SRB法和MTT法抗肿瘤药物筛选结果相关性研究

为了探索抗肿瘤药物体外筛选中磺酰罗丹明B（SRB）法和四甲基偶氮唑盐（MTT）比色法实验结果的相互验证作用,作者以PC-3、BGC-823、Bcap-37细胞系为研究对象,利用SRB法和MTT比色法对供试化合物行体外抑制活性考察,并用SPSS12．0对两种筛选方法得到的实验结果进行差异性分析。结果表明,SRB法和MTT比色法实验结果相关性好,可以相互验证实验结果的准确性和可靠性。     

剑叶龙血树内生放线菌活性菌株的筛选和鉴定

为筛选具有抗菌抗肿瘤活性的药用植物内生菌资源,该文对300余株分离自中国云南西双版纳及越南地区剑叶龙血树的内生放线菌采用琼脂块扩散法进行抗病原细菌活性筛选、平板对峙法进行抗真菌活性筛选、SRB法测定菌株的细胞毒活性及PCR扩增方法筛选非核糖体肽合成酶NRPS基因及聚酮合酶PKS-I、PKS-Ⅱ基因;对得到的优势菌株经8种培养基进行发酵,采用10种病原细菌、3种病原真菌及2种人肿瘤细胞株测试其发酵粗提物的抗菌及抗肿瘤活性以确定最佳发酵培养基;用16S rRNA基因测序方法初步鉴定5株优势菌株的分类地位。结果表明:初筛得到5株抗菌活性、体外细胞毒活性及NRPS、PKS相关基因表达阳性的菌株S01-S05,其中4株(S01-S04)属于链霉菌属、1株(S05)属于类诺卡氏菌属;菌株经不同培养基发酵后产物的抗菌和抗肿瘤活性不同,其中Streptomyces sp. S04在7种培养基中的发酵提取物对8种测试病原菌均有较强抑制作用,且该菌株用Medium C的发酵提取物对人肝癌Hep G2细胞株抑制率达到100%,为剑叶龙血树内生菌活性成分挖掘及新型抗菌药物筛选奠定了基础。     

共附生微生物抗肿瘤活性菌的筛选与鉴定

对分离自海绵和植物组织的一些微生物进行抗肿瘤活性菌株的筛选。采用SRB法对252株微生物菌株的发酵提取物进行了抗肿瘤活性的筛选。结果显示,28%的测试菌株在提取物浓度为100μg/mL时对HeLa细胞的抑制率在50%以上,经复筛确定有6株细菌和1株真菌具有良好且稳定的抗肿瘤活性,其提取物在100μg/mL时对HeLa细胞的抑制率均在80%以上,其中活性最高的两株菌HMJ-390和YX-5对HeLa细胞的IC50分别为40.56μg/mL和5.33μg/mL。经16S rDNA和ITS rDNA序列分析,鉴定HMJ-390为Cellulophagasp.,YX-5为Aspergillussp.。结果表明,作为抗肿瘤药物的潜在来源共附生微生物值得关注。     

MTT比色法抗肿瘤药物筛选实验条件和数据优化探索

MTT比色法是一种重要的体外抗肿瘤药物筛选方法.以PC-3细胞系为研究对象,对影响MTT比色法抗肿瘤药物筛选实验的主要因素一细胞密度、实验操作环节、OD值选取、以及数据的优化处理进行实验探讨.结果表明,当检测化合物6 h～72 h的抑制活性时,种细胞密度2 000个/孔为宜.同时,采用本文所提的实验条件和数据处理办法,可以实现实验结果的准确可靠,3次测试偏差不超过10%.     

