Li You, Zhang Fengli, Qin Zhen, Yang Shang-Tian
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH 43210, USA.
Bioengineering (Basel). 2025 Mar 23;12(4):335. doi: 10.3390/bioengineering12040335.
A fluorescent cell-based assay was developed for the screening of chemicals repressing the expression of human telomerase reverse transcriptase (hTERT). hTERT is reactivated during carcinogenesis and is overexpressed in more than 90% of cancers but is almost silent in normal tissue cells. Because of its critical role in cancer, hTERT is a target in various therapeutic strategies for cancer treatment. In this study, the hTERT promoter was cloned in MCF7 breast cancer cells and used to control the expression of enhanced green fluorescent protein (EGFP). The fluorescence of EGFP indicated the activity of the hTERT promoter, and, in the presence of an hTERT repressor, the EGFP fluorescence signal was reduced as compared to the EGFP fluorescence controlled by the human cytomegalovirus (CMV) promoter, which was not affected by changes in culture conditions and worked as a control. The EGFP reporter cells were cultivated in three-dimensional (3D) microbioreactors to resemble the in vivo tumor physiology and provide in vivo-like responses. The assay's predictability was demonstrated with three known hTERT inhibitors, pristimerin, epigallocatechin gallate, and n-butylidenephthalide, and further evaluated with five widely used anticancer compounds, doxorubicin, cisplatin, paclitaxel, blasticidin, and tamoxifen. The results showed overall accuracy of over 83.3%, demonstrating the feasibility of using the hTERT promoter with EGFP as a reporter for the screening of potential cancer drugs targeting hTERT.
开发了一种基于荧光细胞的检测方法，用于筛选抑制人类端粒酶逆转录酶（hTERT）表达的化学物质。hTERT在致癌过程中被重新激活，在90%以上的癌症中过度表达，但在正常组织细胞中几乎不表达。由于其在癌症中的关键作用，hTERT是各种癌症治疗策略的靶点。在本研究中，hTERT启动子被克隆到MCF7乳腺癌细胞中，并用于控制增强型绿色荧光蛋白（EGFP）的表达。EGFP的荧光表明hTERT启动子的活性，并且，在存在hTERT抑制剂的情况下，与由人类巨细胞病毒（CMV）启动子控制的EGFP荧光相比，EGFP荧光信号降低，CMV启动子不受培养条件变化的影响，用作对照。EGFP报告细胞在三维（3D）微生物反应器中培养，以模拟体内肿瘤生理学并提供类似体内的反应。用三种已知的hTERT抑制剂，原苏木素、表没食子儿茶素没食子酸酯和正丁基苯酞证明了该检测方法的可预测性，并用五种广泛使用的抗癌化合物，阿霉素、顺铂、紫杉醇、杀稻瘟菌素和他莫昔芬进一步评估。结果显示总体准确率超过83.3%，证明了使用以EGFP为报告基因的hTERT启动子筛选靶向hTERT的潜在癌症药物的可行性。
