评价芳香酶活性的两种体外测定法的优缺点:“基于细胞和无细胞测定法”。

IF 1.8 Q3 PHARMACOLOGY & PHARMACY Turkish Journal of Pharmaceutical Sciences Pub Date : 2022-12-21 DOI:10.4274/tjps.galenos.2021.85530
Elif İnce Ergüç, Senem Özcan Sezer, Hande Gürer Orhan
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引用次数: 0

摘要

目的:芳香化酶是一种催化雄激素转化为雌激素的酶。虽然抑制芳香化酶是治疗乳腺癌的一种有效方法,但由于其内分泌干扰/调节作用,也可能产生毒理学后果。在这项研究中,通过测试已知的芳香酶抑制剂,研究了两种体外测定法(无细胞法和基于细胞法)评估芳香酶活性的灵敏度和性能,并对方法进行了部分验证。讨论了这些方法的优缺点。材料与方法:采用两种体外模型评价芳香酶活性;使用荧光底物和重组人酶的无细胞测定法直接测量,以及使用基于细胞的测定法间接评估,在雌激素受体阳性的人乳腺癌细胞(MCF-7 BUS)中,在缺乏雌激素和存在睾丸激素的情况下测定细胞增殖。结果:在无细胞直接测定法中,对照物酮康唑和氨酰硫胺对芳香酶有抑制作用,半数最大抑制浓度(IC50)值与文献相符。在以细胞为基础的间接测量实验中,仅酮康唑剂量依赖性抑制细胞增殖,IC50为3.47 × 10-7 M。两种方法的测定间和测定内的重复性均在可接受的偏差范围内。结论:两种方法均可成功应用。然而,为了在体外评估新化合物潜在的芳香酶活性,似乎更好的方法是分别进行基于细胞和无细胞的实验,这两种实验分别允许中低生物转化和消除细胞毒性潜力。
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Advantages and Disadvantages of Two In Vitro Assays in Evaluating Aromatase Activity: "A Cell-Based and a Cell-Free Assay".

Objectives: Aromatase is an enzyme that catalyzes the conversion of androgens to estrogens. While inhibition of aromatase is a useful approach for treating breast cancer, it may also have toxicological consequences due to its endocrine disrupting/modulating effect. In this study, sensitivity and performance of two in vitro assays -a cell free and a cell-based- for evaluating aromatase activity were investigated by testing known aromatase inhibitors and partial validation of the methods was performed. Advantages and disadvantages of these methods are also discussed.

Materials and methods: Aromatase activity was evaluated via two in vitro models; direct measurement with a cell-free assay using a fluorescent substrate and recombinant human enzyme and indirect evaluation with a cell-based assay where cell proliferation was determined in estrogen receptor positive human breast cancer cells (MCF-7 BUS) in the absence of estrogen and the presence of testosterone.

Results: In the cell-free direct measurement assay, reference compounds ketoconazole and aminoglutethimide have been shown to inhibit the aromatase enzyme with half-maximal inhibitory concentration (IC50) values concordant with literature. In cell-based indirect measurement assay, only ketoconazole dose-dependently inhibited cell proliferation with 3.47 x 10-7 M IC50. Inter-assay and intra-assay reproducibility of both methods was found to be within acceptable deviation levels.

Conclusion: Both methods can be successfully applied. However, to evaluate the potential aromatase activity of the novel compounds in vitro, it seems better to perform both the cell-based and the cell-free assays that allows low-moderate biotransformation and eliminate cytotoxicity potential, respectively.

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CiteScore
3.60
自引率
5.90%
发文量
79
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