RESUMEN

Hypothalamic proopiomelanocortin (POMC) neurons are sensors of signals that reflect the energy stored in the body. Inducing mild stress in proopiomelanocortin neurons protects them from the damage promoted by the consumption of a high-fat diet, mitigating the development of obesity; however, the cellular mechanisms behind these effects are unknown. Here, we induced mild stress in a proopiomelanocortin neuron cell line by inhibiting Crif1. In proopiomelanocortin neurons exposed to high levels of palmitate, the partial inhibition of Crif1 reverted the defects in mitochondrial respiration and ATP production; this was accompanied by improved mitochondrial fusion/fission cycling. Furthermore, the partial inhibition of Crif1 resulted in increased reactive oxygen species production, increased fatty acid oxidation, and reduced dependency on glucose for mitochondrial respiration. These changes were dependent on the activity of CPT-1. Thus, we identified a CPT-1-dependent metabolic shift toward greater utilization of fatty acids as substrates for respiration as the mechanism behind the protective effect of mild stress against palmitate-induced damage of proopiomelanocortin neurons.NEW & NOTEWORTHY Saturated fats can damage hypothalamic neurons resulting in positive energy balance, and this is mitigated by mild cellular stress; however, the mechanisms behind this protective effect are unknown. Using a proopiomelanocortin cell line, we show that under exposure to a high concentration of palmitate, the partial inhibition of the mitochondrial protein Crif1 results in protection due to a metabolic shift warranted by the increased expression and activity of the mitochondrial fatty acid transporter CPT-1.

Asunto(s)

Carnitina O-Palmitoiltransferasa , Proteínas de Ciclo Celular , Ácidos Grasos , Mitocondrias , Animales , Ratones , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Línea Celular , Ácidos Grasos/metabolismo , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Proopiomelanocortina/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo

RESUMEN

Polo-Like Kinases (PLKs) are central players of mitotic progression in Eukaryotes. Given the intimate relationship between cell cycle progression and cancer development, PLKs in general and PLK1 in particular have been thoroughly studied as biomarkers and potential therapeutic targets in oncology. The oncogenic properties of PLK1 overexpression across different types of human cancers are attributed to its roles in promoting mitotic entry, centrosome maturation, spindle assembly and cytokinesis. While several academic labs and pharmaceutical companies were able to develop potent and selective inhibitors of PLK1 (PLK1i) for preclinical research, such compounds have reached only limited success in clinical trials despite their great pharmacokinetics. Even though this could be attributed to multiple causes, the housekeeping roles of PLK1 in both normal and cancer cells are most likely the main reason for clinical trials failure and withdraw due to toxicities issues. Therefore, great efforts are being invested to position PLK1i in the treatment of specific types of cancers with revised dosages schemes. In this mini review we focus on two potential niches for PLK1i that are supported by recent evidence: triple negative breast cancers (TNBCs) and BRCA1-deficient cancers. On the one hand, we recollect several lines of strong evidence indicating that TNBCs are among the cancers with highest PLK1 expression and sensitivity to PLK1i. These findings are encouraging because of the limited therapeutics options available for TNBC patients, which rely mainly on classic chemotherapy. On the other hand, we discuss recent evidence that unveils synthetic lethality induction by PLK1 inhibition in BRCA1-deficient cancers cells. This previously unforeseen therapeutic link between PLK1 and BRCA1 is promising because it defines novel therapeutic opportunities for PLK1i not only for breast cancer (i.e. TNBCs with BRCA1 deficiencies), but also for other types of cancers with BRCA1-deficiencies, such as pancreatic and prostate cancers.

