In vitro characterization and molecular dynamic simulation of shikonin as a tubulin-targeted anticancer agent

Shikonin (SK), a naphthoquinone compound from the purple gromwell, Lithospermum erythrorhizon, possesses a considerable antiproliferative potential. By using a combination of biophysical techniques, cellular assays, immunofluorescence imaging, and molecular dynamic simulation, we identified a possible mechanism of action of SK. SK inhibited the viability of the triple negative breast cancer cells MDA-MB-231 (IC50 of 1 +/- 0.1 mu M), and its inhibitory effect was irreversible. It strongly suppressed the clonogenic and migratory potential of the cells. Although SK did not show any phase-specific inhibition of cell cycle progression, it induced apoptosis as confirmed by annexin-V-based flow cytometry and Western immunoblotting of PARP1. Probing further into its mechanism using a tryptophan-quenching assay, it was found that SK binds the microtubule-building protein tubulin with a dissociation constant (Kd) of 8 +/- 2.7 mu M, without grossly damaging the tertiary structure of the protein. The drug-bound tubulin could not assemble microtubules properly in vitro as confirmed by polymer mass analysis, turbidimetry analysis, and transmission electron microscopy, and in cells, as visualized by immunofluorescence imaging. In cells, SK also suppressed the dynamicity of microtubules as indicated by considerable acetylation of the cellular microtubules. The fine details of tubulin-SK interactions were then elucidated using molecular docking and molecular dynamic simulation. The free energy change of the interaction (Delta Gbind,pred) was found to be -14.60 kcal/mol and the binding involved both the intermolecular van der Waals (Delta Evdw) and the electrostatic (Delta Eele) interactions. Taken together, our data provide evidence for a possible mechanism of action of SK as a tubulin-targeted anticancer agent.

Automated summary

This study investigated the possible mechanism of action of shikonin (SK), a compound from the purple gromwell, in inhibiting the proliferation of triple negative breast cancer cells. SK was found to irreversibly inhibit cell viability and suppress clonogenic and migratory potential. It was also shown to induce apoptosis and bind to the microtubule-building protein tubulin, preventing proper microtubule assembly and affecting microtubule dynamics. Overall, this study provides evidence for SK as a potential tubulin-targeted anticancer agent.