牛至油体外抗肿瘤活性研究

目的观察牛至油对肿瘤细胞株的生长抑制作用。方法采用MTT法检测不同浓度的牛至油对体外培养的多种肿瘤细胞株的生长抑制作用,计算半数抑制浓度(IC50)。结果不同浓度牛至油作用后,人肝癌细胞系HepG2、人子宫颈癌细胞系JTC-26和肺癌细胞系A549出现增殖阻滞。MTT法确定牛至油对肝癌HepG2的IC50为118μg/ml;人子宫颈癌JTC-26的IC50为118μg/ml;肺癌A549的IC50为59μg/ml。结论牛至油具有体外抗肿瘤活性。     

七种中草药提取物抗肿瘤活性部位的筛选研究初报

选取叶下珠、珠子草、千里光、鸦胆子、使君子、冬青和猫爪藤等7种中草药进行提取和初步分离,筛选出有抗肿瘤效果的活性部位,并对其进行初步分析。采用MTT法,以7种中草药醇提物的不同分离部位分别对人肝癌、胃癌、卵巢癌等肿瘤细胞株进行体外增殖抑制作用研究,并以人正常肝细胞株为模型对照跟踪筛选活性部位。结果表明,从7种中草药醇提物中,均获得对肿瘤细胞株具有体外增殖抑制作用的化学分离部位,其中叶下珠和珠子草的乙酸乙酯层和正丁醇层的活性最显著,千里光的乙酸乙酯层、鸦胆子的正丁醇层、使君子和冬青的氯仿层活性次之,猫爪藤氯仿层和冬青的乙酸乙酯层活性较差,其他部位无活性。     

西藏林芝土壤放线菌抗肿瘤活性菌的筛选及鉴定

【目的】筛选并鉴定西藏林芝八一镇土壤中产生抗肿瘤活性物质的放线菌。【方法】用平板稀释法分离放线菌, 用MTT法和纸碟法对放线菌发酵产物进行体外抗肿瘤与抑菌活性检测, 并用多相分类技术对抗肿瘤活性菌株进行鉴定。【结果】共分离出29株放线菌, 得到6个抑制体外肿瘤细胞增殖的活性菌株(20.7%), 同时它们也具有抑菌活性, 其中4株菌的检测样品对Hela细胞的抑制率达80%以上。多相分类研究表明, 6个活性菌株分别隶属于链霉菌属(Streptomyces)的3个已知物种的变种。【结论】林芝八一镇土壤放线菌中蕴藏抗肿瘤活性链霉菌, 是一个潜在的抗肿瘤药用微生物资源。     

植物内生真菌抗肿瘤活性菌株的筛选*

从三尖杉、南方红豆杉及香榧中分离到172株植物内生真菌,利用抗肿瘤体外细胞毒筛选模型(MTT法)对其进行活性检测。结果表明,25株内生真菌(占总分离菌株的14.5%)对KB（人口腔上皮癌）或HL-60（人白血病）细胞具有显著的抑制活性。三尖杉、南方红豆杉及香榧内生真菌的抗肿瘤活性菌株占各供测菌株的3.3%, 19.6%和8.6%。其中7个菌株的细胞毒ID50为1,000以上,占总供测菌株的4.1%。抗肿瘤活性菌株主要分布在拟青霉属及头孢霉属中。     

临床相关毛孢子菌的鉴定及体外药物敏感性研究

目的探讨临床相关毛孢子菌的鉴定方法及对常见抗真菌药物的体外敏感性。方法对48株临床分离的毛孢子菌分别通过形态学、API20C AUX、Vitek 2 Compact及核糖体rDNA ITS序列分析等方法鉴定到种;采用浓度梯度法（E-test）测定氟康唑、伏立康唑、伊曲康唑、两性霉素B及卡泊芬净对48株毛孢子菌的最低抑菌浓度。结果形态学和API20C AUX、Vitek 2 Compact不能准确区分不同种的毛孢子菌,以核糖体rDNA ITS序列分析将48株毛孢子菌鉴定为8个种：阿萨希毛孢子菌,星型毛孢子菌,皮瘤毛孢子菌,真皮毛孢子菌,皮肤毛孢子菌,赖巴克毛孢子菌,T.domesticum,T.jirovecii。体外药敏结果显示：卡泊芬净对毛孢子菌无体外活性,MIC〉32μg/mL;氟康唑和两性霉素B对毛孢子菌活性差,体外活性最好的药物是伏立康唑和伊曲康唑。结论常规方法不易将毛孢子菌准确鉴定到种的水平,ITS序列分析准确快速,可以辅助临床区分难鉴定毛孢子菌。毛孢子菌药敏谱不同于临床常见其他酵母菌,氟康唑和两性霉素B对其活性差,伏立康唑具有良好的体外抗菌活性。     

