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KMID : 0644320000060010067
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Journal of Korean Oriental Oncology
2000 Volume.6 No. 1 p.67 ~ p.79
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Study on Transcriptional Factors Activation Change of HL-60 cell Apoptosis by Hedyotis Diffusa`s Methanol Extract
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Abstract
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Objective : Hedyotis diffusa has been used as an arnicancer agent for several decades in oriental
medicine. We test whether the methanol extract of the herb affects transcriptional
activation factors including NF-κB and AP-l.
Methods :
1. HL-60 cells were treated with various concentrations(from 200 to 50ug/ml) of methanol extract and H_2O extract(200ug/ml)of hedyotis diffusa, After 48h later, the cells were tested for viability by MTT assay.
2. The HL-60 cells were treated with 200ug/ml of methanol extract for the indicated periods. First. Nuclear extracts were isolated and incubated with oligonucleotide probe of NF-κB and AP-l. Second. Nuclear extracts were isolated and reacted with p50, p65. c-rel par-Jun, c-Jun, JunB. JunD antibody on ice for 30min. Finally The cell lnates were prepared and analyzed by westem blotting using anti-Fas, anti-FasL and anti-p53 antibdy.
Results :
l. The methanol extract decreases the viability of human lymphoid origin leukemia HL-60 cells in a dose-depemdemt manner.
2. NF-κB is rapidly activated by the addition of the methanol extract, reaches a peak at 30min and gradually returns to resting level. We confirm tha NF-κB is a heterodimer mainly composed of p65 subunit with c-Rel.
3. Transcriptional activation of AP-1 is detected at 30min and reaches a maximum at lhr after
stimulation of the cells with the methanol extract. AP-l is mainly composed with Jur-D and
partially Jug-B proteins.
4. the methanol extract of Hedyotis diffusa induces the expression of Fas, Fas ligand and p53 proteins of HL-60 cells in a time dependent fashion.
Conclusions : These results suggest that the methanol extract of Hedyotis diffusa exerts anticancer effects to induce the death of human leukomic HL-60 cells via activation of trascriptional factors such a NF-κB and AP-1, increase in expression of Fas mediated signaling proteins, and induction of tumor suppressor gene. p53
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