18 research outputs found
Ocoxin Modulates Cancer Stem Cells and M2 Macrophage Polarization in Glioblastoma
Glioblastoma (GBM) is the most common and devastating primary brain tumor. The presence of cancer stem cells (CSCs) has been linked to their therapy resistance. Molecular and cellular components of the tumor microenvironment also play a fundamental role in the aggressiveness of these tumors. In particular, high levels of hypoxia and reactive oxygen species participate in several aspects of GBM biology. Moreover, GBM contains a large number of macrophages, which normally behave as immunosuppressive tumor-supportive cells. In fact, the presence of both, hypoxia and M2-like macrophages, correlates with malignancy and poor prognosis in gliomas. Antioxidant agents, as nutritional supplements, might have antitumor activity. Ocoxin® oral solution (OOS), in particular, has anti-inflammatory and antioxidant properties, as well as antitumor properties in several neoplasia, without known side effects. Here, we describe how OOS affects stem cell properties in certain GBMs, slowing down their tumor growth. In parallel, OOS has a direct effect on macrophage polarization in vitro and in vivo, inhibiting the protumoral features of M2 macrophages. Therefore, OOS could be a feasible candidate to be used in combination therapies during GBM treatment because it can target the highly resilient CSCs as well as their supportive immune microenvironment, without adding toxicity to conventional treatments.The authors would like to acknowledge Atanasio Pandiella for critical comments on the manuscript, Rosella Galli for donating GBM1, and Jacqueline Gutiérrez, Daniela Moiseev, Daniel Batzan, and Mario Alia for their technical support. RG has been funded by the Fundación Científica Asociación Española Contra el Cáncer. Research has been funded by grants from Fundación Científica Asociación Española Contra el Cáncer (18/006) to JMS; from MINECO: Acción Estratégica en Salud (AES) PI13/01258 to AHL; AES PI17/01621 to JMS; Red Temática de Investigación Cooperativa en Cancer (RTICC) RD12/0036/0027 to AHL, JMS, APN, and PSG; and MINECO-RETOS/FEDER SAF2015-65175 to PSG.S
Targeting the tubulin C-terminal tail by charged small molecules
10 p.-6 fig.The disordered tubulin C-terminal tail (CTT), which possesses a higher degree of heterogeneity, is the target for the interaction of many proteins and cellular components. Compared to the seven well-described binding sites of microtubule-targeting agents (MTAs) that localize on the globular tubulin core, tubulin CTT is far less explored. Therefore, tubulin CTT can be regarded as a novel site for the development of MTAs with distinct biochemical and cell biological properties. Here, we designed and synthesized linear and cyclic peptides containing multiple arginines (RRR), which are complementary to multiple acidic residues in tubulin CTT. Some of them showed moderate induction and promotion of tubulin polymerization. The most potent macrocyclic compound 1f was found to bind to tubulin CTT and thus exert its bioactivity. Such RRR containing compounds represent a starting point for the discovery of tubulin CTT-targeting agents with therapeutic potential.This research was funded by the CAMS Innovation Fund for Medical Sciences (Grant No. 2016-I2M-1-010), State Key Lab Grant type C (Grant No. GTZC201709) (W.-S. F.), Ministerio de Ciencia e Innovación (Grant No. PID2021-123399OB-I00 /AEI/10.13039/501100011033) (M. Á. O.), Ministerio de Ciencia e Innovacion (Grant No. PID2019-104545RB-I00 /AEI/10.13039/501100011033), European Commission-NextGenerationsEU (Regulation EU 2020/2094, through CSIC's Global Health Platform (PTI Salud Global)), Proyecto de Investigación en Neurociencia Fundacion Tatiana Pérez de Guzmán el Bueno 2020 (J. F. D.), the Voelcker Fund (A. R.), the Natural Science Foundation of Guangdong Province of China (Grant No. 2022A1515011419), and the Key Project in Higher Education of Guangdong, China (Grant No. 2022ZDZX2029) (Z. Y.).Peer reviewe
Impact of the clinical context on the 14-3-3 test for the diagnosis of sporadic CJD
