Inhibition of P-glycoprotein-mediated multidrug resistance by unfractionated heparin: A new potential chemosensitizer for cancer therapy

Abstract

Anticoagulant treatment with heparins is frequently used to prevent venous thromboembolism in cancer patient. In the present study, we investigared the ability of unfractionated heparin (UFH) to inhibit P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) on human breast cancer cell line (MDA-MB231) and its doxo-resistant subline. Results were compared to the classic reversing agent, Verapamil (Ver), used, as reference at 50 µM concentration. We analysed the Pgp function by calcein acetoxymethylester (calcein-AM) uptake, a fluorescent marker substrate, before and after in vitro exposure to UFH at clinically achievable dose of 20 U/ml. The mean percentage of calcein-AM retained into cancer cells after 3 and 12 h were 32 ± 10.9 and 45 ± 12.3, respectively, for UFH pretreated cells and 25.3 ± 8.7 and 29.4 ± 10.4, respectively, for Ver pretreated cells when compared to control cells, receiving only medium. Pgp activity was studied by measuring intracellular drug accumulation in doxo-resistant subline, treated (2 h) with either UFH or Ver, prior exposure (2 h) at different doxo concentrations (2, 4 and 8 µM). The mean percentage of remaining intracellular doxo were 55.4 ± 4.5 , 51.4 ± 3.9 and 50 ± 1.8 %, respectively for UFH treated cells, and 44.1 ± 5.8, 39.3 ± 4.4 and 19.4 ± 8.6%, respectively, for Ver treated cells as compared with control cells, receiving only doxo. These results were consistent with the increase of sensitivity to doxo of the same doxo-resistant subline resulting in a 2.2, 2.6 and 2.2-fold increase, respectively, for UFH-doxo combination and 2.2, 2.5 and 2.0-fold respectively, for Ver-doxo combination respect to cells receiving doxo alone, as assessed by MTT test. In conclusion, these findings demonstrate the potentiating effect in vitro of UFH on doxo accumulation and cytotoxicity in MDA-231 cell line and its doxo-resistant subline and suggest that UFH could to be used, as an potential chemosensitizer, in clinical chemotherapy, for increase in vivo, the efficacy of the anticancer treatment.