Development of polyploidization in taxol-resistant human leukemia cells in vitro

The effects of taxol on antitubulin immunofluorescent staining patterns, cellular DNA content, and labeling with [3H]thymidine were measured for the taxol-sensitive HL60 and taxol-resistant K562 cell lines after exposures for 0, 4, 12, and 24 h. Taxol caused a relative increase in the fraction of 4C interphase and metaphase cells in both lines although the 4C interphase accumulation was greater for the resistant K562 line. Of the cells with S-phase DNA content, taxol-treated HL60 cells were less likely to incorporate [3H]thymidine than taxol-treated K562 cells. However, a decrease in percentage of S-phase labeling for both lines relative to control cells was seen. Finally, taxol induced the development of polyploid cells (cells with DNA contents greater than that of the 4C G2-M peak) in the relatively taxol-resistant K562 cells, an effect not seen in the relatively taxol-sensitive HL60 line. After 24 h of taxol exposure 70% of all K562 cells were polyploid while only 8% of the HL60 cells were polyploid. The capacity of K562 cells to generate polyploidy in response to taxol correlated with taxol resistance by previous assay and may be a useful indicator of drug resistance.     

紫杉醇诱导人乳腺癌细胞凋亡过程DNA甲基化水平检测

目的紫杉醇诱导细胞凋亡过程中ＤＮＡ甲基化水平状态及其意义，尚未见报道。方法本文应用反相高效液相色谱法和琼脂糖凝胶电泳法对紫杉醇诱导人乳腺癌细胞（ＢＣａｐ３７）凋亡过程中ＤＮＡ甲基化水平进行分析，并与秋水仙碱、顺铂、鬼臼乙叉甙、阿霉素和放线菌酮处理组进行比较。结果紫杉醇处理ＢＣａｐ３７细胞３小时，ＤＮＡ甲基化水平降低；在处理１２～２４和４８小时时，ＤＮＡ甲基化水平逐渐上升，在４８小时达到最高峰，显著高于对照组（Ｐ＜０．０２）。然而在处理７２小时，ＤＮＡ甲基化水平明显降低，并低于对照组（Ｐ＜０．０５）。除阿霉素处理ＢＣａｐ３７细胞能够使ＤＮＡ甲基化水平降低之外，其余抗癌药处理组细胞ＤＮＡ甲基化水平均有轻度的增高。结论在紫杉醇诱导乳腺癌细胞凋亡过程中，细胞ＤＮＡ甲基化水平增高可能与先前研究发现的腺苷基蛋氨酸合成酶基因表达增高密切相关。本文就ＤＮＡ高甲基化反应在紫杉醇诱导的细胞凋亡中的潜在生物学意义进行了讨论。     

p38丝裂原活化蛋白激酶阻滞剂SB203580对白血病K562细胞周期的作用及机制

 目的　研究p38丝裂原活化蛋白激酶（MAPK）阻滞剂SB203580对K562细胞周期的作用及机制。方法　以反转录-聚合酶链反应（RT-PCR ）和Western blotting方法检测SB203580处理K562细胞后p38、Cyclin D2、Cyclin E、p27 mRNA和蛋白的表达并以流式细胞术（FCM）检测其细胞周期的变化。结果　SB203580处理后K562细胞 p38、Cyclin D2、Cyclin E mRNA和蛋白表达降低;p27 mRNA和蛋白表达增高。G0／G1期细胞增多,S期细胞减少,与用药前相比差异均有统计学意义。结论　SB203580可能通过p38途径,影响细胞周期调控蛋白,最终抑制K562细胞的增生。     

