Revistas
Autores:
Burgos, Miguel; Cavero-Redondo, I.; Álvarez-Bueno, C.; et al.
Revista:
THERAPEUTIC ADVANCES IN MEDICAL ONCOLOGY
ISSN:
1758-8340
Año:
2022
Vol.:
14
Págs.:
17588359211072621
Background: Identification of membrane proteins differentially expressed on tumor cells is a key step in drug development. The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a cell adhesion protein belonging to the immunoglobulin superfamily. Here, we explore the prognostic role CEACAM6 expression on patient outcome in cancer. Methods: A systematic search for studies evaluating the association between tumor expression of CEACAM6 and overall survival (OS) and disease-free survival (DFS) was performed. Hazard ratios (HR) were pooled in a meta-analysis using generic inverse variance and random effect modeling. Subgroup analyses were conducted based on tumor type and method of HR extraction. Results: Sixteen studies met the inclusion criteria. CEACAM6 expression was associated with worse OS [HR = 1.96, 95% confidence interval (CI) = 1.51-2.53], and DFS (HR = 2.49, 95% CI = 2.01-3.07) with subgroup analysis showing no significant differences between disease site subgroups. Conclusions: High expression of CEACAM6 is associated with worse OS and DFS in different malignancies. CEACAM6 is a target for the future development of novel therapeutics.
Autores:
Noblejas-López, M. M.; Nieto-Jiménez, C.; Gálan-Moya, E. M.; et al.
Revista:
JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH
ISSN:
1756-9966
Año:
2021
Vol.:
40
N°:
1
Págs.:
106
Background: Although the anti-HER2 antibody trastuzumab augments patient survival in HER2+ breast cancer, a relevant number of patients progress to this treatment. In this context, novel drug combinations are needed to increase its antitumor activity. In this work, we have evaluated the efficacy of proteolysis targeting chimera (PROTAC) compounds based on BET inhibitors (BETi) to augment the activity of trastuzumab in HER2+ breast cancer models.
Methods: BT474 and SKBR3 HER2+ breast cancer cell lines were used. The effects of trastuzumab and the BET-PROTAC MZ1 either alone or in combination, were evaluated using MTT proliferation assays, three-dimensional invasion and adhesion cultures, flow cytometry, qPCR and Western blot. In vivo studies were carried out in a xenografted model in mice. Finally, a Clariom_S_Human transcriptomic array was applied to identify deregulated genes after treatments.
Results: MZ1 induced a higher antiproliferative effect compared to the BETi JQ1. The combination of MZ1 and -trastuzumab significantly decreased cell proliferation, the formation of three-dimensional structures and cellular invasion compared to either of the drugs alone. Evaluation of apoptosis resulted in an increase of cell death following treatment with the combination, and biochemical studies displayed modifications of apoptosis and DNA damage components. In vivo administration of agents alone or combined, to tumors orthotopically xenografted in mice, resulted in a decrease of the tumor volume only after MZ1-Trastuzumab combination treatment. Results from a transcriptomic array indicated a series of newly described transcription factors including HOXB7, MEIS2, TCERG1, and DNAJC2, that were associated to poor outcome in HER2+ breast cancer subtype and downregulated by the MZ1-trastuzumab combination.
Conclusions: We describe an active novel combination that includes the BET-PROTAC MZ1 and trastuzumab, in HER2+ tumors. Further studies should be performed to confirm these findings and pave the way for their future clinical development.
Revista:
JOURNAL OF CELL SCIENCE
ISSN:
0021-9533
Año:
2021
Vol.:
134
N°:
3
Págs.:
jcs244012
Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile a-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event wasmediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.
Autores:
Nieto-Jiménez, C.; Alcaraz-Sanabria, A.; Martínez-Canales, S.; et al.
Revista:
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
ISSN:
1422-0067
Año:
2020
Vol.:
21
N°:
23
Págs.:
9034
Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.
