三氧化二砷对雄激素非依赖性前列腺癌PC-3细胞株细胞增殖及细胞周期的影响

目的　了解三氧化二砷对雄激素非依赖性前列腺癌PC 3细胞株细胞增殖及细胞周期的影响。方法　以不同浓度的三氧化二砷作用于PC 3细胞 ,采用四甲基偶氮唑蓝 (MTT)法、流式细胞仪、相差显微镜和透射电镜观察细胞增殖状况、细胞周期变化以及细胞形态学的变化。结果　各浓度的三氧化二砷均可抑制PC 3细胞的增殖 ,具有剂量和时间累积效应 (P <0 0 1 )。三氧化二砷可使PC 3细胞生长停滞于G1 期。G1 期细胞数目随三氧化二砷的作用浓度增高而增多。其中3μmol/L、6 μmol/L、1 0 μmol/L的三氧化二砷作用 4 8小时后 ,可见PC 3细胞凋亡 ,分别为 1 1 8%,1 2 7%,2 9 6 %。透射电镜检查 3μmol/L三氧化二砷作用 4 8小时后的PC 3细胞 ,可见明显的凋亡小体。结论　三氧化二砷可抑制PC 3细胞的增殖 ,并具有剂量和时间依赖性 ;抑制细胞增殖与细胞周期阻滞于G1 期有关 ;三氧化二砷并可诱导PC 3细胞凋亡。     

米非司酮对宫颈癌Caski细胞锚定粘附能力的影响

[摘要]目的研究发现,米非司酮对体外人宫颈鳞癌细胞有生长抑制作用,但对其转移能力的影响尚不清楚。本研究的目的在于探讨米非司酮对人宫颈鳞癌Caski细胞粘附力的影响。为临床应用米非司酮治疗宫颈鳞癌提供实验依据。方法体外培养人宫颈鳞癌Caski细胞,应用不同浓度的米非司酮处理Caski细胞,平皿克隆形成试验测定米非司酮对Caski细胞的锚定依赖性生长能力的影响;MTT比色法测定米非司酮对Caski细胞在细胞外基质上的粘附能力的影响。结果①平皿克隆形成试验结果显示：各浓度组的集落抑制率随米非司酮浓度的增加而增加,呈浓度依赖方式（P〈0．05）;②MTT比色法结果显示：米非司酮作用于Caski细胞后,粘附抑制率随米非司酮浓度的增加而增加（P〈0．05）。结论米非司酮能降低Caski细胞锚定依赖性生长及降低在细胞外基质上的粘附能力,且呈浓度依赖方式     

全反式维甲酸对前列腺癌细胞生长及其连接蛋白43表达的影响

目的探讨全反式维甲酸（ATRA）对前列腺癌细胞生长增殖的抑制作用及其与连接蛋白（Cx）43之间的关系。方法不同浓度的ATRA处理体外培养的雄激素非依赖性人前列腺癌细胞株PC3后,分别应用光镜、甲基偶氮唑蓝（MTT）法和集落形成试验分析细胞生长和增殖的抑制情况;蛋白质免疫印迹法（Western blot）检测PC3细胞ATRA作用前后Cx43的表达。结果ATRA浓度为（10^-6～10^-4）mol／L时均可有效抑制PC3细胞的增殖（P〈0．05）,光镜下可见细胞凋亡的特征性改变,抑制呈时间-剂量依赖性。ATRA处理前后PC3的Cx43表达量从无到阳性光带出现,且显色带随ATRA浓度增高而增强。结论ATRA能够抑制前列腺癌细胞的生长和增殖,其机制可能通过上调Cx43的表达来实现的。     

hPARP-1蛋白缺陷细胞株建立及生长特性

目的建立hPARP-1蛋白缺陷细胞株，观察该缺陷所引起的生物学效应。方法用构建的hPARP-1基因反义RNA绿色荧光蛋白真核表达载体（pEGFP-C1-P）转染人胚肺成纤维细胞（HLF），用蛋白免疫印迹法鉴定转染细胞中hPARP-1基因的表达水平。同时观察转染细胞生长形态，绘制生长曲线，用流式细胞术观察细胞周期，软琼脂培养法鉴定转染细胞恶性程度。结果pEGFP-C1-P载体在转染细胞内可较稳定表达，hPARP-1蛋白缺陷细胞株hPARP-1基因的蛋白表达水平比HLF下降了47％，比绿色荧光蛋白载体（HLFC）低45％，HLFC比HLF仅少3％。转染后hPARP-1蛋白缺陷细胞生长形态、生长速度无明显变化，软琼脂培养未见细胞集落。hPARP-1缺陷细胞生长形态无明显变化，细胞倍增时间为0．89d．与HLF细胞0．93d接近，软琼脂培养未见细胞集落。结论成功建立和鉴定了hPARP-1蛋白缺陷细胞株，在正常生长环境中，该缺陷不足以单独引起可观察的生长特性改变。     

