CHIR99021, trough GSK-3ß Targeting, Reduces Epithelioid Sarcoma Cell Proliferation by Activating Mitotic Catastrophe and Autophagy.

Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3ß, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.

Anti-angiogenic effects of crenolanib are mediated by mitotic modulation independently of PDGFR expression.

BACKGROUND: Crenolanib is a tyrosine kinase inhibitor targeting PDGFR-α, PDGFR-ß and Fms related tyrosine kinase-3 (FLT3) that is currently evaluated in several clinical trials. Although platelet-derived growth factor receptor (PDGFR) signalling pathway is believed to play an important role in angiogenesis and maintenance of functional vasculature, we here demonstrate a direct angiostatic activity of crenolanib independently of PDGFR signalling. METHODS: The activity of crenolanib on cell viability, migration, sprouting, apoptosis and mitosis was assessed in endothelial cells, tumour cells and fibroblasts. Alterations in cell morphology were determined by immunofluorescence experiments. Flow-cytometry analysis and mRNA expression profiles were used to investigate cell differentiation. In vivo efficacy was investigated in human ovarian carcinoma implanted on the chicken chorioallantoic membrane (CAM). RESULTS: Crenolanib was found to inhibit endothelial cell viability, migration and sprout length, and induced apoptosis independently of PDGFR expression. Treated cells  showed altered actin arrangement and nuclear aberrations. Mitosis was affected at several levels including mitosis entry and centrosome clustering. Crenolanib suppressed human ovarian carcinoma tumour growth and angiogenesis in the CAM model. CONCLUSIONS: The PDGFR/FLT3 inhibitor crenolanib targets angiogenesis and inhibits tumour growth in vivo unrelated to PDGFR expression. Based on our findings, we suggest a broad mechanism of action of crenolanib.

Non-mitotic effect of albendazole triggers apoptosis of human leukemia cells via SIRT3/ROS/p38 MAPK/TTP axis-mediated TNF-α upregulation.

Albendazole (ABZ) is a microtubule-targeting anthelmintic that acts against a variety of human cancer cells, but the dependence of its cytotoxicity on non-mitotic effect remains elusive. Thus, we aimed to explore the mechanistic pathway underlying the cytotoxicity of ABZ in human leukemia U937 cells. ABZ-induced apoptosis of U937 cells was characterized by mitochondrial ROS generation, p38 MAPK activation, TNF-α upregulation and activation of the death receptor-mediated pathway. Meanwhile, ABZ induced tubulin depolymerization and G2/M cell cycle arrest. ABZ-induced SIRT3 degradation elicited ROS-mediated p38 MAPK activation, leading to pyruvate kinase M2-mediated tristetraprolin (TTP) degradation. Inhibition of TTP-mediated TNF-α mRNA decay elicited TNF-α upregulation in ABZ-treated cells. Either the overexpression of SIRT3 or abolishment of ROS/p38 MAPK activation suppressed TNF-α upregulation and rescued the viability of ABZ-treated cells. In contrast to the inhibition of ROS/p38 MAPK pathway, SIRT3 overexpression attenuated tubulin depolymerization and G2/M arrest in ABZ-treated cells. Treatment with a SIRT3 inhibitor induced TNF-α upregulation and cell death without the induction of G2/M arrest in U937 cells. Taken together, our data indicate that ABZ-induced SIRT3 downregulation promotes its microtubule-destabilizing effect, and that the non-mitotic effect of ABZ largely triggers apoptosis of U937 cells via SIRT3/ROS/p38 MAPK/TTP axis-mediated TNF-α upregulation. Notably, the same pathway is involved in the ABZ-induced death of HL-60 cells.

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets.

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents.

Establishment of a long-term spiral ganglion neuron culture with reduced glial cell number: Effects of AraC on cell composition and neurons.

