The phytochemical, corynoline, diminishes Aurora kinase B activity to induce mitotic defect and polyploidy.

Plants are a rich source for bioactive compounds. However, plant extracts can harbor a mixture of bioactive molecules that promote divergent phenotypes and potentially have confounding effects in bioassays. Even with further purification and identification, target deconvolution can be challenging. Corynoline and acetylcorynoline, are phytochemicals that were previously isolated through a screen for compounds able to induce mitotic arrest and polyploidy in oncogene expressing retinal pigment epithelial (RPE) cells. Here, we shed light on the mechanism by which these phytochemicals can attack human cancer cells. Mitotic arrest was coincident to the induction of centrosome amplification and declustering, causing multi-polar spindle formation. Corynoline was demonstrated to have true centrosome declustering activity in a model where A549 cells were chemically induced to have more than a regular complement of centrosomes. Corynoline could inhibit the centrosome clustering required for pseudo-bipolar spindle formation in these cells. The activity of AURKB, but not AURKA or polo-like kinase 4, was diminished by corynoline. It only partially inhibited AURKB, so it may be a partial antagonist or corynoline may work upstream on an unknown regulator of AURKB activity or localization. Nonetheless, corynoline and acetylcorynoline inhibited the viability of a variety of human cancer derived cell lines. These phytochemicals could serve as prototypes for a next-generation analog with improved potency, selectivity or in vivo bioavailability. Such an analog could be useful as a non-toxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Intrinsic bioactivity of black phosphorus nanomaterials on mitotic centrosome destabilization through suppression of PLK1 kinase.

Although nanomaterials have shown promising biomedical application potential, incomplete understanding of their molecular interactions with biological systems prevents their inclusion into mainstream clinical applications. Here we show that black phosphorus (BP) nanomaterials directly affect the cell cycle's centrosome machinery. BP destabilizes mitotic centrosomes by attenuating the cohesion of pericentriolar material and consequently leads to centrosome fragmentation within mitosis. As a result, BP-treated cells exhibit multipolar spindles and mitotic delay, and ultimately undergo apoptosis. Mechanistically, BP compromises centrosome integrity by deactivating the centrosome kinase polo-like kinase 1 (PLK1). BP directly binds to PLK1, inducing its aggregation, decreasing its cytosolic mobility and eventually restricting its recruitment to centrosomes for activation. With this mechanism, BP nanomaterials show great anticancer potential in tumour xenografted mice. Together, our study reveals a molecular mechanism for the tumoricidal properties of BP and proposes a direction for biomedical application of nanomaterials by exploring their intrinsic bioactivities.

Extract of bulbus of Fritillaria cirrhosa induces spindle multipolarity in human-derived colonic epithelial NCM460 cells through promoting centrosome fragmentation.

Bulbus of Fritillaria cirrhosa D. Don (BFC), an outstanding antitussive and expectorant herbal drug used in China and many other countries, has potential but less understood genotoxicity. Previously, we have reported that aqueous extract of BFC compromised the spindle assembly checkpoint and cytokinesis in NCM460 cells. Here, we found that one remarkable observation in BFC-treated NCM460 cells was multipolar mitosis, a trait classically compromises the fidelity of chromosome segregation. More detailed investigation revealed that BFC-induced spindle multipolarity in metaphases and ana-telophases in a dose- and time-dependent manner, suggesting BFC-induced multipolar spindle conformation was not transient. The frequency of multipolar metaphase correlated well to that of multipolar ana-telophases, indicating that BFC-induced multipolar metaphases often persisted through anaphase. Unexpectedly, BFC blocked the proliferation of binucleated cells, suggesting spindle multipolarity was not downstream of BFC-induced cytokinesis failure. Exposure of BFC to early mitotic cells, rather than S/G2 cells, contributed greatly to spindle multipolarity, indicating BFC might disrupt centrosome integrity rather than induce centrosome overduplication. The immunofluorescence results showed that the centrosomes were severely fragmented by a short-term treatment of BFC and the extent of centrosome fragmentation in early mitotic cells was larger than this in S/G2 cells. Consistently, several genes (e.g. p53, Rb centrin-2, Plk-4, Plk-1 and Aurora-A) involved in regulating centrosome integrity were significantly deregulated by BFC. Together, our results suggest that BFC causes multipolar spindles primarily by inducing centrosome fragmentation. Coupling these results to our previous observations, we recommend the risk/benefit ratio should be considered in the practical use of BFC.

