Berberine Induces Toxicity in HeLa Cells through Perturbation of Microtubule Polymerization by Binding to Tubulin at a Unique Site

Abstract

Berberine has been used traditionally for its diverse pharmacological actions. It exhibits remarkable anticancer activities and is currently under clinical trials. In this study, we report that the anticancer activity of berberine could be partly due to its inhibitory actions on tubulin and microtubule assembly. Berberine inhibited the proliferation of HeLa cells with an IC of 18 μM and induced significant depolymerization of interphase and mitotic microtubules. At its IC, berberine exerted a moderate G2/M arrest and mitotic block as detected by fluorescence-activated cell sorting analysis and fluorescence microscopy, respectively. In a wound closure assay, berberine inhibited the migration of HeLa cells at concentrations lower than its IC, indicating its excellent potential as an anticancer agent. In vitro studies with tubulin isolated from goat brain indicated that berberine binds to tubulin at a single site with a K of 11 μM. Berberine inhibited the assembly of tubulin into microtubules and also disrupted the preformed microtubules polymerized in the presence of glutamate and paclitaxel. Competition experiments indicated that berberine could partially displace colchicine from its binding site. Results from fluorescence resonance energy transfer, computational docking, and molecular dynamics simulations suggest that berberine forms a stable complex with tubulin and binds at a novel site 24 Å from the colchicine site on the β-tubulin. Data obtained from synchronous fluorescence analysis of the tryptophan residues of tubulin and from the Fourier transform infrared spectroscopy studies revealed that binding of berberine alters the conformation of the tubulin heterodimer, which could be the molecular mechanism behind the depolymerizing effects on tubulin assembly.

Cell-specific pattern of berberine pleiotropic effects on different human cell lines

“…Besides the regulation of signal transduction pathways, the direct interaction of berberine with the cellular cytoskeleton may underlie its inhibitory effect on cell migration and cell proliferation. Recently, it has been demonstrated that berberine binds to VASP and inhibits actin polimerization 49 and it also binds to a single site of tubulin, inhibiting microtubule assembly and promoting depolimerization 50 .…”

Berberine, a natural alkaloid sensitizes human hepatocarcinoma to ionizing radiation by blocking autophagy and cell cycle arrest resulting in senescence

“…[ 58,59 ] Although, the mechanisms by which Ber exacerbated radiation mediated G2/M arrest is presently unclear however, a previous report suggests its ability to disrupt mitotic spindle leading to mitotic catastrophe. [ 57 ] It has also been shown earlier that Ber can exert a G2/M arrest via ATM‐Chk1 activation. [ 14 ] As suggested earlier, inhibition of Topoisomerase II activity might result in a mitotic catastrophe.…”

“…A healthy microtubule network is necessary for the completion of autophagic process. [ 56 ] Earlier, Ber has been shown to disrupt microtubule integrity [ 57 ] which might be a causative factor blocked autophagy leading to the accumulation of autophagosomes and lysosomes, although the exact mechanism requires further experimental evidence.…”

Zerumbone, a cyclic sesquiterpene, exerts antimitotic activity in HeLa cells through tubulin binding and exhibits synergistic activity with vinblastine and paclitaxel

“…The reaction mixture was then subjected to centrifugation at 50 000 × g for 1 hour. The supernatant and pellet were collected separately, and the protein concentration in the supernatant was measured using Bradford assay …”