Department of Biology, Faculty of Science, University of Guilan, P.O. Box 41335-1914, Rasht, Iran.
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-154, Tehran, Iran.
Anal Bioanal Chem. 2020 Jan;412(1):149-158. doi: 10.1007/s00216-019-02220-3. Epub 2020 Jan 2.
HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-μ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 μM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-μ was about 8 and 25 μM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.
热休克蛋白 70（HSP70）是一种强大的抗凋亡蛋白，能够阻断细胞凋亡的外在和内在途径。本研究描述了一种快速、敏感且经济的方法，使用荧光素酶作为报告基因来分析凋亡化合物的功能。在该方法中，通过共转化大肠杆菌细胞，将两个表达载体（包含 HSP70 和萤火虫荧光素酶）进行转化。结果发现，经热处理（40-46°C 处理 10 分钟）失活的荧光素酶在室温下大约可以重新激活，并在热失活 60 分钟后恢复到初始活性的 70%。结果表明，作为 HSP70 抑制剂的 VER-155008 和 pifitrin-μ 能够抑制活细胞中热失活荧光素酶的重新激活，其半抑制浓度分别为 124 和 384μM。该方法检测 VER-155008 和 pifitrin-μ 的灵敏度分别约为 8 和 25μM。此外，该报告系统对与 DNA 结合的阿霉素和放线菌素 D 没有反应，我们将这些抗癌化合物用作对照化合物。因此，本研究首次引入了一种基于大肠杆菌细胞中 HSP70 抑制的快速简便的实时荧光素酶报告基因筛选诱导细胞凋亡化合物的方法。
