Growth inhibition and apoptosis induction in human hepatoma cells by tanshinone II A.

In order to study the effect of tanshinone II A on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone II A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6.28 micrograms/ml. After treatment with 1-10 micrograms/ml tanshinone II A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 micrograms/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32 +/- 0.16)%, (3.01 +/- 0.35)%, (3.87 +/- 0.43)%, (6.73 +/- 0.58)% and (20.85 +/- 1.74)% respectively, which were all significantly higher than those in the control group (1.07 +/- 0.13)%. It is concluded that Tanshinone II A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.     

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

Summary:In order to study the effect of tanshinone Ⅱ_A on growth and apoptosis in human hepatomacell line BEL-7402 in vitro,the human hepatoma cell line BEL-7402 was treated with tanshinone Ⅱ_Aat various concentrations for 72 h.Growth suppression was evaluated by MTT assay;apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining(Hoechst33258),transmission electron microscopy(TEM),and DNA agarose gel electrophoresis.Apoptoticrate was quantified by flow cytometry(FCM).The results showed thst Tanshinone Ⅱ_A could inhibitthe growth of hepatoma cells in a dose-dependent manner,with IC_(50) value being 6.28μg/ml.Aftertreatment with 1—10 μg/ml tanshinone Ⅱ_A for 72 h,BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodieswere observed.DNA ladder could be demonstrated on DNA electrophoresis.FCM analysis showedhypodiploid peaks on histogram,and the apoptotic rates at 5     

Effect of tanshinone IIA on the growth behavior of human hepatoma cell line BEL-7402 in vitro and its mechanism]

OBJECTIVE: To investigate the effect of tanshinone IIA on the growth behavior of human hepatoma cell line BEL-7402 in vitro and explore the mechanism. METHODS: Human hepatoma cell line BEL-7402 was exposed to tanshinoneIIA at different concentrations for 72 h, and the suppression of the cell growth was observed under inverted-phase contrast microscope. Apoptosis-related alterations in the cell morphology and biochemistry were examined under fluorescence microscope and transmission electron microscope (TEM) and by DNA agarose gel electrophoresis, and the apoptotic rate was quantified by flow cytometry (FCM). RESULTS: After treatment with 0-10 microg/ml tanshinone IIA for 72 h, the proliferation of BEL-7402 cells was significantly suppressed, and cell apoptosis occurred characterized by cell shrinkage, nuclear chromatin condensation and fragmentation, formation of membrane blebs and apoptotic bodies as observed under fluorescence microscope and TEM. DNA ladder was presented in DNA electrophoresis. FCM analysis yielded the cell apoptotic rates of (20.78+/-2.17) %, (24.64+/-2.07) %, (31.47+/-3.86) %, (43.65+/-4.04) % and (52.36+/-3.75) % at tanshinone IIA concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 microg/ml respectively, all significantly higher than those of the control group [(2.37+/-0.29)%]. CONCLUSION: Tanshinone IIA can inhibit the growth of human hepatoma BEL-7402 cells possibly through the mechanism of apoptosis induction.     

Growth inhibition and apoptosis induction in human hepatoma cells by tanshinone II<Subscript>A</Subscript>

Summary In order to study the effect of tanshinone II_A on growth and apoptosis in human hepatoma cell line BEL-7402in vitro, the human hepatoma cell line BEL-7402 was treated with tanshione I_A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II_A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC₅₀ value being 6.28 μg/ml. After treatment with 1–10 μg/ml tanshione II_A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 μg/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32±0.16)%, (3.01±0.35)%, (3.87±0.43)%, (6.73±0.58)% and (20.85±1.74)% respectively, which were all significantly higher than those in the control group (1.07±0.13)%. It is concluded that Tanshione II_A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition. TANG Zhongzhi, male, born in 1966, Doctor in Charge This project was supported by a grant from Natural Sciences Foundation of Hubei Province (No. 2000J064).     

