Delovanje metformina in 2-deoksi-D-glukoze na celice MDA-MB-231 in modelne limfocite T in vitro

Repas, Jernej (author), Pavlin, Mojca (mentor), Gerger, Armin (comentor)

Presnovne spremembe in izogibanje imunskemu sistemu so temeljne lastnosti raka. Metformin in 2-deoksi-D-glukoza (2DG) sta presnovni učinkovini z različnimi obetavnimi protirakavimi in imunomodulatornimi učinki. Njuna kombinacija povzroči odlepljanje živih celic raka dojke MDA-MB-231 z ohranjeno sposobnostjo proliferacije in vitro. Od podlage neodvisna rast rakavih celic in vitro korelira z metastaziranjem in vivo. Obenem zahteva mitohondrijsko biogenezo, vendar presnovne spremembe pri odlepljanju, ter učinek metformina in 2DG na mitohondrije niso znani. Z metabolomiko smo identificirali presnovne spremembe v celicah MDA-MB-231, tretiranih z metforminom in 2DG. Visoka 2DG in kombinacija metformina in nizke 2DG (metformin+2DG) sta spremenili presnovni profil podobno kot metformin. To ne razloži odlepljanja, ki je posledica zmanjšanja N-glikozilacije proteinov z 2DG in njegovega potenciranja z metforminom. Odlepljene celice so bile mehkejše in presenetljivo presnovno bližje kontroli kot pritrjenim tretiranim celicam. Imele so spremenjen nivo NADPH, glutamina in presnove maščobnih kislin, kar je značilno za rast v odlepljenem stanju. Metformin+2DG in visoka koncentracija 2DG sta povečala mitohondrijsko maso celic raka dojke preko aktivacije mitohondrijske biogeneze in povečane velikosti, ne pa tudi števila mitohondrijev. 2DG in metformin+2DG sta inducirala stres endoplazmatskega retikuluma, ki je bil potencialni sprožilec mitohondrijske biogeneze skupaj z energijsim stresom. Aktivacija z adenozin monofosfatom aktivirane proteinske kinaze je pri tem igrala vlogo, a ni bila zadostna za sprožitev mitohondrijske biogeneze. Metformin in 2DG tako povzročita presnovni stres in mitohondrijske prilagoditve, kar omogoča preživetje rakavih celic ob odlepljanju in energijskem stresu. Vzporedno sta 2DG in metformin+2DG zmanjšala izražanje liganda 1 za receptor programirane smrti 1 (PD-L1) na celicah MDA-MB-231 preko zavrte N-glikozilacije in zmanjšala izražanje receptorja programirane celične smrti 1 (PD-1) na celicah Jurkat. Metformin, visoka koncetracija 2DG in metformin+2DG so zavrli proliferacijo, aktivacijo in izločanje interlevkina 2 preko zmanjšanja proizvodnje adenozin trifosfata, nizka 2DG pa je ohranila izločanje interlevkina 2 in povečala izločanje interferona ?. Nizka koncetracija glukoze je okrepila učinek metformina, medtem kot supra-fiziološka koncentracija glukoze ali eksogeni piruvat nista imela učinka. Presnovne učinkovine delno zavrejo funkcijo celic Jurkat, specifične kombinacije pa bi lahko izboljšale protitumorsko imunost preko osi PD-1/PD-L1.

• Language: Slovenian
• Keywords: metformin, 2-deoksi-D-glukoza, odlepljanje celic, rast v odlepljenem stanju, metabolomika, energijski stres, trojno negativni rak dojke, T celice, mitohondrijska biogeneza, N-glikozilacija proteinov, ER stres, AMPK, os PD-1/PD-L1, nivo glukoze
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2022
• PID: 20.500.12556/RUL-137328
• COBISS.SI-ID: 127802627
• Publication date in RUL: 11.06.2022

REPAS, Jernej, 2022, Delovanje metformina in 2-deoksi-D-glukoze na celice MDA-MB-231 in modelne limfocite T in vitro [online]. Doctoral dissertation. [Accessed 7 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=137328

Secondary language

• Language: English
• Title: Effects of metformin and 2-deoxy-D-glucose on MDA-MB-231 cells and model T lymphocytes in vitro

Abstract

Metabolic alterations and immune evasion are emerging hallmarks of cancer. Metformin and 2-deoxy-D-glucose (2DG) are metabolic drugs with several promising anti-cancer and immunomodulatory effects. Their combination induces detachment of viable MDA-MB-231 breast cancer cells retaining proliferation capacity in vitro. Anchorage-independent growth of cancer cells in vitro is correlated to metastasis formation in vivo and requires mitochondrial biogenesis, but the metabolic changes involved in detachment and the effect of metformin and 2DG on mitochondria are unknown. We used liquid chromatography-mass spectrometry metabolomics to identify alterations in metformin and 2DG treated MDA-MB-231 cells. High concentration of 2DG and the combination of metformin and low concentration of 2DG (metformin+2DG) altered the metabolic profile similarly to metformin. These alterations could not explain detachment, which was actually induced by suppressed protein N-glycosylation by 2DG and its potentiation by metformin. The detached cells were softer and, surprisingly, metabolically closer to control than their attached counterparts. They exhibited altered NADPH, glutamine and fatty acid metabolism characteristic for anchorage-independent growth. Metformin+2DG and high 2DG increased mitochondrial mass in breast cancer cells by activating mitochondrial biogenesis and increasing mitochondrial size, but not their number. 2DG and metformin+2DG induced endoplasmic reticulum stress which could trigger mitochondrial biogenesis together with energy stress. Adenosine monophosphate activated protein kinase activation was also involved but not sufficient for mitochondrial biogenesis. Overall, metformin and 2DG induce metabolic and mitochondrial adaptations enabling breast cancer cell survival in detachment and energy stress. In parallel, 2DG and metformin+2DG reduced programmed death ligand 1 (PD-L1) expression in MDA-MB-231 cells by suppressing N-glycosylation and reduced programmed death 1 (PD-1) expression in Jurkat cells. Metformin, high 2DG and metformin+2DG inhibited Jurkat cell proliferation, activation and interleukin 2 secretion by reducing adenosine triphosphate production, but low 2DG preserved interleukin 2 and boosted interferon γ secretion. Low glucose concentration potentiated the effect of metformin, while supra-physiological glucose levels or exogenous pyruvate had no effect. While metabolic drugs partially inhibit Jurkat function, specific combinations could improve anti-tumor immunity via PD-1/PD-L1 axis inhibition.

• Keywords: metformin, 2-deoxy-D-glucose, cell detachment, metabolomics, energy stress, triple-negative breast cancer, T cells, mitochondrial biogenesis, protein N-glycosylation, ER stress, AMPK, PD-1/PD-L1 axis, anchorage-independence, glucose level