Integrin ¦Â <sub> 1 </ Sub>, The Alpha <sub> 5 </ Sub> Beta <sub> 1 </ Sub>, The Alpha <sub> V </ Sub> And Fn Planted In Ovarian Cancer Abdominal Metastasis The Role And Mechanism

(Part â… )EFFECTS OF INTEGRIN β₁, α₅β₁, α v AND FIBRONECTIN ON ANOIKIS OF OVARIAN CANCERBackground: Anoikis is a form of apoptosis triggered by disruption of cell-extracellular matrix (ECM) contacts. It is often seen in normal epithelial and endothelial cells. The significance of anoikis is that it may prevent the accidentally detached cells from reattaching to new matrices and growing dysplastically. Anoikiscould be a physiological mechanism, allowing the maintenance of tissue cell homeostasis. In contrast, most tumor cells derived from epithelial tissues have lost this characteristic, especially those malignant tumors that are easy to metastasize. The cells detached from tumor do not undergo apoptosis and can colonize elsewhere and grow. Acquisition of cellular resistance to anoikis has been demonstrated to be involved in tumor metastasis. Ovarian cancer cells are liable to detach from tumor early and then to disseminate by implanting onto the peritoneum of the abdominal cavity. Resistance to anoikis may be a key point for ovarian cancer cells' survival in ascites and spreading consequently. Therefore, it is important to elucidate the molecular mechanism of ovarian cancer cells' anoikis resistance. It will help us to understand the molecules and pathways related to this phenomenon, and then take an effective way to block the metastasis.Anoikis and resistance to anoikis is an extremely complex process, and modulated by many factors. Recently, the relationship between integrins and anoikis has interested more and more researchers. Integrins can bind to all kinds of extracellular matrix components, such as fibronectin (FN) and type I/IV collagen. It is reported that interaction between integrin and matrix could protect many cancer cells from anoikis, such as gastric, lung and prostate tumor cells. But as we know, there was few report about the effect of integrins and their ligand on anoikis resistance of ovarian carcinoma cells. So it is considered worthwhile to be explored.Objective: To observe the effects of integrin Pi, a5{3j, av and the soluble fibronectin on survival ability of ovarian cancer cells under anchorage-independent growth. The gene expression of bcl-2> bax and p53, the activity of caspase-3 was determined at the same time. Therefore to explore the role of integrin' s binding with fibronectin in the anoikis process and the associated mechanism.Method: Integrin subunits expression on the human ovarian cancer cell SKOV3-S1 (a high invasive subclone of SKOV3) surface was analyzed by indirect immunofluorescent staining. Anoikis induction model was established with polyhema to prevent cells from adhesion. With Annexin V and PI staining, apoptosis was detected by flow cytometry. The role of different integrin subunits with or without fibronectin during anoikis induction was detected by antibody blocking method. During this anoikis process bcl²> bax and p53 expression were measured by RT-PCR, and the activity of caspase-3 was measured by colorimetric assay.Results: 1. Integrin {3 i, a5p,, a v were commonly expressed in SK0V3-S1 cell by 100%, 98.2%, 100% respectively.2. The soluble fibronectin protected SKOV3-S1 cells fromanoikis by 54.4% and decreased apoptotic cells from 67.3% to 29%. Anti-integrina v blocking antibody resumed the anoikis that was inhibited by fibronectin evidently, and Anti-integrinP i blocking antibody also resumed the anoikis partly. Anti-integrina5 {3 l blocking antibody has no effect on it. Without FN, neither 0 i, a 5 {3 ,, nor av antibody altered anoikis process.3. The treatment of soluble fibronectin increased bcl-2 expression, decreased bax expression, and then increased the ratio of bcl-2/bax. There was no significant change on p53 expression. Anti-integrin a v blocking antibody inhibited this modulation.4. It showed almost no caspase-3 activities in the culture media of SKOV3-S1 cells when they grew attached. Caspase-3 was activated when cells grew in suspension and the absorbency was 0. 284 + 0. 033. The activity of caspase-3 decreased with fibronectin in the culture media and the absorbency was 0. 129 + 0. 011. An elevation of caspase-3 activity was observed when Anti-integrin a v or Pi blocking antibody was added. The absorbency was 0.238 + 0.024, 0.203 + 0.010 respectively.Conclusion: The binding of a v and soluble fibronectin protected SK0V3-S1 cells from anoikis apoptosis, via increasing the ratio of bcl-2/bax, decreasing the activity of caspase-3. This is probably one of the important reasons for ovarian cancer cells'resistance to anoikis.(Part â…¡)EFFECTS OF β₁-INTEGRIN AND FIBRONECTIN ON ADHESION AND INVASION OF OVARIAN CANCER CELLBackground: Invasive growth and distant metastasis is thought to be the most significant biological character of malignant neoplasm. Most patients died of carcinomatosis, instead of primary tumor. Therefore, it' s very important to study the mechanism of tumor metastasis. Ovarian carcinoma is the leading cause of cancer death in female genital tumors, which has a tendency of extensive metastasis and spreading within pelvic and peritoneal cavity. At the moment of diagnosis, 60-70% patients are at advanced-stage with peritoneal metastasis. The survival at 5 years is poor at about 30%. In order to improve the clinical outcome, we should elucidate the molecular mechanism of peritoneal metastasis, find the associated factors, and then improve our methods of ovarian cancer treatment.The incidence of metastasis is a complicate process in which cell adhesion , migration and invasion are closely interconnected with cell-matrix interaction and the associated transmembrane signaling. Intergins, an important family of adhesion molecule in cell surface, mediate cell-extracellular matrix adhesion. Recently, relationship between integrins and tumor metastasis has become a highlight. 