BACKGROUND: Sensorineural deafness is mainly caused by damage to hair cells and degeneration of the spiral ganglion neurons (SGN). Cochlear implants can functionally replace lost hair cells and stimulate the SGN electrically. The benefit from cochlear implantation depends on the number and excitability of these neurons. To identify potential therapies for SGN protection, in vitro tests are carried out on spiral ganglion cells (SGC). NEW METHOD: A glial cell-reduced and neuron-enhanced culture of neonatal rat SGC under mitotic inhibition (cytarabine (AraC)) for up to seven days is presented. Serum containing and neurotrophin-enriched cultures with and without AraC-addition were analyzed after 4 and 7 days. RESULTS: The total number of cells was significantly reduced, while the proportion of neurons was greatly increased by AraC-treatment. Cell type-specific labeling demonstrated that nearly all fibroblasts and most of the glial cells were removed. Neither the neuronal survival, nor the neurite outgrowth or soma diameter were negatively affected. Additionally neurites remain partly free of surrounding non-neuronal cells. COMPARISON WITH EXISTING METHOD: Recent culture conditions allow only for short-term cultivation of neonatal SGC and lack information on the influence of non-neuronal cells on SGN and of direct contact of neurites with test-materials. CONCLUSIONS: AraC-addition reduces the number of non-neuronal cells and increases the ratio of SGN in culture, without negative impact on neuronal viability. This treatment allows longer-term cultivation of SGC and provides deeper insight into SGN-glial cell interaction and the attachment of neurites on test-material surfaces.

Periodic mechanical stress activates EGFR-dependent Rac1 mitogenic signals in rat nucleus pulpous cells via ERK1/2.

The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferation (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic mechanical stress promotes nucleus pulpous cell proliferation in part through the EGFR-Rac1-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

Cell cycle synchronization and BrdU incorporation as a tool to study the possible selective elimination of ErbB1 gene in the micronuclei in A549 cells.

Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1 could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells.

Increased adult hippocampal neurogenesis is not necessary for wheel running to abolish conditioned place preference for cocaine in mice.

Recent evidence suggests that wheel running can abolish conditioned place preference (CPP) for cocaine in mice. Running significantly increases the number of new neurons in the hippocampus, and new neurons have been hypothesised to enhance plasticity and behavioral flexibility. Therefore, we tested the hypothesis that increased neurogenesis was necessary for exercise to abolish cocaine CPP. Male nestin-thymidine kinase transgenic mice were conditioned with cocaine, and then housed with or without running wheels for 32 days. Half of the mice were fed chow containing valganciclovir to induce apoptosis in newly divided neurons, and the other half were fed standard chow. For the first 10 days, mice received daily injections of bromodeoxyuridine (BrdU) to label dividing cells. On the last 4 days, mice were tested for CPP, and then euthanized for measurement of adult hippocampal neurogenesis by counting the number of BrdU-positive neurons in the dentate gyrus. Levels of running were similar in mice fed valganciclovir-containing chow and normal chow. Valganciclovir significantly reduced the numbers of neurons (BrdU-positive/NeuN-positive) in the dentate gyrus of both sedentary mice and runner mice. Valganciclovir-fed runner mice showed similar levels of neurogenesis as sedentary, normal-fed controls. However, valganciclovir-fed runner mice showed the same abolishment of CPP as runner mice with intact neurogenesis. The results demonstrate that elevated adult hippocampal neurogenesis resulting from running is not necessary for running to abolish cocaine CPP in mice.

Dickkopf-3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells.

Dickkopf-3 (Dkk-3) and Dkkl-1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera-2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk-3 but not of Dkkl-1. Ectopic expression of both Dkk-3 and Dkkl-1 induced apoptosis in NT2 cells. Gene silencing of Dkk-3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk-3-silenced cells, this morphological response was not observed in Dkk-3-silenced cells. These findings suggest that Dkk-3 plays a role in the regulation of cell interactions during RA-induced neuronal differentiation.

La apoptosis: un tema de interés para el laboratorio clínico / Apoptosis: a topic of interest for the clinical laboratory

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Surface properties of quantum dots define their cellular endocytic routes, mitogenic stimulation and suppression of cell migration.