Revista:
FOODS
ISSN:
2304-8158
Año:
2020
Vol.:
9
N°:
11
Págs.:
1648
Saffron, as a food colorant, has been displaced by low-cost synthetic dyes. These have unhealthy properties; thus, their replacement with natural food colorants is an emerging trend. Obesity is a worldwide health problem due to its associated comorbidities. Crocetin esters (crocins) are responsible for the red saffron color. Crocetin (CCT) exhibits healthful properties. We aimed to broaden the existing knowledge on the health properties of CCT isolated from saffron, to facilitate its consideration as a healthy natural food colorant in the future. We evaluated the ability of CCT (1 and 5 mu M) to reduce lipid accumulation during the differentiation of 3T3-L1 preadipocytes. Intracellular fat was quantified by Oil Red O staining. CTT cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The number and size of lipid droplets were analyzed using WimLipid software. The expression of adipogenic genes (CCAAT/enhancer-binding protein (C/EBP beta, C/EBP delta, C/EBP alpha), and peroxisome proliferator-activated receptor gamma (PPAR gamma)) was analyzed using quantitative real-time PCR (qRT-PCR). CCT 5 mu M decreased intracellular fat by 22.6%, without affecting viability or lipid droplet generation, via a decrease in C/EBP alpha expression, implicated in lipid accumulation. Thus, CCT is a potential candidate to be included in dietary therapies aimed at reversing adipose tissue accumulation in obesity.
Autores:
Serrano-Oviedo, L.; Nuncia-Cantarero, M.; Morcillo-García, S.; et al.
Revista:
CELLULAR ONCOLOGY
ISSN:
2211-3436
Año:
2020
Vol.:
43
N°:
3
Págs.:
431 - 444
urpose: Triple negative breast cancers (TNBCs) are enriched in cells bearing stem-like features, i.e., cancer stem cells (CSCs), which underlie cancer progression. Thus, targeting stemness may be an interesting treatment approach. The epigenetic machinery is crucial for maintaining the stemness phenotype. Bromodomain and extra-terminal domain (BET) epigenetic reader family members are emerging as novel targets for cancer therapy, and have already shown preclinical effects in breast cancer. Here, we aimed to evaluate the effect of the BET inhibitor JQ1 on stemness in TNBC.
Methods: Transcriptomic, functional annotation and qRT-PCR studies were performed on JQ1-exposed TNBC cells in culture. The results obtained were confirmed in spheroids and spheroid-derived tumours. In addition, limiting dilution, secondary and tertiary tumour sphere formation, matrigel invasion, immunofluorescence and flow cytometry assays were performed to evaluate the effect of JQ1 on CSC features. For clinical outcome analyses, the online tool Kaplan-Meier Plotter and an integrated response database were used.
Results: We found that JQ1 modified the expression of stemness-related genes in two TNBC-derived cell lines, MDA-MB-231 and BT549. Among these changes, the CD44 Antigen/CD24 Antigen (CD44/CD24) ratio and Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) expression level, i.e., both classical stemness markers, were found to be decreased by JQ1. Using a validated spheroid model to mimic the intrinsic characteristics of CSCs, we found that JQ1 decreased surface CD44 expression, inhibited self-renewal and invasion, and induced cell cycle arrest in G0/G1, thereby altering the stemness phenotype. We also found associations between four of the identified stemness genes, Gap Junction Protein Alpha 1 (GJA1), CD24, Epithelial Adhesion Molecule (EPCAM) and SRY-related HMG-box gene 9 (SOX9), and a worse TNBC patient outcome. The expression of another two of the stemness-related genes was found to be decreased by JQ1, i.e., ATP Binding Cassette Subfamily G Member 2 (ABCG2) and RUNX2, and predicted a low response to chemotherapy in TNBC patients, which supports a role for RUNX2 as a potential predictive marker for chemotherapy response in TNBC.