Asunto(s)

Antineoplásicos/uso terapéutico , Proteína BRCA1/deficiencia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Terapia Molecular Dirigida , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Humanos , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Quinasa Tipo Polo 1

RESUMEN

INTRODUCTION: Multiple myeloma (MM) remains incurable due to high rates of relapse after various treatment regimens. WEE1 is a cell cycle related gene that regulates the G2/M checkpoint and promotes cell cycle suspension for consequent DNA repair. To date, there are clinical studies for the evaluation of WEE1 inhibitors in the treatment of solid tumors and studies on cell lines of non-MM hematological tumors. OBJECTIVES: To perform in vitro functional studies to verify the effect of the inhibition of WEE1 on MM cell lines viability and its potential as therapeutic target. MATERIAL AND METHODS: WEE1 expression was evaluated in 22 newly diagnosed MM patients and in four MM cell lines, RPMI-8226, U266 and SKO-007 and SK-MM2, by quantitative real-time PCR (qPCR). After treatment with the WEE1 inhibitor (MK-1775), with or without proteasome inhibitor (bortezomib) pretreatment, we assessed cell viability through Prestoblue functional test, microspheres formation in soft agar, and induction of apoptosis and cell cycle alterations by flow cytometry. RESULTS: All MM cell lines showed WEE1 expression by qPCR. RPMI-8226 and U266 showed a 50% reduction in cell viability after 24 h of incubation with MK-1775, at concentrations of 5 µM and 20 µM, respectively. SKO-007 showed dose and time dependence to this drug. Combination therapy with bortezomib and MK-1775 abolished the formation of soft agar microspheres in the RPMI-8226 cell line (also responsive to the use of both drugs) and U266, but SKO-007 was resistant to all drugs, isolated and combined. However, treatment of bortezomib followed by MK-1775 (sequential treatment) versus bortezomib alone showed statistically significant impact on cell lines total apoptosis: 88.8% vs 74.1% in RPMI-8222 (confirmed by cell cycle experiments); 92.5% vs 86.6% in U266; and 60.2% 30.9% on SKO-007 (p < 0.05). CONCLUSION: The sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in RPMI-8226, U266, and especially SKO-007 cell lines, more efficiently than the use of the same isolated drugs, highlighting its effect in inhibition of proliferation of tumor cells in MM cell lines. Our data suggest that WEE1 can figure as a MM target and that the sequential combination of bortezomib and MK-1775 may be explored in future clinical trials.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bortezomib/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinonas/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/patología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas

RESUMEN

Head and neck cancer (HNSCC) are one of the most common solid malignancies of the world, being responsible for over 350,000 deaths every year. Much of the complications in managing and treating HNSCC advent from the complex genetic and epigenetic landscape of the disease. Emerging information has shown promising results in targeting BRD4, an epigenetic regulator bromodomain that functions as a scaffold for transcription factors at promoters and super-enhancers. Here we show that by disrupting the interaction between BRD4 and histones using the bromodomain inhibitor JQ1, HNSCC cells undergo cell growth arrest followed by cellular senescence. Mechanistically, JQ1 negatively impacted the phosphorylation levels of SIRT1 along with the acetylation levels of mutant p53 (active). In vivo administration of JQ1 resulted in disruption of HNSCC growth along with the activation of cellular senescence, observed by the accumulation of DNA double-strand breaks, p16ink4, accumulation of senescence-associated beta-galactosidase, and loss of phosphorylated Sirt1ser47. Furthermore, we also demonstrate that JQ1 was efficient in reducing the population of cancer stem cells from HNSCC xenografts.

Asunto(s)

Azepinas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Senescencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Epigenoma , Neoplasias de Cabeza y Cuello/patología , Células Madre Neoplásicas/patología , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Animales , Apoptosis , Biomarcadores de Tumor , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Pronóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/secundario , Tasa de Supervivencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

Asunto(s)

Antineoplásicos/farmacología , Quitosano/análogos & derivados , Mitosis/efectos de los fármacos , Morfolinas/farmacología , Nanopartículas/química , Purinas/farmacología , Antineoplásicos/administración & dosificación , Aurora Quinasa B/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Humanos , Células MCF-7 , Mitosis/efectos de la radiación , Morfolinas/administración & dosificación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Purinas/administración & dosificación , Rayos X

RESUMEN

PURPOSE: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. EXPERIMENTAL DESIGN: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. RESULTS: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. CONCLUSIONS: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

Asunto(s)

Proteína BRCA1/deficiencia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Mutaciones Letales Sintéticas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína BRCA2/deficiencia , Proteína BRCA2/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Células Cultivadas , Aberraciones Cromosómicas , Daño del ADN , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1

