cannot undergo a normal process of apoptosis. According to earlier literature, resistance to apoptosis through the Fas receptor pathway coupled with expression of the Fas ligand might enable many cancers to deliver a pre-emptive strike or counterattack against the immune system.The remarkably high incidence of liver metastases in patients with colorectal cancer suggests that the liver provides an environment conducive to the development of metastases. For metastatic tumor cells to become established and develop into overt disease, they must evade immune surveillance and invade and replace local tissue at the secondary site. Several mechanisms are implicated incancer invasion, including release of metalloproteinases that degrade extracellular matrix, secretion of growth factors to promote neovascularization, and changes in adhesion molecules that promote tumor cell migration. However, although the replacement of normal tissue is a prerequisite for tumor growth, the precise mechanisms by which colorectal cancer displaces or destroys resident liver cells are not known. The hepatocytes in the liver appear to be especially susceptible to Fas-mediated death, because injection of anti-Fas antibody induces massive injury to the liver and not elsewhere. Recently, it was found that Fas-ligand (FasL) expressed on the tumor cells plays a pivotal role in colonization of tumor cells in the liver. Yoong et al(2> confirmed that Fas expression is markedly up-regulated on hepatocytes at the margin of colorectal metastases and that many of the hepatocytes are undergoing apoptosis. The elucidation of the interaction of the tumor and its microenvironment has increased surgeons' understanding of the biologic mechanisms mediating metastases formation. The ability to develop effective biologic therapies will depend on understanding the complex interactions among normal cells and tumor cells and on the cytokine cross-talk among all cells within a particular tumor at a particular site.The aim of this study is to study cytokines' influences on hepatic metastasis and invasion of FasL-expressing colorectal carcinoma in vitro.Part IRole of Cytokines in Promoting FasL-expressing Human Colon Cancer Cells' Immune EscapeObjective: To investigate the potential role of FasL expressed on colon carcinoma cells and the effect of endogenous cytokines on tumor cells counterattacking T lymphocyte. Methods: By using immunohistochemical SP method and RT-PCR, the expressions of Fas receptor and Fas ligand in colon carcinoma SW620 cells and Jurkat T lymphocytes were observed so as to supply mRNA expression's and morphological evidence for the functions of Fas receptor and Fas ligand. In an effort to examine the cytotoxicity of effector cells, CytoTox 96? Non-Radioactive Cytotoxicity Assay was adopted to measure LDH releasing value after the SW620 cells were co-cultured with the Jurkat T lymphocytes. Results: In our study, we found that FasL mRNA expression was up-regulated significantly in PMA-plus-Ionomycin-stimulated SW620 cells or TNF-a-stimulated SW620 cells or interleukin-18-stimulated SW620 cells. It was shown that the Fas ligand of colon carcinoma SW620 cells was positive and the positive substances were distributed in the cell membrane and cytoplasm, and the Fas receptor of colon carcinoma SW620 cells was negative. The Fas receptor and the Fas ligand of Jurkat T lymphocytes turned out to be positive. The positive substances were distributed in the cell membrane. PHA-stimulated Jurkat T lymphocytes were co-cultured with PMA-plus-Ionomycin-stimulated (for 48h) SW620 cells or TNF-a-stimulated (for 48h) SW620 cells or interleukin-18-stimulated (for 36h) SW620 cells or SW620 cells (unstimulated) for 4h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat T lymphocytes at effector-to-target ratios of 10:1, 5:1, 2.5:1 and 1.25:1 from the experiment was 74.6%, 40.8%, 32.4%, 10.9% (F=8.19,/> O.05);54.9%, 35.3%, 22.0%, 10.3% (F=ll.12, PO.05);52.5%, 33.1%, 19.8%, 10.1% (F=9.19, P <0.05) and 14.9%, 10.5%, 6.9%, 5.8% (F=3.45, P <0.05) respectively. After theA_/*iilfiii'O/i until flio OT-J A ^p+irviiilo'f^/i Tni"L^?!i+ T 1\rrv??%l^/-\^>\rf oc* -fX*> QVithe cytotoxicity of SW620 cells to PHA-stimulated Jurkat T lymphocytes at effector-to-target ratios of 5:1, 2.5:1 and 1.25:1 from the experiment was 83.9%, 74.1% and 28.5% (F= 137.04, PO.05) respectively. Non-Radioactive Cytotoxicity Assay showed that the apoptotic rate of Jurkat T lymphocytes remarkably increased with the increase of planting concentration of S W620 cells and co-culture time after the SW620 cells were co-cultured with the Jurkat T lymphocytes. The cytotoxicity was significantly enhanced by PMA plus ionomycin or tumor necrosis factor-alpha or interleukin-18. Conclusion: The FasL expressed on human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and facilitate the escape of tumor cells from the host immune system.Part HThe Study on the Relationship between Cytokines and Fas/Fas Ligand Interaction in Human Colorectal Hepatic MetastasesObjective: To investigate the influence of mutual interactions between FasL expressed on colon carcinoma cells and endogenous cytokines on human colon cancer cells' liver metastasis and invasion. Methods: By using immunohistochemical SP method and RT-PCR, the expressions of Fas receptor and Fas ligand in colon carcinoma SW620 cells and Chang liver cells were observed so as to supply mRNA expression's and morphological evidence for the functions of Fas receptor and Fas ligand. In an effort to examine the cytotoxicity of effector cells, CytoTox 96? Non-Radioactive Cytotoxicity Assay was adopted to measure LDHreleasing value after the SW620 cells were co-cultured with the Chang liver cells. Results: In our study, we found that FasL mRNA expression was up-regulated significantly in TNF-a-stimulated SW620 cells or interleukin-18-stimulated SW620 cells. It was shown that the Fas ligand of colon carcinoma SW620 cells was positive and the positive substances were distributed in the cell membrane and cytoplasm, and the Fas receptor of colon carcinoma SW620 cells was negative. The Fas receptor of Chang liver cells turned out to be positive and the positive substances were distributed in the cell membrane, and the Fas ligand of Chang liver cells was negative. IFN-y-stimulated Chang liver cells were co-cultured with TNF-a-stimulated (for 48 h) SW620 cells or interleukin-18-stimulated (for 36 h) SW620 cells or SW620 cells (unstimulated) for 6h, the cytotoxicity of SW620 cells to IFN-Y-stimulated Chang liver cells at effector-to-target ratios of 10:1, 5:1, 2.5:1 and 1.25:1 from the experiment was 73.5%, 53.2%, 24.1%, 10.9% (F=89.73, P <0.05);68.3%, 49.8%, 21.1%, 9.7% (F= 76.87, P <0.05) and 32.7%, 21.8%, 11.1%, 6.7% (F=7.27, P <0.05) respectively. Non-Radioactive Cytotoxicity Assay showed that the apoptotic rate of Chang liver cells remarkably increased with the increase of planting concentration of SW620 cells after the SW620 cells were co-cultured with the Chang liver cells. The cytotoxicity was significantly enhanced by tumor necrosis factor-alpha or interleukin-18. Conclusion: The FasL expressed on human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and enhance liver colonization competence through induction of apoptosis in the Fas-expressing hepatocytes.