Conclusions: We identified a stemness-related gene panel associated with JQ1 and describe how this inhibitor modifies the stemness landscape in TNBC. Therefore, we propose a novel role for JQ1 as a stemness-targeting drug. Loss of the stem cell phenotype via JQ1 treatment could lead to less aggressive and more chemo-sensitive tumours, reflecting a better patient prognosis. Thus, the identified gene panel may be of interest for the clinical management of patients with aggressive TNBC.
Autores:
Nieto-Jiménez, C.; Galán-Moya, E. M. (Autor de correspondencia); Corrales-Sánchez, V.; et al.
Revista:
CANCER LETTERS
ISSN:
1872-7980
Año:
2020
Vol.:
491
Págs.:
50 - 59
The inhibition of bromo- and extraterminal domains (BET) has shown an anti-proliferative effect in triple negative breast cancer (TNBC). In this article we explore mechanisms of resistance to BET inhibitors (BETi) in TNBC, with the aim of identifying novel ways to overcome such resistance. Two cellular models of acquired resistance to the BET inhibitor JQ1 were generated using a pulsed treatment strategy. MTT, colony formation, and cytometry assays revealed that BETi-resistant cells were particularly sensitive to PLK1 inhibition. Targeting of the latter reduced cell proliferation, especially in resistant cultures. Quantitative PCR analysis of a panel of mitotic kinases uncovered an increased expression of AURKA, TTK, and PLK1, confirmed by Western blot. Only pharmacological inhibition of PLK1 showed anti-proliferative activity on resistant cells, provoking G2/M arrest, increasing expression levels of cyclin B, pH3 and phosphorylation of Bcl-2 proteins, changes that were accompanied by induction of caspase-dependent apoptosis. JQ1-resistant cells orthotopically xenografted into the mammary fat pad of mice led to tumours that retained JQ1-resistance. Administration of the PLK1 inhibitor volasertib resulted in tumour regression. These findings open avenues to explore the future use of PLK1 inhibitors in the clinical setting of BETi-resistant patients.
Autores:
Corrales-Sánchez, V.; Noblejas-López, M. M.; Nieto-Jiménez, C.; et al.
Revista:
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
ISSN:
1582-1838
Año:
2020
Vol.:
24
N°:
5
Págs.:
3117 - 3127
Identification of druggable vulnerabilities is a main objective in triple-negative breast cancer (TNBC), where no curative therapies exist. Gene set enrichment analyses (GSEA) and a pharmacological evaluation using a library of compounds were used to select potential druggable combinations. MTT and studies with semi-solid media were performed to explore the activity of the combinations. TNBC cell lines (MDAMB-231, BT549, HS-578T and HCC3153) and an additional panel of 16 cell lines were used to assess the activity of the two compounds. Flow cytometry experiments and biochemical studies were also performed to explore the mechanism of action. GSEA were performed using several data sets (GSE21422, GSE26910, GSE3744, GSE65194 and GSE42568), and more than 35 compounds against the identified functions were evaluated to discover druggable opportunities. Analyses done with the Chou and Talalay algorithm confirmed the synergy of dasatinib and olaparib. The combination of both agents significantly induced apoptosis in a caspase-dependent manner and revealed a pleotropic effect on cell cycle: Dasatinib arrested cells in G0/G1 and olaparib in G2/M. Dasatinib inhibited pChk1 and induced DNA damage measured by pH2AX, and olaparib increased pH3. Finally, the effect of the combination was also evaluated in a panel of 18 cell lines representative of the most frequent solid tumours, observing a particularly synergism in ovarian cancer. Breast cancer, triple negative, dasatinib, olaparib, screening.
Autores:
Noblejas-López, M. M.; Nieto-Jiménez, C.; Burgos, Miguel; et al.