3 iⁱntegrin is widely distributed on the surface ofcells. Fibronectin is the important component of ECM, and can bind with P i-integrin. They play a major role in various fundamental biological processes such as tissue support and connection, morphology maintenance. Furthermore, they can mediate cell adhesion, growth and differentiation , promote cell proliferation and migration, and also mediate signal transduction . It was reported that P i-integrin could mediate ovarian cancer cells adhere to peritoneum. But it' s tangible role in invasive process of ovarian cancer cells and the associated mechanism need to be explored.Objective: To study the role of P i-integrin and fibronectin in adhesion and invasion of ovarian cancer cells and the associated mechanism.Methods: Adhesive and invasive ability of SKOV3-S1 cell were observed with matrigel adhesion test and Boyden chamber assay. The influence of 3 i-integrin was detected by antibody blocking method. Following adhesion to fibronectin or being blocked by anti-integrin 3 i blocking antibody, mRNA expression of MMPs were assayed by reverse transcription polymerasechain reaction.Result: 1.Anti-integrin 3 i blocking antibody significantly inhibited the SKOV3-S1 cells from adhering to the basement membrane matrix by 69.8%. The OD value in MTT test reduced from 0.43 + 0.04 to 0.30 + 0.003.2. Anti-integrin3 j blocking antibody significantly inhibited the SK0V3-S1 cells from invading the basement membrane matrix by 65.7%. In the control group there were 48.4 + 8.8 cells traversed the matrigel-coated membrane in Boyden chamber. When cells were treated with anti-integrin P x blocking antibody, the number of traversed cells was 16.6 + 5.5.3. Fibronectin promoted SK0V3-S1 cells to adhere and spreadrapidly. Reversely, anti-integrinP i blocking antibody inhibited cells from adhering and spreading.4. Fibronectin induced the expression of MMP-2. Anti-integrin 3 i blocking antibody decreased the induction.Conclusion: SKOV3-S1 cells can be induced by the binding of P iⁱntegrin with fibronectin to adhere which causes a series of signal transductions in cells and express MMP-2 gene , accelerates the metastasis of tumor.(Part â…¢)EFFCTS OF FAK ANT I SENSE ODN ON THE EXPRESSION OF MMP-2 INDUCED BY FN IN OVARIAN CANCER CELLBackground: The extracellular matrix is a barrier in the process of cell invasion. To complete the metastasis procedure, the cancer cell must adhere to matrix, secrete protease to hydrolyze it, and then migrate to a new site. A lot of studies have showed that balance between tumor cell and its survival matrix is essential, where it is allowed to grow and form clone. The reorganization of connective tissue includes ECM dehydration and resynthesization. Many proteinases are involved in it. Matrix metalloproteinases (MMPs) can hydrolyze almost all of the ECM components, therefore are thought to be most important. MMP-2 can hydrolyze type IV collagen that is the main ECM component. So it plays a very important role in carcinoma invasion and metastasis. MMP-2 and MMP-9 were viewed in tumor tissue, ascites, and blood of ovarian cancer patients. Research indicated that adhering to FN induced MMP-2 expression in ovarian cancer cells, but the mechanism is still unclear. Focal adhesion kinase (FAK) is a tyrosine proteinase in cytoplasm. It takes part in the form of focal adhesion plaque (FAP). FAK is not only the key proteinase of ECM-integrin signal transduction pathway, but also a convergent point of many signal way. So it acts on many biological activities,and is also correlative with proliferation and metastasis of tumor cell. Up to now, few studies have been addressed about the function of FAK in MMPs expression of ovarian cancer cells.Hence, we set about our research from culturing ovarian cancer cell in vitro. Through experiments, the influence of FN on MMPs' activity was observed. The role of FAK in this signal pathway was studied by antisense oligodeoxynucleotides technology, therefore to explore the mechanism of FN induced MMP-2 expression.Objective: To study the mechanism of MMPs expression in ovarian cancer cells induced by FN through antisense oligodeoxynucleotides technology (ODN).Methods: SKOV3-S1 cell, the high invasive subclone of ovarian cancer SKOV3 cell, was cultured in RPMI 1640 without calf serum. FN worked as extracellular matrix, to induce MMP-2 expression, and then by gelatin zymography, to measure the activity of MMP-2 in the medium in different periods. Antisense ODN was used for blocking the focal adhesion kinase and then to observe the change of MMP-2 activity.Result: 1. No FN, no activity of MMP-2 was observed in the culture medium.2. When FN was used for 4 hours, the activity of MMP-2 appeared, and for 8 hours, the activity of MMP-2 was the highest, and then decreased gradually. The activity of MMP-9 was also seen, but it was weak.3. The activity of MMP-2 was dramatically decreased when FAK antisense ODN was put into the culture medium. The effect was great when the concentration of FAK antisense ODN reached 10-20 u mol/1.Conclusion: SKOV3-S1 cell can be induced by FN to adhere, which causes a series of signal transductions in cells, promotes theexcretion of MMP-2. Perhaps this reaction happens through Grb2/Ras/MAPKs, whose key ingredient is FAK.