RESUMEN

BACKGROUND AND PURPOSE: Over the last decade, the inhibition of PLK1 has proven potent antiproliferative activity in vitro. However, the effectiveness of most synthetic targeted drugs has not yet been translated into clinics. Herein, we investigated the in vitro effects of two second-generation PLK1 inhibitors BI 6727 and GSK461364 in breast cancer cell lines as monotherapy or in combination with other drugs or ionizing radiation. MATERIAL AND METHODS: Cell survival was analyzed through XTT®, clonogenicity and caspase-3 activation assays were also studied, and drug interactions analyzed through a nonlinear regression of a sigmoid doseresponse model. Sensibilization to radiation was assessed through enhancement ratio calculation. RESULTS: Mild effects on the viability of both cell lines tested (MCF-7 and Hs578T) were observed irrespective of the used PLK1 inhibitor. Alternatively, abrogation of PLK1 significantly reduced clonogenicity while effectively sensitized cells to ionizing radiation. Drug interactions showed dissimilar results with antagonistic effects with any drug combination in MCF-7 and clear synergic interactions between both PLK1 inhibitors and cisplatin, temozolomide or doxorubicin in Hs578T, which is TP53 mutated. CONCLUSION: Targeting kinases involved in mitotic checkpoints are expected to prevent mitotic exit and enhance chemosensitization. Nonetheless, despite overexpressing PLK1, in our model, expressive results after its inhibition were only seen through clonogenic assays or when BI 6727 and GSK461364 were combined with ionizing radiation. Disparate responses of cell lines to drug combinations might denote a partial reflection of the substantial differences in the vast spectrum of genetic, biological and epigenetic burden observed in breast cancer. In the near future, individual genomic/proteomic profiling will allow its further classification and will consent the initiation of novel strategies for therapy. Even though the future impact of PLK1-tailored treatment still needs validation, much more pre-clinical and clinical research for this kinase are warranted.

Asunto(s)

Antineoplásicos/farmacología , Bencimidazoles/farmacología , Neoplasias de la Mama/terapia , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pteridinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Tiofenos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/radioterapia , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Células MCF-7 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Quinasa Tipo Polo 1

RESUMEN

AKT3 is one of the major therapeutic targets in melanoma but clinically targeting AKT3 alone seems to be an ineffective therapeutic approach. To identify unique strategies to enhance the efficacy of targeting AKT3, a screen was undertaken where AKT3 was co-targeted with a panel of kinases important in melanoma development. The screen identified WEE1 as the most potent target that when inhibited along with AKT3 would enhance the efficacy of targeting AKT3 in melanoma. RNAi mediated inhibition of AKT3 and WEE1 synergistically inhibited the viability of melanoma cells leading to a 65-75% decrease in tumor development. This approach was effective by mechanistically modulating pathways associated with the transcription factors p53 and FOXM1. Simultaneously regulating the activity of these two transcriptionally driven pathways, cooperatively deregulated cell cycle control and DNA damage repair to synergistically kill melanoma cells. This study uniquely identifies a potential approach to improve the efficacy of targeting AKT3 in melanoma.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Sinergismo Farmacológico , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Melanoma/genética , Melanoma/patología , Ratones , Ratones Desnudos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The Aurora protein kinase (AURKA) and the Polo-like kinase-1 (PLK1) activate the cell cycle, and they are considered promising druggable targets in cancer therapy. However, resistance to chemotherapy and to specific small­molecule inhibitors is common in cancer patients; thus alternative therapeutic approaches are needed to overcome clinical resistance. Here, we showed that the dietary compound resveratrol suppressed the cell cycle by targeting AURKA and PLK1 kinases. First, we identified genes modulated by resveratrol using a genome-wide analysis of gene expression in MDA-MB-231 breast cancer cells. Transcriptional profiling indicated that 375 genes were modulated at 24 h after resveratrol intervention, whereas 579 genes were regulated at 48 h. Of these, 290 genes were deregulated in common at 24 and 48 h. Interestingly, a significant decrease in the expression of genes involved in the cell cycle, DNA repair, cytoskeleton organization, and angiogenesis was detected. In particular, AURKA and PLK1 kinases were downregulated by resveratrol at 24 h. In addition the BRCA1 gene, an AURKA/PLK1 inhibitor, was upregulated at 24 h of treatment. Moreover, two well-known resveratrol effectors, cyclin D1 (CCND1) and cyclin B1 (CCNB1), were also repressed at both times. Congruently, we found that resveratrol impaired G1/S phase transition in both MDA-MB-231 and MCF-7 cells. By western blot assays, we confirmed that resveratrol suppressed AURKA, CCND1 and CCNB1 at 24 and 48 h. In summary, we showed for the first time that resveratrol regulates cell cycle progression by targeting AURKA and PLK1. Our findings highlight the potential use of resveratrol as an adjuvant therapy for breast cancer.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Aurora Quinasa A/antagonistas & inhibidores , Proteína BRCA1/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Estilbenos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Ciclina B1/antagonistas & inhibidores , Ciclina D1/antagonistas & inhibidores , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Células MCF-7 , Análisis de Secuencia por Matrices de Oligonucleótidos , Resveratrol , Transcriptoma/genética , Quinasa Tipo Polo 1