MC002诱导人喉癌细胞Hep-2凋亡及其作用机制

目的与雷公藤内酯醇(PG490)进行比较,观察雷公藤内酯醇衍生物MC002对人喉癌Hep-2细胞体外增殖的抑制作用及其分子机制。方法以MC002和PG490分别处理人喉癌细胞Hep-2,采用噻唑蓝比色法(MTT)检测药物对Hep-2细胞的增殖抑制作用并计算IC50值;利用软琼脂克隆形成实验观察细胞的锚定非依赖性生长能力;选用Hoechst33258染色法观察细胞凋亡的形态学变化;用ROS检测试剂盒分析药物对细胞活性氧水平的影响;以实时荧光定量PCR技术(Realtime-PCR)检测细胞凋亡相关因子Bcl-2及Bax mRNA水平的表达变化。结果 MC002及PG490均对体外培养的人喉癌Hep-2细胞有增殖抑制作用,且具有剂量依赖性,IC50值分别为183.58nmol/L和119.35nmol/L,而且两种药物可以抑制细胞的锚定非依赖性生长能力;此外,MC002及PG490可以降低ROS水平,并且使凋亡相关因子BaxmRNA表达上调,Bcl-2mRNA表达下调。结论 MC002具有与PG490相同的作用,可以抑制人喉癌细胞株Hep-2的生长,诱导人喉癌Hep-2细胞凋亡,其机制可能与调节凋亡相关因子Bcl-2、Bax及ROS的表达有关。     

三氧化二砷对人卵巢癌细胞株A2780细胞增殖的影响

目的探讨三氧化二砷（As2O3）对人卵巢腺癌细胞株A2780细胞体外增殖和细胞周期的影响。方法①体外培养A2780细胞株,采用四唑盐比色法（MTT）测定不同浓度的As2O3对A2780细胞系胞增殖的影响,并绘制剂量-效应曲线。②采用流式细胞仪（FCM）测定细胞周期。结果As2O3有效抑制A2780细胞生长,呈时间及浓度依赖性（P〈0.05）;As2O3可将A2780细胞周期阻滞于G2/M,并呈浓度依赖性（P〈0.05）。结论As2O3抑制体外培养的A2780细胞株生长,并将其阻滞在G2/M期,抑制肿瘤细胞增殖。     

转化生长因子β1对人肝细胞癌HepG-2细胞株增殖的影响

目的 检测转化生长因子TGF-β1对人肝细胞癌HepG-2细胞株增殖的影响,探讨TGF-β1在原发性肝癌发病中的作用.方法 应用噻唑蓝(MTT)比色法检测TGF-β1对人肝细胞肝癌HepG-2细胞株增殖的影响,探讨其调控HepG-2细胞生长的浓度效应和时间效应.结果 TGF-β1对人肝细胞肝癌HepG-2细胞有增殖抑制作用(P＜0.05),且不同浓度TGF-β1(1.0～100.0)μg/L产生的效果不同.结论 TGF-β1对HepG-2细胞的增殖有抑制作用,短时间内在一定浓度范围内呈剂量依赖性.     

白杨素和它的四乙基二磷酸酯对人宫颈癌 Hela细胞增殖和核基质蛋白影响的研究

探讨白杨素和它的四乙基二磷酸酯对人宫颈癌细胞的增殖、分化和核基质蛋白的影响。用终浓度为10μmol/L的白杨素(chrysin,CR)和四乙基白杨素二磷酸酯(tetraethyl bis-phosphoric ester of chrysin,CP)分别处理人人宫颈癌Hela细胞,记录细胞生长数目,进行增殖分化型细胞染色,测定软琼脂集落形成率,应用SDS-PAGE技术分析细胞核基质蛋白成分的变化。CR/CP能明显抑制Hela细胞的增殖,处理组与对照组之间差异有显著性(P<0.05);处理组与对照组比较增殖型细胞减少,分化型细胞数增加,双层软琼脂中细胞集落形成受到明显抑制,对照组集落普遍比药物组集落大,且单个集落中细胞数目多(P<0.05);通过Quantity One软件分析,实验组(CR组与CP组)与对照组(C组)比较,相对分子质量70 0006、8 000、13 000为增加的条带,14 000、17 000、18 000为增强的条带,58 000、43 000是减弱的条带。两实验组相比,以CP组变化显著。CR和CP对Hela细胞具有增殖抑制和诱导分化作用。同时还可以诱导Hela细胞核基质蛋白成分改变,这可能对肿瘤细胞基因表达调控、分化及凋亡起重要作用。     

