CCCP

Background

Carbonylcyanide-3-chlorophenylhydrazone (CCCP) is a protonophore, which causes uncoupling of proton gradient in the inner mitochondrial membrane, thus inhibiting the rate of ATP synthesis. CCCP inhibits STING-mediated IFN-β production via disrupting mitochondrial membrane potential (MMP).

In HL-7702 cells, Mitochondrial inducer CCCP activates mitophagy. CCCP failed to selectively reduce COX6A1 levels^[1]. CCCP alone significantly increased chondrocyte apoptosis treatment with CCCP and eugenol significantly decreased chondrocyte apoptosis, suggesting that eugenol reduces CCCP-induced apoptosis. Our results showed that eugenol significantly the CCCP-induced mitochondrial membrane potential changes^[4].CCCP inhibits activation of STING and its downstream signaling molecules, TBK1 and IRF3, but not STING translocation to the perinuclear region. CCCP down-modulates the STING pathway through DRP1-mediated mitochondria fragmentation^[3].Cultured human osteosarcoma cells were imaged to visualize changes of mitochondrial membrane potential, morphology, and permeability transition. during treatment with a protonophore, CCCP. Cells rapidly exhibited mitochondrial permeability transition and swelling after addition of CCCP, but the swelling subsided within hours, leaving mitochondria that appeared in punctate form^[5]. CCCP -induced mitochondrial fission inhibited IFN-β expression supporting the idea that mitochondrial dynamics modulated activation of STING signaling Mitochondrial dynamics regulated by MFN1 MFN2 and OPA1 mediated fusion and DRP1 and TBC1D15-mediated mitochondrial fission^[8].The mitochondrial homeostasis of a carp fish Megalobrama amblycephala was investigated systematically in a time-course manner by using CCCP. CCCP treatment resulted in the imbalance of mitochondrial homeostasis in Megalobrama amblycephala by promoting mitochondrial oxidative stress, fission and mitophagy, but depressing mitochondrial fusion, biogenesis and function^[9].The proton ionophore CCCP inhibited antigen-stimulated secretion and calcium influx in rat basophilic leukemia cells^[6].The protonophore CCCP suppressed DMXAA-induced type I IFN production through dissipation of mitochondria membrane potential (MMP) ^[7].In combination with starvation and CCCP, damaged mitochondria in GC-1 cell lines overexpressing Spata33 were phagocytosed by autophagosomes, in which damaged mitochondria were degraded^[2].