Revista:
JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH
ISSN:
1756-9966
Año:
2019
Vol.:
38
N°:
1
Págs.:
383
Background Triple negative breast cancer (TNBC) is an incurable disease where novel therapeutic strategies are needed. Proteolysis targeting chimeric (PROTAC) are novel compounds that promote protein degradation by binding to an ubiquitin ligase. In this work, we explored the antitumoral activity of two novel BET-PROTACs, MZ1 and ARV-825, in TNBC, ovarian cancer and in a BET inhibitor resistant model. Methods OVCAR3, SKOV3, BT549, MDA-MB-231 cell lines and the JQ1 resistant cell line MDA-MB-231R were evaluated. MTTs, colony-forming assay, three-dimensional cultures in matrigel, flow cytometry, and western blots were performed to explore the anti-proliferative effect and biochemical mechanism of action of MZ1 and ARV-825. In vivo studies included BALB/c nu/nu mice engrafted with MDA-MB-231R cells. Results The BET-PROTACs MZ1 and ARV-825 efficiently downregulated the protein expression levels of the BET protein BRD4, in MDA-MB-231 and MDA-MB-231R. MZ1 and ARV-825 also showed an antiproliferative effect on sensitive and resistant cells. This effect was corroborated in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR3) cell lines. MZ1 provoked G2/M arrest in MDA-MB-231. In addition, a profound effect on caspase-dependent apoptosis was observed in both sensitive and resistant cells. No synergistic activity was observed when it was combined with docetaxel, cisplatin or olaparib. Finally, in vivo administration of MZ1 rescued tumor growth in a JQ1-resistant xenograft..
Revista:
JOURNAL FOR IMMUNOTHERAPY OF CANCER
ISSN:
2051-1426
Año:
2019
Vol.:
7
N°:
1
Págs.:
111
Background: Dysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study.
Methods: An extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients.
Results: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLC¿2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup.
Conclusions: These data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.
Autores:
Niza, E.; Noblejas-López, M. M. ; Bravo, I.; et al.
Revista:
NANOMATERIALS
ISSN:
2079-4991
Año:
2019
Vol.:
9
N°:
12
Págs.:
1793
Dasatinib (DAS) is a multikinase inhibitor that acts on several signaling kinases. DAS is used as a second-line treatment for chronic accelerated myeloid and Philadelphia chromosome-positive acute lymphoblastic leukemia. The therapeutic potential of DAS in other solid tumours is under evaluation. As for many other compounds, an improvement in their pharmacokinetic and delivery properties would potential augment the efficacy. Antibody-targeted biodegradable nanoparticles can be useful in targeted cancer therapy. DAS has shown activity in human epidermal growth factor receptor 2 (HER2) positive tumors, so conjugation of this compound with the anti-HER2 antibody trastuzumab (TAB) with the use of nanocarriers could improve its efficacy. TAB-targeted DAS-loaded nanoparticles were generated by nanotechnology. The guided nanocarriers enhanced in vitro cytotoxicity of DAS against HER2 human breast cancer cell lines. Cellular mechanistic, release studies and nanoparticles stability were undertaken to provide evidences for positioning DAS-loaded TAB-targeted nanoparticles as a potential strategy for further development in HER2-overexpressing breast cancer therapy.
Autores:
Vicente, M.; Salgado-Almario, J.; Soriano, J.; et al.
Revista:
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
ISSN:
1422-0067
Año:
2019
Vol.:
20
N°:
21
Págs.:
5409
Mitochondria are believed to play an important role in shaping the intracellular Ca2+ transients during skeletal muscle contraction. There is discussion about whether mitochondrial matrix Ca2+ dynamics always mirror the cytoplasmic changes and whether this happens in vivo in whole organisms. In this study, we characterized cytosolic and mitochondrial Ca2+ signals during spontaneous skeletal muscle contractions in zebrafish embryos expressing bioluminescent GFP-aequorin (GA, cytoplasm) and mitoGFP-aequorin (mitoGA, trapped in the mitochondrial matrix). The Ca2+ transients measured with GA and mitoGA reflected contractions of the trunk observed by transmitted light. The mitochondrial uncoupler FCCP and the inhibitor of the mitochondrial calcium uniporter (MCU), DS16570511, abolished mitochondrial Ca2+ transients whereas they increased the frequency of cytosolic Ca2+ transients and muscle contractions, confirming the subcellular localization of mitoGA. Mitochondrial Ca2+ dynamics were also determined with mitoGA and were found to follow closely cytoplasmic changes, with a slower decay. Cytoplasmic Ca2+ kinetics and propagation along the trunk and tail were characterized with GA and with the genetically encoded fluorescent Ca2+ indicator, Twitch-4. Although fluorescence provided a better spatio-temporal resolution, GA was able to resolve the same kinetic parameters while allowing continuous measurements for hours.
