RESUMEN

Microtubules are essential for various cellular processes. The functional diversity of microtubules is attributed to the incorporation of various α- and ß-tubulin isotypes encoded by different genes. In this work, we investigated the functional role of ß4B-tubulin isotype (TUBB4B) in hearing and vision as mutations in TUBB4B are associated with sensorineural disease. Using a Tubb4b knockout mouse model, our findings demonstrate that TUBB4B is essential for hearing. Mice lacking TUBB4B are profoundly deaf due to defects in the inner and middle ear. Specifically, in the inner ear, the absence of TUBB4B lead to disorganized and reduced densities of microtubules in pillar cells, suggesting a critical role for TUBB4B in providing mechanical support for auditory transmission. In the middle ear, Tubb4b-/- mice exhibit motile cilia defects in epithelial cells, leading to the development of otitis media. However, Tubb4b deletion does not affect photoreceptor function or cause retinal degeneration. Intriguingly, ß6-tubulin levels increase in retinas lacking ß4B-tubulin isotype, suggesting a functional compensation mechanism. Our findings illustrate the essential roles of TUBB4B in hearing but not in vision in mice, highlighting the distinct functions of tubulin isotypes in different sensory systems.

Asunto(s)

Cilios , Ratones Noqueados , Tubulina (Proteína) , Animales , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Cilios/metabolismo , Ratones , Citoesqueleto/metabolismo , Cóclea/metabolismo , Microtúbulos/metabolismo

RESUMEN

The Lucena 1 cell line, derived from the human chronic myeloid leukemia cell line K562 under selective pressure of vincristine supplementation, exhibits multidrug resistance (MDR). This study aims to explore and elucidate the underlying mechanisms driving MDR in the Lucena 1 cell line. A proteomic analysis comparing K562 and Lucena 1 revealed qualitative differences, with a focus on the ATP-dependent efflux pump, Translocase ABCB1, a key contributor to drug resistance. Tubulin analysis identified two unique isoforms, Tubulin beta 8B and alpha chain-like 3, exclusive to Lucena 1, potentially influencing resistance mechanisms. Additionally, the association of Rap1A and Krit1 in cytoskeletal regulation and the presence of STAT1, linked to the urea cycle and tumor development, offered insights into Lucena 1's distinctive biology. The increased expression of carbonic anhydrase I suggested a role in pH regulation. The discovery of COP9, a tumor suppressor targeting p53, further highlighted the Lucena 1 complex molecular landscape. This study offers new insights into the MDR phenotype and its multifactorial consequences in cellular pathways. Thus, unraveling the mechanisms of MDR holds promise for innovating cancer models and antitumor targeted strategies, since inhibiting the P-glycoprotein (P-gp)/ABCB1 protein is not always an effective approach given the associated treatment toxicity.

Asunto(s)

Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Proteómica , Humanos , Proteómica/métodos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Células K562 , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Tubulina (Proteína)/metabolismo , Línea Celular Tumoral

RESUMEN

In this study, we have explored the role of the KATNB1 gene, a microtubule-severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co-localize with α-tubulin showing a unique stage-specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured in vitro with an established functional TJ-barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis in vivo was found to block cadmium-induced blood-testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin- and microtubule-based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1) plays a crucial role in BTB and spermatogenesis through its effects on the actin- and microtubule-based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology.

