Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction.
Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis.
Kuo
Chen-Tzu
CT
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Hsu
Ming-Jen
MJ
Chen
Bing-Chang
BC
Chen
Chien-Chih
CC
Teng
Che-Ming
CM
Pan
Shiow-Lin
SL
Lin
Chien-Huang
CH
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000880:Anthraquinones / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3 / Q000096:biosynthesis; D061945:Caspase Inhibitors; D002452:Cell Count; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D024101:Mitochondrial Proteins / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000378:metabolism*; D010616:Phenanthrenes / Q000494:pharmacology*; D051025:bcl-Associated Death Protein / Q000378:metabolism*
C494829:AIFM1 protein, human; D000590:Amino Acid Chloromethyl Ketones; D000880:Anthraquinones; D000972:Antineoplastic Agents, Phytogenic; D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; C494819:BAD protein, human; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D010616:Phenanthrenes; D051025:bcl-Associated Death Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C436544:denbinobin; D051058:Oncogene Protein v-akt; D053148:Caspase 3