植物内生真菌抗肿瘤活性菌株的筛选

从三尖杉、南方红豆杉及香榧中分离到172株植物内生真菌,利用抗肿瘤体外细胞毒筛选模型（MTT法）对其进行活性检测,结果表明,25株内生真菌（占总分离菌株的14.5%)对KB（人口腔上皮癌）或HL-60（人白血病）细胞具有显著的抑制活性。三尖杉、南方红豆杉及香榧内生赵菌的抗肿瘤活性菌株占各供测菌株的3.3%,19.6%和8.6%。其中7个菌株的细胞毒ID50为1,000以上,占总供测菌株的4.1%,抗肿瘤活性菌株主要分布在拟青霉属及头孢霉属中。     

Apoptosis enzyme-linked immunosorbent assay distinguishes anticancer drugs from toxic chemicals and predicts drug synergism

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.     

Toxoplasma gondii: a simple high-throughput assay for drug screening in vitro

Toxoplasma gondii is the etiologic agent of toxoplasmosis. Although the combination of sulfadiazine and pyrimethamine is used as therapy for this disease, these drugs can have serious side effects and its use is limited in pregnancy. Therefore there is a need for new anti-T. gondii drugs in the clinic. Some systems for T. gondii drug screening have been described, but these have limitations and can be difficult. In order to solve these problems, we established a system to screen drugs in vitro that involved using cell viability methods to calculate drug selectivities, which are Trypan blue, [3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuzolium, inner salt] (MTS) method and lactate dehydrogenase (LDH) assay. These assays were simple to establish and perform. The IC₅₀ values calculated from the morphological assay were not significantly different from the EC₅₀ values calculated using the other three methods. In particular, the results of the morphological assay showed a distinct association with the MTS assay (R = 0.9841). These assays could be used for a wide range of applications in the screening of new drugs and may provide an alternative to the techniques currently used to screen for candidate anti-T. gondii compounds in vitro. In this study, we also tested many compounds and identified some that had a good anti-T. gondii effect in vitro based on the MTS assay. This simple and fast system allowed us to determine which compounds to investigate further using in vivo experiments.     

中空纤维测定法在抗肿瘤药物研究中的应用

中空纤维测定法是NCI建立的体内抗肿瘤药物筛选方法。本文介绍了中空纤维测定法的基本实验方法和疗效评判标准,回顾了近年基于中空纤维测定法的抗肿瘤药物方面的研究成果,认为中空纤维测定法不仅适用于药物筛选,在快速研究抗肿瘤药物药效和机制方面也具有重要的应用价值。     

A fluorescence-based high-throughput assay for antimicrotubule drugs

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.     

Comparison of an antimetabolic assay and an antiproliferative assay, both using 3H-thymidine incorporation, to test drug sensitivity of human tumors

Two assays based on the inhibition of 3H-thymidine incorporation into DNA were used to measure either the antimetabolic or the antiproliferative effects of anticancer drugs. A direct comparison of the two assays was made with cell suspensions obtained from 11 ovarian cancers and 22 malignant melanomas. Drugs with different effects on cell cycle phases were tested by both assays, for a total of 53 drug comparisons. When the sensitivity indices specific for each system was used, a significant association (p less than 0.01) was noted between the two assays. The agreement of both assays in defining in vitro sensitivity or resistance was 100% for ovarian cancer. For melanoma, 97% of samples resistant to the antimetabolic assay were also resistant to the antiproliferative assay; whereas, only 45% of samples sensitive to the antimetabolic assay were sensitive to the antiproliferative assay.     