bcl—2基因家族在紫杉醇介导BJAB细胞凋亡中的作用

目的 观察抗微管新药紫杉醇对Ｂ细胞淋巴瘤细胞株ＢＪＡＢ是否具有凋亡诱导作用。并进一步研究ｂｃｌ－２基因家族在此过程中的作用。方法 将不同浓度的紫杉醇作用于ＢＪＡＢ细胞,观察其作用的时间效应及剂量效应;在光镜和电镜下观察其形态变化;用流式细胞仪分析细胞ＤＮＡ含量的改变并做ＤＮＡ片段分析;用免疫组织化学及半定量ＲＴ－ＰＣＲ法观察在紫杉醇作用过程中ｂｃｌ－２基因家族的蛋白及ｍＲＮＡ的变化。结果 紫杉醇能抑制ＢＪＡＢ细胞生长,抑制作用首先表现为Ｇ２／Ｍ期阻滞,一定时间后出现细胞凋亡,并显示剂量和时间效应。在这一过程中,ｂｃｌ－２转录及蛋白表达下降,并出现ｂｃｌ－ｘｓ的转录。结论 紫杉醇可诱导ＢＪＡＢ细胞凋亡,这为其应用于Ｂ细胞淋巴瘤的治疗提供依据。ｂｃｌ－２和ｂｃｌ－ｘｓ参与了紫杉醇介导ＢＪＡＢ细胞凋亡的基因调控。     

Accumulation of cyclin B1, activation of cyclin B1-dependent kinase and induction of programmed cell death in human epidermoid carcinoma KB cells treated with taxol

Cyclin B1 plays a critical role in regulating cell-cycle progression from G₂ through M phase (including exit from M phase). In this study, we investigated the relationship between taxol-induced M-phase arrest, disruption of the cyclin B1-regulation pathway and apoptosis in KB cells. Continuous exposure of KB cells to 0.5 μg/ml taxol caused mitotic arrest and >90% cell death at 48 hr. Mitotic blockade peaked at 24 hr, with 68% of cells in mitosis at that time compared with 3% at baseline, and decreased thereafter. Apoptosis assessed by morphological changes and DNA ladder fragmentation was a later event, peaking at 48 hr (later time points were not studied). Taxol also caused an increase in cyclin B1 accumulation, as assessed by Western blot analysis, and stimulated cyclin B1-dependent kinase. Cyclin B1 accumulation and kinase stimulation peaked at 12 and 24 hr, respectively, at which times they were 5-fold and 90-fold higher than in control untreated cells. These effects decreased thereafter. All taxol-induced cellular effects were abrogated by the protein and RNA synthesis inhibitors cycloheximide and actinomycin D. In contrast, the endonuclease inhibitors aurintricarboxilic acid and zinc markedly inhibited taxol-induced DNA ladder fragmentation without altering taxol-induced cell-cycle arrest, cyclin B1 accumulation, activation of cyclin B1 kinase activity and cytotoxicity. We conclude that taxol-induced stimulation of cyclin B1-dependent kinase activity parallels mitotic arrest, is more pronounced than mitotic arrest and precedes the induction of programmed cell death. Int. J. Cancer 75:925–932, 1998. © 1998 Wiley-Liss, Inc.     

硼替佐米对伊马替尼耐药细胞株K562/G01增殖抑制和诱导凋亡作用的研究

 目的　探讨蛋白酶体抑制剂硼替佐米（BOR）对慢性粒细胞白血病（CML）伊马替尼耐药细胞株K562／G01的增殖抑制和诱导凋亡作用。方法　采用MTT法观察细胞的生长抑制效应;流式细胞术（FCM）检测细胞周期与凋亡。结果　K562／G01细胞对伊马替尼不敏感,伊马替尼对K562／G01和K562细胞的IC50分别是（20.09±1.38）、（0.54±0.13）μmol／L;BOR对K562／G01细胞具有增殖抑制作用,并于48 h时作用效果达高峰,IC50（23.10±2.71）nmol／L,随着BOR浓度和作用时间的增加,FCM检测可见G2／M 期细胞周期阻滞及明显的凋亡峰。结论　BOR对CML伊马替尼耐药细胞株具有增殖抑制和诱导凋亡的作用,其机制可能与细胞周期G2／M 期的阻滞有关。     

维生素E琥珀酸酯诱导耐药白血病K562/ADM细胞凋亡的研究

目的:研究维生素E 琥珀酸酯(vitamin E succinate , VES)对多药耐药白血病K562/ADM细胞的诱导凋亡作用及分子机制。方法: 以体外培养的K562/ADM细胞为研究对象,采用噻唑蓝(methyl thiazolyl tetrazolium, MTT) 比色法检测细胞增殖活性, Wright Giemsa染色、DNA凝胶电泳和流式细胞术(flowcytometry, FCM)检测细胞凋亡;FCM测定细胞Fas、Bcl-2 和p53 蛋白表达水平。结果:VES可显著抑制K562/ADM细胞的生长及增殖,P= 0 .004。光镜下可见K562/ADM细胞呈典型凋亡的形态学改变。DNA凝胶电泳显示典型的凋亡DNA梯形条带。FCM细胞周期分析显示,G1 期阻滞,亚G1 期细胞比例增高,P=0 .005;Fas蛋白表达明显上调,P=0. 002;Bcl -2蛋白表达下调,P=0. 000;p53蛋白表达无明显变化。结论:VES可诱导K562/ADM细胞凋亡,作用机制可能与其上调Fas表达和下调Bcl 2表达有关。     

