In vitro investigation of antitumor effect and mechanism of action of the chalcone analogues on human tumor cells

[J. D. Luković]

129 listova

Uvod: Halkoni su heterociklična jedinjenja koja predstavljaju prekursore u biosintezi flavonoida i drugih biološki aktivnih heterocikličnih jedinjenja. Hemijska struktura halkona, koju karakterišu dva aromatična prstena povezana α, β – nezasićenim karbonilnim sistemom sa tri ugljenikova atoma, omogućava širok spektar bioloških aktivnosti od kojih su najznačajnija antitumorska, antiinflamatorna i antiokidativna dejstva. Cilj: Cilj ovog istraživanja bio je ispitivanje potencijalnog antitumorskog efekta i mehanizma delovanja sedam analoga halkona (H1, H2, H3, H4, H5, H6, H7) na tri različite vrste humanih tumorskih ćelija (HeLa, HCT-116 i MDA-MB-231 ćelijske linije) i na zdrave fibroblaste kao kontrolne ćelije (MRC-5). Kao kontrolne supstance korišćene su cisplatina (cisPt) i dehidrozingeron (DHZ). Metode: Citotoksični efekat analiziran je upotrebom MTT testa nakon 24 i 48 časovne inkubacije tumorskih ćelija različitim koncentracijama ispitivanih halkona H1-H7 i referentnih supstanci (cisPt i DHZ). Tip ćelijske smrti određen je korišćenjem protočne citometrije (flow) korišćenjem bojenja Annexin V-FITC/7-AAD, dok je ekpresija, aktivacija i lokalizacija regulatornih proteina apoptoze određena korišćenjem metoda protočne citometrije i imunofluorescentne mikroskopije. Kao deo ekperimenta, metodom protočne citometrije analiziran je uticaj autofagije na promenu procenta citotoksičnosti ispitivanih halkona. Rezultati: Ispitivani halkoni H1-H7 pokazuju snažnu antitumorsku aktivnost koja je u većem broju parametara efikasnija u odnosu cisplatinu. Takođe, ispitivani halkoni indukuju apoptozu tumorskih ćelija putem aktivacije mitohondrijalnog apoptotskog puta, pri čemu halkoni rezultuju povećanje ekspresije regulatornih apoptotskih proteina u tretiranim tumorskim ćelijama u odnosu na netretirane, kontrolne ćelije. Ispitivani halkoni u određenoj meri indukuju autofagiju u tumorskim ćelijama što doprinosi njihovoj citotoksičnosti.

Introduction: Chalcones represents precursors in the biosynthesis of flavonoids, isoflavonoids and other biologically active heterocyclic compounds. The chemical structure of the chalcones is characterized by two aromatic rings connected by α, β – unsaturated carbonyl system with three carbon atoms. Chemical structure of chalcones is responsible for their antitumor, antiinflammatory and antioxidant effects. Aim: The main goal of this study was examine cytotoxic and apoptotic effect of the seven chalcone analogues (Х1, Х2, Х3, Х4, Х5, Х6, Х7) on three different types of human tumor cells (HeLa - human cervical cancer, HCT-116 - human colon cancer and MDA-MB-231 - human breast adenocarcinoma) and healthy fibroblasts as control cells (MRC-5). Method: The cytotoxic effect was analyzed using the MTT assay after 24 and 48 hour incubation of tumor cells at different concentrations of the tested Х1-Х7 chalcones and reference substances (cisPt and ДХЗ). Cell death type (relative ratio of apoptotic and necrotic cells) was determined using flow cytometry using Annexin V-FITC / 7-AAD staining, while expression, activation and localization of apoptosis regulatory proteins were determined using flow cytometry and immunofluorescence microscopy. As part of the experiment, by flow cytometry was analyzed the contribution of autophagy in the process of apoptosis induced by the tested chalcone analogues was etermined. Results: The examined chalcones Х1-Х7 show strong antitumor activity which is more effective compared to cisplatin. Also, the examined chalcones Х1-Х7 induce cell death via activation of apoptosis in the tumor cells. The investigated chalcones achieve an effective apoptotic effect by activating the mitochondrial apoptotic pathway, resulting increased in expression of regulatory apoptotic proteins in treated tumor cells compared to untreated, control cells. Also, the examined chalcones to induce autophagy in tumor cells, which contributes to their cytotoxicity.

analozi halkona, antitumorski efekat, citotoksičnost, apoptoza, humane tumorske ćelije

chalcone analogues, antitumor effect, cytotoxicity, apoptosis, human tumor cells

577.1:616-006.6(043.3)

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