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

Biochem Pharmacol. 2002 Jan 15;63(2):225-36. doi: 10.1016/s0006-2952(01)00876-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / metabolism
  • Apoptosis*
  • Calcium / metabolism
  • Caspase 1 / metabolism
  • Caspase 3
  • Caspases / metabolism*
  • Catalase / metabolism
  • Cysteine Proteinase Inhibitors / pharmacology
  • Endonucleases / metabolism*
  • Enzyme Activation / drug effects
  • Flavanones*
  • Flavonoids / pharmacology*
  • Free Radical Scavengers / pharmacology
  • HL-60 Cells
  • Humans
  • Hydrogen Peroxide / pharmacology
  • Leukemia, Promyelocytic, Acute
  • Oligopeptides / pharmacology
  • Poly(ADP-ribose) Polymerases / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Reactive Oxygen Species / metabolism*
  • Tumor Cells, Cultured

Substances

  • Cysteine Proteinase Inhibitors
  • Flavanones
  • Flavonoids
  • Free Radical Scavengers
  • Oligopeptides
  • Proto-Oncogene Proteins c-bcl-2
  • Reactive Oxygen Species
  • acetyl-aspartyl-glutamyl-valyl-aspartal
  • Hydrogen Peroxide
  • Catalase
  • Poly(ADP-ribose) Polymerases
  • Endonucleases
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Caspase 1
  • fisetin
  • wogonin
  • Calcium
  • Acetylcysteine