The practical use of quantum dots (QD) as diagnostic, visualizing and therapeutic nano-agents depends on the understanding of fundamental mechanisms of their entrance and trafficking within cells. Here we show that CdSe/ZnS carboxylic-coated QD (COOH-QD) enter fibroblast cells via lipid raft/caveolin-mediated endocytosis, pass early sorting endosomes and accumulate in the multivesicular bodies, but not in the lysosomes. Later phase of their endocytosis leads to the generation of lipid raft/caveolin-dependent endocytosis inhibition that prevents intracellular uptake of new COOH-QD, but not the QD coupled with platelet-derived growth factor BB (PDGF-QD). PDGF-QD enter fibroblasts by the clathrin-mediated endocytosis and undergo similar intracellular trafficking as COOH-QD, yet they accumulate in lysosomes in contrast to COOH-QD. The PDGF-QD activate PDGF receptor-beta and are mitogenic, however, COOH-QD suppress cell migration and chemotaxis. Data show that surface coating of QD with the biologically active proteins redirects their intracellular traffic routes and changes their biological activity.

HSP70 colocalizes with PLK1 at the centrosome and disturbs spindle dynamics in cells arrested in mitosis by arsenic trioxide.

Heat shock protein 70 (HSP70) has been shown to be a substrate of Polo-like kinase 1 (PLK1), and it prevents cells arrested in mitosis by arsenic trioxide (ATO) from dying. Here, we report that HSP70 participates in ATO-induced spindle elongation, which interferes with mitosis progression. Our results demonstrate that HSP70 and PLK1 colocalize at the centrosome in ATO-arrested mitotic cells. HSP70 located at the centrosome was found to be phosphorylated by PLK1 at Ser6³¹ and Ser6³³. Moreover, unlike wild-type HSP70 (HSP70(wt)) and its phosphomimetic mutant (HSP70(SS631,633DD)), a phosphorylation-resistant mutant of HSP70 (HSP70(SS631,633AA)) failed to localize at the centrosome. ATO-induced spindle elongation was abolished in cells overexpressing HSP70(SS631,633AA). Conversely, mitotic spindles in cells ectopically expressing HSP70(SS631,633DD) were more resistant to nocodazole-induced depolymerization than in those expressing HSP70(wt) or HSP70(SS631,633AA). In addition, inhibition of PLK1 significantly reduced HSP70 phosphorylation and induced early onset of apoptosis in ATO-arrested mitotic cells. Taken together, our results indicate that PLK1-mediated phosphorylation and centrosomal localization of HSP70 may interfere with spindle dynamics and prevent apoptosis of ATO-arrested mitotic cells.

In vitro assays predictive of telomerase inhibitory effect of G-quadruplex ligands in cell nuclei.

G-quadruplex-binding and telomerase-inhibiting capacities of G-quadruplex ligands were examined under a cell nuclei-mimicking condition including excess double-stranded DNA (λ DNA) and molecular crowding cosolute (PEG 200). Under the cell nuclei-mimicking condition, a cationic porphyrin (TMPyP4) did not bind to the G-quadruplex despite the high affinity (Ka = 3.6 × 10(6) M(-1)) under a diluted condition without λ DNA and PEG 200. Correspondingly, TMPyP4 inhibited telomerase activity under the diluted condition (IC50 = 1.6 µM) but not under the cell nuclei-mimicking condition. In contrast, the Ka and IC50 values of an anionic copper phthalocyanine (Cu-APC) under the diluted (2.8 × 10(4) M(-1) and 0.86 µM) and the cell nuclei-mimicking (2.8 × 10(4) M(-1) and 2.1 µM) conditions were similar. In accordance with these results, 10 µM TMPyP4 did not affect the proliferation of HeLa cells, while Cu-APC efficiently inhibited the proliferation (IC50 = 1.4 µM). These results show that the cell nuclei-mimicking condition is effective to predict capacities of G-quadruplex ligands in the cell. In addition, the antiproliferative effect of Cu-APC on normal cells was smaller than that on HeLa cells, indicating that the cell nuclei-mimicking condition is also useful to predict side effects of ligands.

Regulation of mitosis by the NIMA kinase involves TINA and its newly discovered partner, An-WDR8, at spindle pole bodies.

The NIMA kinase is required for mitotic nuclear pore complex disassembly and potentially controls other mitotic-specific events. To investigate this possibility, we imaged NIMA-green fluorescent protein (GFP) using four-dimensional spinning disk confocal microscopy. At mitosis NIMA-GFP locates to spindle pole bodies (SPBs), which contain Cdk1/cyclin B, followed by Aurora, TINA, and the BimC kinesin. NIMA promotes NPC disassembly in a spatially regulated manner starting near SPBs. NIMA is also required for TINA, a NIMA-interacting protein, to locate to SPBs during initiation of mitosis, and TINA is then necessary for locating NIMA back to SPBs during mitotic progression. To help expand the NIMA-TINA pathway, we affinity purified TINA and found it to uniquely copurify with An-WDR8, a WD40-domain protein conserved from humans to plants. Like TINA, An-WDR8 accumulates within nuclei during G2 but disperses from nuclei before locating to mitotic SPBs. Without An-WDR8, TINA levels are greatly reduced, whereas TINA is necessary for mitotic targeting of An-WDR8. Finally, we show that TINA is required to anchor mitotic microtubules to SPBs and, in combination with An-WDR8, for successful mitosis. The findings provide new insights into SPB targeting and indicate that the mitotic microtubule-anchoring system at SPBs involves WDR8 in complex with TINA.

Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of adipocyte differentiation in 3T3-L1 cells.

Adipocytes are the key player in adipose tissue inflammation and subsequent systemic insulin resistance and its development involves complex process of proliferation and differentiation of preadipocytes. Fistein, a polyphenol flavonoid, is known to exert anti-inflammatory, anti-carcinogenic and anti-diabetic effects. In this study, we aimed to investigate the effect of fisetin on adipocyte proliferation and differentiation in 3T3-L1 preadipocyte cell line and its mechanism of action. We found that fisetin inhibits adipocyte differentiation in a concentration dependent manner, which were evidenced by Oil Red O staining and the protein expression of mature adipocyte marker genes fatty acid synthase and peroxisome proliferator-activated receptor γ. Moreover, the proliferation of preadipocytes was also markedly suppressed by treatment of fisetin for 24 and 48 h in the differentiation medium. We also found that fisetin inhibition of adipocyte differentiation was largely due to the effect on mitotic clonal expansion. Fisetin suppression of preadipocyte proliferation at early stage of differentiation was accompanied by the changes of expression of a series of cell cycle regulatory proteins. Altogether, our results suggest that the inhibition of adipocyte differentiation by fisetin may be at least in part mediated by cell cycle arrest during adipogenesis.

A natural product inspired tetrahydropyran collection yields mitosis modulators that synergistically target CSE1L and tubulin.

A Prins cyclization between a polymer-bound aldehyde and a homoallylic alcohol served as the key step in the synthesis of tetrahydropyran derivatives. A phenotypic screen led to the identification of compounds that inhibit mitosis (as seen by the accumulation of round cells with condensed DNA and membrane blebs). These compounds were termed tubulexins as they target the CSE1L protein and the vinca alkaloid binding site of tubulin.

Identification of novel mitosis regulators through data mining with human centromere/kinetochore proteins as group queries.

