| Abstract |
The prostate grand is retroperitoneal organ that surrounds the bladder neck and the urethra. Its function is the secretion of the prostatic fluid, which is rich in electrolytes and enzymes, in order to provide a source of energy for the spermatozoa and to increase their mobility. Benign prostatic hyperplasia is the most common benign tumor among men aged above 60. The suggested mechanisms that lead to the development of the disease are the co-operativity between estrogens and androgens and the disruption of the balance between the rate of proliferation and apoptosis of the prostate cells, which is caused by hormonal and developmental factors, as well as by oncogenes. Prostate cancer is the most frequent cancer among men and the second leading cause of death behind lung cancer. Its characteristics are molecular and genetic disorders that cause rapid cellular proliferation and inhibit apoptosis. Genetic background, hormonal disorders, exposure in environmental carcinogens and sexually transmitted diseases contribute significantly to the development of the disease. The subject of this thesis was to determine the role of peptide growth factors and the genes p53, Cyclin D1 και Tpl-2/Cot in the development of both benign and malignant prostatic diseases. For this cause we collected tissue samples from 40 patients with prostate adenocarcinoma, from 40 patients with benign prostatic hyperplasia and from 10 normal donors (collected post-mortem). In the first part of the thesis we studied the expression of peptide growth factors in normal, benign and malignant prostate. Peptide growth factors are regulatory proteins that participate in cellular growth, differentiation and apoptosis. Several oncogenes are in fact growth factors or growth factor receptors. The growth factors studied were: FGF2 (induces proliferation of epithelial and stromal cells and is a powerful angiogenic factor), EGF (its role is important in embryogenesis, in cellular differentiation and in angiogenesis), TGFΒ1 (induces angiogenesis and the formation of the extracellular matrix, represses the immune system and regulates cellular differentiation), VEGF (is involved in the formation of new blood vessels during embryonic development and various other normal and malignant conditions, forming vessels with increased permeability) και IGF1 (induces the proliferation of stromal and epithelial cells). Peptide growth factors are important in normal prostatic development and differentiation, while their presence has been confirmed in prostate cell lines and tumors. Our results showed that growth factors VEGF, EGF and FGF2 are overexpressed in prostate cancer, while growth factors TGFB1 and IGF1 have reduced expression. In prostatic hyperplasia the expression of growth factors FGF2 and EGF is normal, while mRNA downregulation was observed in growth factors VEGF, TGFB1 and IGF1. Statistical analysis revealed that prostate cancer patients with high PSA blood levels downregulate FGF2 (p=0.016). Additionally, cancer patients with low Gleason score (<7) have increased EGF (p=0.035) and IGF1 (p=0.031) mRNA levels. IGF1 levels are also increased in early stage cancers (T1-T2) (p=0.030). In benign prostatic hyperplasia, elder patients have reduced EGF levels (p=0.018), while younger patients overexpress IGF1 (p-0.041). Finally, the co-expression pattern of all five studied peptide growth factors exhibits differences among normal, benign and malignant prostate. These results verify the involvement of growth factors VEGF, FGF2, TGFB1, EGF και IGF1 in the development of benign prostatic hyperplasia and prostate cancer. In the second part of the thesis we studied the expression levels of the genes p53 and Cyclin D1, and correlated their expression with important polymorphisms that these two genes carry. p53 oncosupressor gene is important in several intracellular procedures, including cell cycle arrest, DNA repair and apoptosis. Its importance is great, considering the fact that it is mutated in >50% of human malignancies. p53 has a common polymorphism in codon 72 (Pro to Arg), which alters the biochemical properties of the protein. Our results showed that p53 is overexpressed in prostate cancer, while its levels are downregulated in prostatic hyperplasia. Moreover, the Arg72Pro polymorphism is associated with the development of benign prostatic hyperplasia (p=0.042), but not of prostate cancer. However, patients with prostate cancer that carry the Arg/Arg codon 72 genotype have higher p53 mRNA levels than patients with the other two polymorphic genotypes. Cyclin D1 is a member of the G1 cyclin family, that regulate cell cycle in G1 phase through the molecular pathway of Rb. CCND1 mRNA is alternatively spliced, a procedure that is regulated by a polymorphism (G870A) in a conserved region of exon 4. Our study showed that cyclin D1 is overexpressed in prostate cancer, while it is downregulated in benign prostatic hyperplasia. G870A polymorphism is associated with the development of prostate carcinogenesis (p=0.015), and with early disease onset (p=0.049), but not with the development of prostatic hyperplasia. On the contrary, cyclin D1 expression levels are not affected by the status of the G87A polymorphism. From the above results it is obvious that both p53 and cyclin D1, and their polymorphisms, are implicated in the development of both prostatic disorders. In the second part of the thesis we examined the expression of Tpl-2/Cot kinase. Tpl-2/Cot protooncogene encodes an serine-threonine kinase that is a member of the ΜΑΡΚ family. It activates Erl1/Erk2, JNK and p38MAPK kinases in various cell lines and induces apoptosis through caspases 3 and 9. It also increases the expression of interleukin-2 and TNF-a, activation nuclear transcription factors NFAT and NFkB. Tpl-2/Cot is expressed in most normal tissues in low levels, while it is overexpressed in various malignancies. According to our results, Tpl-2 mRNA were higher in 50% of cancer samples, while its expression was deregulated in benign hyperplasias, since it was overexpressed in 18% of samples and downregulated in 37%. Statistical analysis revealed that Tpl-2 overexpression is observed in prostate cancer patients with age <70 years (p=0.019), while in benign prostatic hyperplasia patients that downregulate Tpl-2 are on average 5 years older that patients with normal Tpl-2 expression (p=0.039). From these results we conclude that Tpl-2 overexpression plays an important role in the development of prostate cancer and contributes to early disease onset. Additionally, deregulation of Tpl-2 expression in benign prostatic hyperplasia samples could be a part of a mechanism by which the prostate cell is lead to hyperplasia. The results of this thesis provide concrete evidence for the involvement of the studied molecules in the development of benign prostatic hyperplasia and prostate cancer, while they could be utilized for early disease diagnosis, correct patient monitoring and treatment.
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Μελέτη των επιπέδων έκφρασης των πεπτιδικών αυξητικών παραγόντων και των γονιδίων P53, CYCLIN D1 και TPL-2/COT στην υπερπλασία και τον καρκίνο του προστάτη