Asunto(s)

Barrera Hematotesticular , Katanina , Células de Sertoli , Animales , Masculino , Células de Sertoli/metabolismo , Ratas , Katanina/metabolismo , Katanina/genética , Barrera Hematotesticular/metabolismo , Citoesqueleto/metabolismo , Ratas Sprague-Dawley , Uniones Estrechas/metabolismo , Espermatogénesis/fisiología , Células Cultivadas , Epitelio Seminífero/metabolismo , Testículo/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo

RESUMEN

We are studying the molecular pathology of a sub-group within schizophrenia (∼ 25 %: termed Muscarinic Receptor Deficit subgroup of Schizophrenia (MRDS)) who can be separated because they have very low levels of cortical muscarinic M1 receptors (CHRM1). Based on our transcriptomic data from Brodmann's area ((BA) 9, 10 and 33 (controls, schizophrenia and mood disorders) and the cortex of the CHRM1-/- mouse (a molecular model of aberrant CHRM1 signaling), we predicted levels of AKT interacting protein (AKTIP), but not tubulin alpha 1b (TUBA1B) or AKT serine/threonine kinase 1 (AKT1) and pyruvate dehydrogenase kinase 1 (PDK1) (two AKTIP-functionally associated proteins), would be changed in MRDS. Hence, we used Western blotting to measure AKTIP (BA 10: controls, schizophrenia and mood disorders; BA 9: controls and schizophrenia) plus TUBA1B, AKT1 and PDK1 (BA 10: controls and schizophrenia) proteins. The only significant change with diagnosis was higher levels of AKTIP protein in BA 10 (Cohen's d = 0.73; p = 0.02) in schizophrenia compared to controls due to higher levels of AKTIP only in people with MRDS (Cohen's d = 0.80; p = 0.03). As AKTIP is involved in AKT1 signaling, our data suggests that signaling pathway is particularly disturbed in BA 10 in MRDS.

Asunto(s)

Proteínas Proto-Oncogénicas c-akt , Receptor Muscarínico M1 , Esquizofrenia , Esquizofrenia/metabolismo , Humanos , Masculino , Femenino , Adulto , Receptor Muscarínico M1/metabolismo , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Lóbulo Frontal/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Animales , Ratones , Tubulina (Proteína)/metabolismo , Ratones Noqueados

RESUMEN

Different wavelengths emitted from light-emitting diodes (LEDs) are known as an influential factor in proliferation and differentiation of various cell types. Since human umbilical cord matrix-derived mesenchymal cells (hUCMs) are ideal tools for human regenerative medicine clinical trials and stem cell researches, in the present study we investigated the neurogenesis effects of single and intermittent green and red LED irradiation on hUCM cells. Exposure of hUCMs to single and intermittent green (530 nm, 1.59 J/cm2) and red (630 nm, 0.318 J/cm2) lights significantly increased the expression of specific genes including nestin, ß-tubulin III and Olig2. Additionally, immunocytochemical analysis confirmed the expression of specific neural-related proteins including nestin, ß-tubulin III, Olig2 and GFAP. Also, alternating exposure of hUCM cells to green and red lights increased the expression of some neural markers more than either light alone. Further research are required to develop the application of LED irradiation as a useful tool for therapeutic purposes including neural repair and regeneration.

Asunto(s)

Diferenciación Celular , Células Madre Mesenquimatosas , Neurogénesis , Cordón Umbilical , Humanos , Células Madre Mesenquimatosas/efectos de la radiación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Diferenciación Celular/efectos de la radiación , Cordón Umbilical/citología , Neurogénesis/efectos de la radiación , Luz , Nestina/metabolismo , Nestina/genética , Células Cultivadas , Neuronas/efectos de la radiación , Neuronas/metabolismo , Neuronas/citología , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos/genética

RESUMEN

Microtubules, polymers of αß-tubulin heterodimers, are essential for various cellular processes. The incorporation of different tubulin isotypes, each encoded by distinct genes, is proposed to contribute to the functional diversity observed in microtubules. However, the functional roles of each tubulin isotype are not completely understood. In this study, we investigated the role of the ß4B-tubulin isotype (Tubb4b) in spermatogenesis, utilizing a Tubb4b knockout mouse model. We showed that ß4B-tubulin is expressed in the germ cells throughout spermatogenesis. ß4B-tubulin was localized to cytoplasmic microtubules, mitotic spindles, manchette, and axonemes of sperm flagella. We found that the absence of ß4B-tubulin resulted in male infertility and failure to produce sperm cells. Our findings demonstrate that a lack of ß4B-tubulin leads to defects in the initial stages of spermatogenesis. Specifically, ß4B-tubulin is needed for the expansion of differentiating spermatogonia, which is essential for the subsequent progression of spermatogenesis.