Development,validation and pilot screening of an in vitro multi-cellular three-dimensional cancer spheroid assay for anti-cancer drug testing

It has been demonstrated that two-dimensional (2D) monolayer cancer cell proliferation assay for anti-cancer drug screening is a very artificial model and cannot represent the characteristics of three-dimensional (3D) solid tumors. The multi-cellular in vitro 3D tumor spheroid model is of intermediate complexity, and can provide a bridge to the gap between the complex in vivo tumors and simple in vitro monolayer cell cultures. In this study, a simple and cost-effective cancer 3D spheroid assay suitable for small molecule anti-cancer compound screening was developed, standardized and validated on H292 non-small lung cancer cell line. A pilot screening with this assay was performed utilizing a compound library consisting of 41 anti-cancer agents. The traditional 2D monolayer cell proliferation assay was also performed with the same cell line and compounds. A correlational study based on the IC₅₀ values from the 2D and 3D assays was conducted. There is low correlation with the two sets of biological data, suggesting the two screening methods provide different information regarding the potency of the tested drug candidates.     

Assessing the data quality in predictive toxicology using a panel of cell lines and cytotoxicity assays

In vitro cell viability assays have a central role in predictive toxicology, both in assessing acute toxicity of chemicals and as a source of experimental data for in silico methods. However, the quality of in vitro toxicity databanks fluctuates dramatically because information they contain is obtained under varying conditions and in different laboratories. The aim of this study was to identify the factors responsible for these deviations and thus the quality of the data extracted for predictive toxicology. Three cell viability assays measuring LDH leakage, WST-1 reduction, and intracellular ATP were compared in an automated environment using four mammalian cell lines: Caco-2, Calu-3, Huh-7, and BHK. Using four standard compounds--polymyxin B, gramicidin, 5-fluorouracil, and camptothecin--a significant lack of sensitivity in LDH assay compared with the other assays was observed. Because the viability IC(50) values for the standards were similar among the cell lines, the biochemical characteristics of different cell lines seem to play only a minor role, with an exception being the hepatocellular Huh-7 cell line. Toxicity assessment of new 1,2,4-triazoles revealed significant differences in their toxic potential, and the results indicate the same sensitivity profile among the assays as observed with the standard compounds. Overall, it can be argued that the assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays.     

Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites

We report two improved assays for in vitro and in vivo screening of chemicals with potential anti-malarial activity against the blood stages of the rodent malaria parasite Plasmodium berghei. These assays are based on the determination of luciferase activity (luminescence) in small blood samples containing transgenic blood stage parasites that express luciferase under the control of a promoter that is either schizont-specific (ama-1) or constitutive (eef1αa). Assay 1, the in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC₅₀ values comparable to those obtained with standard in vitro drug-susceptibility assays. The second assay, the in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1αa promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. Both tests are suitable for use in larger-scale in vitro and in vivo screening of drugs. The standard methodology of anti-malarial drug screening and validation, which includes testing in rodent models of malaria, can be improved by the incorporation of such assays.     

Thymidine incorporation is highly predictive of colony formation and can be used for high-throughput screening

The primary goal of anticancer chemotherapy is to kill cancer cells. Therefore, it is of critical importance that any assay that is used to determine the toxicity of a potential anticancer drug accurately measures viability. While colony formation is widely regarded as the most accurate measure of viability following drug treatment, it is laborious, time consuming, and difficult to carry out with non-adherent cells. For these reasons, it is not suitable for moderate- to high-throughput screening applications. We sought to identify a convenient and reliable assay that would accurately reproduce colony formation results and be amenable to high-throughput applications. Here, we describe a modification of the 3H-thymidine incorporation assay that meets these criteria. The assay can be carried out in 96-well plates with minimal handling of reagents and media. It can be performed with non-adherent and adherent cell lines. Most importantly, LC50 values obtained with this assay show excellent agreement with colony formation results. Taken together, these advantages make the modified 3H-thymidine incorporation assay well suited for high-throughput viability assays in anticancer drug discovery and development.