Integration of RNAi and Small Molecule Screens to Identify Targets for Drug Development.

Cellular models for siRNA and small molecule high-throughput screening have been widely used in the last decade to identify targets for drug discovery. As an example, we present a twofold readout approach based on cell viability and multipolar phenotype. To maximize the discovery of potential targets and at the same time reduce the number of false positives in our dataset, we have combined focused and rationally designed custom siRNA libraries with small molecule inhibitor libraries. Here we describe a cellular model for centrosome amplification as an example of how to design and perform a multiple readout/multiple screening strategy.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

The Noscapine Chronicle: A Pharmaco-Historic Biography of the Opiate Alkaloid Family and its Clinical Applications.

Given its manifold potential therapeutic applications and amenability to modification, noscapine is a veritable "Renaissance drug" worthy of commemoration. Perhaps the only facet of noscapine's profile more astounding than its versatility is its virtual lack of side effects and addictive properties, which distinguishes it from other denizens of Papaver somniferum. This review intimately chronicles the rich intellectual and pharmacological history behind the noscapine family of compounds, the length of whose arms was revealed over decades of patient scholarship and experimentation. We discuss the intriguing story of this family of nontoxic alkaloids, from noscapine's purification from opium at the turn of the 19th century in Paris to the recent torrent of rationally designed analogs with tremendous anticancer potential. In between, noscapine's unique pharmacology; impact on cellular signaling pathways, the mitotic spindle, and centrosome clustering; use as an antimalarial drug and cough suppressant; and exceptional potential as a treatment for polycystic ovarian syndrome, strokes, and diverse malignancies are catalogued. Seminal experiments involving some of its more promising analogs, such as amino-noscapine, 9-nitronoscapine, 9-bromonoscapine, and reduced bromonoscapine, are also detailed. Finally, the bright future of these oftentimes even more exceptional derivatives is described, rounding out a portrait of a truly remarkable family of compounds.

Genistein, isoflavonoids in soybeans, prevents the formation of excess radiation-induced centrosomes via p21 up-regulation.

The centrosome is a cytoplasmic organelle which duplicates once during each cell cycle, and the presence of excess centrosomes promote chromosome instability through chromosome missegregation following cytokinesis. Ionizing radiation (IR) can induce extra centrosomes by permitting the continuation of CDK2/Cyclin-A/E-mediated centrosome duplication when cells are arrested in the cell cycle after irradiation. The work described here shows that, in addition to IR, extra centrosomes were induced in human U2OS and mouse NIH3T3 cells after treatment with agents which include DNA adduct-forming chemicals: benzopyrene (BP), 4-nitroquinoline 1-oxide (4NQO), a DNA cross linker: cis-diamminedichloro-platinum (cisplatin), topoisomerase inhibitors: camptothecin, etoposide, genistein, and ultra-violet light (UV). These agents were divided into two categories with respect to the regulation of p21, which is an inhibitor of CDK2/Cyclin-A/E: specifically, p21 was up-regulated by an IR exposure and treatment with topoisomerase inhibitors. However, UV, BP, 4NQO and cisplatin down-regulated p21 below basal levels. When cells were irradiated with IR in combination with all of these agents, except genistein, enhanced induction of extra centrosomes was observed, regardless of the nature of p21 expression. Genistein significantly suppressed the frequency of IR-induced extra centrosomes in a dose-dependent manner, and 20µg/ml of genistein reduced this frequency to 66%. Consistent with this, genistein substantially up-regulated p21 expression over the induction caused by IR alone, while other agents down-regulated or marginally affected this. This suggests the inhibitory effect of genistein on the induction of extra centrosomes occurs through the inactivation of CDK2/Cyclin-A/E via p21 up-regulation. This hypothesis is supported by the observation that p21 knockdown with siRNA reduced the activity of CDK2/Cyclin-A/E and restored the enhanced effect of a combined treatment with genistein and IR. These results demonstrate the preventive effect of genistein and a crucial role for p21 in IR-induced excess centrosomes.