Asunto(s)

Diferenciación Celular , Ratones Noqueados , Microtúbulos , Espermatogénesis , Espermatogonias , Tubulina (Proteína) , Animales , Masculino , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Espermatogénesis/genética , Ratones , Microtúbulos/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología

RESUMEN

Microtubules, complex cytoskeletal structures composed of tubulin proteins in eukaryotic cells, have garnered recent attention in cardiovascular research. Investigations have focused on the post-translational modifications of tubulin, including acetylation and detyrosination. Perturbations in microtubule homeostasis have been implicated in various pathological processes associated with cardiovascular diseases such as heart failure, ischemic heart disease, and arrhythmias. Thus, elucidating the intricate interplay between microtubule dynamics and cardiovascular pathophysiology is imperative for advancing preventive and therapeutic strategies. Several natural compounds have been identified to potentially modulate microtubules, thereby exerting regulatory effects on cardiovascular diseases. This review synthesizes current literature to delineate the roles of microtubules in cardiovascular diseases and assesses the potential of natural compounds in microtubule-targeted therapies.

Asunto(s)

Enfermedades Cardiovasculares , Microtúbulos , Humanos , Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/tratamiento farmacológico , Animales , Tubulina (Proteína)/metabolismo , Procesamiento Proteico-Postraduccional

RESUMEN

Microtubules (MTs) are widely recognized as targets for cancer therapies. They are directly related to unique mechanical properties, closely dependent on MT architecture and tubulin molecular features. Taxol is known to affect tubulin interactions resulting in the stabilization of the MT lattice, and thus the hierarchical organization stability, mechanics, and function. A deeper understanding of the molecular mechanisms through which taxol modulates intertubulin interactions in the MT lattice, and consequently, its stability and mechanical response is crucial to characterize how MT properties are regulated by environmental factors, such as interacting ligands. In this study, a computational analysis of the effect of taxol on the MT was performed at different scales, combining molecular dynamics simulation, dynamical network analysis, and elastic network modeling. The results show that the taxol-induced conformational differences at the M-loop region increase the stability of the lateral interactions and the amount of surface in contact between laterally coupled tubulins. Moreover, the conformational rearrangements in the taxane binding site result in a different structural communication pattern. Finally, the different conformation of the tubulin heterodimers and the stabilized lateral interactions resulted in a tendency toward higher deformation of the vibrating MT in the presence of taxol. Overall, this work provides additional insights into taxol-induced stabilization and relates the conformational changes at the tubulin level to the MT mechanics. Besides providing useful insights into taxol effect on MT mechanics, a methodological framework that could be used to characterize the effects of other MT stabilizing agents is presented.

Asunto(s)

Microtúbulos , Simulación de Dinámica Molecular , Paclitaxel , Tubulina (Proteína) , Paclitaxel/farmacología , Paclitaxel/química , Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Sitios de Unión , Humanos

RESUMEN

Traumatic spinal cord injury is a major cause of disability for which there are currently no fully effective treatments. Recent studies using epidural electrical stimulation have shown significant advances in motor rehabilitation, even when applied during chronic phases of the disease. The present study aimed to investigate the effectiveness of epidural electric stimulation in the motor recovery of rats with spinal cord injury. Furthermore, we aimed to elucidate the neurophysiological mechanisms underlying motor recovery. First, we improved upon the impact spinal cord injury model to cause severe and permanent motor deficits lasting up to 2 months. Next, we developed and tested an implantable epidural spinal cord stimulator device for rats containing an electrode and an implantable generator. Finally, we evaluated the efficacy of epidural electrical stimulation on motor recovery after spinal cord injury in Wistar rats. A total of 60 animals were divided into the following groups: (i) severe injury with epidural electrical stimulation (injury + stim, n = 15), (ii) severe injury without stimulation (group injury, n = 15), (iii) sham implantation without battery (sham, n = 15), and (iv) a control group, without surgical intervention (control, n = 15). All animals underwent weekly evaluations using the Basso, Beattie, Bresnahan (BBB) locomotor rating scale index, inclined plane, and OpenField test starting one week before the lesion and continuing for eight weeks. After this period, the animals were sacrificed and their spinal cords were explanted and prepared for histological analysis (hematoxylin-eosin) and immunohistochemistry for NeuN, ß-III-tubulin, synaptophysin, and Caspase 3. Finally, NeuN-positive neuronal nuclei were quantified through stereology; fluorescence signal intensities for ß-tubulin, synaptophyin, and Caspase 3 were quantified using an epifluorescence microscope. The injury + stim group showed significant improvement on the BBB scale compared with the injured group after the 5th week (p < 0.05). Stereological analysis showed a significantly higher average count of neural cells in the injury + stim group in relation to the injury group (1783 ± 2 vs. 897 ± 3, p < 0.001). Additionally, fluorescence signal intensity for synaptophysin was significantly higher in the injury + stim group in relation to the injury group (1294 ± 46 vs. 1198 ± 23, p < 0.01); no statistically significant difference was found in ß-III-tubulin signal intensity. Finally, Caspase 3 signal intensity was significantly lower in the stim group (727 ± 123) compared with the injury group (1225 ± 87 p < 0.05), approaching levels observed in the sham and control groups. Our data suggest a regenerative and protective effect of epidural electrical stimulation in rats subjected to impact-induced traumatic spinal cord injury.

Asunto(s)

Modelos Animales de Enfermedad , Plasticidad Neuronal , Ratas Wistar , Traumatismos de la Médula Espinal , Animales , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patología , Ratas , Recuperación de la Función , Terapia por Estimulación Eléctrica/métodos , Sinaptofisina/metabolismo , Tubulina (Proteína)/metabolismo , Espacio Epidural/patología , Médula Espinal/metabolismo , Médula Espinal/patología , Médula Espinal/fisiopatología , Masculino , Caspasa 3/metabolismo , Regeneración Nerviosa , Femenino , Proteínas del Tejido Nervioso , Antígenos Nucleares

RESUMEN

Dynamic properties are essential for microtubule (MT) physiology. Current techniques for in vivo imaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of the AlphaFold2 pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis in Caenorhabditis elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies.

Asunto(s)

Caenorhabditis elegans , Proteínas Fluorescentes Verdes , Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Humanos , Microtúbulos/metabolismo , Ratones , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Ingeniería de Proteínas/métodos , Oocitos/metabolismo

RESUMEN

Dual-target agents have more advantages than drug combinations for cancer treatment. Here, we designed and synthesized a series of novel VEGFR-2/tubulin dual-target inhibitors through a molecular hybridization strategy, and the activities of all the synthesized compounds were tested against tubulin and VEGFR-2. Among which, compound 19 exhibited strong potency against tubulin and VEGFR-2, with IC50 values of 0.76 ± 0.11 µM and 15.33 ± 2.12 nM, respectively. Additionally, compound 19 not only had significant antiproliferative effects on a series of human cancer cell lines, especially MGC-803 cells (IC50 = 0.005 ± 0.001 µM) but also overcame drug resistance in Taxol-resistant MGC-803 cells, with an RI of 1.8. Further studies showed that compound 19 could induce tumor cell apoptosis by reducing the mitochondrial membrane potential, increasing the level of ROS, facilitating the induction of G2/M phase arrest, and inhibiting the migration and invasion of tumor cells in a dose-dependent manner. In addition, compound 19 also exhibits potent antiangiogenic effects by blocking the VEGFR-2/PI3K/AKT pathway and inhibiting the tubule formation, invasion, and migration of HUVECs. More importantly, compound 19 demonstrated favorable pharmacokinetic profiles, robust in vivo antitumor efficacy, and satisfactory safety profiles. Overall, compound 19 can be used as a lead compound for the development of tubulin/VEGFR-2 dual-target inhibitors.

