Quantification of eIF2α Phosphorylation Associated with Mitotic Catastrophe by Immunofluorescence Microscopy.

Mitotic catastrophe is an oncosuppressive mechanism that drives cells toward senescence or death when an error occurs during mitosis. Eukaryotic cells have developed adaptive signaling pathways to cope with stress. The phosphorylation on serine 51 of the eukaryotic translation initiation factor (eIF2α) is a highly conserved event in stress responses, including the one that is activated upon treatment with mitotic catastrophe inducing agents, such as microtubular poisons or actin blockers. The protocol described herein details a method to quantify the phosphorylation of eIF2α by high-throughput immunofluorescence microscopy. This method is useful to capture the 'integrated stress response', which is characterized by eIF2α phosphorylation in the context of mitotic catastrophe.

Clonogenic Assays to Detect Cell Fate in Mitotic Catastrophe.

Mitotic catastrophe (MC) is a cell death modality induced by DNA damage that involves the activation of cell cycle checkpoints such as the "DNA structure checkpoint" and "spindle assembly checkpoint" (SAC) leading to aberrant mitosis. Depending on the signal, MC can drive the cell to death or to senescence. The suppression of MC favors aneuploidy. Several cancer therapies, included microtubular poisons and radiations, trigger MC. The clonogenic assay has been used to study the capacity of single cells to proliferate and to generate macroscopic colonies and to evaluate the efficacy of anticancer drugs. Nevertheless, this method cannot analyze MC events. Here, we report an improved technique based on the use of human colon cancer HCT116 stable expressing histone H2B-GFP and DsRed-centrin proteins, allowing to determine the capacity of cells to proliferate, and to determine changes in the nucleus and centrosomes.

Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumors.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

Discovery of dihydrofuranoallocolchicinoids - Highly potent antimitotic agents with low acute toxicity.

Two series of heterocyclic colchicinoids bearing ß-methylenedihydrofuran or 2H-pyran-2-one fragments were synthesized by the intramolecular Heck reaction. Methylenedihydrofuran compounds 9a and 9h were found to be the most cytotoxic among currently known colchicinoids, exhibiting outstanding antiproliferative activity on tumor cell lines in picomolar (0.01-2.1 nM) range of concentrations. Compound 9a potently and substoichiometrically inhibits microtubule formation in vitro, being an order of magnitude more active in this assay than colchicine. Derivatives 9a and 9h revealed relatively low acute toxicity in mice (LD50 ≥ 10 mg/kg i.v.). The X-Ray structure of colchicinoid 9a bound to tubulin confirmed interaction of this compound with the colchicine binding site of tubulin.

Preferential Killing of Tetraploid Colon Cancer Cells by Targeting the Mitotic Kinase PLK1.

BACKGROUND/AIMS: Chromosomal instability is a well-known factor in the progression of different types of cancer, including colorectal cancer. Chromosomal instability results in severely rearranged karyotypes and aneuploidy. Tetraploidy constitutes an intermediate phase during the polyploidy/aneuploidy cascade in oncogenesis, and tetraploid cells are particularly resistant to chemotherapy. Whether inhibition of the mitotic protein polo-like kinase 1 (PLK1) prevents the survival of tetraploid colon cancer cells is unknown. METHODS: Diploid and tetraploid cells were transfected with siPLK1 or treated with PLK1 inhibitor Bi2536 in combination with spindle poison. Cell toxicity was assessed via crystal violet staining and clonogenic assay. Flow cytometry assessment analyzed numerous cell apoptotic parameters and cell cycle phases. Synergistic activity between Bi2536 and paclitaxel, vincristine or colchicine was calculated using the CompuSyn software. RESULTS: Inhibition or abrogation of PLK1 prevented the survival of colon cancer cells, specifically tetraploid cells. The cell death induced by PLK inhibition was due to mitotic slippage, followed by the activation of the intrinsic pathway of apoptosis. We further demonstrated that co-treatment of the tetraploid colon cancer cells with a PLK1 inhibitor and the microtubule polymerisation inhibitor vincristine or colchicine, but not the microtubule depolymerisation inhibitor paclitaxel, provoked a lethal synergistic effect. CONCLUSION: PLK1 inhibition together with microtubule-targeting chemicals, serve as a potent therapeutic strategy for targeting tetraploid cancer cells.