参考文献:
[1]: Luo P, Yan H, et,al. PLK1 (polo like kinase 1)-dependent autophagy facilitates gefitinib-induced hepatotoxicity by degrading COX6A1 (cytochrome c oxidase subunit 6A1). Autophagy. 2021 Oct;17(10):3221-3237. doi: 10.1080/15548627.2020.1851492. Epub 2020 Dec 14. PMID: 33315519; PMCID: PMC8526032.
[2]: Zhang Y, Xu X, et,al. SPATA33 is an autophagy mediator for cargo selectivity in germline mitophagy. Cell Death Differ. 2021 Mar;28(3):1076-1090. doi: 10.1038/s41418-020-00638-2. Epub 2020 Oct 21. PMID: 33087875; PMCID: PMC7937689.
[3]: Kwon D, Park E, et,al. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) suppresses STING-mediated DNA sensing pathway through inducing mitochondrial fission. Biochem Biophys Res Commun. 2017 Nov 4;493(1):737-743. doi: 10.1016/j.bbrc.2017.08.121. Epub 2017 Aug 30. PMID: 28859978.
[4]: Wu Z, Wang Y, et,al. Eugenol protects chondrocytes and articular cartilage by downregulating the JAK3/STAT4 signaling pathway. J Orthop Res. 2022 Jul 26. doi: 10.1002/jor.25420. Epub ahead of print. PMID: 35880357.
[5]: Minamikawa T, Williams DA, et,al. Mitochondrial permeability transition and swelling can occur reversibly without inducing cell death in intact human cells. Exp Cell Res. 1999 Jan 10;246(1):26-37. doi: 10.1006/excr.1998.4290. PMID: 9882512.
[6]: Mohr FC, Fewtrell C. The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. Multiple effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. J Biol Chem. 1987 Aug 5;262(22):10638-43. PMID: 2440869.
[7]: Prantner D, Perkins DJ, et,al. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) activates stimulator of interferon gene (STING)-dependent innate immune pathways and is regulated by mitochondrial membrane potential. J Biol Chem. 2012 Nov 16;287(47):39776-88. doi: 10.1074/jbc.M112.382986. Epub 2012 Oct 1. PMID: 23027866; PMCID: PMC3501038.
[8]: Kwon D, Park E, et,al.Stimulator of IFN genes-mediated DNA-sensing pathway is suppressed by NLRP3 agonists and regulated by mitofusin 1 and TBC1D15, mitochondrial dynamics mediators. FASEB J. 2017 Nov;31(11):4866-4878. doi: 10.1096/fj.201700328R. Epub 2017 Jul 20. PMID: 28729291.
[9]: Zhang L, Zheng XC, et,al. Carbonyl cyanide 3-chlorophenylhydrazone induced the imbalance of mitochondrial homeostasis in the liver of Megalobrama amblycephala: A dynamic study. Comp Biochem Physiol C Toxicol Pharmacol. 2021 Jun;244:109003. doi: 10.1016/j.cbpc.2021.109003. Epub 2021 Feb 19. PMID: 33617998.

Carbonylcyanide-3-chlorophenylhydrazone (CCCP) 是一种质子载体，可导致线粒体内膜中的质子梯度解偶联，从而抑制 ATP 合成速率。 CCCP 通过破坏线粒体膜电位 (MMP) 抑制 STING 介导的 IFN-β 产生。

在 HL-7702 细胞中，线粒体诱导剂 CCCP 激活线粒体自噬。 CCCP 未能选择性地降低 COX6A1 水平^[1]。单独的 CCCP 显着增加软骨细胞凋亡，CCCP 和丁香酚显着降低软骨细胞凋亡，表明丁香酚减少 CCCP 诱导的细胞凋亡。我们的结果表明，丁香酚可显着改变 CCCP 诱导的线粒体膜电位变化^[4]。CCCP 抑制 STING 及其下游信号分子 TBK1 和 IRF3 的激活，但不抑制 STING 向核周区域的易位。 CCCP 通过 DRP1 介导的线粒体断裂下调 STING 通路^[3]。对培养的人骨肉瘤细胞进行成像，以观察线粒体膜电位、形态和通透性转变的变化。在用质子载体 CCCP 治疗期间。加入 CCCP 后细胞迅速表现出线粒体通透性转变和肿胀，但肿胀在数小时内消退，留下以点状形式出现的线粒体^[5]。 CCCP 诱导的线粒体裂变抑制 IFN-β 表达支持这样的观点:线粒体动力学调节 STING 信号的激活 线粒体动力学受 MFN1 MFN2 和 OPA1 介导的融合以及 DRP1 和 TBC1D15 介导的线粒体裂变^[8]。通过使用 CCCP，以时程方式系统地研究了鲤鱼团头鲂的线粒体稳态。 CCCP 处理通过促进线粒体氧化应激、裂变和线粒体自噬，但抑制线粒体融合、生物发生和功能，导致团头鲂线粒体稳态失衡^[9]。质子离子载体 CCCP 抑制抗原刺激分泌和钙流入大鼠嗜碱性白血病细胞^[6]。质子载体 CCCP 通过耗散线粒体膜电位 (MMP)^[7] 抑制 DMXAA 诱导的 I 型干扰素产生。联合饥饿和 CCCP，过表达 Spata33 的 GC-1 细胞系中受损的线粒体被自噬体吞噬，其中受损的线粒体被降解^[2]。

Protocol