Asunto(s)

Inhibidores de la Angiogénesis , Antineoplásicos , Apoptosis , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Moduladores de Tubulina , Tubulina (Proteína) , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Humanos , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Tubulina (Proteína)/metabolismo , Relación Estructura-Actividad , Proliferación Celular/efectos de los fármacos , Estructura Molecular , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/química , Moduladores de Tubulina/síntesis química , Apoptosis/efectos de los fármacos , Amidas/química , Amidas/farmacología , Amidas/síntesis química , Descubrimiento de Drogas , Animales , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Ratones , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos

RESUMEN

Background: Growing evidence suggests that endometriosis (EMs) is a risk factor for endometriosis-associated ovarian cancer (EAOC). The aim was to identify and validate gene signatures associated with EMs that may serve as potential biomarkers for evaluating the prognosis of patients with EAOC. Methods: The data of EMs and control samples was obtained from GEO database. The weighted gene co-expression network analysis (WGCNA) identified modular genes significantly associated with EMs. The KEGG pathway and GO functional enrichment analyses were also performed. Univariate Cox regression analysis was conducted to screen marker genes associated with the prognosis of EAOC patients. Finally, RT-qPCR and immunohistochemical verified the expression of ADAMTS19 and TUBB in normal ovarian and EAOC tissues, and the biological functions of ADAMTS19 and TUBB were preliminarily explored by CCK8 and Transwell assays. Results: The WGCNA identified 2 co-expression modules, which in total included 615 genes, and 7642 differentially expressed genes (DEGs) were detected thorough analysis of the EAOC dataset. After taking the intersection of 615 modular genes and 7642 DEGs, 214 shared genes were obtained, and univariate COX regression analysis pointed 10 genes associated with the prognosis of EAOC. Moreover, it was demonstrated by RT-qPCR and immunohistochemical staining experiments that ADAMTS19 expression was elevated, while TUBB expression was reduced in EAOC compared with normal ovarian cells and tissues. Finally, cell experiments revealed that ADAMTS19 promoted the proliferation and invasion in EAOC cells, while overexpression of TUBB inhibited these processes. Conclusions: The present study identified and validated new EMs-associated gene markers, which could serve as potential biomarkers for assessing the prognostic risk of EAOC patients. In addition, some of these genes may have significance as novel therapeutic targets and could be used to guide clinical applications.

Asunto(s)

Biomarcadores de Tumor , Endometriosis , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/diagnóstico , Endometriosis/genética , Endometriosis/complicaciones , Endometriosis/patología , Pronóstico , Biomarcadores de Tumor/genética , Proteínas ADAMTS/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proliferación Celular/genética , Adulto , Línea Celular Tumoral

RESUMEN

Despite widespread public interest in the health impact of exposure to microwave radiation, studies of the influence of microwave radiation on biological samples are often inconclusive or contradictory. Here we examine the influence of microwave radiation of frequencies 3.5 GHz, 20 GHz and 29 GHz on the growth of microtubules, which are biological nanotubes that perform diverse functions in eukaryotic cells. Since microtubules are highly polar and can extend several micrometres in length, they are predicted to be sensitive to non-ionizing radiation. Moreover, it has been speculated that tubulin dimers within microtubules might rapidly toggle between different conformations, potentially participating in computational or other cooperative processes. Our data show that exposure to microwave radiation yields a microtubule growth curve that is distorted relative to control studies utilizing a homogeneous temperature jump. However, this apparent effect of non-ionizing radiation is reproduced by control experiments using an infrared laser or hot air to heat the sample and thereby mimic the thermal history of samples exposed to microwaves. As such, no non-thermal effects of microwave radiation on microtubule growth can be assigned. Our results highlight the need for appropriate control experiments in biophysical studies that may impact on the sphere of public interest.

