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Hoechst 33342 induced reactive oxygen species and impaired expression of cytochrome c oxidase subunit 1 leading to cell death in irradiated human cancer cells

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Abstract

Oxidative stress and mitochondrial dysfunction in cancer cells represent features that may be exploited therapeutically. We determined whether minor groove binding ligand Hoechst 33342, known to induce mitochondrial dysfunction via increase in reactive oxygen species (ROS), enhances killing of human head and neck cancer (KB) cells mediated by impaired expression of mitochondrial protein involved in electron transfer. Elevation in ROS generation, increase in ΔΨm, down regulation of cytochrome c oxidase (CO), alteration in expression of antioxidant enzymes viz. Mn-SOD and Catalase, and release of cytochrome c into the cytosol, were observed in time-dependent manner when cells were irradiated (5 Gy) in presence of Hoechst 33342. Persistent increase in ROS observed till 48 h following treatment decreased the clonogenic survival and viability to a large extent via increase in ΔΨm, release of cytochrome c and non-coordinated expression of antioxidant enzymes. Treatment with antioxidants PEG-MnSOD and PEG-catalase inhibited the increase in ROS and loss of cell survival, suggesting the involvement of ROS in the Hoechst 33342-induced cell death. The result demonstrated significant sensitization of cancer cells to radiation-induced toxicity in presence of Hoechst 33342 via increasing ROS to a toxic level and impairing CO expression and antioxidant enzymes. This understanding is expected to benefit both in elucidating the detailed mechanisms of actions of DNA interacting drug and designing better molecules for enhancing radiation-induced cell death among cancer cells.

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Abbreviations

mtDNA:

Mitochondrial DNA

ROS:

Reactive oxygen species

ΔΨm :

Mitochondrial membrane potential

CO:

Cytochrome c oxidase

CO1:

Cytochrome c oxidase sub unit 1

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

H2DCFDA:

2′,7′-Dichlorofluorescein diacetate

TMRM:

Tetramethyl rhodamine methyl ester

JC-1:

5,5′,6,6′-Tetrachloro-1,1′, 3,3′-tetraethylbenzimidazolylcarbocyanine iodide

FCCP:

Carbonyl cyanide p-trifluoromethoxyphenylhydrazone

ETC:

Electron transport chain

HBSS:

Hanks-balanced salts solution

PEG:

Polyethylene glycol

PI:

Propidium iodide

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Acknowledgments

We are thankful to Dr. R.P. Tripathi, Director, Institute of Nuclear Medicine and Allied Sciences for support and constant encouragement. We also thank to Mrs Namita Kalra for help in flow cytometric measurement. This work was carried out as part of a project (INM 301) supported by DRDO from the Ministry of Defense, Government of India. Mr. Mohammad Athar is a recipient of ICMR fellowship.

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Authors and Affiliations

  1. Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Defence R&D Organization, Brig. S. K. Mazumdar Road, Delhi, 110 054, India

    Mohammad Athar, Nabo Kumar Chaudhury & Rajeev Varshney

  2. Department of Biosciences, Jamia Millia Islamia, New Delhi, India

    Mohammad Athar & Mohammad Ejaz Hussain

  3. Defence R&D Organization, Ministry of Defence, Government of India, 339, DRDO Bhawan, Rajaji Marg, New Delhi, 110 105, India

    Rajeev Varshney

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  1. Mohammad Athar
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Correspondence to Rajeev Varshney.

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Athar, M., Chaudhury, N.K., Hussain, M.E. et al. Hoechst 33342 induced reactive oxygen species and impaired expression of cytochrome c oxidase subunit 1 leading to cell death in irradiated human cancer cells. Mol Cell Biochem 352, 281–292 (2011). https://doi.org/10.1007/s11010-011-0764-y

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