In Vitro Anticancer Activities of Anogeissus latifolia,Terminalia bellerica,Acacia catechu and Moringa oleiferna Indian Plants

The present study was designed to evaluate in vitro anti-proliferative potential of extracts from four Indianmedicinal plants, namely Anogeissus latifolia, Terminalia bellerica, Acacia catechu and Moringa oleiferna. Theircytotoxicity was tested in nine human cancer cell lines, including cancers of lung (A549), prostate (PC-3), breast(T47D and MCF-7), colon (HCT-16 and Colo-205) and leukemia (THP-1, HL-60 and K562) by using SRB andMTT assays. The findings showed that the selected plant extracts inhibited the cell proliferation of nine humancancer cell lines in a concentration dependent manner. The extracts inhibited cell viability of leukemia HL-60and K562 cells by blocking G0/G1 phase of the cell cycle. Interestingly, A. catechu extract at 100 μg/mL inducedG2/M arrest in K562 cells. DNA fragmentation analysis displayed the appearance of a smear pattern of cellnecrosis upon agarose gel electrophoresis after incubation of HL-60 cells with these extracts for 24h.     

Antitumor activity and apoptosis induction in human cancer cell lines by Dionysia termeana

Studies have demonstrated that plant extracts possess various biological effects including antitumor activity. In the present study, the antitumor activity of Dionysia termeana, a plant native to Iran, was investigated. Cytotoxic activity of the extract on tumor cell lines using MTT colorimetric assay was determined. Cell cycle analysis by flow cytometry and DNA fragmentation analysis on sensitive cell lines was then carried out. Results obtained indicated that the highest activity of D. termeana was against K562 leukemia cell line with IC50 less than 20 μ g/mL. Fifty-five percent inhibition of Jurkat cells due to exposure to D. termeana was found at 200 μ g/mL of the extract. A549, a lung carcinoma cell, and Fen bladder carcinoma cell line were less affected. In flow cytometry analysis, D. termeana induced apoptosis in the K562 and Jurkat cells. In DNA fragmentation analysis the extract produced ladder formation in both cells. In conclusion, these results indicated that the extract used in this study have antitumor activity through induction of apoptosis particularly in the leukemia cell lines.     

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces growth inhibition and enhances taxol-induced cell death in breast cancer

Purpose

The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) enhances taxol-induced antitumor effects against some human cancer cells. The aim of this study is to investigate whether SAHA can enhance taxol-induced cell death against human breast cancer cells and to illustrate the mechanism in detail.

Methods

A panel of eight human breast cancer cell lines and an immortalized human breast epithelial cell line were used to determine the inhibitory effects of SAHA, taxol, or their combination by MTT assay. The effects of SAHA with or without taxol on cell cycle distributions, apoptosis, and protein expressions were also examined. The inhibitory effects on tumor growth were characterized in vivo in BALB/c nude mice bearing a breast cancer xenograft model.

Results

Taxol-resistant and multi-resistant breast cancer cells were as sensitive to SAHA as taxol-sensitive breast cancer cells. A dose-dependent synergistic growth inhibition was found in all the tested breast cancer cell lines treated with the SAHA/taxol combinations. The synergetic effect was also observed in the in vivo xenograft tumor model. The cell cycle analysis and apoptosis assay showed that the synergistic effects resulted from enhanced G2/M arrest and apoptosis.

Conclusions

SAHA increased the anti-tumor effects of taxol in breast cancer in vitro and in vivo. The combination of SAHA and taxol may have therapeutic potential in the treatment of breast cancer.     

