GC-MS based comparative metabolomies of two breast cancer cell lines treated with nootkatone : master thesis

Proliferacija, diferencijacija i preživljenje pojedinačne stanice u višestaničnom je organizmu pažljivo regulirano, dok u stanicama raka te regulacije nema, one nekontrolirano rastu i dijele se, šire po čitavom tijelu i ometaju funkciju normalnih tkiva i organa. Rak je vodeći uzrok smrti u svijetu. Najvažnije u patologiji raka jest razlikovati benigne (dobroćudne) od malignih (zloćudnih) tumora. Drugi način klasifikacije je prema tipu stanica od kojih je rak sastavljen, prema čemu su glavne podvrste: karcinomi, sarkomi, limfomi, leukemije i mijelomi. Rak dojke je vodeći uzrok smrti povezanih s rakom među ženama. Većina tumora dojke ima značajan broj receptora za estrogen, progesteron i humani epidermalni faktor rasta 2 (HER2) te su klasificirani kao trostruko-pozitivni, odnosno trostruko-negativni. Liječenje može biti lokalno i sistemsko ili najčešće njihovom kombinacijom. Stanična linija je skup stanica, odnosno trajno uspostavljena stanična kultura koja se uzgaja u odgovarajućem mediju u kojem će neograničeno proliferirati pri kontroliranim uvjetima. U ovom diplomskom radu korištene su dvije stanične linije raka dojke, MDA-MB-231 i MCF-7 te biciklički seskviterpen nootkaton. Njegovo antiproliferativno djelovanje na dvije stanične linije raka ispitano je u prethodnom istraživanju korištenjem MTT-testa kojim je dokazano da nootkaton ima dobar sveukupni antiproliferativni učinak. Metabolomika je proučavanje svih metabolita u stanici, tkivu ili organizmu. To uključuje identifikaciju i kvantifikaciju staničnih metabolita. Ovdje je upotrijebljen postupak koji se temelji na gašenju metabolizma stanica te sakupljanju i ekstrakciji metabolita. Primijenjen je na dvije stanične linije raka dojke, MDA-MB-231 i MCF-7 uz dodatak i bez dodatka nootkatona (kontrola). Uzeta je koncentracija nootkatona od 0,5 mmol/L pri kojoj je prethodnim korištenjem MTT-testa nootkaton uspješno ubio 50 % stanica. Nakon sakupljanja i ekstrakcije, metaboliti su analizirani GC-MS metodom koja im ujedno omogućava identifikaciju. Dobiveni su kromatogrami i tablice s određenim podatcima te su uspoređene koncentracije identificiranih metabolita u dvije stanične linije raka dojke i zaključeno je do kojih je metaboličkih promjena došlo.Proliferation, differentitation and survival of individual cells in a multicellular organism is carefully regulated, while in cancer cells there is no such regulation, they grow and divide uncontrollably, spread throughout the body and interfere with the function of normal tissues and organs. Cancer is the leading cause of death in the world. The most important thing in cancer pathology is to distinguish between benign and malignant tumors. Another way of classification is according to the type of cells from which the cancer is composed, according to which the main subtypes are carcinomas, sarcomas, lymphomas, leukemias and myelomas. Breast cancer is the leading cause of cancer-related deaths among women. Most breast tumors have a significant number of receptors for estrogen, progesterone and human epidermal growth factor 2 (HER2) and are classified as triple-positive or triple-negative. Treatment can be local or systemic or most often a combination of them. Cell line is a set of cells that is permanently established cell culture grown in a suitable medium in which it will proliferate indefinitely under controlled conditions. Two breast cancer cell lines, MDA-MB-231 and MCF-7, and the bicyclic sesquiterpene nootkatone were used in this diploma thesis. Its antiproliferative activity on two cancer cell lines was investigated in previous research using the MTT-assay, which proved that nootkatone has a good overall antiproliferative effect. Metabolomics is the study of all metabolites in a cell, tissue or organism. This includes the identification and quantification of cellular metabolites. Here, a procedure based on quenching of cells, harvesting and extraction of metabolites was used. It was applied on two breast cancer cell lines, MDA-MB-231 and MCF-7 with and without the addition of nootkatone (control). Nootkatone concentration of 0,5 mmol/L was taken, at which nootkatone successfully killed 50 % of the cells by previous use of the MTT-assay. After harvesting and extraction, the metabolites were analyzed by the GC-MS method, which also enables their identification. Chromatograms and tables with specific data were obtained, the concentrations of identified metabolites in two breast cancer cell lines were compared and it is concluded which metabolic changes occured