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Cardioprotective effects of Cu^(II)ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1.

ABSTRACT: Cu^(II)ATSM was developed as a hypoxia sensitive positron emission tomography agent. Recent reports have highlighted the neuroprotective properties of Cu^(II)ATSM, yet there are no reports that it confers cardioprotection. We demonstrate that Cu^(II)ATSM activates the redox-sensitive transcription factor Nrf2 in human coronary artery smooth muscle cells (HCASMC) and cardiac myocytes (HCM), leading to upregulation of antioxidant defense enzymes. Oral delivery of Cu^(II)ATSM in mice induced expression of the Nrf2-regulated enzymes in the heart and aorta. In HCASMC, Cu^(II)ATSM increased expression of the Nrf2 stabilizer DJ-1, and knockdown of Nrf2 or DJ-1 attenuated Cu^(II)ATSM-mediated heme oxygenase-1 and NADPH quinone oxidoreductase-1 induction. Pre-treatment of HCASMC with Cu^(II)ATSM protected against the pro-oxidant effects of angiotensin II (Ang II) by attenuating superoxide generation, apoptosis, proliferation and increases in intracellular calcium. Notably, Cu^(II)ATSM-mediated protection against Ang II-induced HCASMC apoptosis was diminished by Nrf2 knockdown. Acute treatment with Cu^(II)ATSM enhanced the association of DJ-1 with superoxide dismutase-1 (SOD1), paralleled by significant increases in intracellular Cu^(II) levels and SOD1 activity. We describe a novel mechanism by which Cu^(II)ATSM induces Nrf2-regulated antioxidant enzymes and protects against Ang II-mediated HCASMC dysfunction via activation of the Nrf2/DJ-1 axis. Cu^(II)ATSM may provide a therapeutic strategy for cardioprotection via upregulation of antioxidant defenses.

SUBMITTER: Srivastava S

PROVIDER: S-EPMC5431352 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Cardioprotective effects of Cu(II)ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1.

Srivastava Salil S Blower Philip J PJ Aubdool Aisah A AA Hider Robert C RC Mann Giovanni E GE Siow Richard C RC

Scientific reports 20161221 1

Cu(II)ATSM was developed as a hypoxia sensitive positron emission tomography agent. Recent reports have highlighted the neuroprotective properties of Cu(II)ATSM, yet there are no reports that it confers cardioprotection. We demonstrate that Cu(II)ATSM activates the redox-sensitive transcription factor Nrf2 in human coronary artery smooth muscle cells (HCASMC) and cardiac myocytes (HCM), leading to upregulation of antioxidant defense enzymes. Oral delivery of Cu<s ...[more]

PMID: 28442712

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Project description:BackgroundDiacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression.MethodsμPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels.ResultsIn vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages.ConclusionIn the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

Project description:Therapies targeting reductive/oxidative (redox) metabolism hold potential in cancers resistant to chemotherapy and radiation. A redox imaging marker would help identify cancers susceptible to redox-directed therapies. Copper(II)-diacetyl-bis(4-methylthiosemicarbazonato) (Cu-ATSM) is a PET tracer developed for hypoxia imaging that could potentially be used for this purpose. We aimed to demonstrate that Cu-ATSM signal is dependent on cellular redox state, irrespective of hypoxia. Methods: We investigated the relationship between 64Cu-ATSM signal and redox state in human cervical and colon cancer cells. We altered redox state using drug strategies and single-gene mutations in isocitrate dehydrogenases (IDH1/2). Concentrations of reducing molecules were determined by spectrophotometry and liquid chromatography-mass spectrometry and compared with 64Cu-ATSM signal in vitro. Mouse models of cervical cancer were used to evaluate the relationship between 64Cu-ATSM signal and levels of reducing molecules in vivo, as well as to evaluate the change in 64Cu-ATSM signal after redox-active drug treatment. Results: A correlation exists between baseline 64Cu-ATSM signal and cellular concentration of glutathione, nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NADH). Altering NADH and NADPH metabolism using drug strategies and IDH1 mutations resulted in significant changes in 64Cu-ATSM signal under normoxic conditions. Hypoxia likewise changed 64Cu-ATSM signal, but treatment of hypoxic cells with redox-active drugs resulted in a more dramatic change than hypoxia alone. A significant difference in NADPH was seen between cervical tumor orthotopic implants in vivo, without a corresponding difference in 64Cu-ATSM signal. After treatment with β-lapachone, there was a change in 64Cu-ATSM signal in xenograft tumors smaller than 50 mg but not in larger tumors. Conclusion: 64Cu-ATSM signal reflects redox state, and altering redox state impacts 64Cu-ATSM metabolism. Our animal data suggest there are other modulating factors in vivo. These findings have implications for the use of 64Cu-ATSM as a predictive marker for redox therapies, though further in vivo work is needed.

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Cardioprotective effects of Cu^(II)ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1.

Publications

Cardioprotective effects of Cu<sup>(II)</sup>ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1.

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