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KMID : 0644320000060010081
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Journal of Korean Oriental Oncology
2000 Volume.6 No. 1 p.81 ~ p.97
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Study of Hedyotis Diffusa Methanol Extract on Anti-tumoral Effect and Mechanism
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Abstract
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Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60.
Methods: Cells were treated with various concentrations (200 to 0.4㎍) and periods (6 to 30 hr) of H_2O and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with 200㎍/ml of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with 200㎍/ml of each extract for 16hr.Then, cells were treated with various doses of each extract for 12 hr and 100㎍/ml of methanol extract for various periods. Lysate from the cells used to measure the activity of caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively, Cells were treated with 200㎍/ml of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with 32^p-ATP and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by suing Phosphoimage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of NF-kB was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at CO_2 incubator for 6 days. The number of colony was cunted under light microscopy (×100).
Results: The death of HL_60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL_60 cells. In addition, it was shown nucleus chromatin condensation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes.
The activity of Caspaxe 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, NF-kB was markedly induced by methanol extract of Hedyotis diffusa.
Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture.
Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator NF-κB, In addition, our results also suggest that methanol exthanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.
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