Analiza antitumorskog delovanja agenasa modifikovanih azot-monoksidom, Sakvinavir-NO i GIT-27NO na kanceru kolona in vitro i in vivo

Abstract

Iz poznate uzročno-posledične veze infekcije i inflamacije, s jedne strane i karcinogeneze i progresije tumora, s druge strane, proistekla je ideja o upotrebi anti-inflamatornih i anti-infektivnih agenasa u terapiji kancera. Međutim, problem gastrotoksičnosti anti-inflamatornih, kao i generalne toksičnosti anti-viralnih lekova uz činjenicu da je reč o agensima niske biološke iskoristljivosti, ozbiljne su barijere u razmatranju njihove primene u terapiji bolesti za koje inicijalno nisu namenjeni. Kako bi se prevazišla farmakološka ograničenja pomenutih jedinjenja, a imajući u vidu poznato svojstvo azot-monoksida (NO) da neutrališe toksičnost supstanci na različitim nivoima, anti-inflamatornom agensu VGX-1027 i anti-retroviralnom agensu sakvinaviru (Saq) adekvatnom hemijskom intervencijom je dodata grupa dizajnirana da donira NO. Prednost novodobijenih jedinjenja GIT-27NO i Saq-NO nad poznatim nesteroidnim anti-inflamatornim agensima modifikovanim kovalentnim vezivanjem NO (NO-NSAID) je odsustvo molekula “nosača”, koji je inače odgovoran za genotoksičnost NO-NSAID. Strukturna promena ova dva jedinjenja doprinela je njihovom snažnom antitumorskom svojstvu, što je potvđeno brojnim in vitro i in vivo studijama, uz umanjenu toksičnost u slučaju GIT-27NO ili potpunom gubitku toksičnosti Saq-NO tretmana.U ovoj studiji je po prvi put ispitano antitumorsko delovanje GIT-27NO i Saq-NO in vitro i in vivo na modelu kancera kolona, jednog od najtežih formi maligniteta. Kako su oba agensa NO-derivati, definisana je uloga NO u njihovom antitumorskom delovanju. Na molekularnom nivou, determinisani su glavni unutarćelijski događaji pokrenuti u odgovoru na tretman pomenutim jedinjenjima. Na kraju, imajući u vidu značaj mikrosredine za rast i progresiju tumora, ispitana je sposobnost GIT-27NO i Saq-NO da povrate osetljivost ćelija kancera kolona na antitumorski imunski odgovor posredovan TRAIL molekulom.

Concept of using anti-inflammatory and anti-infectious agents in cancer therapy is based on a known relationship between inflammation and infection on one side and carcinogenesis and tumor progression on the other side. However, the problem of gastrotoxicity of anti-inflammatory and general toxicity of antiviral drugs, along with their low bioavailability, marks a strong barrier when considering them in therapies for which they were not initially intended for. In order to overcome the pharmacological limits of these compounds, nitric-oxide (NO) with its documented feature to neutralize toxicity of drugs on various levels was added to anti-inflammatory agent VGX-1027 and antiretroviral agent saquinavir (Saq). The advantage of new compounds, GIT-27NO and Saq-NO, over known non-steroid anti-inflammatory agents modified by covalent binding NO moiety (NO-NSAID) is the absence of “carrier” molecule, which is responsible for genotoxicity of NO-NSAID. Structural change in these compounds resulted in potentiation of antitumor action, confirmed by numerous in vitro and in vivo studies, along with reduced toxicity of GIT-27NO or complete loss of toxicity of Saq-NO treatment. In this study, antitumor potential of GIT-27NO and Saq-NO was tested in vitro and in vivo for the first time in colon cancer model, one of the most severe forms of malignancy. As both agents are NO-derivates, the role of NO in their antitumor action was defined. On molecular level, main intercellular events triggered by the treatment were determined. Finally, considering the importance of microenvironment for tumor growth and progression, the ability of GIT-27NO and Saq-NO to re-establish colon cancer cell sensitivity to TRAIL-mediated antitumor immune response was tested. GIT-27NO and Saq-NO reduced the viability of mouse CT26CL25 and human HCT116 colon cancer cell lines, both in vitro and in vivo. The importance of NO release for antitumor action was quite different. While cell viability reduction under GIT-27NO treatment was due to accumulation of high intracellular concentration of NO and consecutively generated oxidative and nitrosative stress, antitumor action of Saq-NO was not mediated by a release of quantitatively relevant amount of this free radical. GIT-27NO induced the accumulation of p53 tumor suppressor, changed pro- and antiapoptotic molecule ratio and triggered mitochondrial membrane depolarization which resulted in cell death via caspase-dependent apoptosis.