姜黄素和顺铂联用对雄激素非依赖性前列腺癌细胞PC-3作用

目的:研究姜黄素和顺铂联用抑制雄激素非依赖性前列腺癌细胞株PC-3增殖的影响.方法:MTT比色法检测细胞生长活性,流式细胞仪测定细胞周期时相变化及检测细胞凋亡,透射电镜观察细胞超微结构变化.结果:姜黄素能显著抑制雄激素非依赖性前列腺癌细胞的体外生长,呈时间与剂量依赖性;细胞周期主要阻滞于G2/M期,部分细胞出现凋亡形态学改变.顺铂和不同浓度姜黄素联合可显著抑制PC-3细胞增殖.姜黄素和顺铂在一定程度上均可诱导PC-3细胞凋亡,两种药物联用后,诱导细胞凋亡作用增强.结论:姜黄素可与顺铂协同抑制人前列腺癌细胞株PC-3的增殖.     

N-乙酰半胱氨酸对结肠癌细胞株H T-29生长及NF-κB p65信号通路的影响

目的探讨N-乙酰半胱氨酸（N-acetylcysteine,NAC）对人结肠腺癌HT-29细胞株增殖作用及NF-κB p65通路影响。方法采用MTT法测定NAC抗HT-29细胞增殖的量效和时效关系,采用免疫细胞化学法探讨对细胞增殖、凋亡及细胞核因子-ΚBp65（NF-κB p65）蛋白表达影响。结果模型对照组HT-29细胞增殖明显,不同剂量NAC对体外培养的结肠腺癌HT-29细胞增殖均具有明显抑制作用,呈剂量和时间依赖性;NAC在0.05-10 mmol.L^-1剂量下,均可使HT-29细胞凋亡率增加,增殖细胞核抗原（PCNA）表达减弱;NF-κB p65蛋白表达均逐渐降低。结论NAC具有HT-29细胞生长抑制作用,机制可能与阻断NF-κB p65通路,抑制氧化损伤有关。     

Discovery and structure-activity relationships of imidazole-containing tetrahydrobenzodiazepine inhibitors of farnesyltransferase

2,3,4,5-Tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepines were found to be potent inhibitors of farnesyltransferase (FT). A hydrophobic substituent at the 4-position of the benzodiazepine, linked via a hydrogen bond acceptor, was important to enzyme inhibitory activity. An aryl ring at position 7 or a hydrophobic group linked to the 8-position through an amide, carbamate, or urea linkage was also important for potent inhibition. 2,3,4, 5-Tetrahydro-1-(1H-imidazol-4-ylmethyl)-7-(4-pyridinyl)-4-[2-(t rifluo romethoxy)benzoyl]-1H-1,4-benzodiazepine (36), with an FT IC(50) value of 24 nM, produced 85% phenotypic reversion of Ras transformed NIH 3T3 cells at 1.25 microM and had an EC(50) of 160 nM for inhibition of anchorage-independent growth in soft agar of H-Ras transformed Rat-1 cells. Selected analogues demonstrated ip antitumor activity against an ip Rat-1 tumor in mice.     

TEL-fusion oncogenic tyrosine kinases determine leukemic cells response to idarubicin

The family of BCR/ABL-related fusion tyrosine kinases (FTKs) is reported to participate in drug resistance in leukemogenesis. Our recent studies revealed a novel potential mechanism of resistance in FTK+ cells underlined by the stimulation of DNA repair. In this work we examined a role of TEL family fusion oncoproteins in the response to idarubicin. We used murine pro-B lymphoid cell line BaF3, and its TEL/ABL, TEL/JAK2 and TEL/PDGFbetaR-transformed clones. The transformed cells, in contrast to their non-transformed counterparts, exhibited resistance to idarubicin in the range 0.01-1 microM. The drug at 0.3 and 1 microM induced DNA damage in the form of strand breaks or/and alkali-labile sites in both transformed and control cells as evaluated by the alkaline Comet assay. The transformed cells removed the damage within 60 min, while the control cells required 120 min to recover. The results obtained suggest that TEL-related FTKs may stimulate the repair of DNA damaged by idarubicin and be relevant to the resistance of the leukemic cells to this drug.     

