| Abstract | Visoke razine reaktivnih kisikovih vrsta (ROS) u stanici izazivaju oksidativni stres koji može dovesti do genomskog oštećenja i nestabilnosti te nekontrolirane proliferacije ili smrti stanica. Stanična linija raka dojke MCF-7 okarakterizirana je slabom genskom ekspresijom te izostankom proteinske ekspresije Sirtuina 3 (Sirt3), mitohondrijske NAD+ ovisne deacetilaze koja kontrolira brojne biološke aktivnosti, a između ostalog i štiti od ROS-a. Stoga se u ovom radu istraživala uloga Sirt3 u MCF-7 stanicama tretiranima rotenonom, inhibitorom mitohondrijskog lanca transporta elektrona i supstancom koja potiče oksidativni stres. Za tu svrhu korištene su transfecirane MCF-7 stanice koje nemaju ili pak imaju stabilnu ekspresiju proteina Sirt3, a istražen je njegov utjecaj na stvaranje ROS-a, starenje, metabolizam, ekspresiju određenih proteina te je istražena uloga u rotenonom-izazvanom oštećenju proteina, lipida i DNA. Za analizu navedenih parametara korištene su sljedeće metode: fluorescencijska analiza količine ROS-a, analiza senescencije, MTT test, Western blot, te metoda lipidne peroksidacije i karbonilacije proteina. Dobiveni rezultati su jasno pokazali da rotenon u MCF-7 stanicama izaziva oksidativni stres na svim analiziranim razinama, te da je Sirt3 stanice zaštitio od oksidativnog stresa na način da je smanjio razinu ROS-a, senescenciju i oštećenje lipida u odnosu na kontrolne stanice. Ipak, Sirt3 nije djelovao zaštitno na proteine i DNA, niti je u značajnoj mjeri spriječio staničnu smrt, što implicira da je aktivirao staničnu zaštitu, ali ne u dovoljnoj mjeri. Nadalje, stanice sa Sirt3 imaju veće oštećenja DNA i lipida što ukazuje da Sirt3 sam ili u kombinaciji s rotenonskim tretmanom ne održava niti poboljšava tumorske karakteristike MCF-7 stanica. Štoviše, s obzirom na faktore u kojima stanice sa ekspresijom Sirt3 pokazuju veću osjetljivost na stres izazvan rotenonom, ovi rezultati upućuju da bi Sirt3 mogao imati ulogu inhibitora rasta tumora, odnosno tumor-supresorsku ulogu u MCF-7 stanicama. |
| Abstract (english) | In the cell, high levels of reactive oxygen species (ROS) cause oxidative stress that can lead to genomic damage and instability and uncontrolled proliferation or cell death. MCF-7 breast cancer cell line is characterized by low gene expression and lack of protein expression of Sirt3, a mitochondrial NAD+ dependent deacetylase that controls many biological activities and, among other things, protects against ROS. Therefore, the role of Sirt3 in MCF-7 cells treated with rotenone, an inhibitor of the mitochondrial electron transport chain, and a causative agent of oxidative stress, was investigated in this thesis. For this purpose, transfected MCF-7 cells with or without stable expression of Sirt3 protein were used, and its effect on ROS generation, aging, metabolism, expression of certain proteins, and role in rotenone-induced damage to proteins, lipids and DNA was explored. The following methods were used to analyze these parameters: fluorescence analysis of ROS with MitosoxRed dye, senescence analysis with SA-β-gal staining, MTT assay, Western blot, and determination of lipid peroxidation and protein carbonylation. The results clearly showed that rotenone in MCF-7 cells induces oxidative stress at all analyzed levels, and that in several cell parameters, Sirt3 protected against oxidative stress (decreased ROS levels, senescence, and lipid damage relative to control cells). However, Sirt3 failed to protect proteins and DNA from damage, and to prevent the cell death, implying that it activated several protective cellular systems, but not sufficiently to fully protect cells. Furthermore, cells with overexpressed Sirt3 showed more DNA and lipid damage, which implies that Sirt3 alone or in combination with rotenone treatment does not maintain or improve the tumor characteristics of the MCF-7 cells. Moreover, given the factors in which Sirt3-overexpressing cells show greater sensitivity towards stress, the possibility to consider Sirt3 as a tumor suppressor in MCF-7 cells is not excluded. |
