| Popis: |
The incidence of malignant melanoma is increasing worldwide and becoming resistant to most chemotherapy and immunotherapy. The arachidonic acid pathway, through COX and LaX enzymes, lead to the formation of a variety of metabolically active products, the eicosanoids, with different roles in carcinogenesis beyond their primary homeostatic functions. Importantly, these enzymes were shown to be overexpressed in melanoma. Thus the aim of this PhD thesis was to determine the effect of COX and LOX inhibitors in melanoma survival and how they may influence melanoma development as well as to unveil pharmacological opportunities when using these drugs in combination with chemotherapeutic ones. For our work we selected the mouse melanoma cell line B16F10 as a convenient cancer experimental model. They are highly resistant to chemotherapy and can be easily used both in vitro and in vivo. The direct in vitro cytotoxic effects of the drugs were assessed in two experimental models (MTT and SRB assays), as well as their influence on COX and LaX enzymes expression, cell cycle and activation of caspases. To check how they modulate melanoma development, in vitro 2-D migration studies and an in vivo lung metastasis experimental model were designed and performed. Our study reports for first time on the expression of COX-1, 5-LOX and 15-LOX-1 enzymes, which was not impaired by the treatments. COX-2 and 12-LOX expression also remained unaffected throughout our experiments. Short term incubations with the drugs failed to inhibit their activity and sometimes even stimulated them. Overall, meloxicam, M-861 and baicalein were the best anticancer drugs when given alone. PD-146176 exerted a strong effect in vitro but this was not observed in vivo. Combination treatments with chemotherapeutic drugs seem to afford better results for the COX inhibitors (meloxicam>ASA) than for the LOX inhibitors (M-861> baicalein> PD-146176>MK-886). In conclusion, our project affords for first time a comprehensive overview of the in vitro and in vivo effects of COX and LaX inhibitors in key aspects of the B16F10 mouse melanoma cell line. As per our preclinical studies and previous clinical studies COX-2 inhibitors -alone or in combination with chemotherapeutic drugs - seem to be superior to LOX inhibitors. Interestingly, the mechanisms of their anticancer effects seem not to be entirely explained by their action upon the enzymes of the arachidonate pathway. |