酒石酸锑钾在诱导人肝癌BEL-7402细胞凋亡中的影响

目的:本研究旨在明确酒石酸锑钾(PAT)在体外对人肝癌BEL7402细胞凋亡的影响及抑癌机制。方法:用PAT以不同浓度、不同时间作用于人肝癌BEL7402细胞,以诱导其凋亡。用MTT比色法观察其细胞毒性,荧光显微镜、透射电镜、TUNEL染色法及流式细胞术(FCM)等方法来检测凋亡,观察其形态学和生化方面的变化。结果:PAT以剂量依赖和时间依赖的方式抑制BEL7402细胞的生长。5～40μmol·L-1的PAT处理48h后,形态学上,肝癌细胞表现为细胞皱缩、核质浓缩、核碎裂、细胞起泡以及凋亡小体形式等凋亡特征的形态学改变。DNA末端原位标记染色法、流式细胞仪均能检测到凋亡细胞。结论:PAT在体外诱导肝癌BEL7402细胞凋亡,能作为一种凋亡诱导剂用于肝癌的治疗。     

羟基磷灰石纳米粒子抑制结肠癌SW-480细胞生长的体外实验

目的:本研究旨在明确羟基磷灰石纳米粒子(HAP)在体外能否诱导人结肠癌SW-480细胞凋亡并从凋亡角度探讨其抑癌机制。方法:用HAP以不同浓度作用于人结肠癌SW-480细胞,以诱导其凋亡。用MTT比色法观察其细胞毒性,荧光显微镜、透射电镜、琼脂糖凝胶电泳法及流式细胞术(FCM)等方法来检测凋亡,观察其形态学和生化方面的变化。结果:HAP以剂量和时间依赖的方式抑制人结肠癌SW-480细胞的生长。25 mg/L,50mg/L HAP作用72 h细胞的生长抑制率分别是49.71%和61.03%。12.5-100 mg/L的HAP处理48 h后,结肠癌细胞表现为细胞皱缩、核质浓缩、核碎裂、细胞起泡以及凋亡小体形式等凋亡特征的形态学改变。流式细胞仪均能检测到凋亡峰,12.5,25,50,100 mg/L HAP作用48 h,凋亡率分别是3.57%,21.45%,37.10%和49.45%。结论:HAP在体外能抑制人结肠癌SW-480细胞增殖并诱导细胞凋亡。     

Effect of lidamycin on telomerase activity in human hepatoma BEL-7402 cells

Objective To investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence. Methods Chromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-β-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA. Results Mitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-β-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells. Conclusion Mitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.     

姜黄素单体影响肝癌BEL-7402细胞凋亡及其周期蛋白D表达的研究

目的 采用细胞学实验观察3种姜黄素单体对肝癌细胞株BEL-7402的抑制增殖及诱导细胞凋亡的作用强度和机制.方法 以四甲基偶氮唑蓝(MTT)、Annexin Ⅴ-FITC双标记及流式细胞术(FCM)实验观察3种姜黄素对肝癌细胞株BEL-7402增殖的抑制作用与细胞周期变化,采用 Western blot实验分析3种姜黄素对肝癌细胞株BEL-7402细胞周期蛋白D表达的影响.结果 (1)3种姜黄素均可通过诱导肝癌细胞凋亡而有效抑制肝癌细胞生长增殖,存在时间与剂量效应,以姜黄素Ⅲ抑制效果最强.(2)3种姜黄素通过影响细胞周期生长的调控信号、降低细胞周期蛋白D表达量致使肝癌细胞停滞于G1/S期.结论 3种姜黄素均可通过抑制肝癌细胞周期蛋白D表达而诱导细胞凋亡,有效地抑制肝癌细胞生长增殖,具有很高的临床治疗价值.     

