Danthron, an anthraquinone derivative, induces DNA damage and caspase cascades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways.
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.
Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan.
Jo-Hua
JH
Chiang
Jai-Sing
JS
Yang
Chia-Yu
CY
Ma
Mei-Due
MD
Yang
Hui-Ying
HY
Huang
Te-Chun
TC
Hsia
Hsiu-Maan
HM
Kuo
Ping-Ping
PP
Wu
Tsung-Han
TH
Lee
Jing-Gung
JG
Chung
D017304:Annexin A5 / Q000737:chemistry; D000880:Anthraquinones / Q000737:chemistry / Q000633:toxicity*; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000633:toxicity*; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism / Q000502:physiology; D053181:Caspase 8 / Q000378:metabolism / Q000502:physiology; D053453:Caspase 9 / Q000378:metabolism / Q000502:physiology; D020169:Caspases / Q000378:metabolism* / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects; D004249:DNA Damage; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D011419:Propidium / Q000737:chemistry; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000378:metabolism* / Q000502:physiology
D017304:Annexin A5; D000880:Anthraquinones; D000972:Antineoplastic Agents, Phytogenic; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D011419:Propidium; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D016650:Fluorescein-5-isothiocyanate; C004315:danthron