Abstract 2143: Mechanistic study of the relative cytotoxicity of doxorubicin loaded nanoparticle formulation compared to free doxorubicin in hepatocellular carcinoma (HCC) cell lines

Abstract

Abstract Background and Aims Efficacy of doxorubicin encapsulated into a biodegradable network (NP) is currently studied in phase III compared to Best Standard of Care in patients with advanced HCC (Relive study). Preclinical studies demonstrated that NP allowed the reversion of chemo-resistance and enhanced cytotoxicity compared to free doxorubicin on resistant human hepatoma and mestastatic liver cells. In this study the mechanism of action of NP in overcoming HCC cellular resistance as well as the mechanisms of cellular uptake were investigated in representative cell lines. Methods Determination of IC20 of NP in the presence of influx drug inhibitors targeting endocytosis (clathrin or caveolae-dependent; sucrose and Genistein respectively), phagocytosis (LY-294002) or macropinocytosis (Cytochalasin D) were performed on two HCC cell lines (HuH7 and HepG2) using a proliferation assay. Each uptake inhibitor was added 30 min before incubation with NP on cultured cells for measurement of proliferation followed by cellular quantification of free doxorubicin by HPLC. The same assays were also performed using Verapamil as an inhibitor of the major multidrug resistance protein P-glycoprotein (P-gp). The P-gp ATPase activity in plasma membrane preparations from vehicle, doxorubicin and NP-treated cells was determined using the Pgp-Glo assay system. Results NP showed lower IC20 than free doxorubicin in HCC proliferation assays. None of the influx inhibitors used in combination with NP impaired the inhibitory effect of NP on the rate of proliferation of HCC cell lines. Similarly, Verapamil did not modulate the cellular potency of NP on the rate of proliferation in these cell lines. Doxorubicin quantification in cellular extracts and supernatant of NP-treated cells confirmed these results. Furthermore, NP had no effect on P-gp ATPase activity in isolated plasma membrane samples. Conclusions All these results demonstrated that NP did not enter into the HCC cell via an active transport mechanism, but by passive diffusion as occurs with free Doxorubicin. The absence of inhibition of the P-gp ATPase by NP implies that the Doxorubicin-nanoparticle conjugate (ion pair) probably ‘hinders’ the doxorubicin from being a substrate for the pump rather than by a direct interaction. The implication of these data on the role of NP in the treatment of HCC will be discussed. Citation Format: Véronique Trochon-joseph, Caroline Lemarchand, Vincent Hayes, Yamina Rayah, Jean-Louis Labernardière, Graham Dixon. Mechanistic study of the relative cytotoxicity of doxorubicin loaded nanoparticle formulation compared to free doxorubicin in hepatocellular carcinoma (HCC) cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2143.