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

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.     

Induction of robust de novo centrosome amplification, high-grade spindle multipolarity and metaphase catastrophe: a novel chemotherapeutic approach

Centrosome amplification (CA) and resultant chromosomal instability have long been associated with tumorigenesis. However, exacerbation of CA and relentless centrosome declustering engender robust spindle multipolarity (SM) during mitosis and may induce cell death. Recently, we demonstrated that a noscapinoid member, reduced bromonoscapine, (S)-3-(R)-9-bromo-5-(4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo-[4,5-g]isoquinoline (Red-Br-nos), induces reactive oxygen species (ROS)-mediated autophagy and caspase-independent death in prostate cancer PC-3 cells. Herein, we show that Red-Br-nos induces ROS-dependent DNA damage that resulted in high-grade CA and SM in PC-3 cells. Unlike doxorubicin, which causes double-stranded DNA breaks and chronic G2 arrest accompanied by ‘templated'' CA, Red-Br-nos-mediated DNA damage elicits de novo CA during a transient S/G2 stall, followed by checkpoint abrogation and mitotic entry to form aberrant mitotic figures with supernumerary spindle poles. Attenuation of multipolar phenotype in the presence of tiron, a ROS inhibitor, indicated that ROS-mediated DNA damage was partly responsible for driving CA and SM. Although a few cells (∼5%) yielded to aberrant cytokinesis following an ‘anaphase catastrophe'', most mitotically arrested cells (∼70%) succumbed to ‘metaphase catastrophe,'' which was caspase-independent. This report is the first documentation of rapid de novo centrosome formation in the presence of parent centrosome by a noscapinoid family member, which triggers death-inducing SM via a unique mechanism that distinguishes it from other ROS-inducers, conventional DNA-damaging agents, as well as other microtubule-binding drugs.     

Paclitaxel-induced aberrant mitosis and mitotic slippage efficiently lead to proliferative death irrespective of canonical apoptosis and p53

Spindle poisons elicit various cellular responses following metaphase arrest, but how they relate to long-term clonogenicity has remained unclear. We prepared several HeLa lines in which the canonical apoptosis pathway was attenuated, and compared their acute responses to paclitaxel, as well as long-term fate, with the parental line. Three-nanomolar paclitaxel induced brief metaphase arrest (<5 h) often followed by aberrant mitosis, and about 90% of the cells of each line had lost their clonogenicity after 48 h of the treatment. A combination of the same concentration of paclitaxel with the kinesin-5 inhibitor, S-trityl-L-cysteine (STLC), at 1 µM led to much longer arrest (～20 h) and predominance of subsequent line-specific responses: mitochondrial outer membrane permeabilization (MOMP) in the apoptosis-prone line, or mitotic slippage without obvious MOMP in the apoptosis-reluctant lines. In spite of this, combination with STLC did not lead to a marked difference in clonogenicity between the apoptosis-prone and -reluctant lines, and intriguingly resulted in slightly better clonogenicity than that of cells treated with 3 nM paclitaxel alone. This indicates that changes in the short-term response within 3 possible scenarios — acute MOMP, mitotic slippage or aberrant mitosis ― has only a weak impact on clonogenicity. Our results suggest that once cells have committed to slippage or aberrant mitosis they eventually undergo proliferative death irrespective of canonical apoptosis or p53 function. Consistent with this, cells with irregular DNA contents originating from mitotic slippage or aberrant mitosis were mostly eliminated from the population within several rounds of division after the drug treatment.     

The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events

Centrosomes of vertebrate cells and spindle pole bodies (SPBs) of fungi were first recognized through their ability to organize microtubules. Recent studies suggest that centrosomes and SPBs also have a function in the regulation of cell cycle progression, in particular in controlling late mitotic events. Regulators of mitotic exit and cytokinesis are associated with the SPB of budding and fission yeast. Elucidation of the molecular roles played by these regulators is helping to clarify the function of the SPB in controlling progression though mitosis.     

Over-expression of Plk4 induces centrosome amplification,loss of primary cilia and associated tissue hyperplasia in the mouse

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development.     

Mechanism,function and regulation of microtubule-dependent microtubule amplification in mitosis

Mitotic spindle mediates the segregation of chromosomes in the cell cycle and the proper function of the spindle is crucial to the high fidelity of chromosome segregation and to the stability of the genome. Nucleation of microtubules (MTs) from centrosomes and chromatin represents two well-characterized pathways essential for the assembly of a dynamic spindle in mitosis. Recently, we identified a third MT nucleation pathway, in which existing MTs in the spindle act as a template to promote the nucleation and polymerization of MTs, thereby efficiently amplifying MTs in the spindle. We will review here our current understanding on the molecular mechanism, the physiological function and the cell-cycle regulation of MT amplification.     

