|M.Sc Student||Mendelsohn Sigal|
|Subject||Involvement of Leptin in Apoptosis of Human Prostate Cancer|
|Department||Department of Medicine||Supervisor||Assistant Professor Ronnie Barkey|
|Full Thesis text - in Hebrew|
Prostate cancer (PCa) progression is known to depend on various hormones and growth factors, but their role and underlying molecular mechanisms remain poorly understood. One of the factors found in various study models to have an effect on growth and proliferation, cell migration and apoptosis, is the adipocyte hormone leptin. The aim of this research was to characterize the effects and mechanism of action of leptin in PCa cell apoptosis.
In view of the complexity of the mechanisms of apoptotic cell death, it was important to determine the effects of leptin on various parameters of serum starvation-induced apoptosis. These were determined in human androgen-dependent LNCaP and PC3/AR PCa cell line models and androgen-independent PC3 and DU145 cell lines. In all four cell lines, leptin enhanced the levels of the apoptotic protein markers caspase 3 and cleaved poly (ADP-ribose) polymerase89 (cPARP89). These stimulatory effects of leptin on caspase 3 and cPARP89 were shown to be time- and dose-dependent in LNCaP cells: the maximal effects were observed with as little as 0.1-1 ng/ml leptin (and essentially maintained at 10 ng/ml) and with an exposure of 6-24 h. Leptin also increased the cleavage of cytokeratin 18 protein, which is an early apoptotic event caused by caspases 9, 3 and 7 and caused DNA fragmentation in all cell lines (measured by Hoechst 33342 DNA staining).
Various kinase inhibitors were used to gain insight into the signaling mechanisms involved in the effects of leptin on apoptosis. In LNCaP cells, inhibition of the Janus kinase 2 (JAK2), p38/c-Jun N-terminal kinase (p38/JNK) or protein kinase C pathways each caused inhibition of the leptin-induced apoptotic proteins. Moreover, leptin increased phosphorylation of JAK2, Akt/protein kinase B and mitogen activated protein kinase (MAPK).
The specificity of leptin effects mediated via its receptor LRb, were investigated using a human leptin mutein (L39A/D40A/F41A), known to act in other systems as a true leptin antagonist. Leptin mutein inhibited the exogenous leptin-induced LNCaP and PC3 cell responses on apoptotic proteins, as well as LNCaP cell signaling. Interestingly, when added without exogenous leptin, the leptin mutein increased the basal apoptotic signals of LNCaP and PC3/AR cells.
The possible involvement of androgen receptors (AR) in leptin-induced apoptosis, was investigated and leptin shown to increase AR protein levels in LNCaP and PC3/AR cells.
In conclusion, the results presented in this research indicate that leptin is clearly involved in activation of apoptosis in human PCa cell lines.