Protoflavonoids from ferns impair centrosomal integrity of tumor cells.

Six protoflavonoids, including two new compounds, were isolated during a large scale screening of fern extracts for original interaction with mitosis. The new compounds isolated from PHEGOPTERIS decursive-pinnata and EQUISETUM fluviatile were 2',3'-dihydroprotogenkwanone (1) and 2',3'-dihydro-2'-hydroxyprotoapigenone (2). Known compounds were: protoapigenone, protogenkwanone, protoapigenin, and 4'- O- ß-D-glucopyranosyl protoapigenin. They showed a cytotoxic activity against HeLa cells at a micromolar level. IC50 values were 2 µM for compound 1 > 10 µM for compound 2, and respectively 2.4, 0.6, > 10 µM for the known compounds. Their cytotoxic effects were associated with phenotypic changes never observed before and characterized by the loss of centrosomal γ-tubulin labelling in both mitotic and interphasic cells.

Pharmacological screening of bryophyte extracts that inhibit growth and induce abnormal phenotypes in human HeLa cancer cells.

Antitumor activities of substances from natural sources apart from vascular plants and micro-organisms have been poorly investigated. Here we report on a pharmacological screening of a bryophyte extract library using a phenotypic cell-based assay revealing microtubules, centrosomes and DNA. Among the 219 moss extracts tested, we identified 41 extracts acting on cell division with various combinations of significant effects on interphasic and mitotic cells. Seven extracts were further studied using a cell viability assay, cell cycle analysis and the phenotypic assay. Three distinct pharmacological patterns were identified including two unusual phenotypes.

The small organic compound HMN-176 delays satisfaction of the spindle assembly checkpoint by inhibiting centrosome-dependent microtubule nucleation.

HMN-176 is a potential new cancer therapeutic known to retard the proliferation of tumor cell lines. Here, we show that this compound inhibits meiotic spindle assembly in surf clam oocytes and delays satisfaction of the spindle assembly checkpoint in human somatic cells by inducing the formation of short and/or multipolar spindles. HMN-176 does not affect centrosome assembly, nuclear envelope breakdown, or other aspects of meiotic or mitotic progression, nor does it affect the kinetics of Spisula or mammalian microtubule (MT) assembly in vitro. Notably, HMN-176 inhibits the formation of centrosome-nucleated MTs (i.e., asters) in Spisula oocytes and oocyte extracts, as well as from isolated Spisula or mammalian centrosomes in vitro. Together, these results reveal that HMN-176 is a first-in-class anticentrosome drug that inhibits proliferation, at least in part, by disrupting centrosome-mediated MT assembly during mitosis.

High-content screening and profiling of drug activity in an automated centrosome-duplication assay.

Maintenance of centrosome number is essential for cell-cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image-based centrosome-duplication assay that is accurate and robust enough for both careful cell-biology studies and high-throughput screening, and employ this assay in a series of chemical-genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform-specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16 320-member library of uncharacterized small molecules, we identified five potent centrosome-duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.

Tobacco BY-2 cell-free extracts induce the recovery of microtubule nucleating activity of inactivated mammalian centrosomes.

The structure and the molecular composition of the microtubule-organizing centers in acentriolar higher plant cells remain unknown. We developed an in vitro complementation assay where tobacco BY-2 extracts can restore the microtubule-nucleating activity of urea-inactivated mammalian centrosomes. Our results provide first evidence that soluble microtubule-nucleating factors are present in the plant cytosolic fraction. The implication for microtubule nucleation in higher plants is discussed.

Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds.

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and gamma-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the gamma-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus gamma-tubulin ring complex (gammaTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the gammaTuRC is required for microtubule nucleation at the centrosome.