Pereyra-Vergara Fernando, Olivares-Corichi Ivonne María, Luna-Arias Juan Pedro, Méndez-Luna David, García-Sánchez José Rubén
Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina del Instituto Politécnico Nacional, Ciudad de México C.P. 11340, Mexico.
Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav-IPN), Ciudad de México C.P. 07360, Mexico.
Int J Mol Sci. 2025 Apr 25;26(9):4102. doi: 10.3390/ijms26094102.
Uncoupling protein 2 (UCP2) plays an important role in normal cells because it mitigates the cytotoxic effect of reactive oxygen species (ROS). However, its overexpression in cancer cells is related to drug resistance and increased cell proliferation due to a decrease in ROS production. In this context, molecules that regulate or block UCP2 have potential as anticancer agents. (-)-Epicatechin, a flavonoid that inhibits cell proliferation, increases ROS, and induces apoptosis in cancerous cells, was evaluated for its effects on gene expression. For this purpose, the real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were performed in MDA-MB-231 and MCF-10A cells to determine the effects of (-)-epicatechin on UCP2 expression. Furthermore, the impact of (-)-epicatechin on cell viability was also determined. To analyze the transcriptional regulation of the gene by (-)-epicatechin, a 5'-region of the human gene (-2093/+297) was amplified, sequenced, cloned, and inserted into a reporter plasmid. To analyze the promoter activity and regulatory motif involved in the effects of (-)-epicatechin, several deletions of the promoter were generated and transfected into MDA-MB-231 and MCF-10A cells. An electrophoretic mobility shift assay (EMSA) was carried out to detect the interaction between DNA and proteins involved in the effect of (-)-epicatechin. The increased expression of the gene in MDA-MB-231 cells was decreased by (-)-epicatechin, and the opposite effect was observed in MCF-10A cells. The promoter region of the human gene (-2093/+297) showed activity, which was decreased by (-)-epicatechin. A sequence of 117 bp located at position -109 b to +8 b has a fragment of 90 bp that is related to the (-)-epicatechin effect. Bioinformatics analysis and EMSA of this sequence revealed the presence of a regulatory site for a protein with zinc fingers. The presence of a response element to (-)-epicatechin in the human promoter revealed that the inhibition of this gene in MDA-MB-231 breast cancer cells occurred at the transcriptional level. In this study, we propose the mechanism of action of (-)-epicatechin that could aid in cancer treatment.
解偶联蛋白2（UCP2）在正常细胞中发挥着重要作用，因为它能减轻活性氧（ROS）的细胞毒性作用。然而，其在癌细胞中的过度表达与耐药性以及由于ROS产生减少导致的细胞增殖增加有关。在这种情况下，调节或阻断UCP2的分子具有作为抗癌剂的潜力。（-）-表儿茶素是一种抑制细胞增殖、增加ROS并诱导癌细胞凋亡的类黄酮，对其对基因表达的影响进行了评估。为此，在MDA-MB-231和MCF-10A细胞中进行了实时定量聚合酶链反应（qRT-PCR）和蛋白质印迹法，以确定（-）-表儿茶素对UCP2表达的影响。此外，还确定了（-）-表儿茶素对细胞活力的影响。为了分析（-）-表儿茶素对该基因的转录调控，扩增、测序、克隆了人类该基因的5'区域（-2093/+297），并将其插入报告质粒。为了分析（-）-表儿茶素作用所涉及的启动子活性和调控基序，对该启动子进行了多次缺失，并转染到MDA-MB-231和MCF-10A细胞中。进行了电泳迁移率变动分析（EMSA）以检测（-）-表儿茶素作用所涉及的DNA与蛋白质之间的相互作用。（-）-表儿茶素降低了MDA-MB-231细胞中该基因的表达增加，而在MCF-10A细胞中观察到相反的效果。人类该基因的启动子区域（-2093/+297）显示出活性，（-）-表儿茶素降低了其活性。位于-109 b至+8 b位置的一段117 bp序列中有一段90 bp的片段与（-）-表儿茶素的作用有关。对该序列的生物信息学分析和EMSA揭示了一个具有锌指结构的蛋白质的调控位点的存在。人类该启动子中存在对（-）-表儿茶素的反应元件，表明在MDA-MB-231乳腺癌细胞中该基因的抑制发生在转录水平。在本研究中，我们提出了（-）-表儿茶素的作用机制，这可能有助于癌症治疗。
