¨P-glycoprotein 1 mediates Galectin-1-induced resistance to doxorubicin in hepatocellular carcinoma cells

CARABIAS P*, BACIGALUPO ML*, OTERO S*, SAFFIOTI N*, ELOLA MT*, WOLFENSTEIN C*, ROSSI JP*, RABINOVICH GA&, ESPELT MV* Y TRONCOSO MF*

Mar del Plata

Congreso; 241. LXI Reunión Científica Anual de la Sociedad de Investigación Clínica (SAIC) Mar del Plata; 2016

Sociedad Argentina de Investigación Clínica

P-glycoprotein 1 mediates Galectin-1-induced resistance to doxorubicin in hepatocellular carcinoma cells Carabias P*, Bacigalupo ML*,Otero S*, Saffioti N*, Elola MT*, Wolfenstein C*, Rossi JP*, Rabinovich GA&,Espelt MV* y Troncoso MF*.*IQUIFIB, Facultad de Farmacia y Bioquímica, UBA.&IByME-CONICET.carabiaspablo@gmail.com Galectin-1 (Gal1), a β-galactoside-binding protein, is overexpressed in hepatocellular carcinoma (HCC) and it is related to tumor aggressiveness. P-glycoprotein 1 (Pgp) is an ATP-dependent drug efflux pump localized in hepatocyte canalicular membrane. Its overexpression in tumor cells decreases intracellular chemotherapeutic drug concentration, bestowing a multidrug resistant phenotype. Previously, we reported that Gal1 overexpression in HCC HepG2 cells (HepG2Gal1) reduces apoptosis induced by the chemotherapeutic drugs camptothecin and doxorubicin (DOX). Also, we described that HepG2Gal1 cells show increased levels of Pgp protein expression.The aim of this work was to determine if Gal1 overexpression reduces intracellular DOX levels in HepG2 cells. Moreover, we confirmed Pgp involvement in Gal1-mediated resistance to DOX-induced death in HepG2 cells.By fluorescence techniqueswe found a significant decrease in intracellular DOX concentration (pmol/µM totalprotein) in HepG2Gal1 cells compared with HepG2 cells (1.2±0.1 vs 1.8±0.1, 30min after treatment; 1.7±0.4 vs 2.9±0.5, 60min;2.3±0.5 vs 4.5±1, 90min; 3.1±0.7 vs 5.6±1.2, 120min).Co-incubation of HepG2 and HepG2Gal1 cells for 24h with DOX (2µM) and verapamil (20µM), a Pgp inhibitor, diminished cell viability (MTT)compared with cells incubated only with DOX (HepG2, 37.4±5.1% vs 54±5.3%; HepG2Gal1, 60.1±3.4% vs 77.8±2.5%). Similarresults were obtained silencing Pgp expression in HepG2Gal1 cells with specificsiRNA (Scr+DOX 61.1±2.7% vs siRNA+DOX 40.8±7.2%). However, probenecid treatment(250µM), a multidrugresistance-associated protein 2 (MRP2) inhibitor, did not changeDOX-treated cell viability (HepG2, 51.5±9.3% vs 53.1±6.7%; HepG2Gal1, 69.0±9.8%vs 78.1±12.3%).In conclusion, Gal1-overexpressing HepG2 cells accumulate lessintracellular DOX, conferring a resistance phenotype. Moreover, Pgp activity inhibition,but not MRP2, or decreasing its expression sensitizes HepG2 cells to DOXtreatment, suggesting the involvement of Pgp in Gal1-induced resistance to DOX.