RESUMEN

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

Asunto(s)

Plaquetas/metabolismo , Neoplasias de la Mama/metabolismo , Catepsina K/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Plaquetas/efectos de los fármacos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Calcio/metabolismo , Catepsina K/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Proteínas Hedgehog/metabolismo , Humanos , Hidrólisis , Ligandos , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Proteolisis , Receptores de Trombina/antagonistas & inhibidores , Trombina/metabolismo , Trombina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/metabolismo

RESUMEN

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Quinasa 1 Relacionada con NIMA , Quinasas Relacionadas con NIMA , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato

RESUMEN

BACKGROUND: Polo-like kinase 1 (PLK1) is a conserved kinase that mediates various mitotic events. Compelling data have repeatedly demonstrated its upregulation in different neoplasia, being frequently associated with poor prognosis. However, in childhood acute lymphoblastic leukemia (ALL), no studies have yet been conducted. PROCEDURE: PLK1 expression and association with biological features were evaluated in 65 consecutively diagnosed childhood ALL samples by quantitative real-time PCR. Moreover, the effects of a specific PLK1 inhibitor, BI 2536, was tested against a panel of nine ALL cell lines at nanomolar concentrations (10, 50, 100 nM). RESULTS: The mRNA expression of PLK1 showed great variability in pediatric ALL, but no difference was evidenced compared to normal bone marrow. Additionally, no association was found between PLK1 mRNA expression with any clinical or biological features. Alternatively, high mRNA expression of PLK1 was present in ALL cell lines. In vitro treatment with BI 2536 strongly diminished growth, while presenting significant reduction in colony formation capacity and increased apoptosis rates. Moreover, strong G2/M arrest was detected suggesting important impaired proliferation after treatment. CONCLUSIONS: PLK1 mRNA expression level is not associated with prognosis in childhood ALL; however, considering the great variability observed in the sample and the in vitro experiments presented herein, BI 2536 treatment might serve as a promising therapeutic to enhance the efficacy of conventional treatment modalities in some childhood ALL cases.

Asunto(s)

Proteínas de Ciclo Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Pteridinas/farmacología , Adolescente , Médula Ósea/metabolismo , Médula Ósea/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/biosíntesis , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Lactante , Células Jurkat , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/biosíntesis , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Estudios Retrospectivos , Tasa de Supervivencia , Quinasa Tipo Polo 1

RESUMEN

BACKGROUND: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. OBJECTIVES: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. METHODS: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. RESULTS: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. CONCLUSIONS: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence.