INDUCTION OF APOPTOSIS OF HUMAN LEUKEMIA CELLS BY α-ANORDRIN

AnordrinisacontraceptiveagentwhichwasfirstdevelopedinChina.Previousstudyshowedthataisomerofanordrin(ANO)exhibitedantitumoreffectbothinvilroandinvivo.l'2Itwasalsofoundthatatsmalldoses,ANQcouldinducedifferentiationofhumanpromyelocyticleukemia.HL-60cells.3However,themechanismofitsaniitumoractivitywasstillnuclear.Inthiswork,wefurtherinvestigatedthetumor-inhibitoryeffectofANOtoexplorethepossiblemechanismsofitsaction.MATERIALSANDMETHODSDrugANOwasproducedbyShanghaiNo.19PharmaceuticalF…     

依托泊甙（VP—16）诱导人肺腺癌细胞凋亡

目的探讨ＶＰ－１６对肺腺癌细胞凋亡的诱导作用。方法采用光镜、电镜、流式细胞仪、凝胶电泳和ＴｄＴ原位末端标记法检测ＶＰ－１６诱导人肺腺癌ＡＧＺＹ－８３－α细胞凋亡规律,并观察放线菌酮对凋亡的抑制程度。结果ＶＰ－１６在显著抑制细胞增殖的同时,能够诱导细胞凋亡,将细胞阻滞于Ｇ２期。放线菌酮对凋亡有显著的抑制作用。结论ＶＰ－１６诱导细胞凋亡,是其杀死肿瘤细胞的重要机制之一。该凋亡具有明显的时间和剂量依赖性。     

辐射诱发肿瘤细胞增殖抑制和凋亡的相关研究

目的 探讨不同辐射敏感性的肿瘤细胞受照后G2/M期阻滞和凋亡发生的关系。为提高肿瘤放射治疗效果提供理论依据。方法 以人早幼粒白血病细胞株HL-60、人T淋巴细胞性白血病细胞系CEM和人红白血病细胞系K562细胞为对象，采用形态学观察、DNA琼脂糖凝胶电泳和流式细胞仪等方法检测细胞周期的改变和凋亡的发生。结果 辐射敏感的HL-60和CEM细胞受照后首先发生G2/M期阻滞，然后在阻滞退出过程中或之后发生凋亡；辐射耐受的K562细胞受照后只发生G2/M期阻滞，不发生凋亡；照射并加入咖啡因（CAF），抑制3种细胞的G2/M期阻滞，促进凋亡；照射并加入佛波酯（TPA），增加HL-60和CEM细胞的G2/M期阻滞，抑制凋。结论 辐射诱发肿瘤细胞的凋亡与G2/M期阻滞有关，用药物调控G2/M期阻滞可影响辐射所致的凋亡。     

紫杉醇诱导胃癌细胞凋亡的实验研究

 目的　探讨紫杉醇对胃癌细胞的诱导凋亡作用及其诱导的胃癌细胞凋亡的周期时相性。方法　用Sub-G1法检测紫杉醇诱导胃癌细胞MKN-28的凋亡,API法检测紫杉醇诱导胃癌细胞凋亡的周期时相性,并分选后激光共聚焦显微镜技术（PSC）观察形态学,MTT法检测紫杉醇对临床胃癌组织细胞的敏感性。结果　Sub-G1法结果显示紫杉醇能诱导胃癌细胞的凋亡,紫杉醇（浓度10 mg／L）诱导胃癌细胞凋亡在10 h后达到高峰;API法检测结果显示紫杉醇诱导胃癌细胞发生凋亡的时相在G2／M期,PSC形态学观察到G2／M期凋亡特征;MTT法结果显示紫杉醇诱导的20例临床胃癌组织标本中,有16例抑制率大于50 %。结论　紫杉醇能诱导胃癌细胞发生凋亡,相对较敏感,其诱导的胃癌细胞凋亡具有周期时相性。     

The importance of drug scheduling and recovery phases in determining drug activity. Improving etoposide efficacy in BCR-ABL-positive CML cells

