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Intracellular protein binding patterns of the anticancer ruthenium drugs KP1019 and KP1339

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Abstract

The ruthenium compound KP1019 has demonstrated promising anticancer activity in a pilot clinical trial. This study aims to evaluate the intracellular uptake/binding patterns of KP1019 and its sodium salt KP1339, which is currently in a phase I–IIa study. Although KP1339 tended to be moderately less cytotoxic than KP1019, IC50 values in several cancer cell models revealed significant correlation of the cytotoxicity profiles, suggesting similar targets for the two drugs. Accordingly, both drugs activated apoptosis, indicated by caspase activation via comparable pathways. Drug uptake determined by inductively coupled plasma mass spectrometry (ICP-MS) was completed after 1 h, corresponding to full cytotoxicity as early as after 3 h of drug exposure. Surprisingly, the total cellular drug uptake did not correlate with cytotoxicity. However, distinct differences in intracellular distribution patterns suggested that the major targets for the two ruthenium drugs are cytosolic rather than nuclear. Consequently, drug–protein binding in cytosolic fractions of drug-treated cells was analyzed by native size-exclusion chromatography (SEC) coupled online with ICP-MS. Ruthenium–protein binding of KP1019- and KP1339-treated cells distinctly differed from the platinum binding pattern observed after cisplatin treatment. An adapted SEC-SEC-ICP-MS system identified large protein complexes/aggregates above 700 kDa as initial major binding partners in the cytosol, followed by ruthenium redistribution to the soluble protein weight fraction below 40 kDa. Taken together, our data indicate that KP1019 and KP1339 rapidly enter tumor cells, followed by binding to larger protein complexes/organelles. The different protein binding patterns as compared with those for cisplatin suggest specific protein targets and consequently a unique mode of action for the ruthenium drugs investigated.

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Abbreviations

GSH:

Glutathione

HMW/LMW:

Ruthenium-content ratio between the high molecular weight fraction and the low molecular weight fraction

ICP-MS:

Inductively coupled plasma mass spectrometry

JC-1:

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

KP1019:

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]

KP1339:

Sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]

PARP:

Poly(ADP-ribosyl)polymerase

PBS:

Phosphate-buffered saline

SEC:

Size-exclusion chromatography

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgments

We are indebted to Vera Bachinger and Maria Eisenbauer for the skilful handling of cell cultures, Elisabeth Rabensteiner, Rosa-Maria Weiss, as well as Christian Balcarek for competent technical assistance, and Irene Herbacek for fluorescence-activated cell sorting analysis. Many thanks go to Rita Dornetshuber, Christian Hartinger, Leonilla Elbling, and Michael Jakupec for inspiring discussions. This work was performed within the Research Platform Translational Cancer Therapy Research Vienna and supported by the Austrian Science Fond grants L212 and L473, by Bürgermeister Fond der Stadt Wien grant 2460, as well as by FFG grant 811591.

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Authors and Affiliations

  1. Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, 1090, Vienna, Austria

    Petra Heffeter, Bihter Atil, Mir Ali Reza Hoda, Ute Jungwirth, Michael Micksche & Walter Berger

  2. Division of Analytical Chemistry, Department of Chemistry, University of Natural Resources and Applied Life Sciences, BOKU, Vienna, Austria

    Katharina Böck & Gunda Koellensperger

  3. Institute for Geological Sciences, University of Vienna, Vienna, Austria

    Wilfried Körner

  4. Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria

    Caroline Bartel & Bernhard K. Keppler

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  1. Petra Heffeter
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  2. Katharina Böck
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  10. Walter Berger
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Correspondence to Walter Berger.

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Heffeter, P., Böck, K., Atil, B. et al. Intracellular protein binding patterns of the anticancer ruthenium drugs KP1019 and KP1339. J Biol Inorg Chem 15, 737–748 (2010). https://doi.org/10.1007/s00775-010-0642-1

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  • DOI: https://doi.org/10.1007/s00775-010-0642-1

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