Doxorubicin—An Agent with Multiple Mechanisms of Anticancer Activity

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  Figure 1. (A) Induction of DNA damage following treatment of cells with doxorubicin (DOX). DOX induces DNA damage through three main mechanisms: the formation of DNA adducts, single-strand break (SSB) induction, and double-strand break (DSB) induction, in which DNA strands remain bound to trapped topoisomerase enzymes through DNA–protein crosslinks (DPCs) and intercalation of DOX in the DNA molecule. (B) Activation of DNA damage response (DDR) pathway. Trapping of topoisomerase enzymes by DOX induces the formation of either SSBs or DSBs. SSBs are detected by poly [ADP-ribose] polymerase 1 (PARP-1), and the ssDNA is covered with replication protein A (RPA). This results in the recruitment of a variety of factors including DNA topoisomerase 2-binding protein 1 (TOPBP-1), RAD9, RAD1, HUS1 interacting nuclear orphan (RHINO), ATR-interacting protein (ATRIP), MRE11–RAD50–NBS1 complexes, and serine/threonine-protein kinase ATR that contribute to the formation of γH2AX protein foci at the sites of damage. DSBs are detected by MRN complexes, which recruit a mediator of DNA damage checkpoint protein 1 (MDC-1) and amplification of the foci formation by ataxia telangiectasia mutated (A-T mutated)…

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  Figure 2. Contribution of DOX to ROS induction and apoptosis. The interaction of DOX with cardiolipin (CL) leads to the elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels, which contribute to DNA damage of nuclear (nDNA) and mitochondrial DNA (mtDNA). DNA damage and mutations in mtDNA encoding proteins of the electron transport chain (ETC) lead to the production of defective ETC components that increase electron leakage and ROS/RNS generation. Alternatively, ROS trigger lipid peroxidation, enhancing ROS pools in the cells. ROS may trigger the activation of the ATM-CHK2-TP53 signaling independently of DNA damage through ROS-mediated ataxia telangiectasia mutated A-T mutated (ATM) dimer formation through modification of enzyme cysteine residues by peroxiredoxin-2 (PRDX2) and thioredoxin-1 (TRX1). Through the P38 protein, ROS may activate the P16 protein, which works as an inhibitor of cyclin-dependent kinases 4/6 (CDK4/CDK6). DOX increases the expression of cellular tumor antigen p53 (TP53) and down-regulates the expression of GATA binding protein 4 (GATA4), which influences the expression of genes under the control of these transcription factors.…

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  Figure 3. The role of DOX in the anti-tumor immune response. (A) DOX induces the apoptotic cell death of cancer cells, contributing to the exposition or release of ATP, calreticulin (CRT), and high mobility group box protein B1 (HMGB1) of cancer cells and promoting their recognition by dendritic cells (DCs) through respective receptors (P2X purinoceptor 7 (P2RX7), CD91, and toll-like receptor 4 (TLR-4)) present on their cellular surface. (B) These events allow the maturation of DCs and the release of immune-stimulating factors such as interleukins (ILs) and tumor-necrosis factor α (TNF-α). These events promote the activation and expansion of T cells. (C) Activated T cells and natural killer (NK) cells recruited to the tumor microenvironment release perforins (PFNs), granzyme A/B (GZMA/B), and interferon γ (IFN-γ), leading to cancer-cell death. (D) DOX induces double-strand break (DSB) formation, contributing to the activation of the ATR/ATM/CHK2 pathway and promoting STAT1/2/3/IRF1-mediated programmed cell death 1 ligand 1 (PD-L1) up-regulation. This confers to the inhibition of immunogenic cell death. (E) ATP released by dying cancer cells activates P2RX7 expressed on surrounding tumor…