Apoptotic cell death by the novel natural compound, cinobufotalin.
Cinobufotalin (CB), one of the bufadienolides prepared from toad venom, was investigated for its cytotoxicity, and the underneath mechanism involved. We primarily utilized DNA fragmentation assay and microscopic observation to assess the effect of various doses of CB in human lymphoma U937 cells. Following that, we investigated other parameters involved in cell death mechanism such as reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and apoptotic proteins activation. HeLa cells were concomitantly used to generalize the data observed. Our results show that CB caused significant DNA fragmentation, decrease of MMP, and an increase in the intracellular Ca(2+) ion and ROS production. In addition, CB induced upregulation of Fas protein, proteolytic activation of cytochrome c, caspase-2, -3, -8 and -9 together with the activation of Bid and Bax. Our findings were further validated using either Fas/FasL antagonist or pan-caspase inhibitor to significantly inhibit CB-induced DNA fragmentation. In our study, we suggest that CB induces caspase dependent cell death in U937 cells, and that Fas plays a role in CB-induced apoptosis. Altogether, our data provides novel insights of the mechanism of action of CB and its potential as a future chemotherapeutic agent.
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan.
Heba
H
Emam
Qing-Li
QL
Zhao
Yukihiro
Y
Furusawa
Alaa
A
Refaat
Kanwal
K
Ahmed
Makoto
M
Kadowaki
Takashi
T
Kondo
D000664:Amphibian Venoms / Q000737:chemistry; D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D002018:Bufanolides / Q000302:isolation & purification / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D006367:HeLa Cells; D006801:Humans; D008516:Medicine, Chinese Traditional; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D053858:Metabolic Networks and Pathways; D017382:Reactive Oxygen Species / Q000378:metabolism; D020298:U937 Cells; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism
D000664:Amphibian Venoms; D000970:Antineoplastic Agents; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002018:Bufanolides; C491901:FAS protein, human; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D020169:Caspases; C063451:cinobufotalin; D002118:Calcium