Cytotoxicity and antimitotic activity of Rhinella schneideri and Rhinella marina venoms.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhinella schneideri and Rhinella marina are toad venoms distributed in different parts of the world, including Brazil, Columbia and amazon. Venoms extracted from different species have many clinical applications such as antimicrobial cardiotonics and treatment of cancer. Aim of the study; In this study, we aim to investigate the effect of venoms extracted from R. schneideri and R. marina on cancer cells and verify possible mechanism of action. MATERIAL AND METHOD: Cytotoxicity analyses was performed using the resazurin reduction assay, where different concentrations of venoms were tested against sensitive CCRF-CEM and P-gp overexpressing ADR/CEM5000 leukemia cells. Programmed cell death was investigated using the flow cytometric annexin V/propidium iodide apoptosis assay. Furthermore, we analyzed flow cytometric cell cycle analyses of CCRF-CEM cells. Effect on tubulin formation was tested using molecular docking and fluorescence microscopy of U2OS-GFP-α-tubulin osteosarcoma cells treated for 24 h with venoms. RESULTS: Cytotoxicity assays revealed a strong activity towards wild-type CCRF-CEM cells (IC50 values of 0.202 ±â€¯0.005 µg/ml and 0.18 ±â€¯0.007 µg/ml for R. schneideri and R. marina, respectively) and multidrug-resistant CEM/ADR5000 cells (IC50 0.403 ±â€¯0.084 µg/ml and 0.32 ±â€¯0.077 µg/ml for R. schneideri and R. marina, respectively). The venoms induced apoptosis as major mechanism of cell death. The venoms induced strong G2/M cell arrest in CCRF-CEM cells. We suggested tubulin as a major target for the venoms. In silico molecular docking of the major constituents of the venoms, i.e. bufalin, marinobufagin, telocinbufagin, hellebrigenin, showed strong binding affinities to tubulin. This result was verified in vitro. The venoms dysregulated microtubule arrangement of U2OS cells expressing GFP-labeled tubulin. Toxicity predictions by QSAR methodology highlighted the toxic features of bufadienolides. CONCLUSION: Our study demonstrated the importance of toad venoms as source of cytotoxic compounds that may serve as lead compounds for the development of novel anticancer drugs.

Chronic Suppression of Hypothalamic Cell Proliferation and Neurogenesis Induces Aging-Like Changes in Sleep-Wake Organization in Young Mice.

Aging is associated with sleep-wake disruption, dampening of circadian amplitudes, and a reduced homeostatic sleep response. Aging is also associated with a decline in hypothalamic cell proliferation. We hypothesized that the aging-related decline in cell-proliferation contributes to the dysfunction of preoptic-hypothalamic sleep-wake and circadian systems and consequent sleep-wake disruption. We determined if cytosine-ß-D-arabinofuranoside (AraC), an antimitotic agent known to suppress hypothalamic cell proliferation and neurogenesis, causes sleep-wake instability in young mice. The sleep-wake profiles were compared during baseline, during 4 weeks of artificial cerebrospinal fluid (aCSF) + 5-bromo-2'-deoxyuridine (BrdU) or AraC+BrdU infusion into the lateral ventricle, and 8 weeks after treatments. The sleep-wake architecture after AraC treatment was further compared with sleep-wake profiles in aged mice. Compared to aCSF+BrdU, 4 weeks of AraC+BrdU infusion significantly decreased (-96%) the number of BrdU+ cells around the third ventricular wall and adjacent preoptic-hypothalamic area and produced a) sleep disruption during the light phase with decreases in non-rapid eye movement (nonREM) (-9%) and REM sleep (-21%) amounts, and increased numbers of shorter (<2 min; 142 versus 98 episodes/12 h) and decreased numbers of longer (>5 min; 19 versus 26 episodes/12 h) nonREM sleep episodes; and b) wake disruption during the dark phase, with increased numbers of shorter (138 versus 91 episodes/12 h) and decreased numbers of longer active waking (17 versus 24 episodes/12 h) episodes. AraC-treated mice also exhibited lower delta activity within nonREM recovery sleep. The sleep-wake architecture of AraC-treated mice was similar to that observed in aged mice. These findings are consistent with a hypothesis that a decrease in hypothalamic cell proliferation/neurogenesis is detrimental to sleep-wake and circadian systems and may underlie sleep-wake disturbance in aging.

Rational design and synthesis of 2-anilinopyridinyl-benzothiazole Schiff bases as antimitotic agents.

Based on our previous results and literature precedence, a series of 2-anilinopyridinyl-benzothiazole Schiff bases were rationally designed by performing molecular modeling experiments on some selected molecules. The binding energies of the docked molecules were better than the E7010, and the Schiff base with trimethoxy group on benzothiazole moiety, 4y was the best. This was followed by the synthesis of a series of the designed molecules by a convenient synthetic route and evaluation of their anticancer potential. Most of the compounds have shown significant growth inhibition against the tested cell lines and the compound 4y exhibited good antiproliferative activity with a GI50 value of 3.8µM specifically against the cell line DU145. In agreement with the docking results, 4y exerted cytotoxicity by the disruption of the microtubule dynamics by inhibiting tubulin polymerization via effective binding into colchicine domain, comparable to E7010. Detailed binding modes of 4y with colchicine binding site of tubulin were studied by molecular docking. Furthermore, 4y induced apoptosis as evidenced by biological studies like mitochondrial membrane potential, caspase-3, and Annexin V-FITC assays.

Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death.

Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

Neurobehavioral and cytotoxic effects of vanadium during oligodendrocyte maturation: a protective role for erythropoietin.

Vanadium exposure has been known to lead to lipid peroxidation, demyelination and oligodendrocytes depletion. We investigated behaviour and glial reactions in juvenile mice after early neonatal exposure to vanadium, and examined the direct effects of vanadium in oligodendrocyte progenitor cultures from embryonic mice. Neonatal pups exposed to vanadium via lactation for 15 and 22 days all had lower body weights. Behavioural tests showed in most instances a reduction in locomotor activity and negative geotaxis. Brain analyses revealed astrocytic activation and demyelination in the vanadium exposed groups compared to the controls. In cell culture, exposure of oligodendrocytes to 300 µM sodium metavanadate significantly increased cell death. Expression of the oligodendrocyte specific proteins, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and oligodendrocyte specific protein (OSP/Claudin) were reduced upon vanadium treatment while simultaneous administration of erythropoietin (EPO; 4-12 U/ml) counteracted vanadium-toxicity. The data suggest that oligodendrocyte damage may explain the increased vulnerability of the juvenile brain to vanadium and support a potential for erythropoietin as a protective agent against vanadium-toxicity during perinatal brain development and maturation.

Evaluation of 2',4'-dihydroxy-3,4,5-trimethoxychalcone as antimitotic agent that induces mitotic catastrophe in MCF-7 breast cancer cells.

We previously reported the synthesis and the anti-proliferative action of 2',4'-dihydroxy-3,4,5-trimethoxychalcone. Here we reported its mechanism of action on MCF-7 cells. The compound induced aberrant spindles, and arrested cells at metaphase/anaphase boundary with accumulation of checkpoint proteins Mad2, Bub1 and BubR1. Live cell imaging revealed that the compound sustained a prolonged mitotic arrest, followed by massive cell death. The results indicate that 2',4'-dihydroxy-3,4,5-trimethoxychalcone exerts its anti-proliferative activity by affecting microtubules and causing mitotic catastrophe, and thus has the potential for antitumor activity.

Concise synthesis and biological evaluation of 2-Aroyl-5-amino benzo[b]thiophene derivatives as a novel class of potent antimitotic agents.

The biological importance of microtubules make them an interesting target for the synthesis of antitumor agents. The 2-(3',4',5'-trimethoxybenzoyl)-5-aminobenzo[b]thiophene moiety was identified as a novel scaffold for the preparation of potent inhibitors of microtubule polymerization acting through the colchicine site of tubulin. The position of the methoxy group on the benzo[b]thiophene was important for maximal antiproliferative activity. Structure-activity relationship analysis established that the best activities were obtained with amino and methoxy groups placed at the C-5 and C-7 positions, respectively. Compounds 3c-e showed more potent inhibition of tubulin polymerization than combretastatin A-4 and strong binding to the colchicine site. These compounds also demonstrated substantial antiproliferative activity, with IC50 values ranging from 2.6 to 18 nM in a variety of cancer cell lines. Importantly, compound 3c (50 mg/kg), significantly inhibited the growth of the human osteosarcoma MNNG/HOS xenograft in nude mice.

Synthesis and evaluation of biphenyl compounds as kinesin spindle protein inhibitors.

Kinesin spindle protein (KSP), an ATP-dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)) assays, combined with fluorescence-assisted cell sorting (FACS) and Western blot studies analyzing cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7, exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.

Synthesis and biological evaluation of novel heterocyclic derivatives of combretastatin A-4.

A novel series of combretastatin A-4 heterocyclic analogues was prepared by replacement of the B ring with indole, benzofurane or benzothiophene, attached at the C2 position. These compounds were evaluated for their abilities to inhibit tubulin assembly: derivative cis3b, having a benzothiophene, showed an activity similar to those of colchicine or deoxypodophyllotoxine. The antiproliferative and antimitotic properties of cis3b against keratinocyte cancer cell lines were also evaluated and the intracellular organization of microtubules in the cells after treatment with both stereoisomers of 3b was also determined, using confocal microscopy.

High-throughput-screening-based identification and structure-activity relationship characterization defined (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole as a highly antimycotic agent nontoxic to cell lines.

Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.

Docking, synthesis, and in vitro evaluation of antimitotic estrone analogs.

