Efekat bioaktivnih supstanci na vijabilnost limfocita hronične limfocitne leukemije

Abstract

Hronična limfocitna leukemija (HLL) nastaje zbog poremećaja ravnoteže između apoptoze i proliferacije B limfocita. Preko 70% supstanci koje se koriste u lečenju tumora su ili supstance prirodnog porekla ili supstance dobijene modifikacijom prirodnih supstanci. Osnovni cilj ovog istraživanja bio je da se ispita efekat dve različite vrste propolisa, hrizina i metanolskih ekstrakata biljaka Ligustrum vulgare i Teucrium pollium i gljiva Phellinus linteus i Cordyceps sinensis na vijabilnost ćelija leukemijskih ćelijskih linija MOLT-4 i JVM-13, na vijabilnost limfocita dobijenih iz krvi pacijenata obolelih od HLL-a, kao i na vijabilnost mononuklearnih leukocita izolovanih iz krvi zdravih osoba (kontrola). Uticaj navedenih bioaktivnih supstanci na vijabilnost ćelija MOLT-4 ćelija i JVM-13 ćelija, HLL limfocita, kao i na vijabilnost zdravih mononuklearnih leukocita je ispitan MTT testom i LDH testom. Tip ćelijske smrti izazvan dejstvom ispitivanih supstanci je određen Annexin V/7AAD testom. Ćelijska lokalizacija i količina proteina uključenih u proces apoptoze Bax, Bcl-2, citohroma s i kaspaze-3 je određena metodama protočne citometrije i fluorescentnom mikroskopijom. Istraživanje je pokazalo da hrizin, metanolski ekstrakti lista i ploda biljke Ligustrum vulgare i biljke Teucrium pollium selektivno smanjuju vijabilnost leukemijskih ćelija a ne utiču na zdrave mononuklearne leukocite. Propolisi i metanolski ekstrakt gljive Cordyceps sinensis smanjuju vijabilnost leukemijskih ćelija, ali utiču na vijabilnost i zdravih mononuklearnih leukocita, a metanolski ekstrakt gljive Phellinus linteus smanjuje vijabilnost ćelija leukemijskih ćelijskih linija MOLT-4 i JVM-13, ali povećava vijabilnost limfocita hronične limfocitne leukemije i zdravih mononuklearnih leukocita. Hrizin, metanolski ekstrakti lista i ploda biljke Ligustrum vulgare i metanolski ekstrakt biljke Teucrium pollium indukuju apoptozu limfocita hronične limfocitne leukemije. Hrizin i metanolski ekstrakt biljke Teucrium pollium povećavaju relativni odnos Bax/Bcl-2, što dovodi do translokacije citohroma s iz mitohondrija u citoplazmu, aktivacije kaspaze-3 i indukcije apoptoze limfocita hronične limfocitne leukemije. Hrizin i metanolski ekstrakt biljke Teucrium pollium indukuju apoptozu HLL limfocita mitohondrijalnim putem in vitro. Iako su dodatna ispitivanja potrebna da bi se precizno rasvetlio kompletan mehanizam uključen u proces apoptoze HLL limfocita koju indukuju hrizin i metanolski ekstrakt biljke Teucrium pollium, ovi agensi imaju obećavajuću ulogu kao budući hemoterapeutici.

Chronic lymphocytic leukemia (CLL) develops due to an imbalance between apoptosis and proliferation of B lymphocytes. More than 70% of compounds used in the treatment of different types of cancers are derived from natural compounds or produced by modification of natural compounds. Therefore, in our investigation we examined the cytotoxicity of two different types of propolis, chrysin, methanol extracts of plants Ligustrum vulgare and Teucrium pollium and fungi Phellinus linteus и Cordyceps sinensis against two leukemia cell lines, MOLT-4 and JVM-13, peripheral blood lymphocytes isolated from CLL patients and peripheral blood mononuclear cells (PBMCs) from healthy individuals (control) in vitro. The effects of these bioactive compounds on viability of MOLT-4 and JVM-13 cell lines, CLL cells derived from CLL patients and PBMCs from healthy subjects were determined by MTT assay and LDH assay. The type of cell death induced by examined compounds was verified by Annexin V/7AAD assay. Intracellular localization and endogenic expression of apoptotic proteins including Bax, Bcl-2, cytochrome c and caspase-3 were determined by flow cytometry and fluorescent microscopy. Our results demonstrated that exposure of MOLT-4 and JVM-13 cell lines and CLL cells to chrysin, leaf and fruit methanol extracts of Ligustrum vulgare and methanol extract of Teucrium pollium, selectively decreased viability of these cells populations, but not in the PBMCs derived from healthy subjects. Both types of propolis and methanol extract of Cordyceps sinensis decreased viability of leukemic cells, but also influenced on viability of PBMCs from healthy subjects. Methanol extract of Phellinus linteus decreased viability of MOLT-4 and JVM-13 cell lines, but increased viability of CLL cells and PBMCs from healthy subjects. Chrysin, leaf and fruit methanol extracts of Ligustrum vulgare and methanol extract of Teucrium pollium induced apoptosis of CLL cells. Our findings demonstrated that chrysin and methanol extract of Teucrium pollium induced the activation of proapoptotic Bax and decrease in the expression of antiapoptotic Bcl-2 protein, induced the release of cytochrome c from mitochondria into cytosol and cleavage/activation of caspase-3, subsequently leading to the activation of apoptosis of CLL cells. Together, these findings suggest that chrysin and methanol extract of Teucrium pollium selectively induce apoptosis of peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia patients via mitochondrial pathway in vitro and that these compounds might have a promising roles as a potential future antileukemic remedies.