doktorska disertacija
Tjaša Vižin (Avtor), Janko Kos (Mentor)

Povzetek

Encimi glikolitične poti že nekaj časa veljajo za večfunkcijske molekule, ki poleg glikolize izvajajo še številne druge regulatorne funkcije. Gama-enolaza (nevronska specifična enolaza) je encim glikolitične poti, ki se prekomerno izraža v nevrogenih in nevroendokrinih tumorjih in je dobro poznan tumorski označevalec. S svojo glikolitično aktivnostjo prispeva k pospešeni aerobni glikolizi, glavnemu viru energije tumorskih celic. Po drugi strani pa lahko preko dodatnega aktivnega mesta na C-koncu molekule deluje kot dejavnik preživetja, kot je bilo dokazano za nevronske celice. To funkcijo uravnava cisteinska proteaza katepsin X, ki odcepi C-končni dipeptid gama-enolaze. Katepsin X je bil potrjen kot pomemben promotor tumorskega procesa in je v napredovanju raka udeležen preko številnih mehanizmov. Obe molekuli sta prisotni v tumorskih celicah, zato lahko njihova interakcija pomembno vpliva na potek bolezni. Namen doktorskega dela je bil prispevati k razumevanju vloge gama-enolaze kot dejavnika preživetja v rakavih celicah in uravnavanja njenih učinkov s katepsinom X ter opredeliti medsebojno odvisnost, povezanost s kliničnimi in patološkimi parametri in uporabnost obeh molekul v diagnozi raka in napovedi izida bolezni pri bolnikih z rakom. V prvem delu smo na dveh adenokarcinomskih (Caco-2, MCF7) in dveh glioblastomskih (U-87 MG, U-251 MG) celičnih linijah raziskali pomen gama-enolaze v prilagoditvi in preživetju tumorskih celic v stresnih pogojih ter njeno uravnavanje s strani katepsina X. Celice smo izpostavili serumskemu stradanju ali gojišču z dodanim mimetikom hipoksije deferoksaminom in s prenosom po westernu ovrednotili spremembe v izražanju necepljene gama-enolaze (z intaktnim C-koncem) v odvisnosti od proteolitične aktivnosti katepsina X. V vseh analiziranih celičnih linijah se je izražanje necepljene gama-enolaze v primerjavi s celokupno gama-enolazo (necepljeno in cepljeno) bistveno bolj povišalo in je bilo povezano z aktivnostjo katepsina X. Posledično je utišanje izražanja gama-enolaze značilno zmanjšalo preživetje celic v stresnih pogojih. Utišanje gena za katepsin X ali inhibicija njegove proteolitične aktivnosti s specifičnim inhibitorjem AMS36 sta vodila v povišano izražanje necepljene gama-enolaze in posledično se je povečalo tudi preživetje celic v stresnih pogojih. To nakazuje, da je funkcija gama-enolaze kot dejavnika preživetja v rakavih celicah povezana z njeno C-končno necepljeno obliko, ki omogoča prilagajanje tumorskih celic na stresne pogoje; katepsin X pa to delovanje uravnava. Poleg tega smo ugotovili, da katepsin X, ki sicer velja za promotorja tumorskega procesa, zavira zaščitno delovanje gama-enolaze v rakavih celicah, kar nakazuje na njegovo večfunkcijsko vlogo pri raku. V drugem delu smo s prenosom po westernu in testi ELISA ovrednotili izražanje necepljene in celokupne gama-enolaze v različnih rakavih celičnih linijah (glioblastomskih: U-87 MG, U-373 MG, U-251 MG; nevroblastomskih: SH-SY5Y; celic raka debelega črevesa: Caco-2; nerakavih celic dojke: MCF 10A; celic raka dojke: MCF7) ter njuno odvisnost od izražanja in aktivnosti katepsina X. Poleg tega smo raziskali klinično uporabnost necepljene in celokupne gama-enolaze v serumu bolnikov z rakom debelega črevesa in danke. Najprej smo pokazali, da se necepljena gama-enolaza različno izraža v rakavih celičnih linijah, pri čemer je bilo njeno izražanje najvišje v tistih celičnih linijah, ki so bile pridobljene iz metastatskih mest (SH-SY5Y, MCF7) ali zelo invazivnih tumorjev (U-87 MG). Izražanje necepljene gama-enolaze je bilo obratno sorazmerno izražanju katepsina X v celicah, ki je bilo najnižje v bolj invazivnih celicah. Izražanje celokupne gama-enolaze se med različnimi celičnimi linijami ni