Characterization and effect of porphyrins on melanoma cells

Melanom je zloćudni tumor kože koji nastaje prilikom maligne transformacije melanocita, a zbog agresivnosti te metastatske prirode predstavlja najsmrtonosniji oblik raka kože. Konvencionalne strategije u liječenju raka poput radioterapije, kemoterapije i biološke terapije često uzrokuju nuspojave te ne mogu nadvladati problem rezistencije tumora i hipoksičnog tumorskog mikrookoliša. Jedno od inovativnih rješenja predstavlja oblik minimalno invazivne i kliničke odobrene fototerapije, fotodinamička terapija. Fotodinamička terapija temelji se na interakciji svjetlosti, fotosenzibilizatora i kisika pri čemu dolazi do fotokemijske reakcije i generiranja reaktivnih kisikovih vrsta koje zatim uzrokuju smrt tumorskih stanica, oštećenje tumorskog krvožilja i potiču imunološki odgovor. Svrha ovog rada bila je ispitati djelotvornost piridilporfirina konjugiranih lipofilnim lancima različitih duljina pri čemu su dva spoja N-oksidirana, a dva N-metilirana. Mjerenjem intenziteta fluorescencije ispitana je količina spojeva koji ulaze u stanice u različitim vremenskim intervalima, fluorescentnom mikroskopijom praćena je lokalizacija porfirina u stanicama te je pomoću MTT testa određena citotoksičnost porfirina u uvjetima normoksije i hipoksije. Rezultati ispitivanja pokazali su kako se spojevi u najvećoj količini nalaze u stanicama nakon 6 h inkubacije te da u većoj količini ulaze u stanice fibroblasta (HDF). Također, pokazano je kako porfirini u većoj količini ulaze u stanice pri 37 ℃ što bi moglo ukazati na ulazak porfirina aktivnim transportom u stanice. Rezultati MTT testa ukazuju na potencijalnu selektivnost porfirina za stanice melanoma (MEWO) u koncentracijama 0.5 i 1 μΜ te pokazuju kako porfirini nisu citotoksični u mraku. Potvrđeni su i rezultati prethodnih ispitivanja gdje je pokazano kako veća lipofilnost spojeva utječe na bolji ulazak u stanicu te da N-oksidirani spojevi potencijalno bolje djeluju u uvjetima hipoksije.Melanoma is a malignant skin tumor that arises during the malignant transformation of melanocytes, and due to its aggressiveness and metastatic nature, it is the deadliest form of skin cancer. Conventional strategies in cancer treatment such as radiotherapy, chemotherapy, and biological therapy often cause side effects and are not able to overcome the problem of tumor resistance and hypoxia in the tumor microenvironment. One of the solutions is photodynamic therapy (PDT), a form of minimally invasive and clinically approved phototherapy. PDT is based on the interaction of light, photosensitizer, and oxygen, whereby a photochemical reaction occurs and the generation of reactive oxygen species is generated, which then causes the death of tumor cells, damages tumor blood vessels, and stimulates the immune response. The aim of this work was to test the effectiveness of porphyrins conjugated with lipophilic chains of different lengths, where two have N-oxidized and two N-methylated pyridyl groups on meso position. The cell uptake of porphyrins at different time points was determined by measuring the fluorescence intensity, fluorescence microscopy was used to examine the localization of porphyrins inside melanoma cell line, and the cytotoxicity of porphyrins was determined under normoxia and hypoxia using the MTT proliferation test. The results showed that the largest amount of the porphyrins was found in the cells after 6 h of incubation, and in larger amount in fibroblasts (HDF) in comparison to melanoma cell line (MeWO). Also, it was shown that porphyrins enter the cells in larger quantities at 37 ℃, which could indicate the entry of porphyrins by active transport into the cells. The results of the MTT test showed the potential selectivity of porphyrins for melanoma cells (MEWO) and show that porphyrins do not show dark toxicity in concentration up to 10 μM. The results of previous tests were also confirmed, showing that the higher lipophilicity of the compounds results in better cellular uptake and that N-oxidized compounds potentially work better in hypoxi