Antitumorski potencijal i uticaj na mitohondrijsku respiraciju L-karnozina i njegovih formulacija primenjenih na kontinuiranim ćelijskim linijama

Abstract

Uvod: Proces karcinogeneze može se javiti u svakoj ćeliji, tkivu ili organu što posledično uzrokuje niz patoloških promena koje rezultuju nastankom velikog broja karcinoma. Zapažanje da mitohondrijska disfunkcija ili nastali defekti mogu biti potencijalni uzrok nastanka tumorskih promena podstaklo je brojne istraživače da polje svog interesovanja usmerene na ispitivanje patogeneze i uloge mitohondrija u nastanku različitih tumora. Karnozin (β-alanil-l- histidin) je prirodni dipeptid prisutan u mnogim tkivima poput mišićnog, plućnog, tkiva oka, zatim u mozgu, bubrezima i jetri. Pored poznatih brojnih uloga koje ispoljava ova supstanca navode se i antioksidativna kao i antitumorska sposobnost koje se ogledaju na više nivoa delovanja. Ove funkcije L-karnozina su ispitivane na mnogobrojnim kontinuiranim ćelijskim linijama kao i životinjskim modelima. Cilj: Doktorska disertacija je koncipirana sa ciljem da se utvrdi antitumorski potencijal čiste supstance L-karnozina kao i preparata Karnozin EXTRA® i Now® L-karnozin u in vitro uslovima na kontinuiranim tumorskim linijama humanog adenokarcinoma dojke (MCF-7) i humanog glioblastoma (U87) anlizirajući uticaj na morfološke karakteristike, proliferativnu sposobnost, eskprimiranost proteina vimentina (VIM) i superoksid-dizmutaze (SOD2), migratornu sposobnost, kao i uticaj na bioenergetski profil. Kako bi se sagledali mogući efekti ovih preparata na organizam čoveka čitav koncept studije sproveden je i na zdravoj ćelijskoj liniji bubrega hrčka (BHK-21/C-13) kao i na ćelijama humanih fetalnih fibroblasta pluća (MRC-5). Materijal i metode: Ispitivanje antitumorskog potencijala sprovedeno je putem opsežnog sagledavanja brojnih mogućih nivoa na kojima karnozin ispoljava svoje delovanje. Ispitivanje morfoloških karakteristika vršeno je nakon standardnog bojenja tumorskih i zdravih ćelija hematoksilinom i eozinom kao i upotrebom faznog kontrasta. Uticaj na proliferativnu sposobnost analiziran je upotrebom MTT testa. Primenom „scratch" eseja praćena je migratorna sposobnost tretiranih ćelija. Nakon imunofluoresentnog bojenja primenom anti-VIM i anti-SOD2 antitela kvantifikovani su rezultati ukupnog intenziteta fluorescencije. I u poslednjem delu eksperimentalne studije vršeno je određivanje parametara mitohondrijske respiracije “intaktnih ćelija”, određivanje koncentracije digitonina potrebne za permeabilizaciju ćelijske membrane i na kraju određivanje parametara mitohondrijske respiracije ćelija nakon permeabilizacije ćelijske membrane. Rezultati: Primena čiste supstance L-karnozina kao i preparata Karnozin EXTRA® i Now® L-karnozin uslovila je izmene morfoloških svojstava u pogledu oblika i veličine, inhibiciju proliferativne sposobnosti tumorskih ćelija kao i inhibiciju migracije ćelija koje ovu sposobnost poseduju (U87). Uticaj na ekprimiranost proteina vimentina u citoplazmi U87 ćelija nakon tretmana preparatom Karnozin EXTRA® bila je evidentna (p<0,0001), dok primena čiste supstance kao i preparata Now® L-karnozin nisu imali efekta. Analizom uticaja ispitivanih preparata na nivo ekspresije oksidativnog (SOD2) markera utvrđeno je da sprovedeni tretmani uzrokuju smanjenje nivoa eksprimiranosti u mitohondrijama MCF-7 ćelija (p<0,0001) dok na U87 ćelijama ne ispoljavaju značajnije promene. Takođe, ustanovljeno je da su sprovedeni tretmani kod ćelija obe tumorske linije doveli do inhibicije produkcije energije putem delovanja na komponente respiratornog lanca mitohondrija (p<0,0001). U pogledu delovanja na zdravim ćelijama sprovedeni tretman čistom supstancom L-karnozin kao i preparatom Now® L-karnozin nije uslovio izmene morfoloških svojstava i proliferativne spososbnosti, dok je preparat Karnozin EXTRA® izazvao suprotne efekate kada je u pitanju ćelijska linija MRC-5 (p<0,0001). Preparat Karnozin EXTRA® dovodi do povećanja ekprimiranosti proteina VIM u svim koncetracijama kao i porasta ekspresije SOD2 markera pri koncentracijama karnozina od 5 i 10 mM kod obe zdrave ćelijske linije (p<0,0001). Tretman preparatom Now® L-karnozin kod BHK-21/C-13 uslovljio je smanjenje dok kod MRC-5 ćelija porast SOD2 markera (p<0,0001). Sprovedeni tretmani pri koncentraciji od 2 i 5 mM karnozina uslovili su povećanje produkcije energije putem stimulacije aktivnosti kompleksa respiratornog lanca mitohondrija kao i maksimalnog kapaciteta elektron transportnog sistema kod BHK-21/C-13 ćelija (p<0,0001), dok je koncentracija od 10 mM karnozina ispoljila inhibitorne efekte (p<0,0001). Inkubacija MRC-5 ćelija sa čistom supstancom L-karnozin kao i preparatom Now® L-karnozin pri svim ispitivanim koncentracijama dovela je do inhibicije aktivnosti kompleksa i respiratornog lanca mitohondrija uz posledično smanjenje vrednosti maksimalnog kapaciteta elektron transportnog sistema, dok je primena preparata Karnozin EXTRA® uzrokovala inhibiciju produkcije energije delujući na svim nivoima respiratornog lanca mitohondrija. Zaključak: Antitumorski potencijal i citotoksični efekti ispitivanih preparata na kontinuiranim tumorskim linijama dokazani su putem delovanja karnozina na više ćelijskih nivoa. Međutim, tačan molekularni mehanizam još uvek je nepoznanica. Rezultati izneti u ovoj doktorskoj disertaciji na tumorskim ali i zdravim ćelijskim linijama govore o potrebi za daljim in vivo ali i kliničkim istaživanjima a sve u svrhu dalekosežnog cilja buduće primene ovih preparata kako u preventivne a tako i u terapijske svrhe oboljenja koja pogađaju humanu populaciju.

