• Natural Product Sciences
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• 제10권1호
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• pp.48-53
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• 2004

Two galloyl monosaccharides, 1,2,6-trigalloylglucose (1, TRgG) and 1,2,3,6- tetragalloylglucose (2, TEgG), were isolated from the stem-bark of Juglans mandshurica. Two galloylglucoses showed cytotoxic effects on human promyelocytic leukemia HL-60 cells. In order to elucidate their mechanism of action, we have investigated the flow cytometric analysis after Annexin V-FITC and PI staining, caspase-3 activity, and internucleosomal DNA fragmentation in HL-60 cells. HL-60 cells treated with both compounds 1 and 2 at 150 and $100\;{\mu}M$, respectively, led to a morphological features of apoptosis, such as plasma membrane blebbing and cell shrinkage. TRgG (1) and TEgG (2) increased the percentage of $FITC^+\;and\;FITC^+PI^+$ cells in flow cytometry after Annexin V-FITC and PI staining. The increase of apoptotic cells was preceded by the activation of caspase-3 reported to play a central role in apoptotic process and inducing internucleosomal DNA fragmentation. TEgG (2) showed to have stronger apoptosis inducing activity in HL-60 cell lines as compared with TRgG (1).

• 동의생리병리학회지
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• 제19권5호
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• pp.1370-1374
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• 2005

The Tosyl-JM3 (TJM3) is a modified compound from one of 1,2,3,4-Tetra- hydroisoquinoline (THIQ) derivatives. The THIQs include potent cytotoxic agents that display a range of anti-tumor activities, antimicrobial activity, and other biological properties. In this study, we investigated the effect of TJM3 on the cytotoxicity, induction of apoptosis in human promyelocytic leukemia cells (HL-60 cells). TJM3 showed a significant cytotoxic activity in HL-60 cells (IC50 = approximately $60{\mu}g/m{\ell}$) after a 24 hr incubation. Treatment of HL-60 cells with TJM3 exhibited several features of apoptosis, including formation of DNA ladders in agarose gel electrophoresis, morphological changes of HL-60 cells with DAPI stain. Here we observed that TJM3 caused a decrease of procaspase-3 protein. Further molecular analysis demonstrated that TJM3 led to cleavage of poly(ADP-ribose) polymerase (PARP) by western blot and increase of hypodiploid (Sub-G1) population in the flow cytometric analysis. In conclusion, these above results indicate that TJM3 dramatically suppresses HL-60 cell growth and induces apoptosis. These data may support a possibility for the use of TJM3 in the prevention and treatment of leukemia.

• 대한한방종양학회지
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• 제6권1호
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• pp.81-97
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• 2000

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 $0.4{\mu}g$) 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 $200{\mu}g/ml$ 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 $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ 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 $200{\mu}g/ml$ 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 using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ 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 counted under light microscopy ($\time100$). 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 and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 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, $NF-{\kappa}B$ 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 $NF-{\kappa}B$, In addition, our results also suggest that methanol 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.

• Archives of Pharmacal Research
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• 제25권4호
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• pp.480-484
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• 2002

Costunolide has been reported to be a cytotoxic and chemopreventive agent. This work investigated the mechanism of the anti proliferative effect of costunolide and determined that it induced differentiation of the human leukemia cell line HL-60. Costunolide exhibited a potent antiproliferative activity against HL-60 cells. It was also found to be a potent inducer of differentiation in human leukemia derived HL-60 cells through the examination of differentiation markers, as assessed by the reduction of nitroblue tetrazolium, the increase in esterase activities and phagocytic activity, morphology change and the expression of CD14 and CD66b surface antigens. These results, accompanied by a decline in the expression of c-myc protein, suggest that costunolide induces differentiation of human leukemia cells to granulocytes and monocytes/macrophages lineage.