In the present study, Autodock 4.0 was employed to discover potential carbonic anhydrase IX inhibitors that are able to interfere with microtubule dynamics by binding to the Colchicine binding site of tubulin. Modifications at position 2' of estrone were made to include moieties that are known to improve the antimitotic activity of estradiol analogs. 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-3-ol-17-one estronem (C9) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C12) were synthesized and tested in vitro. Growth studies were conducted utilizing spectrophotometrical analysis with crystal violet as DNA stain. Compounds C9 and C12 were cytotoxic in MCF-7 and MDA-MB-231 tumorigenic and metastatic breast cancer cells, SNO non-keratinizing squamous epithelium cancer cells and HeLa cells after 48 h exposure. Compounds C9 inhibited cell proliferation to 50% of the vehicle-treated controls from 110 to 160 nm and C12 at concentrations ranging from 180 to 220 nm. Confocal microscopy revealed abnormal spindle morphology in mitotic cells. Cell cycle analysis showed an increase in the number of cells in the G(2) /M fraction after 24 h and an increase in the number of cell in the sub-G(1) fraction after 48 h, indicating that the compounds are antimitotic and able to induce apoptosis.

Synthesis, biological evaluation of 1,1-diarylethylenes as a novel class of antimitotic agents.

The cytotoxic activities of 23 new isocombretastatin A derivatives with modifications on the B-ring were investigated. Several compounds exhibited excellent antiproliferative activity at nanomolar concentrations against a panel of human cancer cell lines. Compounds isoFCA-4 (2 e), isoCA-4 (2 k) and isoNH(2)CA-4 (2 s) were the most cytotoxic, and strongly inhibited tubulin polymerization with IC(50) values of 4, 2 and 1.5 microM, respectively. These derivatives were found to be 10-fold more active than phenstatin and colchicine with respect to growth inhibition but displayed similar activities as tubulin polymerization inhibitors. In addition, cell cycle arrest in the G(2)/M phase and subsequent apoptosis was observed in three cancer cell lines when treated with these compounds. The disruptive effect of 2 e, 2 k and 2 s on the vessel-like structures formed by human umbilical vein endothelial cells (HUVEC) suggest that these compounds may act as vascular disrupting agents. Both compounds 2 k and 2 s have the potential for further prodrug modification and development as vascular disrupting agents for treatment of solid tumors.

Combinatorial strategies by marine cyanobacteria: symplostatin 4, an antimitotic natural dolastatin 10/15 hybrid that synergizes with the coproduced HDAC inhibitor largazole.

Combinatorial biosynthesis meets combinatorial pharmacology, cyanobacterial style: A new antimitotic natural product with features of both dolastatins 10 and 15 was isolated from the same Floridian Symploca sp. sample that produced the histone deacetylase inhibitor largazole. Both agents in combination are more effective in inhibiting cancer cell proliferation than either agent alone.

Antimitotic and antiproliferative activities of chalcones: forward structure-activity relationship.

A series of 59 chalcones was prepared and evaluated for the antimitotic effect against K562 leukemia cells. The most active chalcones were evaluated for their antiproliferative activity against a panel of 11 human and murine cell cancer lines. We found that three chalcones were of great interest as potential antimitotic drugs. In vivo safety studies conducted on one of the most active chalcones revealed that the compound was safe, allowing further in vivo antitumor evaluation.

Effects of anthocyanin-rich extract from red cabbage leaves on meristematic cells of Allium cepa L. roots treated with heavy metals.

The incubation of Allium cepa L. roots in Pb(NO3)2, Cd(NO3)(2)x4H2O or Cr(NO3)(3)x9H2O solution at the concentration of 100 microM lowered the mitotic index (MI) value in meristem by 58%, 39%, 48%, respectively. The proportion of mitotic phases (mainly prophases and telophases) in MI value was also changed. Moreover, mitotic disturbances such as: c-metaphases, sticky and lagging chromosomes, chromosome bridges, binucleate cells, micronuclei, "budding" nuclei and nucleoli partly outside nuclei were induced in the presence of the tested heavy metals, most frequently after Pb treatment. Pre-incubation in the ATH-rich extract from red cabbage leaves caused 2.5%, 1.8% or 1.6% increase in MI value as compared to the meristematic cells of A. cepa L. roots treated only with Cd, Pb, or Cr, respectively. Additionally, the ATH-rich extract was responsible for changing phase index values towards the control level in the material incubated in Pb or Cd. Moreover, the total number of mitotic abnormalities induced by the tested metals was reduced due to the preincubation in the ATH-rich extract, most effectively in the roots treated with Cd (by 2/3) while by half in Cr presence. These data suggest a protective action of the ATH-rich extract from red cabbage leaves against heavy metal toxicity.