Effect of Nardostachyos Rhizoma on Apoptosis, Differentiation and Proliferation in HL-60 cells

Ju Sung-Min,Lee Jun,Choi Ho-Seung,Yoon Sang-Hak,Kim Sung-Hoon,Jeon Byung-Hun The Physiological Society of Korean Medicine and T 2006 동의생리병리학회지 Vol.20 No.1

Nardostachyos Rhizoma (N. Rhizoma) belonging to the family Valerianaceae has been anti-arrhythmic effect, and sedation to the central nerve and a smooth muscle. We reported that the water extract of N. Rhizoma induced apoptotic cell death and differentiation in human promyelocytic leukemia (HL-60) cells. Cytotoxicity of N. Rhizoma was detected only in HL-60 cells (IC50 is about 200 ${\mu}g/ml$). The cytotoxic activity of N. Rhizoma in HL-60 cells was increased in a dose-dependent manner. We used several measures of apoptosis to determine whether these processes were involved in N. Rhizoma-induced apoptotic cell death. The high-dose (200 ${\mu}g/ml$) treatment of N. Rhizoma to HL-60 cells showed cell shrinkage, cell membrane blobbing, apoptotic bodies, and the fragmentation of DNA, suggesting that these cells underwent apoptosis. Treatment of HL-60 cells with N. Rhizoma time-dependently induced activation of caspase-3, caspase-8, and caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase. Also, we investigated the effect of N. Rhizoma on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells was determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide assay, respectively. N. Rhizoma induced the differentiation of HL-60 at the low-dose (100 ${\mu}g/ml$) treatment, as shown by increased expression of differentiation surface antigen CD11b, but not CDl4 and increased reducing activity of NBT. When HL-60 cells were treated with N. Rhizoma at concentration of $50{\mu}g/ml\;and\;100{\mu}g/ml$, NBT-reducing activities induced approximately 1.5-fold and 20.0-fold as compared with the control. In contrast, HL-60 cells treated with the N. Rhizoma-ATRA combination showed markedly elevated levels of 26.3-fold at $50{\mu}g/ml$ N. Rhizoma-0.1 ${\mu}M$ ATRA combination and 27.5-fold at 50 ${\mu}g/ml$ N. Rhizoma-0.2 ${\mu}M$ ATRA combination than when treated with N. Rhizoma alone or ATRA alone. It may be that N. Rhizoma plays important roles in synergy with ATRA during differentiation of HL-60 cells. DNA flow-cytometry indicated that N. Rhizoma markedly induced a G1 phase arrest of HL-60 cells. N. Rhizoma-treated HL-60 cells increased the cell population in G1 phase from 32.71% to 42.26%, whereas cell population in G2/M and S phases decreased from 23.61% to 10.33% and from 37.78% to 33.98%, respectively. We examined the change in the $p21^{WAF1/Cip1}\;and\;p27^{Kip1}$ proteins, which are the CKIs related with the G1 phase arrest. The expression of the CDK inhibitor $p27^{Kip1},\;but\;not\;p21^{WAF1/Cip1}$ were markedly increased by N. Rhizoma. Taken together, these results demonstrated that N. Rhizoma induces apoptotic cell death through activation of caspase-3, and potently inhibits the proliferation of HL-60 cells via the G1 phase cell cycle arrest in association with $p27^{Kip1}$ and granulocytic differentiation induction .

HL60 세포주에서 방사선 조사에 의한 Apoptosis와 세포 주기 관련 유전자의 발현 변화

김진희(Jin Hee Kim),박인규(In Kyu Park) 대한방사선종양학회 1998 Radiation Oncology Journal Vol.16 No.4