Autores:
Garaud, S.; Taher, T. E.; Debant, M.; et al.
Revista:
CELLULAR AND MOLECULAR IMMUNOLOGY
ISSN:
1672-7681
Año:
2018
Vol.:
15
N°:
2
Págs.:
158 - 170
CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.
Autores:
Alcaraz-Sanabria, A.; Nieto-Jiménez, C.; Corrales-Sánchez, V.; et al.
Revista:
MOLECULAR CANCER THERAPEUTICS
ISSN:
1535-7163
Año:
2017
Vol.:
16
N°:
11
Págs.:
2552 - 2562
Ovarian cancer is characterized by frequent mutations at TP53. These tumors also harbor germline mutations at homologous recombination repair genes, so they rely on DNA-damage checkpoint proteins, like the checkpoint kinase 1 (CHEK1) to induce G2 arrest. In our study, by using an in silico approach, we identified a synthetic lethality interaction between CHEK1 and mitotic aurora kinase A (AURKA) inhibitors. Gene expression analyses were used for the identification of relevant biological functions. OVCAR3, OVCAR8, IGROV1, and SKOV3 were used for proliferation studies. Alisertib was tested as AURKA inhibitor and LY2603618 as CHEK1 inhibitor. Analyses of cell cycle and intracellular mediators were performed by flow cytometry and Western blot analysis. Impact on stem cell properties was evaluated by flow cytometry analysis of surface markers and sphere formation assays. Gene expression analyses followed by functional annotation identified a series of deregulated genes that belonged to cell cycle, including AURKA/B, TTK kinase, and CHEK1. AURKA and CHEK1 were amplified in 8.7% and 3.9% of ovarian cancers, respectively. AURKA and CHEK1 inhibitors showed a synergistic interaction in different cellular models. Combination of alisertib and LY2603618 triggered apoptosis, reduced the stem cell population, and increased the effect of taxanes and platinum compounds. Finally, expression of AURKA and CHEK1 was linked with detrimental outcome in patients. Our data describe a synthetic lethality interaction between CHEK1 and AURKA inhibitors with potential translation to the clinical setting.
Autores:
Nieto-Jiménez, C.; Alcaraz-Sanabria, A.; Pérez-Peña, J.; et al.
Revista:
ONCOTARGET
ISSN:
1949-2553
Año:
2017
Vol.:
8
N°:
12
Págs.:
19478 - 19490
Metastatic triple negative breast cancer (TNBC) is an incurable disease with limited therapeutic options, and no targeted therapies available. Triple negative tumors and the basal-like genomic subtype, are both characterized by a high proliferation rate and an increase in cell division. In this context, protein kinases involved in the mitotic formation have a relevant role in this tumor subtype. Recently, Bromodomain and extraterminal domain (BET) inhibitors have shown to be active in this disease by modulating the expression of several transcription factors. In this article, by using an "in silico" approach, we identified genomic functions that can be inhibited pharmacologically in basal-like tumors. Functional annotation analyses identified "cell division" and "regulation of transcription" as upregulated functions. When focus on cell division, we identified the polo-like kinase 1 (PLK) as an upregulated kinase. The PLK inhibitor Volasertib had the strongest anti-proliferative effect compared with other inhibitors against mitotic kinases. Gene expression analyses demonstrated that the BET inhibitor JQ1 reduced the expression of kinases involved in cell division, and synergized with Volasertib in a panel of triple negative cell lines. Combination of both agents augmented cell death. Similarly, combination of both compounds reduced the expression of stem cell markers. Globally, this data demonstrates the synergistic interaction between BET and PLK inhibitors, paving the way for their future clinical development.
