Role Of Tissue Factor In Gastric Cancer And The Underlying Mechanism

[Background]Tissue factor(TF) is the physiological initiator of blood coagulation. TF is a 47KDa membrane glycoprotein, homologous to cytokine receptor. Via interaction with coagulation factor VII /VIIa, TF proceeds in a cascade of extracellular reactions, ultimately resulting in thrombin formation. Tissue factor, a class 2 cytokine receptor, is a transmembrane glycoprotein that consists of three sections: a large extracellular domain, a transmembrane segment, and a cytoplasmic tail . The extracellular domain is important for its haemostatic activity . The transmembrane portion is necessary for stabilization of the molecule and its complex in a favourable position for proteolytic action. The function of the cytoplasmic domain is not yet fully determined. In addition to the complex role in coagulation, TF acts as a signalling receptor and has severalnon-haemostatic actions. TF is involved in the pathophysiology of systemic inflammatory disorders, coagulopathies, atherosclerotic disease, tumour angiogenesis and metastasis.. Recent studies mainly focus on its association with the histologic grade of the tumor. TF is associated with the histologic grading of breast cancer and its micro vessel density, and also related to the histologic grading of small cell lung cancer. Recent study indicated that TF is involved in the tumor angiogenesis, but the underlying mechanism remains unclear. Both coagulation-independent (directly mediated in the signal transduction) and coagulation-dependent (mediated in thrombin formation) mechanisms are involved in the oncogenic blood vessel formation. It is not clear how TF functions. Besides, some of the researches indicated that TF can regulate the tumor growth and its development. Gastric cancer is the most common malignancy in China. Is TF involved in the tumor growth and development? In this research, we study the function of TF involved in the growth and development of gastric cancer, hoping to elucidate the underlying molecular mechanisms and therefore explore the potential factor for gene therapy.[Objective!Study the function of TF involved in the growth and development of gastric cancer, hoping to elucidate theunderlying molecular mechanisms and therefore explore the potential molecule for gene therapy.[Methods]1. The expression of TF and VEGF in malignant tissues of stomach and microvessel density count (MVD) in the tumor tissues were studied by immunohistochemistry.2. The expression of TF in different gastric cancer cell lines was examined by RT-PCR and Western blot.3. The quantity of VEGF in supernatant of the culture medium of different gastric cancer cell lines was studied by ELISA.4. TF highly expressed gastric cancer cell line SGC7901 was transfected with antisense vector and empty vector respectively, (aSGC7901 and nSGC7901). We attained TF extracellular subunit mutation sub cell line and TF intracellular deletion sub cell line, namely mSGC7901 and dSGC7901. Stable transfectants were obtained by G418 screening. mRNA and its corresponding protein in the transfected cells were examined.5. MTT assays were performed to determine the in vitro drug sensitivity of the transfected cells, therefore observe the function of TF mediating the tumor cell growth.6. Using FCM, cell cycle distribution of the transfected cells was studied. The proliferous indexes(PI) were calculated and the growth curves were drawn.7. Expression of VEGF^ TSP and TFPI were studied by Western blot and ELISA; Study the correlation between TF and them and the underlying mechanisms.8. Study the relative invasion index by Transwell room to observe how TF functioned on the invasion ability.9. The transfected cells were respectively implanted subcutaneously into nude mice. The size of tumors formed was measured to observe how TF mediated tumorgenesis and proliferation.10. Microvessel density count (MVD) in the tumor tissues was assessed by immunohistochemistry to observe how TF regulated angiogenesis.[Results]1. The expression of TF in gastric cancer tissues was significantly up-regulated (R0. 05) compared with that in normal gastric tissues and is parallel to the quantity of VEGF and micro vessel density (MVD). Meanwhile, TF highly-expressed gastric cancer tissues have higher metastasis rate than those which expressed low level of TF.2. Among different gastric cancer cell lines, TF was highly expressed in SGC7901 compared to other gastric cancer cell lines and ever-lasting gastric mucosa cells.3. VEGF is most presented in the supertanat of SGC7901 cell culture surum.4. mSGC790K dSGC7901 and aSGC7901 have relatively slow growth compared with their controls.5. mSGC7901 > dSGC7901 and aSGC7901 have decreased proliferatous ability compared with their controls.6. TSP and TFPI expression were upregulated in mSGC790K dSGC7901 and aSGC7901 .There seems no significant difference between the quantity of VEGF in mSGC7901 and the controls, but the quantity of VEGF in dSGC7901 and aSGC7901 supertanat decreased sharply.7. mSGC790K dSGC7901 and aSGC7901 have the decreased in vitro invation ability than the controls.8. In nude mice model, markedly inhibited tumorigenesis and slowed tumor expansion were observed in the experimental group as compared with the controls.9. Decreased microvessel density in and around tumor tissues was observed in mSGC790K dSGC7901 and aSGC7901.[conclusions ]1. TF is highly-expressed in gastric cancer tissue. Meanwhile, it may enhance the expression of VEGF and increase the micro vessel density (MVD).2. The TF expression is parallel to the VEGF expression, MVD and the clinical grading of gastric cancer. (KO. 05). TF may upregulate the transcription of VEGF. By stimulatingthe tumor angiogenesis, VEGF provides the microenvironment for tumor growth and invasion, therefore contributes to the development of gastric cancer.3. The expression of TF is highly related to the metastasis of gastric cancer (RO. 05). In our study, TF highly-expressed gastric cancer tissues have higher metastasis rate than those which expressed low level of TF. However, more work needs to be done to clarify whether TF expression can be a prognostic factor.4. TF proceeds in angiogenesis of gastric cancer by upregulating the expression of VEGF and downregulating the expression of TSP.5. In the process of TF-mediated angiogenesis, the extracellular subunit and the intracellular subunit play different roles. There is no significant difference between the quantity of VEGF in the supertanant of mSGC7901 cell culture and the control group while it significantly decreased in the dSGC7901 group (PCO. 05). This indicated that only the intracellular subunit is fundamental in the regulation of VEGF transcription. MVD in two experimental group both decreased sharply compared to the controls (R0. 05), suggesting there lie different mechanisms of the two subunit regulating the angiogenesis.