Acid-Sensitive Peptide-Conjugated Doxorubicin Mediates the Lysosomal Pathway of Apoptosis and Reverses Drug Resistance in Breast Cancer

The extended use of doxorubicin (DOX) could be limited because of the emergence of drug resistance associated with its treatment. To reverse the drug resistance, two thiol-modified peptide sequences HAIYPRHGGC and THRPPMW­SPVWPGGC were, respectively, conjugated to DOXO-EMCH, forming a maleimide bridge in this study (i.e., T10-DOX and T15-DOX). The structures and properties of peptide–DOX conjugates were characterized using ¹H NMR, ¹³C NMR, mass spectrometry, and high-performance liquid chromatography. Their stability was also evaluated. By using MCF-7/ADR cells as an <i>in vitro</i> model system and nude mice bearing MCF-7/ADR xenografts as an <i>in vivo</i> model, the ability of these novel peptide–DOX conjugates to reverse drug resistance was accessed as compared with free DOX. As a result, the IC₅₀ values for T10-DOX and T15-DOX significantly decreased (31.6 ± 1.6 μM and 27.2 ± 0.8 μM), whereas the percentage of apoptotic cell population increased (35.4% and 39.3%). The <i>in vivo</i> extent of inhibition was more evident in the mice groups treated with peptide–DOX conjugates (59.6 ± 8.99% and 46.4 ± 6.63%), which had DOX primarily accumulated in tumor. These conjugates also showed a longer half-life in plasma and cleared much more slowly from the body. Furthermore, T10-DOX may be more effective than T15-DOX with a higher efficacy and a lower side effect. Most importantly, evidence was provided to support the enhanced intracellular drug accumulation and the induction of lysosomal pathway of apoptosis underlying the drug resistance. As an endosomal/lysosomal marker, cathepsin D permealized the destabilized organelle membrane and was detected in the cytoplasm, leading to the activation of the effector caspase-3 in cell apoptosis. This report is among the first to demonstrate that peptide–DOX-like conjugates promote apoptosis through the initiation of the lysosomal pathway