The influence of monoaza- and hexaaza-crown ethers on the modulation of P-glycoprotein activity

Velik problem u liječenju tumora predstavlja otpornost stanica na kemoterapiju. Najviše istražen mehanizam otpornosti jest povećano izbacivanje lijeka iz stanice pomoću P-glikoproteina (P-gp). P-gp (MDR1, ABCB1) je protein veličine 170 kDa iz obitelji ABC transportera koji sudjeluju u izbacivanju različitih toksina i ksenobiotika te na taj način štite stanicu od štetnih utjecaja. Međutim, P-gp je često pretjerano eksprimiran na površini brojnih tumorskih stanica, uslijed čega dolazi do otpornosti na kemoterapiju i smanjenog odgovora na liječenje. Jedan od smjerova u istraživanju liječenja tumora je usmjeren na sintezu i ispitivanje spojeva koji će djelovati kao inhibitori ove pumpe. U ovome je radu ispitivano djelovanje osam spojeva iz grupe monoaza- (641, 642, 643 i 644) i heksaaza- (1188, 1189, 1191, 1192) krunastih etera, sintetiziranih u laboratoriju Instituta Ruđer Bošković, na aktivnost P-gp-a. Kao model korištene su stanične linije karcinoma ovarija čovjeka bez (A2780) te s povećanom ekspresijom P-gp-a (A2780/Adr). Korištene stanične linije pokazuju otpornost prema različitim supstratima ove pumpe, kao što su doksorubicin i paklitaksel. Ispitivana su antiproliferativna svojstva spojeva pomoću metaboličkog testa metil tiazolil tetrazolij, nakon čega je inhibitorno svojstvo na P-gp pumpu ispitano metodom efluksa rodamina protočnom citometrijom, uz korištenje verapamila, kao pozitivne kontrole. Potencijalna inhibitorna aktivnost spoja s najjačom aktivnosti (644) dodatno je testirana kombiniranim tretmanom s paklitakselom i testinim spojem, prateći eventualno pojačanje djelovanja paklitaksela u otpornim stanicama.The development of drug resistance to chemotherapeutic agents remains one of the primary obstacles in cancer treatment. One of the most studied mechanisms of resistance is the increase of drug efflux by P-glycoprotein (P-gp). P-gp (MDR1, ABCB1) is a 170 kDa protein that belongs to the ATP-binding cassette superfamily of membrane transporters. It is involved in protecting the cell against harmful xenobiotics and other cytotoxic compounds, which it expels out from the cell. However, P-gp is often overexpressed on the surface of numerous tumor cells resulting in the resistance to chemotherapy and reduced response to cancer treatment. One of the approaches in tumor therapy is the development of compounds that inhibit P-gp activity and thus reverse the resistance. The aim of this work was to test the potential P-gp inhibitory activity of eight compounds: monoaza- (641-644) and hexaaza- (1188-1192) crown ethers, synthesized at Ruđer Bošković Institute. We used human ovarian carcinoma cell line A2780 without, as well as A2780/Adr with increased expression of P-gp. The latter cell line is resistant to various P-gp substrates, such as doxorubicin and paclitaxel. The antiproliferative abilities of the compounds were tested using MTT assay, while the inhibitory capacity on the P-gp activity was tested with the rhodamin 123 efflux assay, using flow cytometry and verapamil as a positive control. The potential inhibitory capacity of the compound which showed the highest activity (644) was additionally tested with the combination of paclitaxel and the test compound to prove a potential enhancement of paclitaxel activity in the resistant cells