bistveno razlikovalo. Ugotovili smo, da se gama-enolaza izloča v celični supernatant pretežno v necepljeni obliki in odraža njeno izražanje v celicah. Vrednosti necepljene in cepljene gama-enolaze smo ovrednotili tudi v serumu bolnikov z rakom debelega črevesa in danke. Z imunoprecipitacijo smo preliminarno ugotovili, da je necepljena gama-enolaza prevladujoča oblika tudi v serumu. V 264 serumih bolnikov z rakom debelega črevesa in danke smo s testom ELISA, ki prepoznava samo necepljeno gama-enolazo, in s testom ECLIA (Elecsys NSE Assay, Roche Diagnostics), ki prepoznava celokupno gama-enolazo, izmerili vrednosti obeh. Rezultati so pokazali, da vrednosti različnih oblik gama-enolaze v serumu niso povezane z nobenim kliničnim ali patološkim parametrom. Univariatna analiza preživetja je pokazala, da so višje vrednosti obeh oblik povezane s krajšim preživetjem bolnikov. V skupini bolnikov z metastatskim rakom debelega črevesa in danke (stadij IV), ki niso prejeli paliativne kemoterapije, so bile višje vrednosti obeh oblik značilno povezane s krajšim preživetjem tudi v multivariatni analizi, medtem ko pri bolnikih, ki so prejeli paliativno kemoterapijo, povezave s preživetjem ni bilo. Slednje nakazuje, da vrednosti gama-enolaze v serumu lahko napovejo izid bolezni in odgovor bolnikov z metastatskim rakom debelega črevesa in danke na paliativno kemoterapijo, kar bi bistveno izboljšalo obravnavo bolnikov. Nadaljnje študije na tem področju bi se morale osredotočiti na proučevanje izražanja necepljene gama-enolaze v vzorcih tumorjev, katere bi lahko pokazale dodatne povezave z različnimi kazalci napredovanja raka in omogočile izbor bolnikov z bolj agresivnim tumorskih fenotipom. V zadnjem delu doktorske disertacije smo ovrednotili klinično uporabnost katepsina X v serumu bolnikov z rakom debelega črevesa in danke. V ta namen smo razvili in validirali test ELISA, s katerim smo lahko merili celokupni katepsin X (prokatepsin X in aktivni katepsin X) v serumu. Najprej smo izvedli pilotno klinično študijo, ki je vključevala 77 bolnikov z rakom debelega črevesa in danke, 77 bolnikov, ki so imeli adenome po debelem črevesu, 77 bolnikov brez neoplastičnih sprememb ter 77 zdravih kontrol. Statistično značilnih razlik med posameznimi skupinami nismo opazili, kar pomeni, da celokupni katepsin X nima diagnostične vrednosti, prav tako ni bil povezan z nobenim kliničnim ali patološkim parametrom. Znotraj skupine bolnikov z rakom debelega črevesa in danke pa se je izkazalo, da so višje vrednosti celokupnega katepsina X značilno povezane s krajšim preživetjem bolnikov. Rezultate smo ovrednotili še s potrditveno (264 bolnikov) in validacijsko (460 bolnikov) klinično študijo. Na večjem številu vzorcev se je izkazalo, da so višje vrednosti celokupnega katepsina X značilno povezane s krajšim preživetjem v skupini bolnikov z nemetastatskim rakom (stadiji I-III). Za to skupino je multivariatna Coxova regresijska analiza pokazala močno povezavo med višjimi vrednostmi katepsina X in krajšim preživetjem tistih bolnikov, ki niso prejeli kemoterapije, medtem ko pri bolnikih, ki so prejeli kemoterapijo, razlik v preživetju ni bilo. Vrednosti katepsina X v serumu torej lahko napovejo izid bolezni in odziv na kemoterapijo pri bolnikih z nemetastatskim rakom debelega črevesa in danke, kar uvršča katepsin X med nove obetavne, serumske tumorske označevalce pri bolnikih z rakom debelega črevesa in danke. V okviru doktorskega dela smo prepoznali dodatno vlogo gama-enolaze, ki podpira prilagoditev in preživetje rakavih celic v stresnih pogojih. Razumevanje teh procesov skupaj z zaviranjem njene glikolitične aktivnosti bi predstavljalo nove možnosti pri zdravljenju raka. Prepoznana klinična uporabnost gama-enolaze in katepsina X pa odpira nove možnosti pri odkrivanju in zdravljenju rakavih bolnikov.

