A novel microtubule-modulating noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering

Abstract

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing ‘real’ centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the ‘cytotoxicity-window’ of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some ‘apoptosis-evasive’ cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation.

Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy

“…Treatment of HeLa cells with bromonoscapine 122 and MCF-7 cells with griseofulvin 130 results in upregulation of BubR1, implying a loss of tension across the kinetochore or an absence of attachment to one or more kinetochores. 122 Clearly, in cells with supernumerary centrosomes, microtubule dynamicity promotes transkinetochore tension, which appears necessary for the overall force balance required for centrosome clustering. Mild attenuation of dynamic stability in noncancerous cells appears to be harmless, as griseofulvin and bromonoscapine are apparently nontoxic to healthy cells.…”

“…In recent years, research from a few groups including ours has turned the spotlight onto a property of microtubules that is central to their biological function, viz, their dynamic instability, as a key requirement for effective clustering of supernumerary centrosomes. Treatment of cells exhibiting amplified centrosomes with drugs that gently attenuate microtubule dynamicity, such as griseofulvin 8 or bromonoscapine, 122 induces declustering of supernumerary centrosomes, leading us to postulate that microtubule dynamicity is an important requisite for centrosome clustering. Although centrosome declustering drugs are very different in many respects -for instance, griseofulvin is an antifungal whereas bromonoscapine is a derivative of the antitussive drug, noscapine -the attribute they share is the ability to subtly attenuate microtubule dynamic instability and selectively kill cancer cells.…”

“…131 By contrast, in the absence of clustering, supernumerary centrosomes result in spindle multipolarity, which may cause aneuploidy of a mortally high grade. Alternatively, multipolar cells may arrest in mitosis and succumb to death via other mechanisms, 63,122 as depicted in Figure 4c. Given the lethality of the multipolar state, induction of high-grade spindle multipolarity constitutes a novel chemotherapeutic strategy whose efficacy holds much promise.…”

“…3,4 Supernumerary centrosomes can prove lethal by inducing spindle multipolarity with subsequent anaphase catastrophe 5 or multipolar mitosis. [6][7][8] Much evidence now supports a causative link between CIN and malignant transformation, particularly in multi-step carcinogenesis. [9][10][11] However, dust has not settled on the debate of whether a low level of aneuploidy is merely an outcome of tolerable mitotic errors accrued by cancer cells or whether it is an evolutionarily favored feature selected for conferring some advantage on them.…”

“…In line with this notion, bromonoscapine (also known as EM011), a derivative of the nontoxic, poppyderived antitussive, noscapine, suppresses dynamic instability and centrosome clustering. 122 This drug simply dampens microtubule dynamic instability without causing depolymerization, overpolymerization, or otherwise noticeably impacting microtubule ultrastructure. Like griseofulvin, bromonoscapine causes G2/M arrest followed by apoptosis; however, bromonoscapine-treated cells also succumb to death by another pathway: multipolar mitosis.…”