The impact of glucose and glutamine on the level of phosphoglycerate-dehidrogenase in human malignant cells in vitro

Zloćudni tumori nastaju zbog poremećaja regulacijskih mehanizama, neophodnih za rast i dijeljenje stanice. Nerijetko su im u podlozi nastanka mutacije u različitim genima, najčešće protoonkogenima i tumorsupresorskim genima. Početak zloćudne preobrazbe povezan je s nastankom klona brzoproliferirajućih stanica, na koji se nastavljaju evolucija i selekcija klonova s najsnažnijim replikacijskim potencijalom. Zloćudni tumori posjeduju deset temeljnih obilježja, među kojima je i reprogramirani metabolizam. Metabolička aktivnost stanica zloćudnog tumora promijenjena je u odnosu na zdrave stanice od kojih je tumor nastao, a naročito se intenzivno istražuje u odnosu na metabolizam glukoze i glutamina. Za razliku od zdravih stanica koje svoje metaboličke puteve preusmjeravaju u glikolizu u anaerobnim uvjetima, stanice zloćudnih tumora sklone su metaboliziranju velikih količina glukoze glikolizom u uvjetima u kojima je opskrba kisikom optimalna. Ovaj se fenomen naziva aerobna glikoliza ili Warburgov učinak. Osim glukoze, stanice zloćudnih tumora imaju i veliku potrebu za glutaminom, koji služi kao izvor dušika u biosintetskim reakcijama. Biosinteza i iskorištavanje serina u metabolički reprogramiranim stanicama smatra se iznimno važnim za njihovu proliferaciju. Prvi enzim biosinteze serina je fosfoglicerat dehidrogenaza (PHGDH), čija je povećana ispoljenost, kao posljedica amplifikacije dijela kromosoma, dokazana u nekim vrstama zloćudnih tumora. Cilj ovog istraživanja bio je provjeriti postojanje stanično specifične ispoljenosti PHGDH na razini transkripta (RT-qPCR), proteina i u odnosu na unutarstanični smještaj proteina (Western blot), u odgovoru na dvije vrste gladovanja, u tri linije stanica podrijetlom od tumora glave i vrata, te debelog crijeva. Rezultati pokazuju da se ispoljavanje ciljnog proteina u sve tri linije stanica pojačava u gladovanju. Ova promjena može (FaDu), i ne mora (Detrot562, HT29) biti praćena povećanjem transkripcijske aktivnost gena PHGDH. Neočekivano je otkriveno da u linijama stanica podrijetlom od tumora glave i vrata (FaDu, Detroit562), PHGDH u gladovanju ulazi u jezgru, dok ovaj fenomen izostaje u stanicama podrijetlom od karcinoma debelog crijeva (HT29).The occurrence of malignant tumors is consequential to disturbed regulatory mechanisms that are needed for cellular growth and division. Mutations of protooncogenes and tumor suppressor genes are frequently associated with their genetic background. Initiation of malignant transformation relates to the onset of a highly proliferative clone, while its evolution depends on the selection of clones with the most prominent replicative potential. Reprogrammed metabolism is one of ten basic cancer hallmarks. Metabolic activity of cancer cells is changed with respect to the cells from which specific cancer originates. Currently, the most intense research efforts are focused on the metabolism of glucose and glutamine. Contrary to untransformed cells which utilize glycolytic pathways when deprived of oxygen, malignant cells utilize a high amount of glucose through the same pathway, even when sufficient oxygen is available. This phenomenon is known as aerobic glycolyisis or the Warburg effect. In addition to glucose, malignant cells need glutamine, which they use as a source of nitrogen in biosynthetic reactions. Serine metabolism is considered to be of utmost importance for the proliferation of malignant cells. The first enzyme of serine biosynthesis is phosphoglycerate dehydrogenase (PHGDH). This enzyme is highly expressed in some malignant tumors, as a consequence of chromosome amplification. The objective of this research was to explore the cellular specific expression of PHGDH at the level of the gene transcriptional activity (RT-qPCR), and the level and localization of the protein product, respectively, in three cell lines originating from head and neck malignant tumors and colon carcinoma, exposed to two types of starvation. In all three cell lines, exposure to starvation is associated with an increased level of the PHGDH protein. This change may (FaDu) and may not (Detroit562, HT29) be related to transcriptional activity of the PHGDH gene. During starvation, PHGDH unexpectedly translocates into the nucleus of the head and neck originating cell lines (FaDu, Detroit562), while the effect is absent in colon cancer originating HT29 cells