Inhibition of a mitotic motor protein: where, how, and conformational consequences

We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 A resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg2+ x ADP, reveals that monastrol confers inhibition by "induced-fitting" onto the protein some 12 A away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.     

In vitro effects of 2-methoxyestradiol on MCF-12A and MCF-7 cell growth, morphology and mitotic spindle formation

The influence of 2-methoxyestradiol (2ME) was investigated on cell growth, morphology and spindle formation in a tumorigenic (MCF-7) and non-tumorigenic (MCF-12A) epithelial breast cell line. Inhibition of cell growth was more pronounced in the MCF-7 cells compared to the MCF-12A cells following 2ME treatment. Dose-dependent studies (10(-5)-10(-9) M) revealed that 10(-6) M 2ME inhibited cell growth by 44% in MCF-12A cells and by 84% in MCF-7 cells (p-value < 0.05). 2ME-treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent or less prominent in MCF-12A cells. 2ME had no effect on the length of the cell cycle between S-phase and the time a mitotic peak was reached in either cell line but MCF-7 cells were blocked in mitosis with no statistically significant alterations in the phosphorylation status of Cdc25C. Nevertheless, Cdc2 activity was significantly increased in MCF-7 cells compared to MCF-12A cells (p-value < 0.05). The results indicate that 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest that may result in the induction of apoptosis. The tumorigenic MCF-7 cells were especially sensitive to 2ME treatment compared to the normal MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells.     

Effect of aluminum chloride on mitogenesis,mitosis, and cell cycle in human short-term whole blood cultures: Lower concentrations enhance mitosis

Aluminum, the third most common element in the earth's crust (second to oxygen and silicon) and recently suspected by some investigators to be implicated in Alzheimer disease etiology, has been studied in relation to its effect on mitogenesis, mitosis, and cell cycle. We have observed that 2–4 mM concentrations of AlCl₃ have decreased the number of cells that undergo mitogenesis (PHA-induced blast transformation) and mitosis in human short term whole blood cultures. We have also shown that the rate of the cell cycle was slowed down, i.e., cell cycle time was increased in the presence of AlCl₃. Also, we have demonstrated a reversible effect on aluminum-induced reduced mitotic index in long-term EBV-transformed lymphoblastoid cultures. Although safeguards such as limiting aluminum serum concentrations have been recommended to protect individuals undergoing dialysis, it should be realized that concentration accumulations of aluminum may increase over chronic exposures. Accordingly, if the number of cells stimulated by PHA is reduced in the presence of AlCl₃, there may be a reduction of immune competence, since the degree of PHA stimulation has been used as an indicator of immune response. Similar reductions in mitotic index could affect every tissue involved with cell division. Although it may not be the same for higher concentrations, from our results, we have also shown that decreased mitotic rates were reversible in long-term EBV-transformed lymphoblastoid cultures. Increased numbers of mitoses were observed in human short-term whole blood cultures that were exposed to 2 μM concentrations of aluminum chloride. The concentration is close to those found in normal human serum and within the “safeguard” range recommended for dialysis patients. A similar trend for aluminum sulfate was also observed, while preliminary results for three other aluminum species, lactate, citrate, and maltol, were also reported. Although previous reports have indicated a positive effect of aluminum on mitosis in vitro or in vivo, this is the first such report involving human material. It is clear that higher concentrations of aluminum chloride at 2.0–4.0 mM reversibly inhibit mitosis while more dilute concentrations of 1–2 μM, closer to those found in normal serum, enhance mitosis. The present results, as well as those in the literature, suggest that aluminum may be an essential element in cellular processes for optimal growth, development, and health maintenance. Future research will further test this hypothesis.     

Design,synthesis and biological evaluation of pyrimidine derivatives as novel CDK2 inhibitors that induce apoptosis and cell cycle arrest in breast cancer cells

Cyclin-dependent kinase 2 (CDK2) plays a key role in eukaryotic cell cycle progression which could facilitate the transition from G1 to S phase. The dysregulation of CDK2 is closely related to many cancers. CDK2 is utilized as one of the most studied kinase targets in oncology. In this article, 24 benzamide derivatives were designed, synthesized and investigated for the inhibition activity against CDK2. Our results revealed that the compound 25 is a potent CDK2 inhibitor exhibiting a broad spectrum anti-proliferative activity against several human breast cancer cells. Additionally, compound 25 could block cell cycle at G0 or G1 and induce significant apoptosis in MDA-MB-468 cells. These findings highlight a rationale for further development of CDK2 inhibitors to treat human breast cancer.     

