Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Sci Rep ; 11(1): 23665, 2021 12 08.
Article in English | MEDLINE | ID: mdl-34880347

ABSTRACT

We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core thienoisoquinoline scaffold with several functional groups amenable to modification. Previously we found that sub micromolar concentrations of C75 caused cytotoxicity. We also found that C75 inhibited microtubule polymerization and competed with colchicine for tubulin-binding in vitro. However, here we found that the two compounds synergized suggesting differences in their mechanism of action. Indeed, live imaging revealed that C75 causes different spindle phenotypes compared to colchicine. Spindles remained bipolar and collapsed after colchicine treatment, while C75 caused bipolar spindles to become multipolar. Importantly, microtubules rapidly disappeared after C75-treatment, but then grew back unevenly and from multiple poles. The C75 spindle phenotype is reminiscent of phenotypes caused by depletion of ch-TOG, a microtubule polymerase, suggesting that C75 blocks microtubule polymerization in metaphase cells. C75 also caused an increase in the number of spindle poles in paclitaxel-treated cells, and combining low amounts of C75 and paclitaxel caused greater regression of multicellular tumour spheroids compared to each compound on their own. These findings warrant further exploration of C75's anti-cancer potential.


Subject(s)
Isoquinolines/pharmacology , Microtubules/drug effects , Mitosis/drug effects , Spindle Apparatus/drug effects , Spindle Poles/drug effects , Cell Line, Tumor , Cells, Cultured , Colchicine/pharmacology , Humans , Isoquinolines/chemistry , Microtubules/metabolism , Thiophenes/chemistry
2.
Apoptosis ; 26(5-6): 248-252, 2021 06.
Article in English | MEDLINE | ID: mdl-33870441

ABSTRACT

Mitosis, under the control of the microtubule-based mitotic spindle, is an attractive target for anti-cancer treatments, as cancer cells undergo frequent and uncontrolled cell divisions. Microtubule targeting agents that disrupt mitosis or single molecule inhibitors of mitotic kinases or microtubule motors kill cancer cells with a high efficacy. These treatments have, nevertheless, severe disadvantages: they also target frequently dividing healthy tissues, such as the haematopoietic system, and they often lose their efficacy due to primary or acquired resistance mechanisms. An alternative target that has emerged in dividing cancer cells is their ability to "cluster" the poles of the mitotic spindle into a bipolar configuration. This mechanism is necessary for the specific survival of cancer cells that tend to form multipolar spindles due to the frequent presence of abnormal centrosome numbers or other spindle defects. Here we discuss the recent development of combinatorial treatments targeting spindle pole clustering that specifically target cancer cells bearing aberrant centrosome numbers and that have the potential to avoid resistance mechanism due their combinatorial nature.


Subject(s)
Antineoplastic Agents/therapeutic use , Cell Death/drug effects , Neoplasms/drug therapy , Spindle Poles/drug effects , Antineoplastic Agents/pharmacology , Centrosome/drug effects , Centrosome/metabolism , Drug Combinations , Drug Synergism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Humans , Mitosis/drug effects , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Spindle Poles/metabolism
3.
J Cell Biol ; 220(2)2021 02 01.
Article in English | MEDLINE | ID: mdl-33443571

ABSTRACT

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.


Subject(s)
Centrioles/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Spindle Poles/metabolism , Antigens/metabolism , Cell Cycle Proteins/metabolism , Centrioles/drug effects , HeLa Cells , Humans , Mitosis/drug effects , Models, Biological , Nerve Tissue Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Pyrimidines/pharmacology , Spindle Poles/drug effects , Sulfones/pharmacology , Polo-Like Kinase 1
4.
Sci Rep ; 10(1): 13887, 2020 08 17.
Article in English | MEDLINE | ID: mdl-32807835

ABSTRACT

Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and it inhibits the growth of cells in all kinds of organisms. We recently reported that MG inhibits nuclear division in Saccharomyces cerevisiae. However, the mechanism by which MG blocks nuclear division remains unclear. Here, we show that increase in the levels of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is crucial for the inhibitory effects of MG on nuclear division, and the deletion of PtdIns(3,5)P2-effector Atg18 alleviated the MG-mediated inhibitory effects. Previously, we reported that MG altered morphology of the vacuole to a single swelling form, where PtdIns(3,5)P2 accumulates. The changes in the vacuolar morphology were also needed by MG to exert its inhibitory effects on nuclear division. The known checkpoint machinery, including the spindle assembly checkpoint and morphological checkpoint, are not involved in the blockade of nuclear division by MG. Our results suggest that both the accumulation of Atg18 on the vacuolar membrane and alterations in vacuolar morphology are necessary for the MG-induced inhibition of nuclear division.


