Department of Pharmacy, University of Salerno, I-84084 Fisciano (SA), Italy.
Department of Medical, Oral and Biotechnological Sciences, 'G. d'Annunzio' University of Chieti‑Pescara, I-66100 Chieti, Italy.
Int J Mol Med. 2020 Sep;46(3):1197-1209. doi: 10.3892/ijmm.2020.4669. Epub 2020 Jul 6.
Oxidative stress is widely accepted as a key factor of doxorubicin (Doxo)‑induced cardiotoxicity. There is evidence to indicate that nitrosative stress is involved in this process, and that Doxo interacts by amplifying cell damage. Mitochondrial connexin 43 (mitoCx43) can confer cardioprotective effects through the reduction of mitochondrial reactive oxygen species production during Doxo‑induced cardiotoxicity. The present study aimed to evaluate the involvement of mitoCx43 in Doxo‑induced nitrosative stress. Rat H9c2 cardiomyoblasts were treated with Doxo in the absence or presence of radicicol, an inhibitor of Hsp90, the molecular chaperone involved in Cx43 translocation to the mitochondria that underlies its role in cardioprotection. FACS analysis and RT‑qPCR revealed that Doxo increased superoxide dismutase, and catalase gene and protein expression. As shown by hypodiploid nuclei and confirmed by western blot analysis, Doxo increased caspase 9 expression and reduced procaspase 3 levels, which induced cell death. Moreover, a significant increase in the activation of the NF‑κB signaling pathway was observed. It is well known that the increased expression of inducible nitric oxide synthase results in nitric oxide overproduction, which then rapidly reacts with hydrogen peroxide or superoxide generated by the mitochondria, to form highly reactive and harmful peroxynitrite, which ultimately induces nitrotyrosine formation. Herein, these interactions were confirmed and increased effects were observed in the presence of radicicol. On the whole, the data of the present study indicate that an interplay between oxidative and nitrosative stress is involved in Doxo‑induced cardiotoxicity, and that both aspects are responsible for the induction of apoptosis. Furthermore, it is demonstrated that the mechanisms that further increase mitochondrial superoxide generation (e.g., the inhibition of Cx43 translocation into the mitochondria) significantly accelerate the occurrence of cell death.
氧化应激被广泛认为是多柔比星（Doxo）诱导心脏毒性的关键因素。有证据表明，硝化应激也参与了这一过程，并且 Doxo 通过放大细胞损伤来相互作用。线粒体连接蛋白 43（mitoCx43）可以通过减少 Doxo 诱导的心脏毒性期间线粒体活性氧的产生来发挥心脏保护作用。本研究旨在评估 mitoCx43 在 Doxo 诱导的硝化应激中的作用。用 Doxo 处理大鼠 H9c2 心肌细胞，在没有或存在热休克蛋白 90（Hsp90）抑制剂 Radicicol 的情况下进行处理，Hsp90 是一种分子伴侣，参与 Cx43 向线粒体的易位，这是其发挥心脏保护作用的基础。FACS 分析和 RT-qPCR 显示 Doxo 增加了超氧化物歧化酶和过氧化氢酶基因和蛋白的表达。如低二倍体核所示，并通过 Western blot 分析证实，Doxo 增加了 caspase 9 的表达并降低了 procaspase 3 的水平，从而诱导了细胞死亡。此外，还观察到 NF-κB 信号通路的激活显著增加。众所周知，诱导型一氧化氮合酶的表达增加会导致一氧化氮的过度产生，然后一氧化氮迅速与线粒体产生的过氧化氢或超氧化物反应，形成高度反应性和有害的过氧亚硝酸盐，最终诱导硝基酪氨酸的形成。在这里，这些相互作用得到了证实，并在 Radicicol 的存在下观察到了增强的效果。总的来说，本研究的数据表明，氧化应激和硝化应激之间的相互作用参与了 Doxo 诱导的心脏毒性，并且这两个方面都负责诱导细胞凋亡。此外，研究结果还表明，进一步增加线粒体超氧化物生成的机制（例如，抑制 Cx43 向线粒体的易位）会显著加速细胞死亡的发生。