목 적 : 방사선조사에 의한 apoptosis에서 나타나는 각종 세포주기관련 유전자들의 발현 양상을 RNA와 단백 수준에서 분석하여 방사선조사에 의한 apoptosis에서의 세포주기 조절의 변화를 규명함으로서 방사선치료의 기전에 대한 분자생물학적 이해를 도모하고자 본 연구를 시행하였다. 대상 및 방법 : promyelocytic leukemia 세포주인 HL60 세포주를 배양하여 선형가속기(6MV X-선)로 세포에 8Gy의 방사선을 조사하였다. 조사후 다양한 시간 간격으로 Apoptotic DNAFragmentation Assay법을 이용하여 apoptosis를 확인하고 동시에 세포주기관련 유전자들(cyclin A, cyclin B, cyclin C, cyclin D1, cyclin E, cdc 2, CDK2, CDK4, p16INK4a , p21WAF1 , p27KIP1, E2F, PCNA와 Rb)을 단백질과 RNA 수준에서 분석하기위해 western blot analysis와 반정량적 RT-PCR을 시행하였다. 결 과 : 8 Gy의 방사선 조사에 의해 HL60세포에서 apoptosis가 관찰 되었다. 방사선 조사군에서 cyclin A단백은 조사후 48시간까지 시간이 갈수록 증가하였으며, cyclin E, E2F, CDK2 및 Rb 단백은 증가되었다가 다시 감소를 보였다. Rb단백의 증가는 대부분 비활성형인 ppRb(phosphorylated Rb protein)의 양적변화에 의한 것이었다. cyclin D1, PCNA, CDC2, CDK4, p16INK4a단백은 발현의 차이를 보이지 않았으며 p21WAF1과 p27KIP1 단백은 검출되지 않았다. cyclin A, B, C mRNA는 방사선 조사 직후 감소하였다가 12시간부터 발현이 증가되었으며 cyclin D1 mRNA는 조사후 바로 증가하여 48시간에 다시 감소하였다. cyclin E mRNA는 조사후 시간 이 경과함에 따라 감소하였다. CDK2 mRNA는 3시간째는 감소하다가 6시간부터 많은 증가를 보였으며 CDK4 mRNA는 조사후 6-12시간에 급격한 발현증가를 보였다. p16INK4a RNA는 발현의 변화가 없었으며, p21WAF1 및 p27KIP1 RNA의 발현은 관찰되지 않았다. 결 론 : 이상의 결과로 미루어볼 때, 방사선 조사에 의한 HL60세포의 apoptosis와 세포의 G1/S transition는 밀접한 관계가 있는 것으로 생각되며 Rb단백의 증가와 활성형 Rb단백의 감소 현상도 관련이 있는 것으로 사료된다. 이는 E2F의 비정상적인 과발현 및 cyclin E/CDK2의 발현 증가와 관련이 있는 것으로 추측된다. 또한 p21WAF1 및 p27KIP1는 방사선에 의한 apoptosis에는 관여되지 않는 것으로 사료된다. Purpose : To evaluate changes in expression of cell cycle related genes during apoptosis induced in HL60 cells by X-irradiation to understand molecular biologic aspects in mechanism of radiation therapy. Material and Methods : HL-60 cell line (promyelocytic leukemia cell line) was grown in culture media and irradiated with 8 Gy by linear accelerator (6 MV X-ray). At various times after irradiation, ranging from 3 to 48 hours were analyzed apoptotic DNA fragmentation assay for apoptosis and by western blot analysis and semi-quantitative RT- PCR for expression of cell cycle related genes (cyclin A, cyclin B, cyclin C, cyclin D1, cyclin E, cdc2, CDK2, CDK4, p16INK4a, p21WAF1, p27KIP1, E2F, PCNA and Rb). Results : X-irradiation (8 Gy) induced apoptosis in HL-60 cell line. Cycline A protein increased after reaching its peak 48 h after radiation delivery and cyclin E, E2F, CDK2 and RB protein increased then decreased after radiation. Radiation induced up-regulation of the expression of E2F is due to mostly increase of phosphorylated retinoblastoma proteins (ppRb). Cyclin D1, PCNA, CDC2, CDK4 and p16INK4a protein underwent no significant change at any times after irradiation. There was not detected p21WAF1 and p27KIP1 protein. Cyclin A, B, C mRNA decreased immediately after radiation and then increased at 12 h after radiation. Cyclin D1 mRNA increased immediately and then decreased at 48 h after radiation. After radiation, cyclin E mRNA decreased with the lapse of time. CDK2 mRNA decreased at 3 h and increased at 6h after radiation. CDK4 mRNA rapidly increased at 6 to 12 h after radiation. There was no change of expression of p16INK4a and not detected in expressin of p21WAF1 and p27KIP1 mRNA. Conclusion : We suggest that entry into S phase may contribute to apoptosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced apoptosis of HL60 cells and tosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced apoptosis of HL60 cells and this may be associated with induction of E2F and cyclinE/CDK2. These results support that p21WAF1 and p27KIP1 are not related with radiation induced- apoptosis.

HL-60 백혈령 세포주에서 분화유도물질로 전처치 후의 telomerase 활성

김인호,김숙자,정희정,박성규,이규택,원종호,서원석,백승호,홍대식 大韓血液學會 1999 大韓血液學會誌 Vol.34 No.1

HL-60 세포주에서 myeloblastin mRNA 발현의 하향조절

이대희 고신대학교 의학부 2005 高神大學校 醫學部 論文集 Vol.20 No.1

Background: Recent clinical studies have shown that a high proportion of patients with acute promyelocytic leukemia(APL) achieves complete remission after treatment with all-trans retinoic acid (ATRA). However, most patients who receive continuous treatment with ATRA relapse and develop ATRA-resistant leukmia. In this study, the author investigated the strategies to overcome ATRA resistance of acute promyelocytic leukemia (APL) cells by inducing the differentiation of dendritic cells (DCs) from human leukemic cell lines for the developtment of adoptive immunotherapy. Myeloblastin (mbn) was used as one of the indicators of differentiation in this study. Methods: To study the biochemical and enzymatic charicteristics of the human myeloblastin the enzyme was extracted from human leukocytes and purified by a combination of Ultrogel AcA 54 and Bio-Rex 70 chromatographies. To evaluate the mbn protein expression in cells, anti-mbn antibody was prepared by two-step procedure including ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. HL-60 cell differentiation was induced by the addition of all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA), cholecalcitriol (VD) to the media for 6 days and the expression of mbn mRNA and mbn protein were determined by RT-PCR method and ELISA, respectively. HL-60 cells, K-562 cells, NC-37 and RPMI 7666 cells were cultured in RMPI 1640 supplemented with 10% fetal calf serum for 7 days, with various agents or ligands such as calcium ionophore (CI), Flt3-ligand (FL) and PMA to generate dendritic cells from the cell lines. A portion of ezch cell lines was havested and the rest of them was cultured in the new RPMI 1640 supplemented with 10% fetal calf serum, with Flt3-ligand for 7 days more. RNA was extracted and gene expression from each cell lines was determined by RT-PCR method. The morphology of the cells was evaluated from cytospin slide preparations with Wright's stain. Result: 3.8 mg of proteinase-3 was isolated from 67 ㎎ of leukocyte extract. 6g of anti-mbn polyclonal antibody was raised. PMA induced a significant inhibition of mbn mRNA expression in HL-60 cells. The cells exposed to ATRA or DMSO or VD show a little change in the mbn mRNA expression. Thus the terminal differention of HL-60 cells and K-562 cells by ATRA, DMSO, VD, and hemin was incomplete and a large fraction of the cells was in undifferentiated or premature states. The promyelocytic leukemic cell line HL-60, B lymphoblast cell lines RPMI 7666 and NC-37 could be induced to dendritic cells in vitro. Treatment of HL-60 with PMA resulted in the expression of myeloid-related DC phenotypes, while treatment of RMPI 7666 with FL and treatment of NC-37 with PMA and FL lead to the expression of lymphoid-related DC phenotypes. Conclusion: In conclusion, myeloid-related DC phenotypes and lymphoid-related DC phenotypes can be generated from HL-60, NC-37 RPMI 7666 cell lines, respectively. These DC phenotypes can potentially be used as a cellular leukemia vaccine in vivo or to generate antileukemic T cells in vitro for adoptive immunotherapy.

