Nikulin Sergey, Razumovskaya Alexandra, Poloznikov Andrey, Zakharova Galina, Alekseev Boris, Tonevitsky Alexander
Faculty of Biology and Biotechnologies, Higher School of Economics, Moscow, Russia.
P. A. Hertsen Moscow Oncology Research Center, Branch of the National Medical Research Radiological Center, Ministry of Health of the Russian Federation, Moscow, Russia.
Front Mol Biosci. 2023 Jan 13;10:1075704. doi: 10.3389/fmolb.2023.1075704. eCollection 2023.
Relapse of breast cancer is one of the key obstacles to successful treatment. Previously we have shown that low expression of and genes in breast cancer tissue corresponded to poor prognosis. participates directly in the elongation of polyunsaturated fatty acids (PUFAs) that are considered to play an important role in cancer cell metabolism. Thus, in this work we studied the changes in lipid metabolism in breast cancer cells with reduced expression of either or gene. MDA-MB-231 cells with a stable knockdown of either or gene were used in this study. Transcriptomic and proteomic analysis as well as RT-PCR were utilized to assess gene expression. Content of individual fatty acids in the cells was measured with HPLC-MS. HPLC was used for analysis of the kinetics of PUFAs uptake. Cell viability was measured with MTS assay. Flow cytometry was used to measure activation of apoptosis. Fluorescent microscopy was utilized to assess accumulation of ROS and formation of lipid droplets. Glutathione peroxidase activity was measured with a colorimetric assay. We found that the knockdown of IGFBP6 gene led to significant changes in the profile of fatty acids in the cells and in the expression of many genes associated with lipid metabolism. As some PUFAs are known to inhibit proliferation and cause death of cancer cells, we also tested the response of the cells to single PUFAs and to combinations of docosahexaenoic acid (DHA, a n-3 PUFA) with standard chemotherapeutic drugs. Our data suggest that external PUFAs cause cell death by activation of ferroptosis, an iron-dependent mechanism of cell death with excessive lipid peroxidation. Moreover, both knockdowns increased cells' sensitivity to ferroptosis, probably due to a significant decrease in the activity of the antioxidant enzyme GPX4. Addition of DHA to commonly used chemotherapeutic drugs enhanced their effect significantly, especially for the cells with low expression of gene. The results of this study suggest that addition of PUFAs to the treatment regimen for the patients with low expression of and genes can be potentially beneficial and is worth testing in a clinically relevant setting.
乳腺癌复发是成功治疗的关键障碍之一。此前我们已经表明，乳腺癌组织中 和 基因的低表达与预后不良相关。 直接参与多不饱和脂肪酸（PUFAs）的延长，而多不饱和脂肪酸被认为在癌细胞代谢中起重要作用。因此，在这项工作中，我们研究了 或 基因表达降低的乳腺癌细胞中脂质代谢的变化。本研究使用了稳定敲低 或 基因的MDA-MB-231细胞。利用转录组学和蛋白质组学分析以及逆转录聚合酶链反应（RT-PCR）来评估基因表达。用高效液相色谱-质谱联用仪（HPLC-MS）测量细胞中单个脂肪酸的含量。高效液相色谱用于分析多不饱和脂肪酸摄取的动力学。用MTS法测量细胞活力。流式细胞术用于测量细胞凋亡的激活。荧光显微镜用于评估活性氧（ROS）的积累和脂滴的形成。用比色法测量谷胱甘肽过氧化物酶活性。我们发现，敲低胰岛素样生长因子结合蛋白6（IGFBP6）基因导致细胞中脂肪酸谱以及许多与脂质代谢相关基因的表达发生显著变化。由于已知一些多不饱和脂肪酸可抑制癌细胞增殖并导致其死亡，我们还测试了细胞对单一多不饱和脂肪酸以及二十二碳六烯酸（DHA，一种n-3多不饱和脂肪酸）与标准化疗药物组合的反应。我们的数据表明，外源性多不饱和脂肪酸通过激活铁死亡（一种依赖铁的细胞死亡机制，伴有过度的脂质过氧化）导致细胞死亡。此外，两种基因敲低均增加了细胞对铁死亡的敏感性，这可能是由于抗氧化酶谷胱甘肽过氧化物酶4（GPX4）的活性显著降低。将DHA添加到常用化疗药物中可显著增强其效果，尤其是对于 基因低表达的细胞。本研究结果表明，将多不饱和脂肪酸添加到 和 基因低表达患者的治疗方案中可能具有潜在益处，值得在临床相关环境中进行测试。