Asunto(s)

Daño del ADN/genética , Disqueratosis Congénita/genética , Fibroblastos/metabolismo , Ribosomas/fisiología , Telómero/genética , Proteína p53 Supresora de Tumor/genética , Biomarcadores/metabolismo , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Senescencia Celular/fisiología , Disqueratosis Congénita/metabolismo , Disqueratosis Congénita/patología , Fibroblastos/citología , Perfilación de la Expresión Génica , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G 2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and pre-clinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Quinasa Tipo Polo 1

RESUMEN

Despite efforts to improve surgical, radiologic, and chemotherapeutic strategies, the outcome of patients with glioblastoma (GBM) is still poor. Polo-like kinase 1 (PLK1) is a serine/threonine kinase that plays key roles in cell cycle control and has been associated with tumor growth and prognosis. Here, we aimed at testing the radiosensitizing effects of the PLK1 inhibitor BI 2536 on eight GBM cell lines. For cell cycle analysis, T98G, U251, U343 MG-a, LN319, SF188, U138 MG, and U87 MG cell lines were treated with 10, 50, or 100 nM of BI 2536 for 24 hours. In addition, cell cultures exposed to BI 2536 50 nM for 24 hours were irradiated with γ-rays from (60)Cobalt source at final doses of 2, 4, and 6 Gy. Combinatorial effects were evaluated through proliferation and clonogenic capacity assays. Treatment with BI 2536 caused mitotic arrest after 24 hours, and increased apoptosis in GBM cells. Moreover, our results demonstrate that pretreatment with this drug sensitized six out of seven GBM cell lines to different doses of γ-irradiation as shown by decreased growth and abrogation of colony-formation capacity. Our data suggest that PLK1 blockage has a radiosensitizing effect on GBM, which could improve treatment strategies for this devastating tumor.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pteridinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de la radiación , Línea Celular Tumoral , Glioblastoma/enzimología , Glioblastoma/patología , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Quinasa Tipo Polo 1

RESUMEN

Cervical adenocarcinoma is one of the most common gynecological malignancies. Despite the improvements in multimodality treatment, advanced disease is still associated with a significantly poor prognosis making the search for more effective therapeutic agents imperative. BI 2536, an unambiguous inhibitor of Polo-like kinase 1 (PLK1), has shown anticancer activity in a variety of tumor cell types. Herein, we present more evidence of the antiproliferative effects of this drug on HeLa cells. Nanomolar concentrations (10-100 nmol/l) of the drug significantly decreased cell proliferation and clonogenic capacity. Our results also demonstrate that inhibition of PLK1 promoted G2/M arrest and resulted in a dramatic increase in the mitotic index after 24 h of treatment. Apoptosis onset was evinced by the accumulation of a sub-G1 population as well as by a significant increase in caspase-3 activity at longer periods of exposure. Taken together, our results reinforce the prospect of directing against PLK1 as a potential therapeutic target to be evaluated in different preclinical models for cervical carcinoma.

Asunto(s)

Antineoplásicos/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pteridinas/metabolismo , Apoptosis , Ciclo Celular/efectos de los fármacos , Femenino , Células HeLa , Humanos , Quinasa Tipo Polo 1

RESUMEN

Chromosomal instability is a key feature in cancer progression. Recently we have reported that BRCA1 regulates the transcription of several genes in prostate cancer, including ATM (ataxia telangiectasia mutated). Although it is well accepted that ATM is a pivotal mediator in genotoxic stress, it is unknown whether ATM transcription is regulated during the molecular response to DNA damage. Here we investigate ATM transcription regulation in human prostate tumor PC3 cell line. We have found that doxorubicin and mitoxantrone repress ATM transcription in PC3 cells but etoposide and methotrexate do not affect ATM expression. We have demonstrated that BRCA1 binds to ATM promoter and after doxorubicin exposure, it is released. BRCA1 overexpression increases ATM transcription and this enhancement is abolished by BRCA1 depletion. Moreover, BRCA1-BRCT domain loss impairs the ability of BRCA1 to regulate ATM promoter activity, strongly suggesting that BRCT domain is essential for ATM regulation by BRCA1. BRCA1-overexpressing PC3 cells exposed to KU55933 ATM kinase inhibitor showed significant decreased ATM promoter activity compared to untreated cells, suggesting that ATM transcriptional regulation by BRCA1 is partially mediated by the ATM kinase activity. In addition, we have demonstrated E2F1 binding to ATM promoter before and after doxorubicin exposure. E2F1 overexpression diminishes ATM transcription after doxorubicin exposure which is impaired by E2F1 dominant negative mutants. Finally, the co-regulator of transcription CtIP increases ATM transcription. CtIP increases ATM transcription. Altogether, BRCA1/E2F1/CtIP binding to ATM promoter activates ATM transcription. Doxorubicin exposure releases BRCA1 and CtIP from ATM promoter still keeping E2F1 recruited and, in turn, represses ATM expression.