HL-60 세포주에서 myeloblastin mRNA 발현의 하향조절

이대희 고신대학교(의대) 고신대학교 의과대학 학술지 2005 고신대학교 의과대학 학술지 Vol.20 No.1

Background : Recent clinical studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieves complete remission after treatment with all-trans retinoic acid (ATRA). However, most patients who receive continuous treatment with ATRA relapse and develop ATRA-resistant leukemia. In this study, the author investigated the strategies to overcome ATRA resistance of acute promyelocytic leukemia (APL) cells by inducing the differentiation of dendritic cells (DCs) from human leukemic cell lines for the development of adoptive immunotherapy. Myeloblastin (mbn) was used as one of the indicators of differentiation in this study. Methods : To study the biochemical and enzymatic charicteristics of the human myeloblastin the enzyme was extracted from human leukocytes and purified by a combination of Ultrogel AcA 54 and Bio-Rex 70 chromatographies. To evaluate the mbn protein expression in cells, anti-mbn antibody was prepared by two-step procedure including ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. HL-60 cell differentiation was induced by the addition of all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA), cholecalcitriol (VD) to the media for 6 days and the expression of mbn mRNA and mbn protein were determined by RT-PCR method and ELISA, respectively. HL-60 cells, K-562 cells, NC-37 and RPMI 7666 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum for 7 days, with various agents or ligands such as calcium ionophore (CI), Flt3-ligand (FL) and PMA to generate dendritic cells from the cell lines. A portion of each cell lines was harvested and the rest of them was cultured in the new RPMI 1640 supplemented with 10% fetal calf serum, with Flt3-ligand for 7 days more. RNA was extracted and gene expression from each cell lines was determined by RT-PCR method. The morphology of the cells was evaluated from cytospin slide preparations with Wright's stain. Results : 3.8 mg of proteinase-3 was isolated from 67 mg of leukocyte extract. 6g of anti-mbn polyclonal antibody was raised. PMA induced a significant inhibition of mbn mRNA expression in HL-60 cells. The cells exposed to ATRA or DMSO or VD show a little change in the mbn mRNA expression. Thus the terminal differention of HL-60 cells and K-562 cells by ATRA, DMSO, VD, and hemin was imcomplete and a large fraction of the cells was in undifferentiated or premature states. The promyelocytic leukemic cell line HL-60, B lymphoblast cell lines RPMI 7666 and NC-37 could be induced to dendritic cells in vitro. Treatment of HL-60 ith PMA resulted in the expression of myeloid-related DC phenotypes, while treatment of RPMI 7666 with FL and treatment of NC-37 with PMA and FL lead to the expression of lymphoid-related DC phenotypes. Conclusion : In conclusion, myeloid-related DC phenotypes and lymphoid-related DC phenotypes can be generated from HL-60, NC-37, and RPMI 7666 cell lines, respectively. These DC phenotypes can potentially be used as a cellular leukemia vaccine in vivo or to generate antileukemic T cells in vitro for adoptive immunotherapy.

Dimethylsulfoxide (DMSO) induces downregulation of heme oxygenase-1 (HO-1) in HL-60 cells: involvement of HO-1 in HL-60 cell differentiation

( Eun Mi Noh ),( Dong Hyu Cho ),( Young Rae Lee ),( Young Ju Jeong ),( Jong Hyeon Kim ),( Hee Suk Chae ),( Jin Ny Park ),( Won Seok Jung ),( Sung Joo Park ),( Jong Suk Kim ) 생화학분자생물학회 2011 BMB Reports Vol.44 No.11

Heme oxygenase-1 (HO-1), an inducible enzyme with broad tissue expression, is wel1-regulated in response to hematopoietic stress and preserves vascular homeostasis. We investigated the involvement of HO-1 in HL-60 cell differentiation. Dimethyl sulfoxide (DMSO) completely decreased HO-1 expression in a time-dependent manner, but clearly induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression. Interestingly, zinc protoporphyrin (ZnPP), a strong inhibitor of HO-1, induced HL-60 cell differentiation. In contrast, treatment with cobalt protoporphyrin (CoPP), an activator of HO-1, decreased CD11b expression. Additionally, ZnPP downregulated HO-1 protein expression in HL-60 cells, whereas CoPP induced upregulation. These results suggest that HO-1 might have a negative function in DMSO-induced HL-60 cell differentiation. This study provides the first evidence that HO-1 plays an important role in DMSO-induced HL-60 cell differentiation. [BMB reports 2011; 44(11): 753-757]

HL-60세포에서 disulfiram의 항암작용과 미토콘드리아 안정성에 대한 연구

신효원,한용,주홍구,Shin, Hyowon,Han, Yong,Joo, Hong-Gu 대한수의학회 2019 大韓獸醫學會誌 Vol.59 No.4

Disulfiram (DSF) is a member of the dithiocarbamate family that can bind copper. Recent studies have shown that DSF has anti-cancer activities, but the mechanism has not been clarified. Therefore, it is important to study the action mechanism of DSF to maximize its anticancer effects. A human leukemia cell line, HL-60, was used in this study. HL-60 cells were treated with DSF and the cellular metabolic activity was measured. DSF increased the cell death of HL-60 cells in annexin V-fluorescein isothiocyanate/propidium iodide staining analysis. In addition, DSF decreased the mitochondrial membrane potential (MMP) of the HL-60 cells. The cytotoxicity of DSF on HL-60 cells was observed at 0.4 μM. Interestingly, the reduction of MMP by DSF was recovered by N-acetyl-L-cysteine, an inhibitor of reactive oxygen species (ROS) production. This suggests that the decrease in MMP by DSF is closely related to the production of ROS in HL-60 cells, which indicates the relationship between the apoptosis of HL-60 cells by DSF and the role of the mitochondria. This study provides clinicians and researchers with valuable information regarding the anti-cancer activity of DSF in terms of the action mechanism.

초음파 병행 추출을 이용한 마황과 복분자, 당귀 분획물의 면역활성 조절 효과

김정화,김대호,유진현,김철희,권민철,성낙술,이승은,이현용 한국약용작물학회 2005 한국약용작물학회지 Vol.13 No.4

당귀, 복분자, 마황의 추출공정 요인별 [추출온도 (40℃, 60℃, 100℃), 초음파 병행 (처리조건 : 초음파별) 추출]에 따른 추출물들의 분획물들의 추출수율 증진 및 생산성 향상이 가능한 공정 개발을 위하적 공정 요인에 따라 추출과 분획을 실시하였다. 그 중 모든 분획 추출물 들 중에서 60℃ 온도에서 40kHz의 초음파 병행 추출한 복분자 water 분획층 수율이 59.63%로 다른 추출 공정에 비하여 가장 높게 나타났으며, 당귀 water 분획층의 수율은 50.17%로 나타나 water 분획층의 수율이 가장 높게 나타났으며, butanol, ethyl acetate, chloroform 분획층의 순서로 수율이 나타난 것을 확인 할 수 있었다. 이것으로 60℃의 40 kHz 초음파 병행 추출 공정이 생산성이 가장 우수한 것으로 나타났다. 면역활성 증진 효과에서 인간의 면역세포인 B, T 세포에 대한 생육 활성도를 각 공정별 추출물을 이용하여 측정한 결과 60℃의 40 kHz의 초음파 병행 추출물의 water 분획물에서 180% 이상의 높은 생육 활성도를 나타내었고, butanol 분획물에서는 160% 이상의 높은 생육 활성도를 나타내었다. Cytokines 분비량은 면역 세포에 대한 활성 실험에서 효과가 가장 높게 나타났던 추출공정인 60℃의 40 kHz 초음파 병행 추출물의 water 분획층과 butanol 분획층을 이용하여 분획물 실험을 행하였다. B세포에서의 IL-6 분비량에 복분자 water 분획충이 1.0 g/l에서 5일째 17×10-4 ph/cell로 가장 높은 증진 효과를 나타내었으며, butanol 분획층이 16.8×10-4 pg/cell의 증진효과를 보였고, 마황과 당귀 water 분획층이 각각 16.5×10-4 pg/cell, 16.5×10-4 pg/cell의 증진효과를 보였다 B 세포에서의 TNF-α분비량은 복분자 water 분획층이 1.0 g/l에서 5일째 18×10-4 pg/cell로 가장 증진 효과가 크게 나타났다. 이것은 세포의 생육이 시간에 따라 증가됨으로서 그에 따른 cytokines의 분비량도 시간에 따라 증가하는 것으로 사료된다. 추출공정 요인인 온도와, 초음파를 달리하여 그것의 분획물인 water 분획층과 butanol 분획층에 대한 T 세포에서의 cytokine 분비량을 측정한 결과 온도의 변화를 준 추출공정에서의 IL-6분비량은 60℃에서 가장 좋은 분비량을 나타낸 것으로 복분자가 18.5×10-4 pg/cell로 가장 높은 증진 효과를 나타내었다. 마황과 당귀 또한 60℃로 추출하였을 때, 1.08배, 1.04배 높은 분비량을 나타내었다. 또한 초음파의 주파수의 변화를 주어 추출한 것으로 T세포의 TNF-α 분비량을 측정한 결과 40 kHz의 초음파 병행 추출한 복분자 water 분획층이 1.0 g/l 농도에서 20.4×10-4 pg/cell로 60 kHz로 병행 추출한 복분자 water 분획층의 19.5×10-4 pg/cell보다 높은 분비량을 나타내었다. 당귀와 마황에서도 40 kHz의 초음파로 병행 추출한 것이 더 높은 분비량을 나타내었다. NK-cell의 활성도를 공정 요인별로 측정한 결과 가장 좋은 활성도를 나타낸 것으로는 60℃에서 40 kHz의 초음파로 병행 추출한 복분자 water 분획층 이 배양 6일째 날 1.0 g/l의 농도에서 97%, butanol 분획층은 93%의 활성증진을 보여 초음파 병행 추출하지 많은 것과 다른 온도 조건에 비해 높은 생육 활성도를 나타내었다. 