In vitro evaluation of synergistic cytotoxic effects of tyrosine kinases and O-GlcNAc transferase inhibitors in cancer cell lines

N-acetilglukozaminilacija in fosforilacija sta medsebojno povezani dinamični post-translacijski modifikaciji, ki sta vključeni v regulacijo številnih celičnih procesov. N-acetilglukozaminilacijo uravnavata dva encima, in sicer O-β-N-acetilglukozaminil transferaza (OGT), ki pripenja, in O-β-N-acetilglukozaminil hidrolaza (OGA), ki odstranjuje N-acetilglukozamine iz tarčnega mesta proteina. Po drugi strani pa je proces fosforilacije uravnavan z več encimi, med katerimi je tudi družina tirozin kinaz. Le-te so pomembne tarče v rakavih obolenjih, saj so se zaviralci tirozin kinaz izkazali kot uspešen terapevtski pristop. Znano je, da lahko kinaze vplivajo na aktivnost OGT in obratno – OGT lahko vpliva na aktivnost kinaz. Na podlagi tega smo sklepali, da lahko zaviralci OGT posredno vplivajo na delovanje tirozin kinaz. Našo domnevno smo preverjali na izbranih rakavih celičnih linijah, na katerih smo vrednotili citotoksične vplive zaviralcev OGT, zaviralcev tirozin kinaz ter njihove kombinacije. Določili smo srednjo inhibitorno vrednost (IC50) za posamezno celično vrsto in izbrane zaviralce tirozin kinaz (regorafenib, sorafenib, imatinib, ibrutinib, dasatinib) ter pri tem ugotovili, da se vrednosti nahajajo v različnih koncentracijskih območjih. Rezultat je bil pričakovan, saj smo uporabili različne vrste celičnih linij, katerih preživetje je različno odvisno od tarčnih tirozin kinaz. Podatki so prav tako pokazali, da dodatek zaviralca OGT ojača delovanje regorafeniba na celicah HAP-1 in AMO-1. V nadaljevanju smo nato proučevali sinergistične učinke kombinacije zaviralca OGT in regorafeniba na živost, stopnjo proliferacije in celični cikel rakave celične linije AMO-1. Pokazali smo, da izbrana kombinacija zaviralca tirozin kinaze in OGT statistično značilno zniža živost in stopnjo proliferacije celic AMO-1, vendar nima signifikantnega učinka na razporeditev celic v posamičnih fazah celičnega cikla. Preliminarni rezultati so prav tako nakazali, da proženje celične smrti najverjetneje ne poteka preko aktivacije programirane celične smrti (t.j. apoptoze), saj nismo uspeli določiti povišanja deleža zgodnjih apoptotičnih celic. Rezultati kažejo, da je lahko sočasna uporaba TKI in zaviralcev OGT obetaven in učinkovit pristop proženju celične smrti v nekaterih vrstah rakavih celic, a so potrebne nadaljnje študije, ki bi osvetlile dogajanje na molekularnem nivoju.O-GlcNAcylation and phosphorylation are dynamic post-translational modifications, which are involved in regulation of many cellular processes and have an extensive cross talk. O-GlcNAcylation is regulated by two enzymes: O-GlcNAc transferase (OGT), which attaches O-GlcNAc, and O-GlcNAcase (OGA), which removes it from the protein target site. Phosphorylation, on the other hand, is regulated by several enzymes including the tyrosine kinase family, which are important targets for cancer treatment. Namely, tyrosine kinase inhibitors have shown to be a successful therapeutic approach in treatment of malignancies. It is known that kinases can affect OGT activity and vice versa – OGT can affect kinase activity. Based on this, we conclude that OGT inhibitors may indirectly affect the activity of tyrosine kinases. Our hypothesis was tested on selected cancer cell lines, on which we evaluated the cytotoxic effects of OGT inhibitors, tyrosine kinase inhibitors and their combinations. The mean inhibitory value (IC50) of selected tyrosine kinase inhibitors (regorafenib, sorafenib, imatinib, ibrutinib, dasatinib) for each cell type was determined. We observed that IC50 values differed between cell lines and between the types of inhibitor used. This result was expected since we used different cell types whose survival distinctively depends on specific tyrosine kinases. Data also revealed that the addition of an OGT inhibitor enhances the action of regorafenib on HAP-1 and AMO-1 cell lines. Next, we examined whether these observed synergistic effects on cytotoxicity, after co-treatment of AMO-1 cells with an OGT inhibitor and regorafenib, are also reflected on the level of cell viability, proliferation rate and the distribution of cells within the cell cycle phases. We confirmed that the combination of regorafenib and the OGT inhibitor causes a synergistic reduction of the cell viability and inhibits the proliferation rate of AMO-1 cells. However, this combination did not have a significant effect on cell distribution in individual phases of the cell cycle. Preliminary results also indicate that cell death is most likely not triggered through the activation of the programmed cell death pathways (i.e. apoptosis), as we were unable to determine a time-dependent increase of early apoptotic cells. Based on these results, we provide evidence that co-treatment with TKI and OGT inhibitors may be a promising and effective approach to effectively trigger cell death in some types of cancer cells. Nevertheless, further studies are needed to shed light on the underlying molecular mechanisms as well use more relevant experimental models (e.g. 3D cell cultures)