Proučevanje sinergističnega vpliva zaviralca O-GlcNAc-transferaze in regorafeniba na celično proliferacijo mielomske celične linije AMO-1

Kočevar, Lucija (author), Gobec, Martina (mentor), Weiss, Matjaž (co-mentor)

Fosforilacija in N-acetilglukozaminilacija sta pogosti in zelo pomembni posttranslacijski modifikaciji, udeleženi v številnih celičnih procesih, kot so celična delitev, celična signalizacija, transkripcija proteinov, celična smrt. Še več, poleg direktne vpletenosti v potek celičnih procesov je med njima prisoten dinamičen medsebojni vpliv. Spremembe pri uravnavanju ene ali druge modifikacije lahko iztirijo potek omenjenih celičnih procesov in vodijo v razvoj različnih bolezni. Iniciacija in potek obeh procesov sta zato strogo regulirana s strani encimov, in sicer potek fosforilacije nadzorujejo encimi imenovani kinaze, N acetilglukozaminilacija pa poteka pod nadzorom N acetil β glukozaminil transferaze (OGT) in N acetil β glukozaminil hidrolaze (OGA). Zaviranje delovanja kinaz je uspešen pristop pri terapiji rakavih obolenj. Zaradi medsebojnega vpliva fosforilacije in N-acetilglukozaminilacije smo sklepali, da bi med zaviralci kinaz in zaviralci OGT lahko prišlo do sinergističnih toksičnih učinkov na proliferacijo rakavih celic. V ta namen smo izvedli eksperiment na mielomski celični liniji AMO 1, ki smo jo izpostavili kombinaciji regorafeniba (zaviralec kinaz) in OSMI 1 (zaviralec OGT). Preverili smo vpliv teh učinkovin na metabolno aktivnost celične linije in ugotovili, da je v primeru sočasne uporabe zaviralcev metabolna aktivnost statistično značilno nižja, kot če celice izpostavimo zaviralcema posamično. To zmanjšanje bi lahko bilo posledica zmanjšane živosti celic ali zavrtja celične proliferacije. Ugotovili smo, da učinkovini sinergistično vplivata na zavrtje celične proliferacije, prav tako pa se sinergizem kaže pri živosti celic, kjer kombinacija učinkovin signifikantno zmanjša živost celične linije AMO 1. Poskušali smo določiti, katera vrsta celične smrti poteče, vendar nam je uspelo dokazati le, da ne pride do apoptoze, ampak gre za druge mehanizme proženja celične smrti. Ker smo opazili tudi vidne učinke na celično proliferacijo, smo naprej raziskali učinke spojin na nivoju celičnega cikla. Pokazali smo, da kombinacija učinkovin značilno zniža delež celic v fazi S, pri čemer pa prihaja do zastoja celic predvsem v fazi G1 in tudi v fazi G2 celičnega cikla. Ugotovili smo, da se sinergizem kaže že na nivoju ciklinov in od ciklinov odvisnih kinaz, ki nadzorujejo potek in napredovanje celičnega cikla. S tem smo dokazali, da imata regorafenib in OSMI-1 sinergistično delovanje na mielomsko celično linijo AMO-1 in bi v prihodnje lahko predstavljala smotrno kombinacijo v protitumorni terapiji.

• Language: Slovenian
• Keywords: fosforilacija, N-acetilglukozaminilacija, sinergizem, regorafenib, OSMI 1, mielomska celična linija AMO 1, proliferacija, celični cikel
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-136377
• Publication date in RUL: 27.04.2022

Secondary language

• Language: English
• Title: Evaluation of the synergistic effects of O-GlcNAc transferase inhibitor and regorafenib on the proliferation of the myelomic cell line AMO-1

Abstract

Phosphorylation and O GlcNAcylation are common and very important posttranslational modifications, being a part of many cellular processes, such as cell division, cell signaling, protein transcription and cell death. Even more, besides their direct involvement in cellular processes it is known, there is an extensive cross-talk between the two. Dysregulation of any of the two modifications can couse disruptions in cellular processes mentioned above resulting in development of many diseases. Both modifications are therefore tightly regulated by regulatory enzymes, phophorylation by kinases and O GlcNAcylation by O linked N-acetylglucosaminyl transferase (OGT) and O linked N-acetylglucosaminyl hydrolase (OGA). Inhibition of kinases shows great success in cancer therapy. Extensive cross-talk between phosphorylation and O GlcNAcylation gives us hope for synergistic effects of kinase inhibitors and OGT inhibitors on proliferation of cancer cells. We carried out an experiment on myelomic cell line AMO 1, that we treated with regorafenib (kinase inhibitor) and OSMI 1 (OGT inhibitor). We found the metabolic activity of the cell line treated with a combination of both drugs is significantly lower compared to metabolic activity of cell lines treated with one drug or the other. This could be caused by decreased cell viabilty or low cell proliferation rate. We confirmed synergistic effects of regorafenib and OSMI 1 on lower cell proliferation rate and we also found a significant drop in cell viabilty of AMO 1 cell line treated with combination of both drugs. We tried to determine the type of cell death caused by the two inhibitors but with lack of evidence we only managed to prove that apoptosis is not the type of cell death that occurs. Due to obvious effects on cell proliferation our next step was to establish what happens during the cell cycle. The combination of substances significantly lowered the population of cells in the S phase of cell cycle and increased the cell populations in G2 and mostly G1 phase of cell cycle. Further we showed the synergistic effects on cyclins and cyclin dependent kinases which play an important role in regulation and promotion of cell cycle. Obtained results confirmed that regorafenib and OSMI 1 have synergistic effects on myeloma cell line AMO 1 and are therefore a sensible combination for antitumor therapy.

• Keywords: phosphorylation, O GlcNAcylation, synergy, regorafenib, OSMI 1, myelomic cell line AMO 1, proliferation, cell cycle.