Asunto(s)

Microtúbulos , Microondas , Microtúbulos/efectos de la radiación , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Temperatura

RESUMEN

3-Nitrotyrosine (3-NT), a byproduct of oxidative and nitrosative stress, is implicated in age-related neurodegenerative disorders. Current literature suggests that free 3-NT becomes integrated into the carboxy-terminal domain of α-tubulin via the tyrosination/detyrosination cycle. Independently of this integration, 3-NT has been associated with the cell death of dopaminergic neurons. Given the critical role of tyrosination/detyrosination in governing axonal morphology and function, the substitution of tyrosine with 3-NT in this process may potentially disrupt axonal homeostasis, although this aspect remains underexplored. In this study, we examined the impact of 3-NT on the axons of cerebellar granule neurons, which is used as a model for non-dopaminergic neurons. Our observations revealed axonal shortening, which correlated with the incorporation of 3-NT into α-tubulin. Importantly, this axonal effect was observed prior to the onset of cellular death. Furthermore, 3-NT was found to diminish mitochondrial motility within the axon, leading to a subsequent reduction in mitochondrial membrane potential. The suppression of syntaphilin, a protein responsible for anchoring mitochondria to microtubules, restored the mitochondrial motility and axonal elongation that were inhibited by 3-NT. These findings underscore the inhibitory role of 3-NT in axonal elongation by impeding mitochondrial movement, suggesting its potential involvement in axonal dysfunction within non-dopaminergic neurons.

Asunto(s)

Axones , Mitocondrias , Tirosina , Tirosina/análogos & derivados , Tirosina/metabolismo , Animales , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Axones/metabolismo , Tubulina (Proteína)/metabolismo , Células Cultivadas , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Cerebelo/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Ratones

RESUMEN

BACKGROUND: Thiostrepton (TST) is a known inhibitor of the transcription factor Forkhead box M1 (FoxM1) and inducer of heat shock response (HSR) and autophagy. TST thus may be one potential candidate of anticancer drugs for combination chemotherapy. METHODS AND RESULTS: Immunofluorescence staining of mitotic spindles and flow cytometry analysis revealed that TST induces mitotic spindle abnormalities, mitotic arrest, and apoptotic cell death in the MDA-MB-231 triple-negative breast cancer cell line. Interestingly, overexpression or depletion of FoxM1 in MDA-MB-231 cells did not affect TST induction of spindle abnormalities; however, TST-induced spindle defects were enhanced by inhibition of HSP70 or autophagy. Moreover, TST exhibited low affinity for tubulin and only slightly inhibited in vitro tubulin polymerization, but it severely impeded tubulin polymerization and destabilized microtubules in arrested mitotic MDA-MB-231 cells. Additionally, TST significantly enhanced Taxol cytotoxicity. TST also caused cytotoxicity and spindle abnormalities in a Taxol-resistant cell line, MDA-MB-231-T4R. CONCLUSIONS: These results suggest that, in addition to inhibiting FoxM1, TST may induce proteotoxicity and autophagy to disrupt cellular tubulin polymerization, and this mechanism might account for its antimitotic effects, enhancement of Taxol anticancer effects, and ability to overcome Taxol resistance in MDA-MB-231 cells. These data further imply that TST may be useful to improve the therapeutic efficacy of Taxol.

