CCCP
(Synonyms: 羰基氰化氯苯腙,Carbonyl cyanide 3-chlorophenylhydrazone; Carbonyl Cyanide m-Chlorophenylhydrazone) 目录号 : GC14727
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。
Cas No.:555-60-2
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
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- Purity: >99.50%
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| Cell experiment [1]: | |
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Cell lines |
HL-7702 cells |
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Preparation Method |
HL-7702 cells were treated with DMSO, gefitinib, CCCP for 24 h or starved with HBSS for 3 h. |
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Reaction Conditions |
20 µM CCCP for 24 h |
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Applications |
Mitochondrial inducer carbonyl cyanide m-chlorophenizone (CCCP) activates mitophagy. CCCP failed to selectively reduce COX6A1 levels. |
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References: [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. |
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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].
References:
[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]。
| Cas No. | 555-60-2 | SDF | |
| 别名 | 羰基氰化氯苯腙,Carbonyl cyanide 3-chlorophenylhydrazone; Carbonyl Cyanide m-Chlorophenylhydrazone | ||
| 化学名 | (3-chlorophenyl)carbonohydrazonoyl dicyanide | ||
| Canonical SMILES | ClC1=CC(N/N=C(C#N)/C#N)=CC=C1 | ||
| 分子式 | C9H5ClN4 | 分子量 | 204.62 |
| 溶解度 | ≥ 20.5mg/mL in DMSO | 储存条件 | Store at 2-8°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 4.8871 mL | 24.4355 mL | 48.8711 mL |
| 5 mM | 0.9774 mL | 4.8871 mL | 9.7742 mL |
| 10 mM | 0.4887 mL | 2.4436 mL | 4.8871 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Current mechanistic insights into the CCCP-induced cell survival response
Biochem Pharmacol2018 Feb;148:100-110.PMID: 29277693DOI: 10.1016/j.bcp.2017.12.018
The ring-substituted derivatives of carbonyl cyanide phenylhydrazone, CCCP and FCCP, are routinely used for the analysis of the mitochondrial function in living cells, tissues, and isolated mitochondrial preparations. CCCP and FCCP are now being increasingly used for investigating the mechanisms of autophagy by inducing mitochondrial degradation through the disruption of the mitochondrial membrane potential (ΔΨm). Sustained perturbation of ΔΨm, which is normally tightly controlled to ensure cell proliferation and survival, triggers various stress pathways as part of the cellular adaptive response, the main components of which are mitophagy and autophagy. We here review current mechanistic insights into the induction of mitophagy and autophagy by CCCP and FCCP. In particular, we analyze the cellular modifications produced by the activation of two major pathways involving the signaling of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the transcription factor EB (TFEB), and discuss the contribution of these pathways to the integrated cellular stress response.
CCCP induces autophagy in an AMPK-independent manner
Biochem Biophys Res Commun2011 Dec 16;416(3-4):343-8.PMID: 22119190DOI: 10.1016/j.bbrc.2011.11.038
AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKα1/α2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFP-LC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKα1/α2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy.
CCCP activation of the reconstituted NaK-pump
J Membr Biol1990 Aug;117(2):153-61.PMID: 2170657DOI: 10.1007/BF01868682
In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na+ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15 degrees C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (Vm), we postulated that a Vm build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K+ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331-355). Although cytoplasmic K+ accelerated the change of ADP- and K(+)-sensitive EP (E*P) to K(+)-sensitive ADP-insensitive EP (E2P), CCCP did not complete with cytoplasmic K+ when cytoplasmic Na+ was saturated. When the PLs were phosphorylated with 20 microM ATP and 20 microM palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E*P content and decreased the ADP-sensitive K(+)-insensitive EP (E1P). The results described above suggest that CCCP affects the E1P to E*P change in the E1P----E*P----E2P conversion and that this reaction step is inhibited by Vm.
A bifunctional fluorescent sensor for CCCP-induced cancer cell apoptosis imaging
Chem Commun (Camb)2020 Oct 21;56(82):12423-12426.PMID: 32936131DOI: 10.1039/d0cc04200e
The detailed mechanism and the extent of pH/SO2 changes during apoptosis remain unknown. The developed sensor NPCF for SO2 and pH dual detection illustrates that SO2 can reduce the inflammation caused by LPS and the acidification of the environment. The levels of SO2 and pH change during carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis.