A novel contribution of the β-galactoside-binding protein Galectin-1 to hepatocellular carcinoma cell drug resistance

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

Congreso; III INTERNATIONAL CONGRESS IN TRANSLATIONAL MEDICINE; 2016

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. This high mortality is related to treatment failures due to increased resistance to therapeutic drugs. 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 localized in cell membranes. Its overexpression in tumor cells decreases intracellular chemotherapeutic drug concentration leading to a multidrug resistant phenotype. Previously, we reported that Gal1 overexpression in HCC HepG2 cells (HepG2Gal1) induces epithelial-mesenchymal transition, a key process in cancer metastasis. Also, we described that Gal1 protects HepG2 cells from cell death induced by transforming growth factor β1 (TGF-β1), a cytokine present in tumor microenvironment.The aim of this work was to determine if Gal1 overexpression confers HCC cell drug resistance.We observed that HepG2Gal1 cells exhibited increased viability (MTT) compared with control cells incubated for 24h with 1µM camptothecin (CPT) (58.5% vs 49.9%) or 5µM doxorubicin (DOX) (62.5% vs 47.6%). This result was confirmed using Hoechst staining and analyzing nuclear morphology by fluorescence microscopy. HepG2Gal1 cells showed a lower percentage of apoptotic nuclei respect to control cells incubated with CPT (1µM, 17.4±7.1% vs 53.2±21.9% 48h) or DOX (5µM, 28.5±5.6% vs 45.1±5.4% 48h).By fluorescence techniques we found a significant decrease in intracellular DOX concentration (pmol/µM total protein) 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). Western Blot analysis showed increased Pgp protein levels in HepG2Gal1 cells compared with control cells (237% vs 100%). Further, we examined Pgp involvement in Gal1-mediated resistance to DOX by loss of function techniques. Co-incubation of HepG2 and HepG2Gal1 cells for 24h with DOX (2µM) and verapamil (20µM), a Pgp inhibitor, diminished cell viability 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%). Similar results were obtained silencing Pgp expression in HepG2Gal1 cells with specific siRNA (Scr+DOX 60.0±1.8% vs siRNA+DOX 44.2±6.1%). However, probenecid (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%).In conclusion, Gal1 protects HepG2 cells from CPT and DOX-induced cell death. Also, HepG2Gal1 cells accumulate less intracellular DOX likely due to the increased Pgp levels, thus conferring a resistance phenotype. Moreover, Pgp activity inhibition, but not MRP2, or decreasing its expression sensitizes HepG2 cells to DOX treatment, suggesting the involvement of Pgp in Gal1-induced resistance to DOX.