BACKGROUND: The 14-3-3 test appears to be a valuable aid for the clinical diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) in selected populations. However, its usefulness in routine practice has been challenged. In this study, the influence of the clinical context on the performance of the 14-3-3 test for the diagnosis of sCJD is investigated through the analysis of a large prospective clinical series. METHODS: Six hundred seventy-two Spanish patients with clinically suspected sCJD were analyzed. Clinical classification at sample reception according to the World Health Organization's (WHO) criteria (excluding the 14-3-3 test result) was used to explore the influence of the clinical context on the pre-test probabilities, and positive (PPV) and negative (NPV) predictive values of the 14-3-3 test. RESULTS: Predictive values of the test varied greatly according to the initial clinical classification: PPV of 98.8%, 96.5% and 45.0%, and NPV of 26.1%, 66.6% and 100% for probable sCJDi (n = 115), possible sCJDi (n = 73) and non-sCJDi (n = 484) cases, respectively. According to multivariate and Bayesian analyses, these values represent an improvement of diagnostic certainty compared to clinical data alone. CONCLUSION: In three different contexts of sCJD suspicion, the 14-3-3 assay provides useful information complementary to clinical and electroencephalographic (EEG) data. The test is most useful supporting a clinical impression, whilst it may show deceptive when it is not in agreement with clinical data
IDP-410: a novel therapeutic peptide that alters N-MYC stability and reduces angiogenesis and tumor progression in glioblastomas
13 p.-7 fig.Glioblastomas (GBMs) are the most frequent and highly aggressive brain tumors, being resistant to all cytotoxic and molecularly targeted agents tested so far. There is, therefore, an urgent need to find novel therapeutic approaches and/or alternative targets to bring treatment options to patients. Here, we first show that GBMs express high levels of N-MYC protein, a transcription factor involved in normal brain development. A novel stapled peptide designed to specifically target N-MYC protein monomer, IDP-410, is able to impair the formation of N-MYC/MAX complex and reduce the stability of N-MYC itself. As a result, the viability of GBM cells is compromised. Moreover, the efficacy is found dependent on the levels of expression of N-MYC. Finally, we demonstrate that IDP-410 reduces GBM growth in vivo when administered systemically, both in subcutaneous and intracranial xenografts, reducing the vascularization of the tumors, highlighting a potential relationship between the function of N-MYC and the expression of mesenchymal/angiogenic genes. Overall, our results strengthen the view of N-MYC as a therapeutic target in GBM and strongly suggest that IDP-410 could be further developed to become a first-in-class inhibitor of N-MYC protein, affecting not only tumor cell proliferation and survival, but also the interplay between GBM cells and their microenvironment.This work was supported by Ministerio de Economía y Competitividad: (Acción Estratégica en Salud) and FEDER funds: PI16/01550 to JMS, by “Asociación Española contra el Cancer” grants: INVES192GARG to RG, GCTRA16015SEDA to JMS, and IDEAS20095SÁNC to PSG, and by Ministerio de Ciencia, Innovación y Universidades and FEDER funds: RTI2018-093596 to PSG. MGA was supported by the Young Employment Initiative (Comunidad de Madrid).Peer reviewe
Apolipoprotein E genotyping method by real time PCR, a fast and cost-effective alternative to the TaqMan and FRET assays
The apolipoprotein E gene (APOE) polymorphism genotyping has an allegedly important predictive value
for coronary heart disorders and Alzheimer’s disease. We developed a simple, fast, cost-effective and
suited for high-throughput protocol for determining APOE genotypes by Real Time PCR monitored by
SYBR® Green. The method is based on differential amplification by allele-specific primers. These primers
have variations in their 3 -end nucleotides such that are specific for one of the two variants in each polymorphic
position. By this protocol,we obtained a 100% concordance with the APOE genotypes determined
by sequencing analysis. The main advantages of this method are its relative simplicity and the reduced
cost compared to other methodologies, such as the TaqMan® and FRET assays.Fundingwas provided by grants FIS 05/0912 from the Ministerio
de Sanidad, CIEN Foundation and the Spanish CIBERNED network.Peer reviewe
BMP and WNT signalling cooperate through LEF1 in the neuronal specification of adult hippocampal neural stem and progenitor cells.
