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

Abstract

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 (1)H NMR, (13)C NMR, mass spectrometry, and high-performance liquid chromatography. Their stability was also evaluated. By using MCF-7/ADR cells as an in vitro model system and nude mice bearing MCF-7/ADR xenografts as an in vivo 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 in vivo 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.

An albumin-based tumor-targeted oxaliplatin prodrug with distinctly improved anticancer activity in vivo

“…14–16 One of the most elegant and promising ways to exploit the EPR effect is the use of human serum albumin (HSA) as a carrier. 17,18 Abraxane®, 19,20 an albumin-bound nanoparticle formulation of paclitaxel, which was approved for metastatic breast cancer, non-small cell lung cancer (NSCLC) and pancreas adenocarcinoma, as well as the doxorubicin-based drug aldoxorubicin, 21 which is currently in clinical phase III studies, are two successful examples. Aldoxorubicin possesses a maleimide moiety, which is known to rapidly bind to the free thiol group (cysteine at position 34) 22 of albumin exploiting it as a carrier without the use of exogenous compounds usually used for nanoformulations.…”

Doxorubicin conjugated with a trastuzumab epitope and an MMP-2 sensitive peptide linker for the treatment of HER2-positive breast cancer

“…Furthermore, intermediate products N -Fmoc-DOX (calcd, 766.2; found, 790.7 [M + Na] + ) and N -Fmoc-DOX-14- O -hemiglutarate (calcd, 880.2; found, 902.5 [M + Na] + ) were also monitored and their molecular weight agreed with the theoretical weights (data not shown) (Soudy et al., 2013 ). Given that these molecular ion peaks cannot offer the structural information for molecules (Sheng et al., 2015 ), tandem mass spectrometry and collision-induced dissociation (CID) were performed, and the product ion spectrum was resolved based on the similarity between the peptide-drug conjugate fragmentation and fragmentation of the individual peptide and conjugated drug. As shown in Figure 2(C) , some characteristic sequence-specific b ions and y ions resulting from the cleavage of the peptide backbone in the positive ion mode were observed.…”

“…To date, various linkages, such as hydrazone, oxime and ester bonds were employed to form a DOX-peptide conjugate (Schlage et al., 2011 ). Among these linkages, a Michael-addition of a thiol to a maleimide is one of the most common classes in conjugation (Sann, 2006 ), which has also been used in our previous study (Sheng et al., 2015 ; Yuan et al., 2016 ). Theoretically, this reaction is specific and efficient.…”

Transferrin receptor-targeted pH-sensitive micellar system for diminution of drug resistance and targetable delivery in multidrug-resistant breast cancer

“…It has been widely proved that TfRs are overexpressed on various types of malignant cells, including the MCF-7/Adr cells used in this study. 21,23 Here, we investigated the TfR expression on MCF-7/Adr cells by immunofluorescence (as shown in Figure 5A). The large amount of FITC-labeled secondary antibody (green) on MCF-7/Adr cells confirmed the TfR expression.…”

“…21 Recently, it has been demonstrated that the emergence of drug resistance is partially associated with further overproduction of TfR, which therefore could render it a potential target for MDR tumors. 22 Sheng et al 23 have demonstrated that TfR ligand and doxorubicin (DOX) conjugate exhibited the ability to target drug-resistant tumors. Recently, HAIYPRH, a TfR ligand (7pep), has been screened through phage display.…”