绿脓杆菌制剂对人肝癌细胞株BEL-7402的杀伤效应

目的探索绿脓杆菌制剂对人肝癌细胞BEL-7402的杀伤效应。方法采用MTT法检测不同浓度的绿脓杆菌制剂对人肝癌细胞BEL-7402增殖的作用,同时利用电子显微镜、透射电镜观察细胞BEL-7402的形态学变化。结果MTT检测表明：绿脓杆菌制剂为10×107/mL、5×107/mL、2.5×107/mL时对肝癌细胞生长杀伤作用与对照组比较差异有高度统计学意义（P〈0.01）;电子显微镜、透射电镜观察发现肝癌细胞BEL-7402于12、24 h出现凋亡形态学改变,40 h形态学表现为凋亡与坏死并存。结论绿脓杆菌制剂对肝癌细胞BEL-7402生长有抑制作用,诱导细胞凋亡及坏死可能是主要作用机制。     

四种检测细胞凋亡方法的比较

目的：比较４种细胞凋恨检测方法的优缺点。方法：以ｂｕｆａｌｉｎ诱导ＨＬ６０细胞凋亡为例,选择４例方法即电镜（ＥＭ）、ＤＮＡ电泳、原位缺口标记法（ＴＵＮＥＬ）和流式细胞术（ＦＣＭ）检测细胞凋亡,并对凋亡率进行了比较。结果：ＥＭ和ＤＮＡ电泳技术是定性说明凋亡的可靠方法,而ＴＵＮＥＬ和ＦＣＭ擅长于定量检测凋亡细胞,ＥＭ、ＴＵＮＥＬ和ＦＣＭ检测的凋亡率具有显相关性。结论：研究细胞凋亡时宜选用既特异、灵敏     

亚砷酸体外对人肝癌细胞株BEL-7402影响的初步研究

目的 体外培养人肝癌细胞株BEL－7402,从多个角度探讨三氧化二砷（As2O3）的抗肿瘤作用及其机制。方法 应用倒置相差显微镜、电子显微镜、透谢电镜、流式细胞仪,分别对不同浓度加药组及对照组BEL－7402细胞的存活。形态学改变,细胞DNA含量的分布进行了观察和测定。结果 0.5、1、2μmol/L As2O3均能抑制人肝癌细胞株BEL－7402细胞的生长增殖。流式细胞仪分析显示,加药组在G1期细胞前均出现亚二倍体峰,且G0／G1期细胞减少,S期细胞增多;电镜下,对照组细胞核质比大、核大、核膜有明显切迹,0.5μmol/L As2O3组细胞核质比减少、核变圆、胞浆内出现分化良好的细胞器, 0.5、1、2μmol/L As2O3组均可见细胞膜完整、核固缩、凋亡小体形成。结论 三氧化二砷不仅抑制人肝癌细胞增殖,而且诱导细胞凋亡。     

丹参酮ⅡA抑制HepG2细胞生长及诱导其凋亡的实验研究

目的:研究丹参酮ⅡA对人肝癌细胞HepG2的生长抑制作用和凋亡诱导作用.方法:以0μg/mL丹参酮ⅡA作阴性对照,MTT法检测0.5～10.0 μg/mL丹参酮ⅡA作用人肝癌细胞HepG2 24,48,72 h的生长抑制率;HT33258荧光染色、琼脂糖凝胶电泳、流式细胞仪检测不同浓度丹参酮ⅡA作用HepG2细胞72 h后的细胞凋亡.结果:0.5～10.0 μg/mL丹参酮ⅡA均能抑制人肝癌细胞HepG2生长,并有明显的时间和剂量依赖性;在24,48,72 h的半数抑制浓度分别为14.7,7.4,3.9 μg/mL;经丹参酮ⅡA作用后,荧光染色可以观察到典型的凋亡细胞形态特征;琼脂糖凝胶电泳结果显示除1.0 μg/mL组外均可见明显的凋亡细胞形成的梯状条带;流式细胞仪检测不同浓度丹参酮ⅡA作用72 h后的细胞凋亡率分别为20.32%±2.16%,28.01%±2.35%,33.87%±3.43%,46.73%±4.08%和57.85%±3.74%,与对照组比较差异均有统计学意义(P＜0.05).结论:丹参酮ⅡA在体外能明显抑制人肝癌细胞HepG2生长,抑制其生长的机制可能是诱导细胞凋亡.     

