Emodin azide methyl anthraquinone derivative triggers mitochondrial-dependent cell apoptosis involving in caspase-8-mediated Bid cleavage

AMAD, an emodin azide methyl anthraquinone derivative, was extracted from the nature giant knotweed rhizome of traditional Chinese herbs. Here, we investigated the anticancer activities and signaling pathways implicated in AMAD-induced apoptosis in human breast cancer cell lines MDA-MB-453 and human lung adenocarcinoma Calu-3 cells. AMAD was found to have a potent cytotoxic effect on both cell lines. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. Moreover, this apoptotic induction was associated with a collapse of the mitochondrial membrane potential and activated caspases (cysteine aspartase) cascade involving in caspase-8, caspase-9, caspase-3, and poly(ADP-ribose) polymerase cleavage in a concentration-dependent manner. It was noteworthy that AMAD also effectively cleaved Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, and induced the subsequent release of cytochrome c from mitochondria into the cytosol. Furthermore, suppression of caspase-8 activity with Z-IETD-FMK partially inhibited release of cytochrome c and Bid cleavage induced by AMAD, whereas exposure to Z-LETD-FMK, a caspase-9 inhibitor, had no effect. Additionally, there was significant change in other mitochondrial membrane proteins triggered by AMAD, such as Bcl-xl and Bad. It was intriguing that AMAD decreased the generation of reactive oxygen species in both cell lines. DNA-binding assay exhibited apoptosis induced by AMAD was not involved in intercalating to DNA. Taken together, these data suggested that AMAD induced apoptosis via a mitochondrial pathway involving caspase-8/Bid activation in both cell lines.