Efekat ekskretorno-sekretornog antigena mišićnih larvi Trichinella spiralis i antihelmintika mebendazola na ćelije melanoma u uslovima in vitro i in vivo

Abstract

Melanom je vrlo agresivna forma tumora kože, koja je u većini sluĉajeva rezistentna na konvencionalnu hemioterapiju. Zbog toga melanom predstavlja veliki izazov za istraživanje novih terapijskih pristupa. Rezultati, dobijeni na animalnim modelima, ukazuju da helminti ili njihovi ekskretorno-sekretorni (ES) produkti mogu da modulišu imuni sistem domaćina, i tako dovedu do smanjivanja intenziteta ili spreĉavanja razvoja autoimunih, alergijskih i malignih bolesti. Sa druge strane, lekovi koji su odobreni za razliĉite indikacije ali ne i za terapiju kancera, a koji poseduju prethodno neprepoznat citotoksiĉni efekat prema malignim ćelijama, mogli bi se brzo naći u primeni i za ovu novu indikaciju. Rezultati dobijeni u in vitro i in vivo uslovima pokazuju da bi jedan od takvih lekova mogao biti mebendazol. Mebendazol je antihelmintik, pripada grupi benzimidazola i široko se koristi u veterinarskoj i humanoj medicini. U ovom radu posebnu pažnju usmerili smo na analizu dejstva ES produkata mišićnih, infektivnih larvi (L1) Trichinella spiralis i mebendazola na melanomske ćelije I) In vitro ispitivanja: a) Ispitivali smo efekat ES L1 antigena i mebendazola na preživljavanje melanomskih ćelija. Humane (Fem-X) i mišje (B16) melanomske ćelije gajene su u jednom sloju i posle 20h tretirane sa po pet rastućih koncentracija ispitivanih supstanci (ES L1 antigen: 12.5–200 μg/ml, i mebendazol: 0.007-2.5μM) u kompletnom hranljivom medijumu. Kontrola se sastojala od ćelija gajenih u samom medijumu. Preživljavanje ćelija je odreĊivano korišćenjem metode redukcije dimetiltiazol difenil tetrazolijum bromida (MTT test) 72 sata nakon dodavanja antigena. Dobijeni rezultati ukazuju da ES L1 sntigen T. spiralis na statistiĉki znaĉajan naĉin ispoljava blago smanjenje preživljavanja kod obe melanomske linije. Mebendazol je, takoĊe, doveo do smanjenja preživljavanja kod melanomskih ćelija. Kod obe ispitivane supstance ustanovljeno je da je jaĉi efekat prema humanim melanomskim ćelijama nego prema mišjim, ali to nije bilo statistiĉki znaĉajno...

Melanoma is a very aggressive form of skin cancer. It is in most cases resistant to conventional chemotherapy. Because of that it represents a great challenge for investigation of new therapeutic approaches. It is well known that co infection with different pathogens, including helminths and in particular Trichinella spiralis (T. spiralis), can ameliorate (alter) the progression of different diseases (autoimmune, allergic) including malignant one. Drugs approved for indications other than cancer that possess previously unrecognized cytotoxicity toward malignant cells could be rapidly used for this new indication. Data suggest that one of drugs with such indication could be mebendazole. Benzimidazole anthelmintics including mebendazole are widely used in human and veterinary medicine. We studied the in vivo and in vitro effect of: a) T. spiralis excretory-secretory muscle larvae (ES L1) antigen and b) mebendazole on the melanoma cells. I) In vitro studies: a) We investigated the effect of ES L1 antigen and mebendazole on the survival of melanoma cells. Human melanoma (Fem-x), and mouse melanoma (B16) tumor cells were cultured as a monolayer and after 20h treated with five different concentrations of investigated compounds (ES L1 antigen: 12.5–200 μg/ml, and mebendazole: 0.007-2.5μM) in complete nutrient medium. Control consisted of cell cultivated in medium alone. Cell survival was determined 72 h later by using reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT test). Obtained results indicated that T. spiralis ES L1 antigen, in a statistically significant manner, exerted a mild survival inhibition of target tumor cell lines. Mebendazole exerted strong inhibition of the survival. With both compounds stronger effect was determined toward human melanoma tumor cells compared with the effect on mouse melanoma cells. b) Also, we investigated the effect of ES L1 antigen (50-200 μg/ml) and mebendazole (1.25-5μM) on the apoptosis of mouse B16 melanoma cells. Apoptosis was measured after cultivation of the cells for 72h with ES L1 antigen, and for 24h with mebendazole...