榄香烯对人肝癌细胞7402、宫颈癌细胞Hela的凋亡诱导作用及下调Bcl—2蛋白表达

目的 研究中药榄香烯对人肝癌细胞7402、宫颈癌细胞Hela的体外抑瘤效应及其作用机制。方法 应用MTT比色法观察榄香烯对细胞的生长抑制作用;荧光显微镜和透射电镜观察细胞结构的改变;DNA凝胶电泳和流式细胞术等分析细胞凋亡、周期变化及Bcl-2蛋白表达情况。结果 榄香烯对7402和Hela细胞有较强的体外增殖抑制作用。榄香烯能诱导7402和Hela细胞凋亡,同时伴随有Bcl-2蛋白表达下调。结论 榄香烯对7402、Hela细胞有较强的抗瘤效应,其可能与Bcl-2蛋白下调引起的细胞凋亡有关。     

In vitro study on the morphology of human blood dendritic cells and LPAK cells inducing apoptosis of the hepatoma cell line

Objective To observe in vitro effects and morphological changes of human peripheral blood dendritic cells (DCs) on the ability of lymphokine and phytohaemagglutinin um (PHA) activated killer (LPAK) cells to induce apoptosis of the human hepa toma cell line (BEL-7402, B).Methods Experimental groups were divided into LD group （DCs+L+B）, L gro up （L+B）, D group （DCs+B） and B group. The methods of neutral red u ptake, ordinary light microscopy, electron microscopy, TDT mediated X-dUTP nick end labeling (TUNEL) were used.Results The difference between the D group and the B group was not distinct (P&gt;0.05 ). The difference between the LD group and the L group was distinct, with DCs+ LPAK &gt;LPAK (P&lt;0.01) in cytotoxity. Apoptotic cells were TUNEL positive in light microscopy, and apoptotic nuclei were stained yellow brown and dar k brown, with size and shape varying from cell to cell. Ultrastructural cha nge in apoptotic tumor cells comprised of compaction and condensation of nuclear chromatin, and condensation of cytoplasm and apoptotic bodies. At the sam e time, LPAK cells manifested the characteristics of autophagic apoptosis, and t here were some autophagic bodies in it. Conclusions The combination of human blood DCs and LPAK cells could induce apoptosis of BEL -7402 cells effectively, with some LPAK cells manifesting the characteristics o f autophagic apoptosis.     

4HPR对人宫颈癌细胞凋亡的影响

目的 研究Ｎ-(4-羟基苯基)维生素甲酰胺（4HPR）对人宫颈癌细胞凋亡的影响。方法 应用光学显微镜、激光共聚焦显微镜、电子显微镜、荧光显微镜从细胞形态学方面观察4HPR对人宫颈癌细胞凋亡的影响;应用流式细胞仪、细胞DNA琼脂糖凝胶电泳法分析4HPR诱导人宫颈癌细胞凋亡的细胞特征、生物化学特征及凋亡细胞百分率。结果 电子显微镜和激光共聚焦显微镜镜下可见细胞固缩,核膜扭曲,核染色体聚集成块并靠近核膜等凋亡细胞特征;光学显微镜和荧光显微镜下可见凋亡小体;细胞DNA被降解,在琼脂糖凝胶电泳中呈现典型的“阶梯状”图谱;流式细胞仪检测结果显示二倍体核型的特征,在DNA直方图上,G1峰左侧出现亚二倍体细胞群的峰型;凋亡百分率结果显示4HPR可诱导宫颈癌细胞凋亡,且呈时间和浓度依赖性（P＜0.01）。结论 4HPR可诱导人宫颈癌细胞凋亡, 且呈时间和浓度依赖性。     