Asunto(s)

Proteína BRCA1/metabolismo , Proteínas Portadoras/metabolismo , Factor de Transcripción E2F1/metabolismo , Proteínas Nucleares/metabolismo , Antibióticos Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/química , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Reparación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Doxorrubicina/farmacología , Endodesoxirribonucleasas , Humanos , Morfolinas/farmacología , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Pironas/farmacología , Transcripción Genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-ß-actin enhancer promoter control] gene plasmid (50 ng/µL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 µm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.

Asunto(s)

Animales Modificados Genéticamente , Bovinos/embriología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Clonación de Organismos/métodos , Fertilización In Vitro , Inhibidores de Proteínas Quinasas/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Bovinos/genética , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos , Femenino , Fertilización In Vitro/efectos de los fármacos , Fertilización In Vitro/veterinaria , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Lactonas/farmacología , Masculino , Sesquiterpenos/farmacología , Transgenes/genética

RESUMEN

Osteosarcoma is the most common primary malignant tumor of bone, which frequently occurs in the second decade of life. Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with chemoresistant or metastatic tumors is still poor. Therefore, there is a need for the development of more efficient therapeutic agents. BI 2536, an innovative selective inhibitor of Polo-like kinase 1, has shown anticancer potential promoting mitotic arrest and apoptosis in a variety of tumor cells, including osteosarcoma. Here, we present more evidence of the antiproliferative effects of BI 2536 on HOS and MG-63 osteosarcoma cell lines. Our results showed that nanomolar concentrations (10, 50, and 100 nmol/l) of the drug significantly decreased cell proliferation and clonogenic capacity, inducing mitotic arrest and aneuploidy. Interestingly, although BI 2536 mediated a moderate increase of apoptosis after 48 h in HOS cells, no increased caspase-3 activity was detected for MG-63 cells. In contrast to previous studies, we show that perturbation of normal mitotic progression by BI 2536 in these osteosarcoma cell lines results in caspase-independent mitotic catastrophe followed by necrosis. Our findings reinforce the likelihood of directing against Polo-like kinase 1 as a therapeutic option in the treatment of osteosarcoma.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pteridinas/farmacología , Antineoplásicos/farmacología , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Inestabilidad Cromosómica , Relación Dosis-Respuesta a Droga , Humanos , Mitosis/efectos de los fármacos , Osteosarcoma/genética , Quinasa Tipo Polo 1

RESUMEN

Inhibition of tyrosine kinase enzyme WEE1 is an important step for the treatment of cancer. The bioactivities of a series of WEE1 inhibitors have been previously modeled through comparative molecular field analyses (CoMFA and CoMSIA), but a two-dimensional image-based quantitative structure-activity relationship approach has shown to be highly predictive for other compound classes. This method, called multivariate image analysis applied to quantitative structure-activity relationship, was applied here to derive quantitative structure-activity relationship models. Whilst the well-known bilinear and multilinear partial least squares regressions (PLS and N-PLS, respectively) correlated multivariate image analysis descriptors with the corresponding dependent variables only reasonably well, the use of wavelet and principal component ranking as variable selection methods, together with least-squares support vector machine, improved significantly the prediction statistics. These recently implemented mathematical tools, particularly novel in quantitative structure-activity relationship studies, represent an important advance for the development of more predictive quantitative structure-activity relationship models and, consequently, new drugs.

Asunto(s)

Antineoplásicos/química , Carbazoles/química , Carbazoles/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirroles/química , Pirroles/farmacología , Algoritmos , Antineoplásicos/farmacología , Inteligencia Artificial , Proteínas de Ciclo Celular/metabolismo , Humanos , Concentración 50 Inhibidora , Análisis de los Mínimos Cuadrados , Análisis Multivariante , Proteínas Nucleares/metabolismo , Análisis de Componente Principal , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-Actividad Cuantitativa , Análisis de Regresión