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Identifier

000417888

Title

Ο ρόλος των αγγειογενών και αγγειοστατικών C-X-C χημειοκινών στην ανάπτυξη του καρκίνου του παχέος εντέρου

Alternative Title

The role of angiogenic and angiostatic C-X-C chemiokines in the development of colon cancer

Author

Εμμανουήλ, Γιώργος

Thesis advisor

Κουρούμαλης, Ηλίας

Reviewer

Θερμού, Κυριακή
Κουτρουμπάκης, Ιωάννης
Νότας, Γεώργιος
Μουζάς, Ιωάννης
Τζαρδή, Μαρία
Κοφτερίδης, Διαμαντής
Ρωμανός, Ιωάννης

Abstract

Colorectal cancer is the second leading etiology of cancer death in Western countries as almost half of the patients die of metastatic disease after curative surgery despite adjunct chemotherapy. In the development and the metastasis of the tumor CXC chemokines are possibly the proteins with the most devastating influence upon these procedures. Following this statement the main goal of our study was to quantify the amount of 3 angiodrastic of these chemokines and one pro-angiogenic cytokine produced by cancer tissue compared to normal mucosa from the same patient. In addition we studied the same chemokine production by two well characterized colon adenocarcinoma cell lines. The CXC chemokines we chose to study belong to the CXC family and two of them, CXCL8 and CXCL6, bear the ELR aminocid motif, the presence of which is considered to confer pro-tumoral and angiogenic properties. The third CXC chemokine, CXCL4, without this ELR motif is believed to be angiostatic, although its role in the development of the tumor appears quite controversial in many studies. Methods The measurement of the quantity of these 4 angiodrastic cytokines was done in two separate tissue samples from cancer and normal tissue obtained from the same patient. Results were expressed as pg chemokine / ng tissue protein. In addition to these angiodrastic cytokines several other clinicopathological factors (Ki67, p53, p21, bcl2, EGFR and MLH1) were semi-quantified and the possible association of these parameters with chemokine levels was investigated. Moreover the colon cancer cell lines, HT-29 and Caco2 were cultured for 24 hours in the presence of human serum, either from cancer patients or from healthy individuals. The effect of addition to the incubation medium Fetal Bovine Serum (FBS) was also investigated. Tissue chemokines were detected by Western-Blot Analysis and quantified by ELISA. The clinicopathological indeces were detected and semi-quantified Immunohistochemically. The results were statistically analyzed by the Wilcoxon test. Culture supernatants from five different culture experiments were studied. Cells were incubated with serum (human from five cancer patients and five healthy individuals and FBS. As controls incubations with only culture medium (McCoys for HT-29 and MEM for Caco2) were run so as their basal chemokine production could be identified. All cultures were run on six-well culture plates for 24 hours and the time points at which we measured the chemokine production were : 2, 4, 6, 12 and 24 hours. The quantification of chemokines in the supernatants was done by ELISA and statistical analysis was done using the one-way ANOVA and General Linear Model for repeated measures tests. Results There was a significant increase of CXCL6 (p=0.005) and VEGF (p=0.003) in cancerous tissue compared to normal. Patients with lower levels of CXCL4 and CXCL8 appeared to live significantly longer. Moreover patients with loss of EGFR expression showed higher levels of CXCL8 while p21 loss was associated with increased levels of CXCL6. Chemokine levels were not correlated with TNM or Duke’s classification. A strong p53 expression was accompanied by decreased survival.In the culture studies of HT-29 and Caco2 cell lines we demonstrated that colonic epithelial cells are potent producers of angiodrastic chemokines.HT29 and Caco2 cells produce all four chemokines under basal conditions and over 24 hours after incubation with human serum or FBS. However the production is not uniform for all chemokines .The secretion response of these two cell lines is completely different. HT29 produce more IL-8 and VEGF irrespective of serum incubation while Caco2 cells are relatively unresponsive. The opposite is true for CXCL6 and CXCL4. Moreover HT29 cells produce more IL-8 and VEGF over 24 hours when incubated with cancer serum. On the other hand Caco2 cells produce more CXCL4 when incubated with cancer serum. Conclusions 1) The angiogenic factors CXCL6 and VEGF are increased in colorectal cancer tissue with no association with the clinical stage of the disease or survival. 2) However increased levels of tissue CXCL8 and CXCL4 are associated with poor survival. 3) Strong expression of p53 is found in patients with poor survival. 4) Culture studies imply that cancer cells are producers of all four cytokines studied but the response of the two cell lines is different. CXCL8 and VEGF seem to be similarly regulated in a different way than CXCL4 and CXCL6. 5) Inclusion of Fetal Bovine Serum in the incubation medium strongly influences the results.

Language

Greek

Subject

Caco-2

Cell lines

HT-29

Human colon cancer

Χημειοκίνες

Issue date

2018-07-18

Collection

School/Department--School of Medicine--Department of Medicine--Doctoral theses