Ras inhibition results in growth arrest and death of androgen-dependent and androgen-independent prostate cancer cells

Prostate cancer is one of the most frequently diagnosed cancers in human males. Progression of these tumors is facilitated by autocrine/paracrine growth factors which activate critical signaling cascades that promote prostate cancer cell growth, survival and migration. Among these, Ras pathways have a major role. Here we examined the effect of the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS), on growth and viability of androgen-dependent and androgen-independent prostate cancer cells. FTS downregulated Ras, inhibited signaling to Akt and reduced the levels of cell-cycle regulatory proteins including cyclin D1, p-RB, E2F-1 and cdc42 in LNCaP and PC3 cells. Consequently the anchorage-dependent and anchorage-independent growth of LNCaP and PC3 cells were inhibited. FTS also induced apoptotic cell death which was inhibited by the broad-spectrum caspases inhibitor, Boc-asp-FMK. Our study demonstrated that androgen-dependent and androgen-independent prostate cancer cells require active Ras for growth and survival. Ras inhibition by FTS results in growth arrest and cell death. FTS may be qualified as a potential agent for the treatment of prostate cancer.     

Bovine lactoferrin stimulates anchorage-independent cell growth via membrane-associated chondroitin sulfate and heparan sulfate proteoglycans in PC12 cells

Bovine lactoferrin (bLf) is an iron-binding secretory protein present in breast milk, mucosal secretions, and the secondary granules of neutrophils. Although bLf has multiple functions, including antimicrobial and immunomodulatory activities, its effect on neuronal cells is not fully understood. We report that bLf prevents cell adhesion of PC12 cells and allows them to be cultivated in suspension. PC12 cells normally adhere well to plastic culture plates and show anchorage-dependent cell growth, but we found that soon after adding bLf, they detach from culture plates and begin to grow in suspension. When bLf was removed from the medium, the cells began to re-adhere to the plates. Thus, bLf inhibits cell adhesion and stimulates anchorage-independent growth in PC12 cells. On the other hand, bLf-induced cell suspension growth was not observed when cells were grown on a laminin matrix, suggesting that bLf does not affect integrin-mediated cell adhesion on a laminin matrix. Treatment of cells with heparin or chondroitin sulfate A or C inhibited bLf-induced growth in cell suspension. Furthermore, pretreatment of cells with heparinase and/or chondroitinase prevented direct binding of bLf to the cell membrane. These results suggest that bLf binds to the membrane of PC12 cells via membrane-associated proteoglycans and leads to anchorage-independent growth.     

Anicemycin, a new inhibitor of anchorage-independent growth of tumor cells from Streptomyces sp. TP-A0648

The anchorage-independence of cells is closely related to their tumorigenicity. In the screening of inhibitors of anchorage-independent growth of tumor cells, anicemycin was isolated from the fermentation broth of an actinomycete strain TP-A0648. The producing strain was isolated from a leaf of Aucuba japonica collected in Toyama, Japan and identified as Streptomyces sp. based on the taxonomic data. The structure of anicemycin was elucidated as a new analog of spicamycin by NMR and MS analysis. Anicemycin inhibited the anchorage-independent growth of the human ovary cancer SKOV-3 cells with an IC50 of 0.015 microM about three times more potently than their anchorage-dependent growth.     

氨基胍对人翼状胬肉成纤维细胞增殖影响的体外试验研究

目的:观察氨基胍(AG)对体外培养的人翼状胬肉成纤维细胞增殖的影响。方法:取人翼状胬肉标本进行体外贴壁细胞常规培养,传代至第3～5代细胞用于试验,以每孔1×104个细胞接种于培养板中,加入不同浓度(250、500、750、1000μmol.L-1)的AG作为AG组,另设不加AG的为对照组,检测作用24、48、72h后各孔的吸光度,并计算细胞增殖抑制率,每个时间点、每个浓度均设6个复孔。结果:人翼状胬肉成纤维细胞增殖抑制率随AG浓度的增大和作用时间的延长明显升高(P<0.05)。结论:AG在受试浓度和受试时间范围内,对体外培养的人翼状胬肉成纤维细胞具有抑制作用且呈浓度、时间依赖性。     

Cytotoxicity and apoptotic inducibility of Vitex agnus-castus fruit extract in cultured human normal and cancer cells and effect on growth