Ključne besede

rak (medicina);adenokarcinom;glioblastom;celične linije;gama-enolaza;katepsin X;tumorski označevalci;rakave celice;preživetje;stresni pogoji;utišanje genov;diagnostika;zdravljenje;disertacije;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [T. Vižin]
UDK: 577.15:616-006(043.3)
COBISS: 279301888 Povezava se bo odprla v novem oknu
Št. ogledov: 17
Št. prenosov: 3
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: ǂThe ǂrole of gamma-enolase in cancer and its regulation by proteolytic enzymes
Sekundarni povzetek: Enzymes of the glycolytic pathway have been considered as multifunctional molecules that besides its glycolytic activity perform various additional regulatory functions. Gamma-enolase (neuron-specific enolase) is an enzyme of the glycolytic pathway that is overly-expressed in tumours of neurogenic and neuroendocrine origin and is a well-known tumour marker. As an enzyme of the glycolytic pathway it contributes to accelerated aerobic glycolysis, the main energy source of tumour cells. On the other hand, gamma-enolase might act as a pro-survival factor, as has been demonstrated for neuronal cells. This function is achieved through an additional active site localized at the C-terminal end of the molecule and cathepsin X, a cysteine protease, was shown to regulate this function by proteolytic cleavage of the C-terminal dipeptide. Cathepsin X has been confirmed as an important promoter of tumour progression, which participates in a variety of cancer related mechanisms. Both molecules are present in tumour cells; therefore their interaction might have a significant impact on the disease course. The purpose of the doctoral thesis was to contribute to the understanding of the role of gamma-enolase as a survival factor in cancer cells and the regulation of its effects by cathepsin X and to define the interplay, relation with clinical and pathological parameters and utility of both molecules in diagnosis and prognosis of cancer. In the first part we investigated the importance of gamma-enolase in adaptation and survival of tumour cells exposed to stressful conditions and its regulation by cathepsin X. Two adenocarcinoma (Caco-2, MCF7) and two glioblastoma (U-87 MG and U-251 MG) cell lines were exposed to serum starvation or media with hypoxia mimetic desferrioxamine. Using western blot we evaluated the changes in expression of uncleaved gamma-enolase (with intact C-terminus) in relation to cathepsin X proteolytic activity. Uncleaved gamma-enolase, rather than total (including uncleaved and cleaved forms) gamma-enolase was significantly up-regulated and related to cathepsin X proteolytic activity in all analysed cell lines. Consequently, gamma-enolase silencing led to significantly lower cell survival in stressful conditions. Cathepsin X silencing or specific inhibition with AMS36 significantly increased uncleaved gamma-enolase expression and consequently, promoted cell survival in stressful conditions. The results suggest that gamma-enolase function as a survival factor in cancer cells is related to its intact C-terminal end, promoting adaptation of tumour cells to stressful conditions; and cathepsin X regulates this function. Cathepsin X, which has been considered as a promoter of tumour processes, here suppresses the protective function of gamma-enolase, which suggests its multi-function role in cancer. In the second part, using Western blot and ELISA, we evaluated the expression of uncleaved and total gamma-enolase in different tumour cell lines (glioblastoma: U-87 MG, U-373 MG, U-251 MG; neuroblastoma: SH-SY5Y; colon cancer: Caco-2, human breast fibrocystic disease cells: MCF 10A; breast cancer cells: MCF7) and their relation to cathepsin X expression and activity. In addition, we investigated the clinical applicability of both forms in sera from patients with colorectal cancer. We have shown that uncleaved gamma-enolase is differentially expressed in the analysed cell lines with the highest levels in those cell lines, which were derived from metastatic sites (SH-SY5Y, MCF7) or highly invasive tumours (U-87 MG). Uncleaved gamma-enolase expression was inversely related to cathepsin X expression in cells, the latter being the lowest in more invasive cells. Total gamma-enolase expression did not differ among the analysed cell lines. Gamma-enolase is excreted into the cell supernatant predominantly as uncleaved form reflecting its expression in cells. Uncleaved and total gamma-enolase levels were evaluated in serum from colorectal cancer. A preliminary immunoprecipitation showed uncleaved gamma-enolase to be the predominant form in serum. Using ELISA, which specifically recognizes uncleaved gamma-enolase and ECLIA (Elecsys NSE assay, Roche Diagnostics), which recognizes total gamma-enolase we measured the values of both forms in sera from 264 patients with colorectal cancer. Results showed no association with clinical or pathological parameters. However, univariate survival analysis of the entire set showed that higher levels of both forms associated with shorter patient survival. Additionally, in multivariate analysis for the group of patients with metastatic colorectal cancer (stage IV) who did not receive palliative chemotherapy, higher levels of both forms significantly associated with shorter survival, while in patients who received palliative chemotherapy, no relation to survival could be observed. The latter suggests that gamma-enolase levels in serum can predict the disease course and response of patients with metastatic colorectal cancer to palliative chemotherapy, which may lead to significant improvements in patients’ treatment. Future studies should focus on examining the expression of uncleaved gamma-enolase in tumour samples, which might provide additional relations to various indicators of cancer progression allowing selection of patients with more aggressive tumour phenotype. In the last part of the thesis we evaluated diagnostic applicability of cathepsin X in serum from patients with colorectal cancer. We developed and validated an ELISA assay which detects total cathepsin X (procathepsin X and active cathepsin X) in serum. First, we conducted a pilot clinical study that included 77 patients with colorectal cancer, 77 patients with adenomas of the colon, 77 patients without neoplastic changes and 77 healthy controls. No statistically significant differences between the groups were observed, which means that total cathepsin X has no diagnostic value. Also, no significant association to any clinical or pathological parameter could be detected. Within the group of patients with colorectal cancer, higher levels of total cathepsin X significantly associated with shorter patients’ survival. These results were further evaluated by a confirmatory (including 264 patients) and validation (including 460 patients) clinical study. A larger cohort of patients provided additional evidence that higher total cathepsin X levels significantly associate with shorter survival of patients with non-metastatic cancer (stages I-III). For this group, multivariate Cox's regression analysis showed a strong association of higher cathepsin X levels to shorter survival of patients who did not receive adjuvant chemotherapy, while in patients who received chemotherapy, no difference could be detected. Values of cathepsin X in sera could therefore predict the disease course and response to chemotherapy in patients with non-metastatic colorectal cancer. The latter classifies cathepsin X as a new promising blood-based tumour marker in colorectal cancer. In the doctoral thesis we have identified an additional role of gamma-enolase, which supports the adaptation and survival of cancer cells exposed to stressful conditions. Understanding these processes along with inhibiting gamma-enolase glycolytic activity represents new possibilities in cancer therapy. The recognized clinical utility of gamma-enolase and cathepsin X opens up new possibilities in diagnosis and treatment of cancer patients.
Vrsta dela (COBISS): Doktorsko delo/naloga
Komentar na gradivo: Univ. v Ljubljani, Fak. za farmacijo
Strani: 200 str.
ID: 17675852