14 Pages, 8 Figures. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-27581-0.Neuronal production from neural stem cells persists during adulthood in the subgranular zone of the hippocampal dentate gyrus. Extracellular signals provided by the hippocampal microenvironment regulate the neuronal fate commitment of the stem cell progeny. To date, the identity of those signals and their crosstalk has been only partially resolved. Here we show that adult rat hippocampal neural stem and progenitor cells (AH-NSPCs) express receptors for bone morphogenetic proteins (BMPs) and that the BMP/P-Smad pathway is active in AH-NSPCs undergoing differentiation towards the neuronal lineage. In vitro, exposure to the BMP2 and BMP4 ligands is sufficient to increase neurogenesis from AH-NSPCs in a WNT dependent manner while decreasing oligodendrogenesis. Moreover, BMP2/4 and WNT3A, a key regulator of adult hippocampal neurogenesis, cooperate to further enhance neuronal production. Our data point to a mechanistic convergence of the BMP and WNT pathways at the level of the T-cell factor/lymphoid enhancer factor gene Lef1. Altogether, we provide evidence that BMP signalling is an important regulator for the neuronal fate specification of AH-NSPCs cultures and we show that it significantly cooperates with the previously described master regulator of adult hippocampal neurogenesis, the WNT signalling pathway.This work was supported by the Spanish Ministerio de Economía y Competitividad (grant number SAF2015-70433-R).Peer reviewe
Ocoxin Modulates Cancer Stem Cells and M2 Macrophage Polarization in Glioblastoma
Glioblastoma (GBM) is the most common and devastating primary brain tumor. The presence of cancer stem cells (CSCs) has been linked to their therapy resistance. Molecular and cellular components of the tumor microenvironment also play a fundamental role in the aggressiveness of these tumors. In particular, high levels of hypoxia and reactive oxygen species participate in several aspects of GBM biology. Moreover, GBM contains a large number of macrophages, which normally behave as immunosuppressive tumor-supportive cells. In fact, the presence of both, hypoxia and M2-like macrophages, correlates with malignancy and poor prognosis in gliomas. Antioxidant agents, as nutritional supplements, might have antitumor activity. Ocoxin® oral solution (OOS), in particular, has anti-inflammatory and antioxidant properties, as well as antitumor properties in several neoplasia, without known side effects. Here, we describe how OOS affects stem cell properties in certain GBMs, slowing down their tumor growth. In parallel, OOS has a direct effect on macrophage polarization in vitro and in vivo, inhibiting the protumoral features of M2 macrophages. Therefore, OOS could be a feasible candidate to be used in combination therapies during GBM treatment because it can target the highly resilient CSCs as well as their supportive immune microenvironment, without adding toxicity to conventional treatments
Design, Synthesis, and in vitro Evaluation of Tubulin-Targeting Dibenzothiazines with Antiproliferative Activity as a Novel Heterocycle Building Block
We prepared a series of free NH and N-substituted dibenzonthiazines with potential anti-tumor activity from N-aryl-benzenesulfonamides. A biological test of synthesized compounds (59 samples) was performed in vitro measuring their antiproliferative activity against a panel of six human solid tumor cell lines and its tubulin inhibitory activity. We identified 6-(phenylsulfonyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 6-tosyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide as the best compounds with promising values of activity (overall range of 2–5.4 μM). Herein, we report the dibenzothiazine core as a novel building block with antiproliferative activity, targeting tubulin dynamics.Fil: Guerra, Walter Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Lucena Agell, Daniel. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Hortigüela, Rafael. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Rossi, Roberto Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Diaz, J. Fernando. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Padrón, Jose M.. Universidad de la Laguna. Departamento de Química Orgánica. Instituto Universitario de Bio-Orgánica "Antonio González"; EspañaFil: Barolo, Silvia Maricel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; Argentin
Probing a distinct druggable tubulin binding site with gatorbulins 1–7, their metabolic and physicochemical properties, and pharmacological consequences
13 p.-8 fig.-7 tab.Microtubules, consisting of α/β-tubulin heterodimers, are prime targets for anticancer drug discovery.
Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh
pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards
GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2–7 (1b–1g) of this highly modified
cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and β-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK.This work was supported by the National Institutes of Health, NCI grant R01CA172310 (H.L.), the Debbie and Sylvia DeSantis Chair professorship(H.L.) and Ministerio de Ciencia e Innovación PID2021-123399OB-I00 (M.A.O.) PID2019-104545RB-I00 (J.F.D.). We thank Ganadería Fernando Díaz for calf brains supply.Peer reviewe
Synthesis and Structure-Activity Relationship Studies of Dioxane- and Oxathiane-Based Analogs of (–)-Zampanolide
We have prepared a series of analogs of the complex marine macrolide (–)-zampanolide, which incorporate a dioxane-, oxathiane-, or oxathiane-dioxide ring in place of the natural tetrahydropyran moiety and we have determined their microtubule-binding affinity and antiproliferative activity against human cancer cells. The synthesis of these analogs was based on a convergent strategy with a HWE-based macrocyclization and a stereoselective aza-aldol reaction as key steps. The microtubule-binding affinity and cellular potency of the dioxane- and oxathiane-based analogs with a natural (Z,E)-sorbamide-based side chain were essentially indistinguishable from those of natural (–)-zampanolide; changing the configuration of the sorbamide unit from Z,E to E,E resulted in a slight loss in activity. In contrast, the presence of an oxathiane-dioxide ring caused a steep decrease in microtubule-binding and a significant loss in growth inhibitory activity. In addition, a substantial loss in potency was observed against a multidrug-resistant, P-glycoprotein-overexpressing cell line, while no such effect was found for the dioxane- or oxathiane-based analogs. A high-resolution X-ray crystal structure of the complex between beta-tubulin and dioxane-zampanolide was obtained, which showed that this compound, like natural (–)-zampanolide, induces helical structuring of the M-loop