그리고 당귀 분획물 또한 60℃, 40 kHz의 초음파 추출물의 water 분획층과 butanol 분획층에서 각 92%와 80%의 활성 증진을 나타내 다른 추출 공정보다 높은 활성을 나타내었다. 이것으로 알 수 있듯이 생체 방어 기능을 증진 시킬 수 있는 최적의 추출공정으로는 60℃에서 40 kHz의 초음파를 병행하여 추출하는 것으로 사료된다. 세포 분화활성도의 측정 결과는 복분자 water분획층이 가장 높은 세포 분화 활성도를 나타내었고, 또한 복분자와 마황의 분획물이 crude 상태의 초음파 병행 추출물에 비해 최고 20% 이상의 분화 활성도를 나타내는 것을 확인 할 수 있었다. This study was performed to examine immuno-regulatory activities of Ehpedrae Sinica STAPF, Rubus Coreanus Miq. and Angelica gigas Nakai extracts in conbination with ultrasonification. The extract yields of plants were the highest in the extraction system of 60℃ and 40 kHz of ultrasonification. The immune cell growth ratio of human immune B and T cells was increased compared to other fractions by the water fraction of the plants at 60℃ and 40 kHz. The water fractions of the plants at 60℃ and 40 kHz increased the specific secretion of IL-6 and TNF-α of human immune B and T cells compared to other fractions of the plants. The water fraction of Ehpedrae Sinica STAPF among the plants was observed to show the highest specific secretion of IL-6 and TNF-α. Also, NK-92 MI cells growth was increased in adding the water fractions of the plants at 60℃ and 40 kHz. The water fraction of Ehpedrae Sinica STAPF among the plants showed the highest in NK-92 MI cell growth ratio. The differentiation activity of the HL-60 cells significantly increased in adding the water fraction of Ehpedrae Sinica STAPF compared to other fractions of the plants. These results suggest that the water fractions of the plants in extraction system of temperature 60℃ and ultrasonification 40 kHz have marked useful immuno-stimulatory activities.

Capsaicin potentiates 1,25-dihydoxyvitamin D_3-and all-trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells

Kang, So N.,Chung, Su W.,Kim, Tae S. 전남대학교 약품개발연구소 2001 약품개발연구지 Vol.10 No.-

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D_3[I,25-(OH)_2,D_3] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin. an active component of the red pepper of the genus Capsocten, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5-30 ㎍/ml capsaicin for 72h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5nM 1,25-(OH)_2, D_3 or 50nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)_2D_3 and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)_2D_3- and all-trans retinoic acid-treated HL-60 cells. In addition. inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, l-(5-isoquinolinesulfonyl)-2_-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1.25-(OH)_2D_3 or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)_2 D_3- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin

백화사설초 메탄올 추출물에 의한 HL-60 세포(細胞) 고사과정(枯死過程)에서의 cell cycle 관련인자(關聯因子)의 활성변화(活性變化) 연구(硏究)

한세희,이종범,문구,문석재,원진희,박래길,이종덕,Han, Se-Hee,Lee, Jong-Bum,Moon, Gu,Moon, Suk-Jae,Won, Jin-Hee,Park, Lae-Gil,Lee, Jong-Deok 대한암한의학회 2000 大韓癌韓醫學會誌 Vol.6 No.1

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.