Povezave med lipidnimi kapljicami in mehanizmi uravnavanja membranske sestave pri feroptozi

Vončina, Kaja (author), Petan, Toni (mentor)

Preoblikovanje membranskih lipidov in s tem membran je pomembno za učinkovito prilagajanje rakavih celic okolju. Pomembno vlogo pri uravnavanju maščobnokislinske sestave membran imata aciltransferazi AGPAT3 in LPCAT3, ki vplivata na vsebnost polinenasičenih maščobnih kislin v fosfolipidih in s tem uravnavata občutljivost celic za feroptozo. Ni pa znano, kako je delovanje AGPAT3 in LPCAT3 povezano s presnovo lipidnih kapljic (LK), ki služijo tudi kot skladišče različnih maščobnih kislin, shranjenih v obliki nevtralnih lipidov. V tem delu smo v celičnem modelu agresivnega raka dojke s tehnikami RNA interference v kombinaciji s farmakološko in presnovno modulacijo LK ter feroptotskih obrambnih mehanizmov preučevali vlogo AGPAT3 in LPCAT3 pri občutljivosti celic za feroptozo v povezavi s homeostazo lipidov. V celicah smo sprožili feroptotski stres z dodatkom RSL3, inhibitorja glutation peroksidaze 4, medtem ko smo na biogenezo, količino in sestavo LK vplivali z dodatkom inhibitorjev diacilglicerol aciltransferaz 1 in 2 (DGATi) ter dokozaheksaenojske kisline (DHA). Ugotovili smo, da utišanje AGPAT3 zmanjša občutljivost celic za feroptozo, inducirano z RSL3, kar se kaže v zmanjšanem deležu mrtvih celic in nižji lipidni peroksidaciji. Utišanje LPCAT3 ni vplivalo za feroptozo, ki smo jo inducirali le z RSL3, vendar se je občutljivost izrazito zvišala, ko smo dodali še DGATi. Pri obeh utišanjih smo opazili kopičenje nevtralnih lipidov v LK in negativen vpliv na viabilnost celic. Ugotovili smo tudi, da visoka raven feroptotskega stresa vpliva na količino proteinov, vpletenih tako v presnovo LK kot v preoblikovanje membran, vključno z AGPAT3, ACSL3, ACSL4 in lipazo ATGL. Naše ugotovitve ponujajo nov vpogled v povezave med procesi, ki uravnavajo sestavo membran, presnovo LK in feroptozo, ter prispevajo k boljšemu razumevanju mehanizmov, ki uravnavajo občutljivost rakavih celic MDA-MB-231 na lipidno peroksidacijo in feroptotski stres.

• Language: Slovenian
• Keywords: feroptoza, rak dojke, lipidne kapljice, lipidni metabolizem, preoblikovanje membran, maščobne kisline, AGPAT3, LPCAT3
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [K. Vončina]
• Year: 2026
• PID: 20.500.12556/RUL-184969
• UDC: 618.19-006(043.2)
• COBISS.SI-ID: 285383171
• Publication date in RUL: 19.07.2026

Secondary language

• Language: English
• Title: Interactions between lipid droplets and mechanisms regulating membrane composition in ferroptosis

Abstract

Membrane lipid remodeling is important for the efficient adaptation of cancer cells to their environment. The acyltransferases AGPAT3 and LPCAT3 play a crucial role in regulating the fatty acid composition of membranes, influencing the content of polyunsaturated fatty acids in phospholipids and thereby regulating cell sensitivity to ferroptosis. However, it remains unknown how the activity of AGPAT3 and LPCAT3 is linked to the metabolism of lipid droplets (LDs), which also serve as dynamic reservoirs for various fatty acids stored in the form of neutral lipids. In this work, we investigated the role of AGPAT3 and LPCAT3 in sensitivity to ferroptosis in connection with lipid homeostasis in a cell model of aggressive breast cancer using RNA interference techniques combined with pharmacological and metabolic modulation of LDs and ferroptotic defense mechanisms. Ferroptotic stress was induced by supplementing cells with RSL3, a glutathione peroxidase 4 inhibitor, while the biogenesis, quantity and composition of LDs were modulated by diacylglycerol acyltransferase 1 and 2 inhibitors (DGATi) and docosahexaenoic acid (DHA). We found that AGPAT3 silencing reduces cell sensitivity to RSL3-induced ferroptosis, as reflected by a decreased percentage of dead cells and lower lipid peroxidation. Silencing of LPCAT3 did not affect ferroptosis induced by RSL3 alone, but this sensitivity increased when DGATi were also added. In both silencing conditions, we observed an accumulation of neutral lipids in LDs and a negative impact on cell viability. We also found that high levels of ferroptotic stress affect the abundance of proteins involved in both LD metabolism and membrane remodeling, including AGPAT3, ACSL3, ACSL4 and the lipase ATGL. Our findings offer new insights into the connections between the processes regulating membrane composition, LD metabolism and ferroptosis, contributing to a better understanding of the mechanisms that regulate the sensitivity of MDA-MB-231 cancer cells to lipid peroxidation and ferroptotic stress.

• Keywords: ferroptosis, breast cancer, lipid droplets, lipid metabolism, membrane remodeling, fatty acids, AGPAT3, LPCAT3