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1.
Cell Rep Med ; 5(6): 101593, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38843842

ABSTRACT

Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated in vivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.


Subject(s)
Aging , Cyclic AMP-Dependent Protein Kinases , Diet, Ketogenic , Long-Term Potentiation , Memory , Proteome , Signal Transduction , Animals , Cyclic AMP-Dependent Protein Kinases/metabolism , Aging/physiology , Aging/metabolism , Diet, Ketogenic/methods , Proteome/metabolism , Mice , Male , Memory/physiology , Long-Term Potentiation/physiology , Mice, Inbred C57BL , Hippocampus/metabolism , Synapses/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Neuronal Plasticity/physiology , Phosphorylation
2.
Aging Cell ; 22(5): e13814, 2023 05.
Article in English | MEDLINE | ID: mdl-36973898

ABSTRACT

Age is the main risk factor for the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction, and cognitive disabilities in humans, primates, rodents, and invertebrates. Necroptosis mediates degeneration of injured axons, but whether necroptosis triggers neurodegeneration and cognitive impairment along aging is unknown. Here, we show that the loss of the necroptotic effector Mlkl was sufficient to delay age-associated axonal degeneration and neuroinflammation, protecting against decreased synaptic transmission and memory decline in aged mice. Moreover, short-term pharmacologic inhibition of necroptosis targeting RIPK3 in aged mice, reverted structural and functional hippocampal impairment, both at the electrophysiological and behavioral level. Finally, a quantitative proteomic analysis revealed that necroptosis inhibition leads to an overall improvement of the aged hippocampal proteome, including a subclass of molecular biofunctions associated with brain rejuvenation, such as long-term potentiation and synaptic plasticity. Our results demonstrate that necroptosis contributes to age-dependent brain degeneration, disturbing hippocampal neuronal connectivity, and cognitive function. Therefore, necroptosis inhibition constitutes a potential geroprotective strategy to treat age-related disabilities associated with memory impairment and cognitive decline.


Subject(s)
Necroptosis , Neurodegenerative Diseases , Humans , Mice , Animals , Aged , Proteomics , Rejuvenation , Aging/physiology , Brain , Memory Disorders
3.
EMBO J ; 41(22): e111952, 2022 11 17.
Article in English | MEDLINE | ID: mdl-36314651

ABSTRACT

Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.


Subject(s)
Aging , Brain , Protein Serine-Threonine Kinases , Unfolded Protein Response , X-Box Binding Protein 1 , Animals , Mice , Aging/genetics , Brain/metabolism , Endoplasmic Reticulum Stress/genetics , Protein Serine-Threonine Kinases/genetics , Proteomics , Signal Transduction/physiology , X-Box Binding Protein 1/genetics , X-Box Binding Protein 1/metabolism
4.
Angew Chem Int Ed Engl ; 60(20): 11278-11282, 2021 05 10.
Article in English | MEDLINE | ID: mdl-33751770

ABSTRACT

The scalable synthesis of the oxaquinolizidine marine natural product desmethylxestospongin B is based on the early application of Ireland-Claisen rearrangement, macrolactamization, and a late-stage installation of the oxaquinolizidine units by lactam reduction. The synthesis serves as the source of material to investigate calcium signaling and its effect on mitochondrial metabolism in various cell types, including cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Biological Products/pharmacology , Inositol 1,4,5-Trisphosphate Receptors/antagonists & inhibitors , Mitochondria/drug effects , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Humans , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mitochondria/metabolism , Molecular Structure
5.
Sci Signal ; 13(640)2020 07 14.
Article in English | MEDLINE | ID: mdl-32665411

ABSTRACT

Spontaneous Ca2+ signaling from the InsP3R intracellular Ca2+ release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca2+ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP3R activity or mitochondrial Ca2+ uptake increased α-ketoglutarate (αKG) abundance and the NAD+/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)-to-mitochondria Ca2+ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca2+ inhibited αKGDH activity and increased NAD+, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca2+ from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca2+ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca2+ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.


Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/metabolism , Mitochondria/metabolism , Neoplasms/metabolism , Oxidative Phosphorylation , Cell Line, Tumor , Cell Survival , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/pathology , Humans , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/genetics , Neoplasms/pathology
6.
Mol Oncol ; 14(2): 347-362, 2020 02.
Article in English | MEDLINE | ID: mdl-31788944

ABSTRACT

Endothelin-1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin-1 rely on its activation by endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N-terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R ) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self-renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2-dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC-like traits. Therefore, phospho-ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.


Subject(s)
Carcinogenesis/metabolism , Colorectal Neoplasms/metabolism , Endothelin-Converting Enzymes/metabolism , Neoplastic Stem Cells/metabolism , Animals , Carcinogenesis/genetics , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Casein Kinase II/metabolism , Cell Line, Tumor , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Endothelin-Converting Enzymes/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Mutation , Naphthyridines/pharmacology , Neoplasm Invasiveness/genetics , Neoplasm Metastasis/genetics , Phenazines/pharmacology , Phosphorylation , Prognosis , Protein Stability , Recombinant Proteins , Up-Regulation , Xenograft Model Antitumor Assays
7.
Biochem Pharmacol ; 171: 113714, 2020 01.
Article in English | MEDLINE | ID: mdl-31738894

ABSTRACT

Brain tumours are among the deadliest tumours being highly resistant to currently available therapies. The proliferative behaviour of gliomas is strongly influenced by ion channel activity. Small-conductance calcium-activated potassium (SK/KCa) channels are a family of ion channels that are associated with cell proliferation and cell survival. A combined treatment of classical anti-cancer agents and pharmacological SK channel modulators has not been addressed yet. We used the gold-derivative auranofin to induce cancer cell death by targeting thioredoxin reductases in combination with CyPPA to activate SK channels in neuro- and glioblastoma cells. Combined treatment with auranofin and CyPPA induced massive mitochondrial damage and potentiated auranofin-induced toxicity in neuroblastoma cells in vitro. In particular, mitochondrial integrity, respiration and associated energy generation were impaired. These findings were recapitulated in patient-derived glioblastoma neurospheres yet not observed in non-cancerous HT22 cells. Taken together, integrating auranofin and SK channel openers to affect mitochondrial health was identified as a promising strategy to increase the effectiveness of anti-cancer agents and potentially overcome resistance.