化癥胶囊方剂诱导前列腺癌细胞系PC-3凋亡的实验研究

目的：明确化癥胶囊方剂对前列腺癌细胞PC-3的诱导凋亡作用。方法：PC-3细胞与适宜浓度化癥胶囊方剂共同孵育于1640培养液中,采用一系列细胞凋亡定性、定量检测方法研究化癥胶囊方剂诱导的PC-3细胞凋亡,如行DNA凝胶电泳、流式细胞仪DNA含量分析以检测凋亡。结果：化癥胶囊方剂可诱导PC-3细胞发生凋亡特征性的生化改变,如DNA提取及琼脂糖凝胶电泳分析显示,化癥胶囊方剂可诱导PC-3细胞染色体DNA片段化,形成凋亡特征性的“DNA梯状(DNA ladder)”电泳图谱;流式细胞仪检测可见有低G1期细胞出现。在试验范围内凋亡比率与药物浓度呈一定相关。结论：化癥胶囊方剂可诱导PC-3细胞凋亡,其作用与药物浓度呈一定相关性。     

白杨素敏化rhsTRAIL诱导人肝癌Bel-7402细胞凋亡

目的:研究白杨素(ChR)是否具有增敏重组人可溶性肿瘤坏死因子相关凋亡诱导配体(rhsTRAIL)诱导人肝癌Bel-7402细胞凋亡作用。方法:体外培养人肝癌Bel-7402细胞。碘化丙啶(PI)染色流式细胞术(FCM)分析细胞死亡率。DNA琼脂糖凝胶电泳确证诱导细胞凋亡作用。Western Bloting检测细胞DR5蛋白的表达。结果:ChR40μmol/L、rhsTRAIL 100ng/mL以及两者合用的细胞死亡率分别为4.91%±0.38%、5.89%±0.39%和28.7%±2.50%。ChR(40μmol/L)联合rhsTRAIL(100ng/mL)处理48小时,人肝癌Bel-7402细胞展示出典型DNA梯形条带图谱。Western Blot分析结果发现:白杨素以浓度和时间依赖的方式上调Bel-7402细胞DR5表达。结论:亚细胞毒性浓度的白杨素具有敏化rhsTRAIL诱导人肝癌Bel-7402细胞凋亡作用。     

紫杉醇、5-氟脲嘧啶抑制肝癌细胞BEL-7402生长和诱导凋亡的比较

OBJECTIVE: To investigate the effect of paclitaxel and 5-flurouracil (5-Fu) on growth inhibition and apoptosis of human hepatoma BEL-7402 cells. METHODS: Growth inhibition of BEL-7402 cells treated with paclitaxel and 5-Fu, respectively, was measured by ATP-tumor chemosensitivity assay (ATP-TCA), and the cell cycle kinetics and apoptosis were analyzed by flow cytometry and microscopic examination. RESULTS: BEL-7402 cells were highly sensitive to paclitaxel with growth inhibition observed in both dose- and time-dependent manners (IC(50)=5.58 x 10(-7) mol/L). Paclitaxel induced significantly higher rate of cell apoptosis than the control group (P<0.05) but significantly lower rate than that induced by 5-Fu (P<0.01). Necrosis was observed predominantly in paclitaxel-treated cells whereas 5-Fu caused mainly cell apoptosis (P<0.05). Levels of apoptosis increased in proportion to the decrement of paclitaxel concentration but directly proportional to increment of 5-Fu concentration. CONCLUSIONS: Paclitaxel and 5-Fu are effective in inducing growth inhibition and apoptosis of BEL-7402 cells. While 5-Fu causes mainly apoptosis in hepatoma cells, the anticancer mechanism of paclitaxel is predominantly through induction of necrosis.