magistrski študijski program Laboratorijska biomedicina
Suzana Semič (Avtor), Sergej Pirkmajer (Mentor), Mojca Pavlin (Komentor)

Povzetek

Uvod: Rak je med vodilnimi vzroki smrti. Pri ženskah je najpogostejši rak dojke, ki predstavlja 21 % primerov raka. Presnovne motnje, kot sta debelost in sladkorna bolezen tipa II, so pomemben dejavnik tveganja za razvoj raka dojke. Po drugi strani pa so presnovne spremembe ena temeljnih značilnosti rakavih celic. Učinkovine, ki vplivajo na presnovo, bi torej lahko bile uporabne pri zdravljenju raka dojke. Med takšnimi učinkovinami je tudi metformin, ki se sicer uporablja za zdravljenje sladkorne bolezni. Namen: Nedavno je bilo ugotovljeno, da metformin, ki inhibira kompleks I dihalne verige, v kombinaciji z 2-deoksiglukozo (2-DG), ki inhibira glikolizo, v celični kulturi povzroči odlepljanje rakavih celic. V nasprotju s splošno sprejetim prepričanjem, da so odlepljene celice mrtve, te celice v velikem deležu ostanejo žive in sposobne za nadaljnjo proliferacijo. Namen našega dela je bil preveriti, ali na celice raka dojke podobno kot metformin delujejo tudi drugi inhibitorji dihalne verige. Kot poskusni model smo uporabili celice MDA-MB-231, celično linijo trojno negativnega raka dojke. Hipoteze: (1) Za preživetje pritrjenih in odlepljenih celic MDA-MB-231 je glikoliza pomembnejša od oksidativne fosforilacije; (2) Sočasno tretiranje z metforminom in inhibitorjem glikolize 2-DG ima podoben učinek na celice MDA-MB-231 kot sočasno tretiranje z drugimi inhibitorji dihalne verige in inhibitorjem glikolize 2-DG; (3) Hipoksija inducira izražanje HIF-1α v pritrjenih in odlepljenih celicah MDA-MB-231; (4) Inhibicija glikolize z 2-DG poslabša preživetje pritrjenih in odlepljenih celic MDA-MB-231 v pravi hipoksiji ne pa v kemični hipoksiji. Metode: Število in preživetje celic MDA-MB-231 smo ocenjevali z barvilom propidijev jodid in metodo pretočne citometrije, nastajanje reaktivnih kisikovih zvrsti smo ocenjevali z barvilom CM-H2DCFDA in metodo pretočne citometrije, izražanje proteinov in signalne poti pa smo ocenjevali z odtisom western. Rezultati: Pri uporabi različnih inibitorjev energijske presnove je bilo od 20 do 95 % odlepljenih MDA-MB-231 celic živih. Preživetje celic je bilo manjše pri inhibiciji glikolize z 2-DG kot pri inhibiciji dihalne verige z metforminom ali inhibitorji dihalne verige, kot so antimicin A, natrijev azid in oligomicin. Inhibicija dihalne verige je pomembno zmanjšala preživetje celic MDA-MB-231 šele, ko smo celice gojili v odsotnosti glukoze. To nakazuje, da imajo celice MDA-MB-231 funkcionalno dihalno verigo, vendar je njihovo preživetje bolj odvisno od glikolize kot od oksidativne fosforilacije. S tem smo potrdili prvo hipotezo. Sočasno tretiranje z 2-DG in metforminom ali 2-DG in drugimi inhibitorji dihalne verige je imelo podoben učinek na odlepljenje živih celic MDA-MB-231. Ta rezultat podpira drugo hipotezo. Pri pritrjenih celicah MDA-MB-231 je hipoksija (1 % O2) inducirala izražanje transkripcijskega dejavnika HIF-1α. V odlepljenih celicah je bilo izražanje HIF-1α manjše tako v normoksičnih kot tudi hipoksičnih razmerah. Ti rezultati delno podpirajo tretjo hipotezo. Ugotovili smo tudi, da inhibicija glikolize z 2-DG poslabša preživetje celic MDA-MB-231 tako v pravi kot v kemični hipoksiji, inducirani s CoCl2, kar ne podpira naše četrte hipoteze. Zaključki: Inhibicija dihalne verige in/ali glikolize povzroči odlepljanje živih rakavih celic. Odlepljene rakave celice se po svojih funkcijskih lastnostih razlikujejo od prilepljenih celicah. Pri preučevanju modulatorjev energijske presnove in njihovih učinkov na rakave celice je pomembno, da se v analizo vključijo tako prilepljene kot odlepljene celice.

