V drugem delu smo se osredotočili na to, ali lahko z uporabo antioksidantov preprečimo celično smrt sproženo s H2O2. Uporabili smo dva antioksidanta – NAC in vitC, ki imata terapevtski potencial [76,77]. NAC lovi proste radikale z direktno interakcijo z ROS preko redoks potenciala tiolne skupine ali pa indirektno z višanjem ravni glutationa v celici [37]. VitC pa je donor elektronov in zato lahko reducira spojine z neparnimi elektroni, kot so recimo nekateri ROS (superoksid in hidroksilni radikal) [95].
Najprej smo poskusili z dodatkom antioksidantov NAC in vitC izboljšati viabilnost celic divjega tipa in celic z izbitim genom za stefin B po tretiranju s H2O2 (slika 10). Dodatek NAC je viabilnost celic z izbitim genom za stefin B izboljšal približno za 25 %. Pri celicah divjega tipa pa ni bilo signifikantne spremembe v smrtnosti ob dodatku NAC.
Presenetljivo pa je bilo, da se je delež smrtnosti celic po tretiranju z vitC in H2O2 povišal v primerjavi s celicami tretiranimi zgolj s H2O2.
42
V naslednjem koraku smo preverili, kako bo dodatek antioksidantov NAC in vitC vplival na pH lizosomov, nastanek ROS in oksidacijo kardiolipina. Dodatek NAC in vitC pri celicah divjega tipa nista vplivala na pH (slika 11). Je pa dodatek vitC statistično značilno povečal nastanek ROS (slika 12) in oksidacijo kardiolipina (slika 13). Dodatek NAC je pri celicah divjega tipa zmanjšal nastanek ROS (slika 12), pri oksidaciji kardiolipina pa ni bilo večjih sprememb (slika 13). Pri celicah z izbitim genom za stefin B je NAC zmanjšal nastanek ROS (slika 12), oksidacijo kardiolipina (slika 13) in spremembo v pH (slika 11). VitC pa na pH v lizosomih ni vplival, je pa statistično značilno povečal nastanek ROS (slika 12) in oksidacijo kardiolipina (slika 13).
Na podlagi rezultatov lahko sklepamo, da antioksidant NAC ščiti celice z izbitim genom za stefin B pred celično smrtjo zaradi ROS. NAC je sintetični prekurzor znotrajceličnega cisteina in glutationa, zato najverjetneje pride do njegovega učinka zaradi lastnosti direktnega lovljenja prostih radikalov in zaradi posrednega višanja ravni glutationa v celicah [112]. Možno je, da je NAC zmanjšal poškodbe lizosomov pri celicah z odsotnim stefinom B s tem, da je preprečil Fentonovo reakcijo v lizosomih. V lizosomih namreč poteka fentonova reakcija, kjer nastanejo zelo reaktivni hidroksilni radikali, ki so pogosto tudi vzrok lipidne peroksidacije [88]. Rezultati vitC so bili presenetljivi, saj je prišlo do poslabšanja deleža celične smrti in poškodb organelov. Možno je, da je do tega prišlo zato, ker vitamin C v mediju že ob nizki koncentraciji železa ali druge kovine lahko oksidira in generira H2O2 [95]. Z dodatkom kelatorja železa bi zato lahko povečali učinkovitost vitC. Prav tako, če bi celice tretirali s H2O2 in kelatorjem železa, verjetno ne bi prišlo do nastanka hidroksilnega radikala v lizosomih, kar bi lahko že izboljšalo preživetje celic v kolikor je ravno nastanek le-tega pomemben pri indukciji celične smrti.
Poleg dodatka kelatorja železa pa bi lahko še optimizirali koncentracijo vitC. Znano je namreč, da je učinek vitC odvisen od njegove koncentracije. Pri nizkih koncentracijah deluje kot antioksidant, pri visokih koncentracijah pa ima oksidativni učinek [36].
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6 Zaključek
Tekom opravljanja naše raziskave smo potrdili, da je pri primarnih MMTV-PyMT tumorskih celicah z izbitim genom za stefin B prišlo do signifikantno višjega deleža smrti kot pri celicah divjega tipa. Hipotezo, da gre pri oksidativnem stresu sproženem s H2O2
za apoptozo, smo ovrgli. Potrdili smo spremembe lizosomov in mitohondrijev, najverjetneje zaradi povečanega nastanka ROS. Naši eksperimenti so namreč pokazali, da odsotnost stefina B vodi do povečanega nastanka ROS, kar pripelje preko oksidacije kardiolipina do celične smrti. Kljub temu, da pride do spremembe pH kislih veziklov, pa najverjetneje v celični smrti sproženi s 3 mM H2O2 katepsini nimajo glavne vloge.
Pogosto pa pri oksidativnem stresu pride do kombinacije različnih oblik celične smrti in ne moremo govoriti le o eni. Najverjetneje pride v celicah z odsotnim stefinom B do aktivacije NLRP3 inflamasoma in od ROS-odvisne celične smrti (npr. feroptoza), kar bi lahko potrdili v nadaljevanju raziskovalne naloge.
Predvidevali smo, da bosta antioksidanta NAC in vitC izboljšala občutljivost celic na oksidativni stres. To hipotezo smo delno potrdili, saj je NAC viabilnost celic izboljšal, vitC pa še poslabšal. Potrdili pa smo hipotezo, da bo NAC boljši antioksidant pri zmanjšanju vpliva H2O2 kot vitC. Preglednica 9 prikazuje pregled prvotno zastavljenih hipotez ter njihovo končno ovrednotenje.
Preglednica 9: Pregled ovrednotenja zastavljenih hipotez.
hipoteza ovrednotenje
Celice z izbitim genom za stefin B so bolj občutljive na
tretiranje s H2O2 kot celice divjega tipa. potrjena H2O2 sproži apoptozo v izbranem celičnem modelu. ovržena H2O2 povzroči poškodbe celičnih komponent. potrjena Antioksidanta NAC in vitC preprečita celično smrt in
omejita poškodbe sprožene s H2O2. delno potrjena NAC bo bolj učinkovito zmanjšal vpliv H2O2 kot vitC.
potrjena
Rezultati odpirajo nova vprašanja in možnosti za nove raziskave. Zanimivo bi bilo narediti poskuse še z nižjo koncentracijo H2O2, da bi preverili, če v takšnih pogojih pride do apoptoze. Nadalje bi lahko še potrdili, če katepsini ostanejo ujeti v lizosomih ali pa pride do izlitja le-teh v citosol. Z uporabo specifičnih inhibitorjev bi lahko preverili, katero izmed drugih oblik celične smrti smo sprožili s H2O2 in če ima NLRP3 inflamasom pomembno vlogo. Zanimivo bi bilo preveriti tudi stres v ER, ki ga tudi povezujejo s celično smrtjo.
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