Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca(2+)-dependent endonuclease.
Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H(2)O(2) (200 microM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.
Lee
Woan Rouh
WR
Institute of Medical Sciences, School of Medicine, Taipei Medical University, 250 Wu-Hsing Street, ROC, Taipei, Taiwan.
Shen
Shing Chuan
SC
Lin
Hui Yi
HY
Hou
Wen Chi
WC
Yang
Ling Ling
LL
Chen
Yen Chou
YC
D000111:Acetylcysteine / Q000378:metabolism; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004720:Endonucleases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D044950:Flavanones; D005419:Flavonoids / Q000494:pharmacology*; D044948:Flavonols; D016166:Free Radical Scavengers / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015473:Leukemia, Promyelocytic, Acute; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D015853:Cysteine Proteinase Inhibitors; D044950:Flavanones; D005419:Flavonoids; D044948:Flavonols; D016166:Free Radical Scavengers; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D006861:Hydrogen Peroxide; D002374:Catalase; D011065:Poly(ADP-ribose) Polymerases; D004720:Endonucleases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; C017875:fisetin; C085514:wogonin; D002118:Calcium; D000111:Acetylcysteine