Mitochondria-targeted DZ-1-dihydroartemisinin conjugate induces Bax/Bak-independent apoptotic cell death in colorectal carcinoma cells

Artemisinin and its derivatives possess anti-malarial as well as anti-cancer properties against various malignancies. However, their clinical application as anti-cancer agents is limited by several factors, most notably a lack of tumor selectivity. To address this limitation, we developed a mitochondria-targeted DZ-1-DHA conjugate with potential imaging applications by conjugating dihydroartemisinin (DHA), the most potent artemisinin derivative, to the DZ-1 dye, generating DZ-1-DHA. This study evaluates the cytotoxic effects and mitochondrial mechanisms underlying DZ-1-DHA–induced cell death. HCT116 and BxPC-3 cells were treated with increasing concentrations of DZ-1-DHA and assessed for cytotoxicity using the trypan blue exclusion assay. SW48 colorectal carcinoma cells were additionally evaluated by trypan blue dose-response analysis and immunoblotting. Primary non-malignant colon cells were compared with HCT116 cells by trypan blue analysis and immunoblotting, and LC₅₀ values were determined to calculate a selectivity index. Apoptosis in HCT116 cells was evaluated by TUNEL staining, Annexin V–FITC/PI analysis, and immunoblotting for apoptotic pathway activation. Mitochondrial membrane potential and oxidative stress were evaluated using JC-1, DCFDA, and MitoSOX. Bid⁻/⁻, Bax⁻/⁻Bak⁻/⁻ HCT116 cells were used to examine the requirement for canonical mitochondrial apoptosis regulators, while caspase-3-deficient and caspase-3-reconstituted MCF-7 cells were used to assess caspase-3 dependency. DZ-1-DHA induced strong, dose-dependent cytotoxicity in HCT116 human colorectal carcinoma, BxPC-3 pancreatic adenocarcinoma cells, and SW48 human colon adenocarcinoma cells. In HCT116 cells, DZ-1-DHA reduced cell viability in a concentration-dependent manner, with an IC₅₀ value of 14.46 µM after 24 h of treatment, significantly reducing cell viability compared to control ( p < 0.001), whereas DHA showed only modest effects ( p < 0.05). DZ-1-DHA also induced PARP-1 and caspase cleavage, DNA fragmentation, mitochondrial oxidative stress, and loss of mitochondrial membrane potential. Extended trypan blue analysis yielded LC₅₀ values of 52.89 µM in primary normal colon cells from a single donor and 13.17 µM in HCT116 cells, corresponding to a selectivity index of 4.02. DZ-1-DHA-induced cell death persisted in Bax⁻/⁻Bak⁻/⁻ HCT116 cells. Ferrostatin-1 attenuated DZ-1-DHA-induced lipid peroxidation and significantly reduced cell death, supporting a contribution of ferroptosis-associated oxidative mechanisms. DZ-1-DHA induces mitochondria-associated, ROS-driven apoptotic cell death in HCT116 cells that persists in the absence of Bid and Bax/Bak. Experiments in caspase-3-deficient and caspase-3-reconstituted MCF-7 cells further support an important role for caspase-3 in DZ-1-DHA-induced apoptotic signaling. DZ-1-DHA also induces lipid peroxidation and ferroptosis-associated molecular changes. These findings identify DZ-1-DHA as a mechanistically distinct in vitro lead compound warranting further preclinical investigation, including in vivo efficacy, pharmacokinetic, and toxicological studies.