Asunto(s)

Autofagia , Proteína Forkhead Box M1 , Paclitaxel , Huso Acromático , Tioestreptona , Tubulina (Proteína) , Humanos , Paclitaxel/farmacología , Tioestreptona/farmacología , Línea Celular Tumoral , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Proteína Forkhead Box M1/metabolismo , Autofagia/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Femenino , Sinergismo Farmacológico , Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Mitosis/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Células MDA-MB-231

RESUMEN

Herein, we designed and synthesized a series of novel 2-methylthieno [3,2-d]pyrimidine analogues as tubulin inhibitors with antiproliferative activities at low nanomolar levels. Among them, compound DPP-21 displayed the most potent anti-proliferative activity against six cancer cell lines with an average IC50 of ∼6.23 nM, better than that of colchicine (IC50 = 9.26 nM). DPP-21 exerted its anti-cancer activity by suppressing the polymerization of tubulin with an IC50 of 2.4 µM. Furthermore, the crystal structure of DPP-21 in complex with tubulin was solved by X-ray crystallography to 2.94 Å resolution, confirming the direct binding of DPP-21 to the colchicine site. Moreover, DPP-21 arrested the cell cycle in the G2/M phase of mitosis, subsequently inducing tumor cell apoptosis. Additionally, DPP-21 was able to effectively inhibit the migration of cancer cells. Besides, DPP-21 exhibited significant in vivo anti-tumor efficacy in a B16-F10 melanoma tumor model with a TGI of 63.3 % (7 mg/kg) by intraperitoneal (i.p.) injection. Notably, the combination of DPP-21 with NP-19 (a PD-L1-targeting small molecule inhibitor reported by our group before) demonstrated enhanced anti-cancer efficacy in vivo. These results suggest that DPP-21 is a promising lead compound deserving further investigation as a potential anti-cancer agent.

Asunto(s)

Antineoplásicos , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Pirimidinas , Tiofenos , Moduladores de Tubulina , Humanos , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/química , Moduladores de Tubulina/síntesis química , Proliferación Celular/efectos de los fármacos , Animales , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Ratones , Tiofenos/química , Tiofenos/farmacología , Tiofenos/síntesis química , Estructura Molecular , Relación Dosis-Respuesta a Droga , Apoptosis/efectos de los fármacos , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Descubrimiento de Drogas , Tubulina (Proteína)/metabolismo , Línea Celular Tumoral , Inmunoterapia , Ratones Endogámicos C57BL , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/química , Inhibidores de Puntos de Control Inmunológico/síntesis química , Melanoma/tratamiento farmacológico , Melanoma/patología , Modelos Moleculares

RESUMEN

A series of novel 2-substituted 2, 3-dihydroquinazolin-4(1H)-one derivatives were designed, synthesized and estimated for their in vitro antiproliferative activities against HepG2, U251, PANC-1, A549 and A375 cell lines. Among them, compound 32 was the most promising candidate, and displayed strong broad-spectrum anticancer activity. The mechanism studies revealed that compound 32 inhibited tubulin polymerization in vitro, disrupted cell microtubule networks, arrested the cell cycle at G2/M phase, and induced apoptosis by up-regulating the expression of cleaved PARP-1 and caspase-3. Furthermore, molecular docking analysis suggested that compound 32 well occupied the binding site of tubulin. In addition, compound 32 exhibited no significant activity against 30 different kinases respectively, indicating considerable selectivity. Moreover, compound 32 significantly inhibited the tumour growth of the HepG2 xenograft in a nude mouse model by oral gavage without apparent toxicity. These results demonstrated that some 2-substituted 2, 3- dihydroquinazolin-4(1H)-one derivatives bearing phenyl, biphenyl, naphthyl or indolyl side chain at C2-position might be potentially novel antitumor agents as tubulin polymerization inhibitors.