Introduction: The process of carcinogenesis may be developed in any cell, tissue or organ, which consequently causes a series of pathological changes resulting in different cancers. Mitochondrial dysfunction or defects could be a potential cause of tumour changes, which has encouraged numerous researchers to focus their field of interest on examining the pathogenesis and role of mitochondria in the development of various tumours. Carnosine (β- alanyl-l-histidine) is a natural dipeptide present in many tissues such as muscles, lungs, eyes, brain, kidneys and liver. In addition to the well-known numerous roles, there are also an antioxidant and antitumour potential of L-carnosine, which are reflected on several levels of action. These functions of L-carnosine have been investigated on numerous continuous cell lines as well as on animal models. The aim: This thesis was designed to determine the antitumour potential of the pure substance L-carnosine as well as the supplements Karnozin EXTRA® and Now® L-carnosine in vitro on continuous tumour cell lines of human breast adenocarcinoma (MCF-7), and human glioblastoma (U87), analyzing the effects on morphological features, proliferative capacity, vimentin (VIM), and superoxide dismutase (SOD2) protein expression, migratory ability, as well as the effect on their bioenergetic profile. To understand the possible effects of these formulations, the entire concept of the study were carried out on the healthy hamster kidney cell line (BHK-21/C-13), and on human fetal lung fibroblast cells (MRC-5). Material and methods: The evaluation of antitumour potential was conducted by analyzing a number of possible levels on which carnosine exerts its action. Morphological changes were analyzed after standard staining of tumor and healthy cells with hematoxylin and eosin, and by using phase contrast. The proliferative capacity was analyzed using the MTT test. The migratory ability of the treated cells was monitored by "scratch" assay. Using immunofluorescent staining of cells with anti-VIM and anti-SOD2 antibodies, the total fluorescence intensity was determined. In the last part of the experimental study we analyzed the "intact cells" respiration, digitonin concentration required for cell membrane permeabilization, and activities of mitochondrial respiratory complexes. Results: Application of the pure substance of L-carnosine, and the Karnozin EXTRA® and Now® L-carnosine resulted in changes in morphological characteristics in terms of shape and size. Inhibition of the proliferative capacity of both tumour cell lines and migration of U87 cells were also presented. The effect on the VIM expression in the cytoplasm of U87 cells after treatment with the Karnozin EXTRA® was evident (p<0.0001), while the application of the pure substance and the Now® L-carnosine had no effect. Analysis of the oxidative (SOD2) marker level revealed that the treatments caused a decrease in the MCF-7 cells (p<0.0001), while no significant changes were observed in U87 cells. Also, it was established that the treatments of both tumour lines led to the inhibition of energy production by acting on the components of the mitochondrial respiratory chain. Regarding the effect on healthy cells, the treatment with the pure substance of Lcarnosine, and Now® L-carnosine did not change the morphology and proliferative capacity, while the Karnozin EXTRA® caused the opposite effects on the MRC-5 cell line. Treatment with Karnozin EXTRA® led to an increase in the expression of VIM at all concentrations (p<0.0001), as well as an increase in the SOD2 expression at the carnosine concentrations of 5 and 10 mM in both healthy cell lines (p<0.0001). Treatment with Now® Lcarnosine in BHK-21/C-13 cells decreased, while in MRC-5 cells increased SOD2 expressions (p<0.001). Treatments carried out at concentrations of 2 and 5 mM of carnosine led to an increase in energy production by stimulating the activity of the mitochondrial respiratory chain complex as well as the maximum capacity of the electron transport system in BHK-21/C-13 cells (p<0.0001), while the concentration of 10 mM carnosine exhibited inhibitory effects (p<0.0001). Incubation of MRC-5 cells with the pure substance Lcarnosine as well as the preparation Now® L-carnosine at all tested concentrations led to the inhibition of the activity of complex I of the mitochondrial respiratory chain with a consequent decrease in the value of the maximum capacity of the electron transport system, while the application of the preparation Karnozin EXTRA® caused the inhibition of energy production by acting on all levels of the mitochondrial respiratory chain (p<0.001). Conclusion: The antitumour potential and cytotoxic effects of the testedLcarnosine formulations on continuous tumour cell lines were proven through the its action on several cellular levels. However, the exact molecular mechanism is still unraveled. The results presented in this thesis on tumour and healthy cell lines indicate for further in vivo and clinical studies, all for the far-reaching goal of the future application of these formulations for both preventive and supportive treatment of diseases that affect the human population.