Subject(s)
Autophagy-Related Proteins/metabolism , Cell Membrane/metabolism , Cell Nucleus Division/drug effects , Membrane Proteins/metabolism , Pyruvaldehyde/pharmacology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Alleles , Autophagy-Related Proteins/genetics , Cell Membrane/drug effects , Membrane Proteins/genetics , Microtubules/drug effects , Microtubules/metabolism , Mutation/genetics , Phosphatidylinositol Phosphates/pharmacology , Phosphorylation/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae Proteins/genetics , Spindle Poles/drug effects , Spindle Poles/metabolism , Vacuoles/drug effects
5.
Sci Rep ; 8(1): 12791, 2018 08 24.
Article in English | MEDLINE | ID: mdl-30143724

ABSTRACT

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.


Subject(s)
Centrioles/metabolism , Germ Cells/metabolism , Oocytes/metabolism , Animals , Calcium-Binding Proteins/metabolism , Centrioles/drug effects , Centrosome/drug effects , Centrosome/metabolism , Female , Germ Cells/cytology , Germ Cells/drug effects , Green Fluorescent Proteins/metabolism , Metaphase/drug effects , Mice , Microtubules/drug effects , Microtubules/metabolism , Nocodazole/pharmacology , Oocytes/cytology , Oocytes/drug effects , Oogonia/cytology , Oogonia/drug effects , Oogonia/metabolism , Ovary/embryology , Spindle Apparatus/drug effects , Spindle Apparatus/metabolism , Spindle Poles/drug effects , Spindle Poles/metabolism , Tubulin/metabolism
6.
ACS Chem Biol ; 11(6): 1544-51, 2016 06 17.
Article in English | MEDLINE | ID: mdl-27121275

ABSTRACT

The genetic integrity of each organism is intimately tied to the correct segregation of its genome during mitosis. Insights into the underlying mechanisms are fundamental for both basic research and the development of novel strategies to treat mitosis-relevant diseases such as cancer. Due to their fast mode of action, small molecules are invaluable tools to dissect mitosis. Yet, there is a great demand for novel antimitotic compounds. We performed a chemical genetic suppression screen to identify compounds that restore spindle bipolarity in cells treated with Monastrol, an inhibitor of the mitotic kinesin Eg5. We identified one compound-MAC1-that rescued spindle bipolarity in cells lacking Eg5 activity. Mechanistically, MAC1 induces the formation of additional microtubule nucleation centers, which allows kinesin Kif15-dependent bipolar spindle assembly in the absence of Eg5 activity. Thus, our chemical genetic suppression screen revealed novel unexpected insights into the mechanism of spindle assembly in mammalian cells.


Subject(s)
Antimitotic Agents/pharmacology , Cyclohexanols/pharmacology , Kinesins/antagonists & inhibitors , Oximes/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Spindle Poles/drug effects , Thiones/pharmacology , A549 Cells , Humans , Kinesins/metabolism , Microtubule-Organizing Center/metabolism , Microtubules/metabolism , Tubulin/metabolism
7.
Proc Natl Acad Sci U S A ; 111(29): 10416-21, 2014 Jul 22.
Article in English | MEDLINE | ID: mdl-24972791

ABSTRACT

We demonstrate the use of surface-enhanced Raman spectroscopy (SERS) as an excellent tool for identifying the binding site of small molecules on a therapeutically important protein. As an example, we show the specific binding of the common antihypertension drug felodipine to the oncogenic Aurora A kinase protein via hydrogen bonding interactions with Tyr-212 residue to specifically inhibit its activity. Based on SERS studies, molecular docking, molecular dynamics simulation, biochemical assays, and point mutation-based validation, we demonstrate the surface-binding mode of this molecule in two similar hydrophobic pockets in the Aurora A kinase. These binding pockets comprise the same unique hydrophobic patches that may aid in distinguishing human Aurora A versus human Aurora B kinase in vivo. The application of SERS to identify the specific interactions between small molecules and therapeutically important proteins by differentiating competitive and noncompetitive inhibition demonstrates its ability as a complementary technique. We also present felodipine as a specific inhibitor for oncogenic Aurora A kinase. Felodipine retards the rate of tumor progression in a xenografted nude mice model. This study reveals a potential surface pocket that may be useful for developing small molecules by selectively targeting the Aurora family kinases.


Subject(s)
Drug Discovery/methods , Molecular Dynamics Simulation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Spectrum Analysis, Raman , Animals , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase A/chemistry , Aurora Kinase A/metabolism , Aurora Kinase B/antagonists & inhibitors , Aurora Kinase B/chemistry , Aurora Kinase B/metabolism , Binding, Competitive/drug effects , Cell Cycle/drug effects , Cell Death/drug effects , Disease Progression , Dose-Response Relationship, Drug , Felodipine/chemistry , Felodipine/pharmacology , HeLa Cells , Humans , Kinetics , Mice , Mice, Nude , Neoplasms/pathology , Reproducibility of Results , Spindle Poles/drug effects , Spindle Poles/metabolism , Surface Properties
SELECTION OF CITATIONS
SEARCH DETAIL
...