Asunto(s)

Antineoplásicos , Apoptosis , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Ratones Desnudos , Polimerizacion , Quinazolinonas , Moduladores de Tubulina , Tubulina (Proteína) , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Animales , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Proliferación Celular/efectos de los fármacos , Ratones , Relación Estructura-Actividad , Quinazolinonas/farmacología , Quinazolinonas/química , Quinazolinonas/síntesis química , Polimerizacion/efectos de los fármacos , Apoptosis/efectos de los fármacos , Estructura Molecular , Simulación del Acoplamiento Molecular , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Línea Celular Tumoral , Ratones Endogámicos BALB C

RESUMEN

Almost half of all medicines approved by the U.S. Food and Drug Administration have been found to be developed based on inspiration from natural products (NPs). Here, we report a novel strategy of scaffold overlaying of scaffold-hopped analogs of bioactive flavones and isoflavones and installation of drug-privileged motifs, which has led to discovery of anticancer agents that surpass the functional efficiency of the original NPs. The analogs, 2,3-diaryl-pyridopyrimidin-4-imine/ones were efficiently synthesized by an approach of a nitrile-stabilized quaternary ammonium ylide as masked synthon and Pd-catalyzed activation-arylation methods. Compared to the NPs, these NP-analogs exhibited differentiated functions; dual inhibition of human topoisomerase-II (hTopo-II) enzyme and tubulin polymerization, and pronounced antiproliferative effect against various cancer cell lines, including numerous drug-resistant cancer cells. The most active compound 5l displayed significant inhibition of migration ability of cancer cells and blocked G1/S phase transition in cell cycle. Compound 5l caused pronounced effect in expression patterns of various key cell cycle regulatory proteins; up-regulation of apoptotic proteins, Bax, Caspase 3 and p53, and down-regulation of apoptosis-inhibiting proteins, BcL-xL, Cyclin D1, Cyclin E1 and NF-κB, which indicates high efficiency of the molecule 5l in apoptosis-signal axis interfering potential. Cheminformatics analysis revealed that 2,3-diaryl-pyridopyrimidin-4-imine/ones occupy a distinctive drug-relevant chemical space that is seldom represented by natural products and good physicochemical, ADMET and pharmacokinetic-relevant profile. Together, the anticancer potential of the investigated analogs was found to be much more efficient compared to the original natural products and two anticancer drugs, Etoposide (hTopo-II inhibitor) and 5-Flurouracile (5-FU).

Asunto(s)

Antineoplásicos , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Flavonoides/química , Flavonoides/farmacología , Flavonoides/síntesis química , Iminas/química , Iminas/farmacología , Iminas/síntesis química , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/síntesis química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/química , Moduladores de Tubulina/síntesis química , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/farmacología

RESUMEN

A progressive aggregation of Tau proteins in the brain is linked to both Alzheimer's disease (AD) and various Tauopathies. This pathological process can be enhanced by several substances, including heparin. However, very little if anything is known about molecules that can inhibit the aggregation of Tau isoforms. In this study, we examined the effect of phosphatidylserines (PSs) with various lengths and saturations of fatty acids (FAs) on the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N-terminal inserts that enhance binding of Tau to tubulin. We found that PS with unsaturated and short-length FAs inhibited Tau aggregation and drastically lowered the toxicity of Tau oligomers that were formed in the presence of such phospholipids. Such an effect was not observed for PS with fully saturated long-chain FAs. These results suggest that a short-chain irreversible disbalance between saturated and unsaturated lipids in the brain could be the trigger of Tau aggregation.

Asunto(s)

Fosfolípidos , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/química , Humanos , Fosfolípidos/química , Fosfolípidos/metabolismo , Agregado de Proteínas/efectos de los fármacos , Heparina/química , Heparina/farmacología , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química

RESUMEN

ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.

Asunto(s)

Proteínas de Ciclo Celular , Centrosoma , Microtúbulos , Proteínas del Tejido Nervioso , Células-Madre Neurales , Neurogénesis , Animales , Microtúbulos/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Neurogénesis/genética , Células-Madre Neurales/metabolismo , Centrosoma/metabolismo , Proliferación Celular , Movimiento Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Tubulina (Proteína)/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética