P-glycoprotein 1 mediates Galectin-1-induced resistance to doxorubicin in hepatocellular carcinoma (HCC) cells

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

Mar del Plata

Congreso; P-glycoprotein 1 mediates Galectin-1-induced resistance to doxorubicin in hepatocellular carcinoma (HCC) cells; 2016

Sociedad Argentina de Investigación Clínica (SAIC)

Galectin-1 (Gal1), a β-galactoside-binding protein, is overexpressed in HCC and it is related to tumor aggressiveness. P-glycoprotein 1 (Pgp), also known as multidrug resistance protein 1 (MDR1), is an ATP-dependent drug efflux pump. Its overexpression in tumor cells decreases intracellular chemotherapeutic drug concentration, showing a multidrug resistant phenotype. Previously, we reported that Gal1 overexpression in human 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.Our aim was to evaluate if Gal1 overexpression reduces intracellular DOX levels in HepG2 cells, and to confirm Pgp involvement on Gal1-mediated resistance to DOX-induced cell death.By fluorescence techniques we found a significant decrease in intracellular DOX concentration (pmol/µM total protein, min after treatment) in HepG2Gal1 cells compared with HepG2 cells (1.2±0.1 vs 1.8±0.1, 30; 1.7±0.4 vs 2.9±0.5, 60; 2.3±0.5 vs 4.5±1, 90; 3.1±0.7 vs 5.6±1.2, 120).Co-incubation of HepG2 or HepG2Gal1 cells for 24h with DOX (2µM) and verapamil (20µM), a Pgp inhibitor, diminished cell viability (MTT) compared with cells incubated only with DOX, respectively (HepG2, 37.4±5.1% vs 54±5.3%; HepG2Gal1, 60.1±3.4% vs 77.8±2.5%). Similar results were obtained silencing Pgp expression in HepG2Gal1 cells with siRNA (Scr+DOX 61.1±2.7% vs siRNA+DOX 40.8±7.2%). However, probenecid treatment (250µM), a multidrug resistance-associated protein 2 (MRP2) inhibitor, did not change DOX-treated cell viability (HepG2, 51.5±9.3% vs 53.1±6.7%; HepG2Gal1, 69.0±9.8% vs 78.1±12.3%).Thus, Gal1-overexpressing HepG2 cells accumulate less intracellular DOX, showing a resistant phenotype. Moreover, Pgp activity inhibition, but not MRP2, or Pgp expression silencing sensitizes HepG2 cells to DOX, suggesting the involvement of Pgp in Gal1-mediated resistance to DOX-induced cell death.