Characterization of the ANKRD1 gene and the effect of violacein on rhabdomyosarcoma cells in vitro

[E. N. Milošević]

78 str.

Biologija - Molekularna biologija / Biology - Molecular biology

Rabdomiosarkomi (RMS) su najčešći maligni tumori mekog tkiva kod dece i adolescenata. Zbog uzrasta pacijenata i agresivnosti tumora, lečenje predstavlja veliki izazov. Da bi sе doprinelо identifikaciji novih terapeutskih targeta i terapeutika za RMS, u ovoj disertaciji je istražen ANKRD1 (eng. ankyrin repeat domain 1), označen kao potencijalni marker za diferencijalnu dijagnostiku, i antitumorski potencijal prirodnog pigmenta violaceina, koristeći tri ćelijske linije RMS. Svaka linija je eksprimirala različite količine ANKRD1 proteina, iako je nivo transkripta bio sličan. Imunocitohemijom je otkrivena jedarna i citoplazmatska lokalizacija ANKRD1 u svim linijama. Tačkasti obrazac bojenja ANKRD1 u jedrima RD i HS-729 ćelija poklapao se sa markerom Kahalovih tela. Povećanje ekspresije ANKRD1 nije bilo moguće, verovatno usled proteazomalne degradacije. Ovo ukazuje na štetne efekte povećanja ekspresije ANKRD1 na ćelije RMS, sugerišući opravdanost daljih istraživanja njegove uloge u patogenezi RMS i razmatranja kao terapetskog targeta. Violacein je pokazao citotoksični efekat na ćelije RMS, indukujući apoptozu, dok nije imao značajan uticaj na preživljavanje neizmenjenih ćelija i embriona zebrica pri koncentracijama toksičnim za tumorske ćelije. Uticao je i na migratorni potencijal ćelija RMS. Tokom tretmana se zadržavao na površini ćelija, a njegovo dejstvo nije zavisilo od oksidativnog stresa. Dodavanje violaceina je povećalo toksičnost doksorubicina prema ćelijama RMS. Ovo istraživanje doprinosi boljem razumevanju funkcije ANKRD1 u RMS i ukazuje na potencijal violaceina kao dodatne terapijske opcije za lečenje ovog tipa tumora.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Due to the age of patients and the tumor aggressiveness, treatment presents a significant challenge. To contribute to the identification of new therapeutic targets and treatment options for RMS, we investigated ANKRD1 (ankyrin repeat domain 1), marked as a potential differential diagnostic marker, and the antitumor potential of natural pigment violacein, using three RMS cell lines. Each line expressed a different amount of ANKRD1 protein, although the transcript levels were similar. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined lines. The punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with the marker of Cajal bodies. Overexpression of ANKRD1 was not achieved in the cells, likely due to proteasomal degradation. The unsuccessful overexpression of ANKRD1 indicates its detrimental effects on RMS cells, suggesting the need to investigate its role in RMS pathogenesis and potential as a therapeutic target. Violacein demonstrated a cytotoxic effect on RMS cells, inducing apoptosis, while it did not significantly affect the survival of non-malignant cells and zebrafish embryos at concentrations toxic to tumor cells. Violacein also affected the migratory potential of RMS cells. It did not penetrate the cells but remained on their surface, and its action was independent of oxidative stress. The addition of violacein increased the toxicity of doxorubicin to RMS cells. This study contributes to a better understanding of the ANKRD1 function in RMS and highlights the potential of violacein as an additional therapeutic agent for this tumor.

ANKRD1, rabdomiosarkom, violacein, Kahalova tela, proteazomalna degradacija

ANKRD1, rhabdomyosarcoma, violacein, Cajal bodies, proteasomal degradation

577.2:616‑006.36(043.3)

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