• Toxicological Research
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• 제25권4호
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• pp.181-188
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• 2009

Uncontrolled cell growth and increased cell proliferation are major features of cancer that are dependent on the stable structure and dynamics of the cytoskeleton. Since stable cytoskeleton structure and dynamics are partly regulated by myosin light chain kinase (MLCK), many current studies focused on MLCK inhibition as a chemotherapeutic target. As a potent and selective MLCK inhibitor, ML-7 [1-(5-iodonaphthalene-1-sulfonyl)-1 H-hexahydro-1,4-diazapine hydrochloride] is a promising candidate for an anticancer agent, which would induce apoptosis as well as prevents invasion and metastasis in certain types of cancer cells. This study assessed cytotoxic effects of ML-7 against HL-60 cells and therapeutic efficacy of ML-7 as a potential antileukemia agent. Trypan-blue exclusion assays showed dose- and time- dependent decreases in ML-7 treated HL-60 cells (p<0.05). Comet assays revealed a significant increase in DNA damage in HL-60 cells after treatment with $40{\mu}M$ ML-7 for 2h. Sub-G1 fractions, analyzed by flow cytometry increased in a dose-dependent manner, suggesting that ML-7 can induce apoptotic cell death in HL-60 cells. ML-7 was selectively cytotoxic towards HL-60 cells; not affecting normal human lymphocytes. That selective effect makes it a promising potential anti-leukemia agent. In addition, anticancer efficacy of ML-7 in combination with flavonoids (genistein or quercetin) or anticancer drugs (cisplatin or Ara-C) against HL-60 cells was assessed. Combination of ML-7 with flavonoids increased the anti-cancer effect of ML-7 to a greater extent than combination with the anticancer drugs. This implies that ML-7 in combination with flavonoids could increase the efficacy of anticancer treatment, while avoiding side effects cansed by conventional anticancer drug-containing combination chemotherapy.

• 동의생리병리학회지
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• 제19권3호
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• pp.772-778
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• 2005

The chloromethyl-2-dihydroxyphosphinyl-6,7-dimethoxy-1,2,3,4-tetrahydro- isoquinoline (CDDT) is a newly synthesized derivative from 1,2,3,4-Tetra- hydroisoquinoline (THIQ). The THIQs include potent cytotoxic agents that display a range of antitumor activities, antimicrobial activity, and other biological properties. In this study, we investigated the effect of CDDT on the cytotoxicity, induction of apoptosis in human promyelocytic leukemia cells (HL-60 cells). CDDT showed a significant cytotoxic activity in HL-60 cells ($IC_{50}$ = approximately $37\;{\mu}g/ml$) at a 24 hr incubation. Treatment of HL-60 cells with CDDT displayed several features of apoptosis, including formation of DNA ladders in agarose gel electrophoresis, morphological changes of HL-60 cells with DAPI stain. Here we observed that CDDT caused activation of caspase-3, caspase-8, and caspase-9. The most efficacious time on the activation of caspases-3 was achieved at 12 hr. Further molecular analysis demonstrated that CDDT led to cleavage of poly(ADP-ribose) polymerase (PARP), increase of hypodiploid (Sub-G1) population in the flow cytometric analysis. In conclusion, these above results indicate that CDDT dramatically suppresses HL-60 cell growth by activation of caspase-3 with caspase-8, -9 activity. These data may support a pivotal mechanism for the use of CDDT in the prevention and treatment of leukemia.

• 동의생리병리학회지
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• 제19권3호
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• pp.677-683
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• 2005

The aim of this study was to investigate the apoptotic effect and its mechanism on Radix Aconiti (RA) extract in HL-60 human leukemia cell line. RA extract induced apoptosis as confirmed by discontinuous fragmentation of DNA. To clarify the mechanisms on RA extract-induced apoptosis, we examined the caspase-3, -8 enzyme activity and protein levels including Fas, FasL in HL-60 cells. Treatment with RA extracts resulted in the increase of caspase-3 enzyme activity in a time and dose-dependent manners, which was accompanied by the cleavage of poly-(ADP-ribose) polymerase (PARP). This activation of caspase-3 enzyme resulted from cleavage of procaspase-8, which was followed by increases of FasL, Fas protein expression in RA extracts-treated HL-60 cells. In conclusion, RA extract induced apoptosis of HL-60 human leukemia cell line. This results suggest that the apoptotic mechanisms of RA extract on HL-60 cells involved in FasL, Fas activation, procaspase-8 cleavage, activation of caspase-3 and cleavage of PARP. Collectively, these results suggest that RA may be a valuable agent as a anti-cancer drug.

