Insights into P-Glycoprotein Inhibitors: New Inducers of Immunogenic Cell Death

Abstract

Doxorubicin is a strong inducer of immunogenic cell death (ICD), but it is ineffective in P-glycoprotein (Pgp)-expressing cells. Indeed, Pgp effluxes doxorubicin and impairs the immunesensitizing functions of calreticulin (CRT), an "eat-me" signal mediating ICD. It is unknown if classical Pgp inhibitors, designed to reverse chemoresistance, may restore ICD. We addressed this question by using Pgp-expressing cancer cells, treated with Tariquidar, a clinically approved Pgp inhibitor, and <i>R</i>-3 compound, a <i>N</i>,<i>N</i>-bis(alkanol)amine aryl ester derivative with the same potency of Tariquidar as Pgp inhibitor. In Pgp-expressing/doxorubicin-resistant cells, Tariquidar and <i>R</i>-3 increased doxorubicin accumulation and toxicity, reduced Pgp activity, and increased CRT translocation and ATP and HMGB1 release. Unexpectedly, only <i>R</i>-3 promoted phagocytosis by dendritic cells and activation of antitumor CD8⁺T-lymphocytes. Although Tariquidar did not alter the amount of Pgp present on cell surface, <i>R</i>-3 promoted Pgp internalization and ubiquitination, disrupting its interaction with CRT. Pgp knock-out restores doxorubicin-induced ICD in MDA-MB-231/DX cells that recapitulated the phenotype of <i>R</i>-3-treated cells. Our work demonstrates that plasma membrane-associated Pgp prevents a complete ICD notwithstanding the release of ATP and HMGB1, and the exposure of CRT. Pharmacological compounds reducing Pgp activity and amount may act as promising chemo- and immunesensitizing agents.