K562 leukaemic cells are known to be less sensitive to etoposide than other cell lines, despite having similar topo II mRNA levels and cleavable complex formation. We have investigated the effect of etoposide schedule on cell cycle distribution, apoptosis and p21(waf1) and cdk1(p34) status in two bcr-abl-positive chronic myeloid leukaemia (CML) cell lines (K562 and KU812) and two small cell lung cancer (SCLC) cell lines (H69 and GLC4). During a continuous 5-day exposure, the SCLC cell lines showed a time and concentration-dependent loss of cell viability, with an initial block in the G2/M phase of the cell cycle followed by apoptosis. In contrast, the two CML cell lines showed no significant apoptosis or loss of viability after a similar block in G2/M. However, when K562 or KU812 cells were placed in drug-free medium following a 3-day drug exposure there was marked, concentration-dependent apoptosis (% apoptosis after release at 1 microM etoposide in K562, 10% at 24 h, 30% at 48 h). Our data also show that p21(waf1) does not increase after etoposide treatment in either H69 or GLC4 (both with mutated-p53). Although K562 and KU812 cells are null-p53, the arrest in G2/M during drug exposure was associated with increased p21(waf1) and a decrease in cdk1 (both P<0.001 compared with controls). Upon release of these cells from drug-medium, p21(waf1) gradually returned to control levels, which was associated with an easing of the block at G2/M and an induction of apoptosis. This study highlights the importance of cell cycle regulatory proteins in drug sensitivity and resistance, and suggests that in cells such as K562 and KU812, a pulsed schedule may be more active than a single prolonged exposure.     

Microtubule changes and cytotoxicity in leukemic cell lines treated with taxol

Taxol, a diterpenoid plant product that enhances the polymerization of tubulin, is currently entering clinical trials in the treatment of human leukemia. In order to develop an in vitro assay to predict tumor sensitivity to taxol, human leukemic cell lines were exposed to clinically achievable concentrations of taxol for relevant exposure periods. Changes in microtubules visualized by indirect immunofluorescence were compared to drug sensitivity measured by a clonogenic assay. Taxol produced either multiple mitotic asters in G2/M or microtubule bundling throughout the cell cycle. In cells that were relatively resistant to taxol, microtubule bundling was reversible while microtubule bundling in relatively sensitive cells persisted in the presence or absence of taxol. In contrast, aster formation was unrelated to cytotoxicity in any cell line. In the future, these microtubule effects may be useful in predicting the chemotherapeutic efficacy of taxol.     

Anti-proliferative and apoptotic effects of celecoxib on human chronic myeloid leukemia in vitro

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anti-cancer effects of celecoxib is not fully understood. There is little data on the potential role of COX-2 in lymphoma pathogenesis. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of celecoxib on human chronic myeloid leukemia cell line, K562 and other hematopoietic cancer cell lines like Jurkat (human T lymphocytes), HL60 (human promyelocytic leukemia) and U937 (human macrophage). Treatment of these cells with celecoxib (10-100 microM) dose-dependently, reduced cell growth with arrest of the cell cycle at G0/G1 phase and induction of apoptosis. Further mechanism of apoptosis induction was elucidated in detail in K562 cell line. Apoptosis was mediated by release of cytochrome c into the cytoplasm and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). This was followed by DNA fragmentation. The level of anti-apoptotic protein Bcl-2 was decreased without any change in the pro-apoptotic Bax. Celecoxib also inhibited NF-kB activation. Celecoxib thus potentiates apoptosis as shown by MTT assay, cytochrome c leakage, PARP cleavage, DNA fragmentation, Bcl-2 downregulation and possibly by inhibiting NF-kB activation.     

Taxol sensitizes human astrocytoma cells to radiation.

Taxol is a chemotherapeutic drug which acts by stabilizing microtubules, preventing normal mitosis and resulting in a block of the cell cycle at G2 and M. The drug is isolated from the yew, Taxus sp. L., and is currently being evaluated in a series of Phase II and Phase III clinical trials. Taxol blocks cells in the most radiosensitive phases of the cell cycle and thus could act as a cell cycle-specific radiosensitizer. We report the results of combined taxol-radiation exposures in the human Grade III astrocytoma cell line, G18. Taxol is a potent inhibitor of G18 cell division; a concentration of 10 nM is cytostatic for a cell population observed for at least two doubling times. Cell survival curves for G18 cells showed a significant concentration-dependent interaction between taxol and radiation. Treatment of G18 cells with a fixed taxol concentration and radiation dose showed the interaction to be dependent on the duration of taxol exposure and consequently the fraction of cells in the G2 or M phase of the cell cycle. The sensitizer enhancement ratio for 10 nM taxol at 10% survival is 1.8 and, for 1 nM taxol, it is 1.2. These results suggest that appropriate combinations of taxol have a more than additive interaction in human tissue culture and may have a role in clinical protocols.