DZ-1-Artesunate Induces Apoptosis Via a Bid-, Bax-, and Bak-Independent Caspase-3 Activation Pathway

Artesunate (ART), a well-established antimalarial agent, has demonstrated promising anticancer activity in both in vitro and in vivo studies. Despite its potential, clinical application of ART in oncology is limited by modest efficacy and dose-limiting toxicity, likely due to nonspecific accumulation in normal tissues. Targeted delivery strategies are therefore essential to enhance therapeutic selectivity and reduce off-target effects. To this end, ART has been conjugated with targeting molecules such as aptamers, dyes, and polymers. DZ-1, a heptamethine cyanine dye, selectively accumulates in cancer cells through overexpression of organic anion transporting polypeptides (OATPs), offering a promising vehicle for tumor-specific delivery. In this study, we evaluated the anticancer efficacy and underlying mechanisms of a novel conjugate of DZ-1 and ART (DZ-1-ART) in three human cancer cell lines: colon cancer (HCT116), pancreatic cancer (BxPC-3), and breast cancer (MCF-7). DZ-1-ART induced time-dependent cytotoxicity across all tested cancer cell lines. Mechanistically, DZ-1-ART localized to both mitochondria and lysosomes, but functional studies indicated that lysosomes were not essential for its pro- apoptotic activity. Instead, DZ-1-ART triggered mitochondria-mediated apoptosis via a Bid-, Bax-, and Bak-independent pathway, leading to caspase-3 activation and cell death. Our findings demonstrate that DZ-1-ART undergoes intracellular trafficking through lysosomes and mitochondria, but induces apoptosis primarily through a mitochondrial, Bid-Bax/Bak- independent, caspase-3-dependent pathway. These results support the development of DZ-1- ART as a tumor-targeted anticancer agent with potential applications in theranostics.