BACKGROUND: Proteins functioning in the same biological pathway tend to be transcriptionally co-regulated or form protein-protein interactions (PPI). Multiple spatially and temporally regulated events are coordinated during mitosis to achieve faithful chromosome segregation. The molecular players participating in mitosis regulation are still being unravelled experimentally or using in silico methods. RESULTS: An extensive literature review has led to a compilation of 196 human centromere/kinetochore proteins, all with experimental evidence supporting the subcellular localization. Sixty-four were designated as "core" centromere/kinetochore components based on peak expression and/or well-characterized functions during mitosis. By interrogating and integrating online resources, we have mined for genes/proteins that display transcriptional co-expression or PPI with the core centromere/kinetochore components. Top-ranked hubs in either co-expression or PPI network are not only enriched with known mitosis regulators, but also contain candidates whose mitotic functions are not yet established. Experimental validation found that KIAA1377 is a novel centrosomal protein that also associates with microtubules and midbody; while TRIP13 is a novel kinetochore protein and directly interacts with mitotic checkpoint silencing protein p31(comet). CONCLUSIONS: Transcriptional co-expression and PPI network analyses with known human centromere/kinetochore proteins as a query group help identify novel potential mitosis regulators.

Actualización del manejo de extravasaciones de agentes citostáticos / Update in the management of extravasations of cytocytostatic agent

Objetivo Presentar las novedades descritas hasta la actualidad del manejo específico de las extravasaciones de los agentes citostáticos una vez extravasados. MétodoSe realizó una búsqueda en PubMed, Medline e IDIS-Iowa para identificar los trabajos redactados en inglés y/o español que describieron novedades de las medidas específicas para el manejo de sus extravasaciones. Se revisaron también las referencias incluidas en estos trabajos, así como fuentes terciarias recientes relacionadas con oncología o citostáticos. La búsqueda abarcó el periodo comprendido entre 1997 y 2010.ResultadosÚnicamente 22 agentes citostáticos cuentan con algún tipo de medida específica como tratamiento de su extravasación. Se presentan esta medidas en función del citostático de interés, clasificado según su grupo farmacológico. Conclusiones A pesar de que hasta ahora no hay un consenso general en el tratamiento específico de los agentes citostáticos al extravasarse, esta revisión recopila y esquematiza la información publicada actualmente para que pueda servir a cualquier centro sanitario nacional donde se prescriban, manipulen o administren fármacos citostáticos (AU)

Objective To present current developments in the specific management of extravasations of antineoplastic agents after the extravasation. Method We conducted a search in PubMed, Medline and IDIS-Iowa to identify papers written in English or Spanish that described new specific measures for the management of extravasations. We also reviewed the references given in these papers and recent tertiary sources related to oncology or cytostatic agents. The search covered the period between 1997 and 2010.ResultsThere are only specific measures for the treatment of extravasations of 22 cytostatic agents. These measures are presented for each cytostatic agent, according their drug group. Conclusions Although currently there is no general consensus on the specific management of antineoplastic agents after extravasation, this review outlines the information collected and published so far, so that it may be of use to any national health centre where cytostatic drugs are prescribed, handled or administered(AU)

CERT depletion predicts chemotherapy benefit and mediates cytotoxic and polyploid-specific cancer cell death through autophagy induction.

Chromosomal instability (CIN) has been implicated in multidrug resistance and the silencing of the ceramide transporter, CERT, promotes sensitization to diverse cytotoxics. An improved understanding of mechanisms governing multidrug sensitization might provide insight into pathways contributing to the death of CIN cancer cells. Using an integrative functional genomics approach, we find that CERT-specific multidrug sensitization is associated with enhanced autophagosome-lysosome flux, resulting from the expression of LAMP2 following CERT silencing in colorectal and HER2(+) breast cancer cell lines. Live cell microscopy analysis revealed that CERT depletion induces LAMP2-dependent death of polyploid cells following exit from mitosis in the presence of paclitaxel. We find that CERT is relatively over-expressed in HER2(+) breast cancer and CERT protein expression acts as an independent prognostic variable and predictor of outcome in adjuvant chemotherapy-treated patients with primary breast cancer. These data suggest that the induction of LAMP2-dependent autophagic flux through CERT targeting may provide a rational approach to enhance multidrug sensitization and potentiate the death of polyploid cells following paclitaxel exposure to limit the acquisition of CIN and intra-tumour heterogeneity.