Abstract
Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of ∼2 μM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) chloride (RPC2) retards or arrests microtubule motion as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins. Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase, suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy, these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an associative binding constant of 4.8 × 106 M−1 for RPC2 to preformed microtubules and support a 1 : 1 RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is co-localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule stabilizing agent and sharing many similarities with cells treated with paclitaxel.
Highlights
Microtubules (MTs) play an essential role in mitosis, cellular structure, and trafficking, as well as offer a promising target for innovative chemotherapeutic agents.[1,2] MTs are composed of ab-tubulin heterodimers that undergo highly regulated and dynamic bouts of polymerization and depolymerization
As Microtubule stabilizing agents (MSAs) activity is observed for RPC2 in vitro, the presumption is that the same is occurring in vivo, which is most strongly supported by the movie data, as the interpretation of the MT structure in xed cells is o en ambiguous
The combination of the live cell movies, xed cell MT structure, cytotoxicity, sub-cellular localization, in vitro MT binding, polymerization, and TEM data build a convincing case that RPC2 enters cells, binds, and disrupts the normal MT network by stabilizing the MTs present
Summary
Microtubules (MTs) play an essential role in mitosis, cellular structure, and trafficking, as well as offer a promising target for innovative chemotherapeutic agents.[1,2] MTs are composed of ab-tubulin heterodimers that undergo highly regulated and dynamic bouts of polymerization and depolymerization. Microtubule targeting drugs disrupt this ‘dynamic instability’ by inhibiting or promoting polymerization and interfere with mitosis and other essential cellular processes, leading to apoptosis.[3,4] Agents that inhibit tubulin polymerization, such as nocodazole (NCZ), vincristine, and colchicine, are known as microtubule destabilizing agents (MDAs) and are used therapeutically.[5,6] Microtubule stabilizing agents (MSAs), more recently discovered, inhibit MT depolymerization. The rst identi ed MSA, paclitaxel (PTX, Taxol), was discovered in 1979,7 and approved for clinical use in 1993 for treatment of solid
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