Ključne besede

rak dojk;energijska presnova;metformin;2-deoksi-D-glukoza;odlepljene celice;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [S. Semič]
UDK: 616-006:611.69(043.3)
COBISS: 4457585 Povezava se bo odprla v novem oknu
Št. ogledov: 382
Št. prenosov: 77
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: ǂThe ǂeffect of modulators of energy metabolism on survival of brest cancer cells in vitro
Sekundarni povzetek: Background: Cancer is one of the leading causes of mortality. Among women the most common cancer is breast cancer, which represents 21 % of all cancers. Metabolic disorders, such as obesity and type 2 diabetes mellitus, are important risk factors for development of breast cancer. On the other side, metabolic changes are one of the hallmarks of cancer cells, so the substances that affect metabolism, could be used in treatment of breast cancer. One of those substances is metformin, which is used for the treatment of diabetes. Aim: It was recently discovered that metformin, which inhibits complex I of respiratory chain in combination with 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, causes detachment of cancer cells in cell culture. In contrary with general accepted belief, that detached cells are dead, high percent of those cells are viable and able of continuing proliferation. Aim of our work was, to find out if other inhibitors of respiratory chain work on breast cancer cells, like metformin. We used MDA-MB-231 cell, the cell line of triple negative breast cancer as our experimental model. Hypotheses: (1) For survival of floating and attached MDA-MB-231 cells, glycolysis is more important than oxidative phosphorylation. (2) Combined treatment with metformin and glycolysis inhibitor 2-DG has a similar effect on MDA-MB-231 cells as combination of other inhibitors of the respiratory chain and 2-DG. (3) Hypoxia induces expression of HIF-1α in floating and attached MDA-MB-231 cells. (4) Glycolysis inhibition with 2-DG impair survival of floating and attached MDA-MB-231 cells in real hypoxia, and not in chemically-induced hypoxia. Methods: We evaluated the cell number and survival of MDA-MB-231 cells with propidium iodide staining and the method of flow cytometry. For detection of reactive oxygen species, we used CM-H2DCFDA staining and the method of flow cytometry. Expression of proteins and signal pathways were evaluated with western blot. Results: The analysis of the floating MDA-MB-231 cells has shown that 20 to 95 % of cells remain viable when different metabolism inhibitors are used. Survival of cells was lower with inhibition of glycolysis with 2-DG, than with inhibition of respiratory chain with metformin or others respiratory chain inhibitors. Inhibition of respiratory chain has significantly lowered the survival of MDA-MB-231 cells only when the cells were grown in the absence of glucose, indicating that MDA-MB-231 cells have a functional respiratory chain, but their survival is more dependent on glycolysis than oxidative phosphorylation. We thus confirmed our first hypothesis. Combined treatment with 2-DG and metformin or 2-DG and other mitochondrial respiratory chain inhibitors, like antimycin A, sodium azide and oligomycin, had a similar synergic effect on detached living MDA-MB-231 cells. This result support hypothesis two. In attached MDA-MB-231 cells hypoxia (1 % O2) induced expression of transcription factor HIF-1α, while in floating MDA-MB-231 cells HIF-1α expression was reduced in both normoxia and hypoxia. This result partially confirmed our third hypothesis. We also found that the inhibition of glycolysis with 2-DG reduced survival of attached MDA-MB-231 cell in real and chemically-induced hypoxia, induced with CoCl2, which does not support our fourth hypothesis. Conclusions: Inhibition of the respiratory chain and/or glycolysis cause the living cancer cell to detach. Functional characteristic in floating cancer cells are different than in attached cancer cells. While studying metabolism modulators and their effect on cancer cells, it is important that we analyze both, floating and attached cells.
Sekundarne ključne besede: Dojke;
Vrsta dela (COBISS): Magistrsko delo/naloga
Komentar na gradivo: Univ. Ljubljana, Fak. za farmacijo
Strani: VIII, 56 f.
ID: 12040866