Indole derivatives as multifunctional drugs: Synthesis and evaluation of antioxidant,photoprotective and antiproliferative activity of indole hydrazones

Two series of indole derivatives 4–17, 20–22 were easily prepared and assayed for their radical-scavenging ability. Arylidene-1H-indole-2-carbohydrazones showed different extent antioxidant activity in DPPH, FRAP and ORAC assays. Good antioxidant activity is related to the number and position of hydroxyl groups on the arylidene moiety as well as to the presence of methoxy or 4-(diethylamino) group. On the contrary low antioxidant activity is showed by the isomeric 1H-indol-2-yl(methylene)-benzohydrazides. Furthermore, hydrazones 4–17 showed photoprotective capacities with satisfactory in vitro SPF as compared to the commercial PBSA sunscreen filter. The indole 16 and 17, showing the best antioxidant and photoprotective profile, were included in different formulation and their topical release was evaluated. Varying the formulation composition, it was possible to optimize skin adsorption and solubility of the active indole in the formulation. The antiproliferative effect of the hydrazones 4–17 was tested on human erythroleukemia K562 and melanoma Colo-38 cells. Hydrazones 11, 16 and 17 showed growth inhibition at sub micromolar concentrations on both cell lines. These results indicate indole hydrazones as potential multifunctional molecules especially in the treatment of neoplastic diseases being the good antioxidant properties of 16 and 17 correlated to their high antiproliferative activity.     

Synthesis and characterization of new copper thiosemicarbazone complexes with an ONNS quadridentate system: cell growth inhibition, S-phase cell cycle arrest and proapoptotic activities on cisplatin-resistant neuroblastoma cells

Two new copper thiosemicarbazone complexes with an ONNS quadridentate system were synthesized and evaluated for anticancer activity on cisplatin-resistant neuroblastoma cells. Among these two copper complexes, the substituted 8-hydroxyquinoline-2-carboxaldehyde–4,4-dimethyl-3-thiosemicarbazide (CuHQDMTS) exhibited stronger cell growth inhibition activity than the unsubstituted copper 8-hydroxyquinoline-2-carboxaldehyde thiosemicarbazide complex (CuHQTS). Both CuHQTS and CuHQDMTS showed dose-dependent cell growth inhibition, cell cycle arrest and apoptosis induction activities on the SK-N-DZ neuroblastoma cells. Increased expression of p53 protein molecules was detected in the SK-N-DZ cells treated with CuHQTS. The data obtained in this study suggest that CuHQDMTS and CuHQTS hold potential as new, effective drugs for treatment of refractory neuroblastoma in children.     

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks,HR and NHEJ

Chloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto- and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci and displayed co-staining with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G₁ but increased significantly in S phase and remained at a high level in G₂, where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O⁶-chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O⁶-chloroethylguanine-triggered cross-links, which give rise to DSBs in the treatment cell cycle, and that HR, but not NHEJ, is the major route of protection against this group of anticancer drugs. Base excision repair had no significant impact on ACNU-induced cytotoxicity.     

6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐l‐rhamnopyranosylcatalpol and verbascoside: Cytotoxicity,cell cycle kinetics,apoptosis, and ROS production evaluation in tumor cells

The aim of this study was to evaluate the impact that 6‐O‐(3″, 4″‐di‐O‐trans‐cinnamoyl)‐α‐ l ‐rhamnopyranosylcatalpol (Dicinn) and verbascoside (Verb), two compounds simultaneously reported in Verbascum ovalifolium, have on tumor cell viability, apoptosis, cell cycle kinetics, and intracellular reactive oxygen species (ROS) level. At 100 µg/mL and 48 hours incubation time, Dicinn and Verb produced good cytotoxic effects in A549, HT‐29, and MCF‐7 cells. Dicinn induced cell‐cycle arrest at the G₀/G₁ phase and apoptosis, whereas Verb increased the population of subG₁ cells and cell apoptosis rates. Furthermore, the two compounds exhibited time‐dependent ROS generating effects in tumor cells (1‐24 hours). Importantly, no cytotoxic effects were induced in nontumor MCF‐10A cells by the two compounds up to 100 µg/mL. Overall, the effects exhibited by Verb in tumor cells were more potent, which can be correlated with its structural features, such as the presence of phenolic hydroxyl groups.     

A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells

In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here. The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints. We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells. IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels).