Uticaj etil estra cikloheksil analoga etilendiamin dipropanske kiseline na ćelije melanoma miša in vitro i in vivo

Abstract

Melanom je visoko agresivni maligni tumor kože. Incidenca melanoma se značajno povećala u poslednjih 30 godina, stope morataliteta su visoke, a i pored napretka u hemio-/imuno-/ i radioterapiji odgovor pacijenata sa uznapredovalim stadijumom bolesti je svega 15-25%. Veliki napori se ulažu u otkrivanje, sintezu, karakterizaciju i ispitivanje mehanizma delovanja novih jedinjenja - potencijalnih antitumorskih agenasa. Među njima se izdvajaju derivati etilendiamin dipropanske kiseline, posebno etil estar cikloheksil analog etilendiamin dipropanske kiseline (EE), čiji antimelanomski efekat do sada nije detaljnije objašnjen. Cilj ovog rada je bio da se ispita citotoksični potencijal EE na ćelijskom i animalnom modelu melanoma kao i molekularni mehanizam dejstva koji EE pokreće. U eksperimentima su korišćene ćelijske linije mišjeg melanoma (B16), mišjih makrofaga (RAW264.7), humanog melanoma (518A2), humanih plućnih fibroblasta (MRC-5) i humanih keratinocita (HaCaT). Vijabilitet ćelija je ispitan testom aktivnosti kisele fosfataze, sulforodamin B testom, MTT testom i bojenjem tripan plavim, dok je integritet ćelijske membrane utvrđen testom oslobađanja laktat dehidrogenaze. Molekularni mehanizam dejstva EE na melanom miša je ispitan utvrđivanjem produkcije slobodnih kiseoničnih radikala, depolarizacije mitohondrija, aktivacije kaspaza, eksternalizacije fosfatidil serina, fragmentacije DNK i prisustva kiselih vezikula u ćelijama, korišćenjem odgovarajućih fluorohroma i analizom na protočnom citofluorimetru. Nivoi proteina značajnih za apoptozu i autofagiju su mereni imunoblot metodom. Morfološke karakteristike ćelija su ispitane fluorescentnom i transmisionom elektronskom mikroskopijom. U in vivo eksperimentima miševima soja C57Bl/6 je indukovan primarni i metastatski melanom injektiranjem B16 ćelija supkutano, odnosno intravenski. Sistemska toksičnost EE je ispitana praćenjem funkcije kostne srži, jetre i bubrega. Ispitan je efekat EE na progresiju supkutanog melanoma u pogledu veličine, a zatim su tumori izolovani i u tumorskom tkivu analizirana ekspresija gena i količina proteina od značaja za apoptozu i autofagiju metodom kvantitativne reakcije lančanog umnožavanja sa reverznom transkripcijom, odnosno metodom imunoblota...

Melanoma is a highly aggressive skin malignant tumor. The incidence of melanoma is increasing for the last 30 years, mortality is high, and despite all the improvements in chemo-/immuno- and radiotherapy the response of patients with advanced disease is as low as 15-25 %. Great efforts are put into the discovering, synthesis, characterization and investigation of molecular mechanisms of action of these compounds - potential antitumor agents. Promising new potential cytotoxic agents are derivatives of ethylene diamine dipropanoic acid, specifically ethyl ester cyclohexyl analog of ethylene diamine dipropanoic acid (EE). Its antimelanoma effect was not previously investigated and thus the aim of this research was to assess cytotoxic potential and molecular mechanism of EE's action using cell and animal melanoma models. Mouse melanoma (B16), mouse macrophages (RAW264.7), human melanoma (518A2), human pulmonary fibroblast (MRC-5) and human keratinocyte (HaCaT) cell lines were used in the experiments. Cell viability was assessed using acid phosphatase, sulphorhodamin B, MTT and trypan blue assays, whereas lactate dehydrogenase release assay was used for measuring cell membrane integrity. Molecular mechanism of action was investigated by measuring the production of reactive oxygen species, mitochondrial membrane depolarization, caspase activation, phosphatidyl serine externalization, DNA fragmentation, and number of acidic vesicles, using appropriate fluorochromes followed by flow cytometry analysis. Levels of apoptosis- and autophagy- related proteins were measured using immunoblot. Cell morphology was investigated using fluorescent and transmission electron microscopy. C57Bl/6 mice were used for in vivo experiments. Primary and metastatic melanoma were established by injecting B16 cells subcutaneously and into the tail vein of the mice. The bone marrow, liver, and kidney function were assessed for systemic toxicity of EE. The effect of EE on growth of subcutaneous melanoma was observed, tumors isolated, and apoptosis- and autophagy- related gene and protein expression in the tumor tissue was assessed using reverse transcriptase quantitative polymerase chain reaction and immunoblot, respectively...