Molekulski mehanizmi delovanja antitumorskog agensa iz grupe sintetskih tubulizina, tubugi 1, na odabranim model sistemima melanoma

Abstract

Tubulizini su sekundarni metaboliti miksobakterija koji svoju antitumorsku aktivnost ostvaruju narušavanjem organizacije deobnog vretena. U ovoj studiji ispitivan je antitumorski potencijal sintetskog analoga tubulizina, tubugi 1, in vitro i in vivo na modelu mišjeg i humanog melanoma. Tubugi 1 je pokazao selektivnost prema malignom fenotipu. U mišjim B16 ćelijama, tubugi 1 je indukovao atipičnu apoptozu bez eksternalizacije fosfatidilserina (PS), što je pripisano očuvanosti lipida membrane u uslovima intenzivnog oksidativnog stresa. Iako PS ima ključnu ulogu u uklanjaju apoptotskih ćelija, ovo se nije odrazilo na fagocitnu aktivnost makrofaga in vitro. Delotvornost eksperimentalnog agensa potvrdjena je in vivo. Smanjeni volumen tumora je, pored direktnog uticaja na maligne ćelije, rezultat i indukcije citotoksičnog fenotipa, kao i očuvane fagocitne sposobnosti makrofaga. Sa druge strane, kod A-375 ćelija tubugi 1 je indukovao mitotsku katastrofu, morfološki manifestovanu mikronukleacijama, a biohemijski – aktivacijom kaspaze 2 i nukleusnog faktora κB (NF-κB). Opisani fenomen bio je praćen intenziviranom, ali prolaznom autofagijom citoprotektivnog karaktera. Sa jenjavanjem autofagnog procesa, jačala je apoptoza praćena porastom proapoptotskog indeksa i aktivnosti kaspaze 3. Svi opisani događaji u potpunosti su korelirali sa dinamičnim promenama u signalnim putevima uključenim u ćelijsku deobu i smrt – mitogenom-aktivirane protein kinaze (MAPK) i fosfatidilinozitol 3-kinaze (PI3K/Akt). U celini, rezultati ove doktorske disertacije ukazaju na značajan antitumorski potencijal sintetskog derivata prirodnih tubulizina, tubugi 1, pokazan na modelu mišjeg i humanog melanoma.

Tubulysins are secondary metabolites of myxobacteria that exert their antitumor activity by disrupting the organization of the mitotic spindle. In this study, the antitumor potential of synthetic analog of tubulysins, tubugi 1, was tested in mouse and human melanoma model in vitro and in vivo. Tubugi 1 showed selectivity for the malignant phenotype. In murine B16 cells, tubugi 1 induced atypical apoptosis without phosphatidylserine (PS) externalization, which was attributed to membrane lipid preservation under conditions of intense oxidative stress. Although PS plays a key role in apoptotic cell removal, this did not affect macrophage phagocytic activity in vitro. The efficacy of the experimental agent was confirmed in vivo. In addition to its direct effect on malignant cells, reduced tumor volume is ascribed to established cytotoxic phenotype, as well as the preserved phagocytic ability of macrophages. On the other hand, in A-375 cells, tubugi 1 induced a mitotic catastrophe, manifested morphologically by micronuclei formation, and biochemically by activation of caspase 2 and the nuclear factor κB (NF-κB). The described phenomenon was accompanied by intense, but transient autophagy showing cytoprotective features. With the depletion of the autophagic process, apoptosis increased, followed by an increase in proapoptotic index and caspase 3 activity. All of the events described correlated completely with the dynamic changes in signaling pathways involved in cell division and death – mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K/Akt). Finally, the results of this doctoral dissertation pointed to the important antitumor potential of the synthetic derivative of natural tubulysins, tubugi 1, in the mouse and human melanoma model.