• Parasites, Hosts and Diseases
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• 제33권3호
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• pp.173-178
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• 1995

톡소포자충(RH strain) tachyzoltes를 감마선에 조사하여 인체 백혈병 세포인 HL-60 세포와 정상 마우스의 복강 대식세포에 감염시킨 다음 충체 증식 능력에 어떠한 변화가 오는지 $^3H-uracil$ 흡수시험을 통하여 알아보았다 감마선 조사량은 30, 70, 100및 300 Gy로 하였고 tachyzoite감염은 시켰으나 감마선을 조사하지 않은 비조사군(NI군)과 tachyzoites를 넣지 않고 숙주세포만을 배양한 비감염군을 각각 두었다. 3H-uracil 흡수시험 결과 12-24시간 후 대식세포의 NI 군은 2,190-4,787(countperminute. 이하 같음), HL-60세포의 NI 군은 2,967-8,254의 높은 흡수량을 보였으나. 감마선 조사군은 대식세포 감염시 381-703(100 Gy조사군) 및 218-408(300 Gy조사군), HL-60 세포 감염시 1,911-2,618(30 Gy), 1,253-1,384(70 Gy), 1,013-1,090(100 Gy) 및 483-588(300 Gy)의 흡수량을 각각 보였다. 충체 증식에 대한 억제율은 300 Gy 조사시 12-24시간에 대식세포의 경우 90-92%. HL-60 세포의 경우 80-94%이었다. 이상과 같이 톡소포자충 RH tachyzoites가 감마선 조사 후 세포내 분열증식 능력에 치명적인 영향을 받는다는 것을 uracil 흡수 시험법으로 확인하였다.

• 대한한방종양학회지
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• 제6권1호
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• pp.99-111
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• 2000

Objectives: Hedyotis diffusa is used to treat cancer in traditional Korea Medicine. So this study was carried out to examine the expression of cell cycle related genes in HL-60 cells undergoing apoptosis by the methanol extract of Hedyotis diffusa. Methods: 1. HL-60 cells were treated with various concentrations (from 200 to $50{\mu}g/ml$)of methanol extract and H20 extract ($200{\mu}g/ml$) of hedyotis diffusa. After 48 h later, the cells were tested for viability by MTT assay. 2. The HL-60 cells were treated with $200{\mu}g/ml$ of methanol extract for the indicated periods. The whole cell lysates were prepared and analyzed by western blotting using anti-p53 antibody. 3. The nuclear extract were prepared and analyzed by western blotting using anti-p21 antibody, anti-p27 antibody, anti-cyclin A antibody, anti-cyclin E antibody and anti-CDK2 antibody. Results: 1. The methanol extract of Hedyotis diffusa induced the death of HL-60 cells in a dose dependent manner. 2. The methanol extract of Hedyotis diffusa makedly decreased the level of p21/Cipl and cyclin A in a time dependent manner. 3. The methanol extract of Hedyotis diffusa markedly increased the level of p27/Kipl and cyclin E in a time dependent manner. 4. The methanol extract of Hedyotis diffusa markedly did not affect the level of CDK2. Conclusions: These results provide evidence that expression of cell cycle related genes in HL-60 cells undergoing apoptosis by the methanol extract of Hedyotis diffusa mainly results from decreased level of p21/Cipl and increased level of p27/Kipl of the cell cycle related genes.

• 동의생리병리학회지
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• 제19권3호
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• pp.633-639
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• 2005

The composition of Hwoangbaec-tang has been traditionally used in Korea to treat cancer. Hwoangbaec-tang I is the water extracts prepared from Angelica dahurica, Fritillariae verticillata, Ailanthus altissima, Viscum coloratun, Scutellaria Radix, Ginseng Radix, Astragalus membranaceus, and Glycyrrhizae Radix. Hwoangbaec-tang II also is the water extracts prepared from Ginseng Radix, Astragalus membranaceus, and Glycyrrhizae Radix. The anti-leukemic effects of human promyelocytic leukaemia (HL-60 cells) by Hwoangbaec-tang I or II was accessed by propidium iodide staining flow cytometric analysis, and apoptosis-inducing activity was further confirmed by a nuclear morphological change, a ladder pattern of DNA fragmentation, and an activation of caspase-3 and 9. Hwoangbaec-tang I was found to induce the apoptosis of HL-60 cells via caspase-3 and 9 pathway. In the other side, Hwoangbaec-tang II was found to inhibit the growth of HL-60 cells by inducing these cells to differentiate toward granulocytes. These results indicate that the different anti-leukemic effects of Hwoangbaec-tang in HL-60 cells can be induce the apoptosis or differnetiation of HL-60 cells in Hwoangbaec-tang composition dependent manner.