A crude extract was prepared with ethanol from dried ripened Vitex agnus-castus fruits growing in Israel (Vitex extract). Cytotoxicity of the extract against human uterine cervical canal fibroblast (HCF), human embryo fibroblast (HE-21), ovarian cancer (MCF-7), cervical carcinoma (SKG-3a), breast carcinoma (SKOV-3), gastric signet ring carcinoma (KATO-III), colon carcinoma (COLO 201), and small cell lung carcinoma (Lu-134-A-H) cells was examined. After culture for 24 h (logarithmic growth phase) or 72 h (stationary growth phase), the cells were treated with various concentrations of Vitex extract. In both growth phases, higher growth activity of cells and more cytotoxic activity of Vitex extract were seen. The cytotoxic activity against stationary growth-phase cells was less than that against logarithmic growth-phase cells. DNA fragmentation of Vitex extract-treated cells was seen in SKOV-3, KATO-III, COLO 201, and Lu-134-A-H cells. The DNA fragmentation in Vitex extract-treated KATO-III cells was inhibited by the presence of the antioxidative reagent pyrrolidine dithiocarbamate or N-acetyl-L-cysteine (NAC). Western blotting analysis showed that in Vitex extract-treated KATO-III cells, the presence of NAC also inhibited the expression of heme oxygenase-1 and the active forms of caspases-3, -8 and -9. It is concluded that the cytotoxic activity of Vitex extract may be attributed to the effect on cell growth, that cell death occurs through apoptosis, and that this apoptotic cell death may be attributed to increased intracellular oxidation by Vitex extract treatment.     

应用苏木素法测定非单体中药提取物对细胞增殖的影响

目的建立测定非单体中药提取物对贴壁细胞增殖影响的方法。方法分别用苏木素染色法、MTT比色法及CCK-8比色法测定HUVEC细胞44 h的增殖活性,并用苏木素法、MTT比色法和CCK-8比色法检测金荞麦提取物对M21细胞增殖的影响,葡萄籽原花青素(GSP)对HUVEC细胞增殖的影响,并对不同方法进行比对。结果苏木素法检测HUVEC细胞44 h的增殖结果与CCK-8和MTT比色法结果具有一致性。在测定非单体中药提取物对贴壁细胞增殖影响方面,苏木素法与CCK-8以及MTT比色法相比具有更好的重复性和准确性。结论苏木素染色法测定非单体中药提取物对贴壁细胞增殖的影响具有稳定、简便和经济等优点。     

Enhancement of gallic acid-induced human pulmonary fibroblast cell death by N-acetyl cysteine and L-buthionine sulfoximine

Gallic acid (GA) has various biological properties including anti-cancer effect. However, little is known about the toxicological effect of GA in primary normal cells. Here, we evaluated the effects of GA on human pulmonary fibroblast (HPF) cells in relation to reactive oxygen species (ROS) and glutathione (GSH). GA inhibited the growth of HPF cells at 24 hours in a dose-dependent manner. GA also induced HPF cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). GA increased ROS levels including O(2)(?-) and GSH-depleted cell numbers in HPF cells at 24 hours. Treatment with 2 mM N-acetyl-cysteine (NAC) intensified growth inhibition and death in GA-treated HPF cells. NAC decreased ROS levels and increased GSH depletion in these cells. Treatment with 10 μM L-buthionine sulfoximine (BSO) also enhanced growth inhibition and death in GA-treated HPF cells. BSO increased ROS levels and GSH depletion in these cells. In conclusion, GA-induced HPF cell death was accompanied by ROS increase and GSH depletion. The changes of ROS and GSH levels by NAC and BSO appeared to affect cell growth and death in GA-treated HPF cells.     

Anicequol,a novel inhibitor for anchorage-independent growth of tumor cells from Penicillium aurantiogriseum Dierckx TP-F0213

A novel inhibitor for anchorage-independent growth of tumor cells was isolated from the culture broth of a fungal strain. The producing strain TP-F0213 was identified as Penicillium aurantiogriseum Dierckx based on the taxonomic study. The compound designated anicequol was obtained by solvent extraction, HP-20 and silica gel chromatographies and recrystallization. The planar structure was elucidated by NMR analysis to be 16-acetoxy-3,7,11-trihydroxyergost-22-en-6-one. The absolute configuration was determined by the X-ray analysis of 3,7-bis-p-bromobenzoyl derivative. The carbon skeleton of anicequol has the same absolute configuration as ergostane and the configurations of substituents are 3beta, 5alpha, 7beta, 11beta, 16beta and 24S. Anicequol inhibited the anchorage-independent growth of human colon cancer DLD-1 cells with the IC50 of 1.2 microM whereas the IC50 against anchorage-dependent growth was 40 microM.