Department of Medical Chemistry, Medical University of Gdansk, Debinki 1, 80-211, Gdansk, Poland.
Department of Biophysics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany.
Sci Rep. 2021 Jan 15;11(1):1616. doi: 10.1038/s41598-020-80816-x.
Osteosarcoma (OS) is one of the most malignant tumors of childhood and adolescence. Research on mitochondrial dynamics (fusion/fission) and biogenesis has received much attention in last few years, as they are crucial for death of cancer cells. Specifically, it was shown that increased expression of the cytoplasmic dynamin-related protein 1 (Drp1) triggers mitochondrial fission (division), which activates BAX and downstream intrinsic apoptosis, effectively inhibiting OS growth. In the presented study, human OS cells (metastatic 143B OS cell line) were incubated with 2-methoxyestradiol (2-ME) at both physiologically and pharmacologically relevant concentrations. Cell viability was determined by the MTT assay. Confocal microscopy and western blot methods were applied to examine changes in Drp1 and BAX protein levels. Mitochondrial Division Inhibitor 1, MDIVI-1, was used in the study to further examine the role of Drp1 in 2-ME-mediated mechanism of action. To determine quantitative and qualitative changes in mitochondria, electron microscopy was used. 2-ME at all used concentrations increased mitochondrial fission and induced autophagy in OS cells. At the concentration of 1 µM 2-ME increased the area density of mitochondria in OS cells. Subsequent, upregulated expression of Drp1 and BAX proteins by 2-ME strongly suggests the activation of the intrinsic apoptosis pathway. We further observed 2-ME-mediated regulation of glycolytic state of OS cells. Therefore, we suggest that changes of mitochondrial dynamics may represent a novel mechanism of anticancer action of 2-ME. This finding may open new approaches to improve the efficacy of chemotherapy in the treatment of OS, however, it has to be confirmed by in vivo studies.
骨肉瘤（OS）是儿童和青少年中最恶性的肿瘤之一。近年来，人们对线粒体动力学（融合/裂变）和生物发生的研究引起了广泛关注，因为它们对癌细胞的死亡至关重要。具体来说，已经表明细胞质动力相关蛋白 1（Drp1）的表达增加会触发线粒体裂变（分裂），从而激活 BAX 和下游内在凋亡，有效地抑制 OS 的生长。在本研究中，用 2-甲氧基雌二醇（2-ME）孵育人骨肉瘤细胞（转移性 143B OS 细胞系），浓度分别为生理相关和药理相关浓度。通过 MTT 测定法测定细胞活力。应用共聚焦显微镜和western blot 方法来检测 Drp1 和 BAX 蛋白水平的变化。在研究中使用线粒体分裂抑制剂 1（MDIVI-1）进一步研究 Drp1 在 2-ME 介导的作用机制中的作用。为了确定线粒体的定量和定性变化，使用了电子显微镜。所有使用浓度的 2-ME 均增加了 OS 细胞中的线粒体裂变并诱导自噬。在 1µM 2-ME 的浓度下，OS 细胞中线粒体的面积密度增加。随后，2-ME 上调 Drp1 和 BAX 蛋白的表达强烈提示内在凋亡途径的激活。我们进一步观察到 2-ME 介导的 OS 细胞糖酵解状态的调节。因此，我们认为线粒体动力学的变化可能代表 2-ME 抗癌作用的一种新机制。这一发现可能为改善骨肉瘤治疗中的化疗效果开辟新途径，但仍需通过体内研究来证实。
