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Title: Evaluation of the influence of transcription factors PPAR ligands on melanoma cells

Abstract:

Peroxisome proliferator-activated receptors (PPAR) belong to a superfamily of nuclear transcription factors involved in the regulation of gene expression by direct interaction with DNA. Three types of PPAR proteins are currently known: PPARa, PPARδ/β and PPARγ. In spite of great similarity of their protein structure, subtle differences in amino acid sequence cause alterations in affinity of ligands for specific receptor types, and even selective binding of some of them the ligands only to one type of receptor. Among synthetic ligands of nuclear receptors, fibrates are strong agonists of PPAR. They are therapeutics that lower the concentration of plasma triglycerides concentration and correct lipoprotein level. Fenofibrate is a member of this group of compounds. Selective ligands of PPARδ/β include: L-165041, GW-501516 and carbacycline. Synthetic ligands of PPARγ are medications able to increase tissue sensitivity to insulin, belonging to thiazolidinedione group (glitazones) e.g. ciglitazone. They lower plasma glucose level in humans, and also reduce the amount of circulating free fatty acids. In recent years many interesting reports on anticancer potential of PPAR ligands were published. Melanoma still remains one of the cancers lacking due characterization in terms of molecular features. The reports published so far indicate that changes in the level and activity of transcription factors: MITF, NFkB, AP-1, Notch, CREB, Ets-1, β-katenine/LEF/TCF, STAT, RAR and RXR affect melanoma progression. These transcription factors influence cell proliferative abilities, invasivnes and metastatic potential. Retinoid receptors RAR and RXR are co-receptors of PPAR transcription factors. Unlike retinoid receptors, that are relatively well characterized in the skin, melanocytes and melanoma, the role of PPAR in melanoma progression is much less known. What is more, the published data are often contradictory. The aim of this study was to determine whether PPAR ligands influence proliferation, apoptosis and expression of selected proteins that are significant for malignant melanoma progression, migration and angiogenesis. In order to verify the working hypothesis of anticancer activity of PPAR, mRNA and protein expression for three receptors: PPAR α, β/δ, γ in four melanoma cell lines derived from different stages of its development was examined. The effect of different ligands on transcriptional activity of PPAR and the ability to bind active factors to peroxisome proliferator response element, PPRE was also analyzed. The influence of synthetic ligands of individual PPAR on melanoma cells proliferation, and expression of cell cycle regulatory proteins, such as p21, cyclin D1, c-Myc, NFkB was determined. In addition, the effect of PPAR ligands on the secretion of proteins significant for cancer growth (VEGF, MMP-2 and 9, MIA) and the potential of PPAR ligands for induction of programmed cell death, i.e. apoptosis, was investigated. The study was conducted on four cell lines derived from different stages of melanoma development: WM35 – from primary site, horizontal growth phase (RGP), WM9 – from metastatic site in lymph nodes, WM239A – from metastatic site in the skin, and A375P – from malignant solid tumor in lungs. The study employed commonly accepted analytical techniques, including classical biochemical methods (spectrophotometry, spectrofluorymetry, centrifugation, in native and denaturing condition electrophoresis of proteins, electrophoresis of DNA zymography, Western blot, chromatography), cell biology techniques (monolayer cell cultures, staining techniques, microscopic analysis) and molecular biology tools (DNA and RNA isolation, RT-PCR, electrophoresis of PCR products, lipotransfection of cells using specific vectors, transcriptional activity testing, gel shift assay). It was shown that melanoma cells expressed all three forms of active PPARs. ; PPAR ligands at concentrations used in these experiments (ciglitazon - 5, 10, 15 μM, fenofibrat - 5, 10, 15 μM, karbacyklin - 0.1, 1.0, 5.0 μM, linoleic acid - 15, 20, 30 μM and 9-cis-retinoic acid - 0.1, 0.3, 1.0) decreased proliferation of melanoma cells, however, these changes were in long time reversible in our experimental setup. None of PPARs ligands under investigation induced apoptosis in studied cell lines. The results of the present work suggest that PPARα may play an important part in the control of melanoma growth. This was evidenced by the change in cell proliferation rate and in the secretion of MMPs, VEGF and, importantly, MIA and NFκB as a result of activation of this receptor by a specific ligand. The proteins MIA and NFκB are prognostic markers of melanoma progression in vivo. PPARγ, despite its influence on melanoma cell proliferation, did not prove to be a universal „controller” of the mechanisms significant for neoplastic growth. It was also demonstrated that PPARβ/δ, in the cell lines examined in this study, played a role of a “proliferation controller”. Since the study showed that the increased expression of PPARδ/β protein was correlated with the elevated malignancy of the cell line, it can be hypothesized that the receptor and its ligand carbacycline decreased growth potential of melanoma cells. The obtained results indicate an important function of PPARs in the control of proliferation both directly by influencing certain cell cycle proteins and via antagonizing NFκB action. They are also a good point for continuation of in vivo studies on the role of PPAR in biology of the melanoma.

Level of degree:

2 - studia doktoranckie

Degree discipline:

onkologia ; biologia molekularna

Degree grantor:

Wydział Lekarski

Promoter:

Piotr Laidler

Date issued:

2009

Format:

application/pdf

Identifier:

oai:dl.cm-uj.krakow.pl:933

Call number:

ZB-110918

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tylko w bibliotece

Location of original object:

Biblioteka Medyczna Uniwersytetu Jagiellońskiego - Collegium Medicum

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Last modified:

Sep 16, 2019

In our library since:

Nov 21, 2012

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http://dl.cm-uj.krakow.pl:8080/publication/933

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ZB-110918 Sep 16, 2019
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