Anthraquinones have been shown to induce apoptosis in different types of
tumor cells, but the mechanisms of
danthron-induced cytotoxicity and apoptosis in human
gastric cancer cells have not been adequately explored. This study investigated the roles of
caspase cascades, ROS, DNA damage, mitochondrial disruption, and Bax and Bcl-2
proteins in
danthron-induced apoptosis of SNU-1 human
gastric cancer cells, a commonly used cell culture system for in vitro studies. Cells were incubated with different concentrations of
danthron in a time- and/or dose-dependent manner. Cell morphological changes (shrinkage and rounding) were examined by a phase-contrast microscope, whereas cell viability and apoptotic populations were determined by flow cytometric analysis using
propidium iodide (PI) and
annexin V-FITC staining. The fluorescent
DAPI nucleic acid stain and Comet assay were applied to detect
danthron-induced
chromatin condensation (an apoptotic characteristic) and DNA damage. Increasing the levels of
caspase-3, -8, and -9 activities was involved in
danthron-induced apoptosis, and they could be attenuated by inhibitors of specific
caspases, indicating that
danthron triggered the
caspase-dependent apoptotic pathway. Further studies with flow cytometric analyses indicated that cellular levels of ROS, cytosolic Ca(2+), and mitochondrial permeability transition (MPT) pore opening were increased, but the level of mitochondrial membrane potential (ΔΨ(m)) was decreased. Also, the ratio of Bax/Bcl-2 levels and other proapoptotic
proteins associated with modulating the ΔΨ(m) were up-regulated. Apoptotic signaling was also stimulated after exposure to
danthron and determined by Western blotting and real-time PCR analyses. In summary, it is suggested that
danthron-induced apoptotic cell death was involved in mitochondrial depolarization, which led to release of
cytochrome c,
apoptosis-inducing factor (AIF), and
endonuclease G (Endo G) and caused the activation of
caspase-9 and -3 in SNU-1 human
gastric cancer cells.