Revista:
AMINO ACIDS
ISSN:
1438-2199
Año:
2011
Vol.:
40
N°:
2
Págs.:
641 - 651
Astrocytes are glial cells in the central nervous system (CNS) that play key roles in brain physiology, controlling processes, such as neurogenesis, brain energy metabolism and synaptic transmission. Recently, immune functions have also been demonstrated in astrocytes, influencing neuronal survival in the course of neuroinflammatory pathologies. In this regard, PKCepsilon (PKC¿) is a protein kinase with an outstanding role in inflammation. Our previous findings indicating that PKC¿ regulates voltage-dependent calcium channels as well as morphological stellation imply that this kinase controls multiple signalling pathways within astrocytes, including those implicated in activation of immune functions. The present study applies proteomics to investigate new protein targets of PKC¿ in astrocytes. Primary astrocyte cultures infected with an adenovirus that expresses constitutively active PKC¿ were compared with infection controls. Two-dimensional gel electrophoresis clearly detected 549 spots in cultured astrocytes, and analysis of differential protein expression revealed 18 spots regulated by PKC¿. Protein identification by mass spectrometry (nano-LC-ESI-MS/MS) showed that PKC¿ targets molecules with heterogeneous functions, including chaperones, cytoskeletal components and proteins implicated in metabolism and signalling. These results support the notion that PKC¿ is involved in astrocyte activation; also suggesting that multiple astrocyte-dependent processes are regulated by PKC¿, including those associated to neuroinflammation.
Revista:
JOURNAL OF NEUROSCIENCE RESEARCH
ISSN:
0360-4012
Año:
2010
Vol.:
88
N°:
5
Págs.:
1094 - 1105
Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress-dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase-11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress-dependent astrocyte death in ischemic conditions. According to our results, caspase-11 is up-regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER-stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase-11 mRNA levels and luciferase activity driven by a caspase-11 promoter, indicating that caspase-11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress-associated CHOP in caspase-11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase-11. In turn, caspase-11 suppression with siRNA rescued astrocytes from OGID- and ER stress-induced death, supporting the idea that caspase-11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase-1 and caspase-3 significantly reduced astrocyte death, which indicates that these proteases ...
Revista:
AMINO ACIDS
ISSN:
0939-4451
Año:
2010
Vol.:
38
N°:
4
Págs.:
1131 - 1143
Synapsin 2 proteins are key elements of the synaptic machinery and still hold the centre stage in neuroscience research. Although fully sequenced at the nucleic acid level in mouse and rat, structural information on amino acid sequences and post-translational modifications (PTMs) is limited. Knowledge on protein sequences and PTMs, however, is mandatory for several purposes including conformational studies and the generation of antibodies. Hippocampal proteins from rat and mouse were extracted, run on two-dimensional gel electrophoresis and multi-enzyme digestion was carried out to generate peptides for mass spectrometrical analysis [nano-LC-ESI-(CID/ETD)-MS/MS]. As much as 12 synapsin 2 proteins (6 alpha and 6 beta isoforms) in the mouse and 13 synapsin 2 proteins (6 alpha and 7 beta isoforms) were observed in the rat. Protein sequences were highly identical to nucleic acid sequences, and only few amino acid exchanges probably representing polymorphisms or sequence conflicts were detected. Mouse and rat synapsins 2a differed in three amino acids, while rat and mouse synapsins 2b differed in two amino acids. As much as 13 phosphorylation sites were determined by MS/MS data in rat and mouse hippocampus and 5 were verified by phosphatase treatment. These findings are important for interpretation of previous results and design of future studies on synapsins.