Subject(s)
Auranofin/pharmacology , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Neuroblastoma/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Small-Conductance Calcium-Activated Potassium Channels/agonists , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Auranofin/administration & dosage , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Synergism , Glioblastoma/drug therapy , Glioblastoma/pathology , Humans , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , Small-Conductance Calcium-Activated Potassium Channels/metabolism , Spheroids, Cellular/drug effects , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Thioredoxin-Disulfide Reductase/metabolism
8.
Open Biol ; 7(11)2017 11.
Article in English | MEDLINE | ID: mdl-29118270

ABSTRACT

The hallmark of high-risk human papillomavirus (HR-HPV)-related carcinogenesis is E6 and E7 oncogene overexpression. The aim of this work was to characterize epithelial oral and cervical cancer cells that express HR-HPV E6 and E7 oncoproteins. Transcriptomic assay using DNA microarrays revealed that PIR gene expression was detected in oral cells in an HR-HPV E6/E7-dependent manner. In addition, PIR was overexpressed in HPV-positive SiHa and Ca Ski cells, whereas it was undetectable in HPV-negative C33A cells. The PIR expression was dependent on functional HR-HPV E6 and E7 oncoproteins even though the E7 oncoprotein had higher activity to induce PIR overexpression in comparison with E6. In addition, using an siRNA for PIR silencing in oral cells ectopically expressing HR-HPV E6/E7, there was a significant increase in E-cadherin transcripts and a decrease in Vimentin, Slug, Zeb and Snail transcripts, suggesting that HR-HPV-induced PIR overexpression is involved in epithelial-mesenchymal transition. Furthermore, migration of PIR-silenced cells was significantly decreased. Finally, using inhibitors of some specific pathways, it was found that EGFR/ERK and PI3 K/AKT signalling pathways are important for E7-mediated PIR overexpression. It can be concluded that PIR gene expression is highly dependent on the expression of HR-HPV oncoproteins and is important for EMT regulation.


Subject(s)
Carrier Proteins/genetics , Nuclear Proteins/genetics , Oncogene Proteins, Viral/metabolism , Papillomaviridae/metabolism , Carrier Proteins/metabolism , Cell Line, Tumor , Cervix Uteri/cytology , Cervix Uteri/virology , Dioxygenases , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Mitogen-Activated Protein Kinase 3/metabolism , Mouth Mucosa/cytology , Mouth Mucosa/virology , Nuclear Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism , Up-Regulation , Vimentin/genetics , Vimentin/metabolism
9.
J Cell Biochem ; 117(2): 334-43, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26138431

ABSTRACT

Oncogenic kinase Aurora A (AURKA) has been found to be overexpresed in several tumors including colorectal, breast, and hematological cancers. Overexpression of AURKA induces centrosome amplification and aneuploidy and it is related with cancer progression and poor prognosis. Here we show that AURKA phosphorylates in vitro the transcripcional co-repressor Ski on aminoacids Ser326 and Ser383. Phosphorylations on these aminoacids decreased Ski protein half-life. Reduced levels of Ski resulted in centrosomes amplification and multipolar spindles formation, same as AURKA overexpressing cells. Importantly, overexpression of Ski wild type, but not S326D and S383D mutants inhibited centrosome amplification and cellular transformation induced by AURKA. Altogether, these results suggest that the Ski protein is a target in the transformation pathway mediated by the AURKA oncogene.


Subject(s)
Aurora Kinase A/metabolism , Cell Transformation, Neoplastic/metabolism , DNA-Binding Proteins/physiology , Proto-Oncogene Proteins/physiology , Amino Acid Sequence , Animals , Centrosome/metabolism , Gene Expression , HEK293 Cells , Humans , MCF-7 Cells , Mice , Molecular Sequence Data , NIH 3T3 Cells , Phosphorylation , Protein Processing, Post-Translational , Spindle Apparatus/metabolism
10.
Oncotarget ; 6(40): 42749-60, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26543229

ABSTRACT

Endothelin-converting enzyme-1c (ECE-1c) is a membrane metalloprotease involved in endothelin-1 synthesis, which has been shown in vitro to have a role in breast, ovary and prostate cancer cell invasion. N-terminal end of ECE-1c displays three putative phosphorylation sites for the protein kinase CK2. We studied whether CK2 phosphorylates N-terminal end of ECE-1c as well as whether this has a role in migration and invasion of colon cancer cells. CK2 phosphorylated the N-terminal end of ECE-1c and this was precluded upon inhibition of CK2. Inhibition also led to diminished protein levels of both endogen ECE-1 or GFP-fused N-terminal end of ECE-1c in 293T embryonic and DLD-1 colon cancer cells, which highlighted the importance of this motif on UPS-dependent ECE-1c degradation. Full-length ECE-1c mutants designed either to mimic or abrogate CK2-phosphorylation displayed increased or decreased migration/invasion of colon cancer cells, respectively. Moreover, ECE-1c overexpression or its silencing with a siRNA led to increased or diminished cell migration/invasion, respectively. Altogether, these data show that CK2-increased ECE-1c protein stability is related to augmented migration and invasion of colon cancer cells, shedding light on a novel mechanism by which CK2 may promote malignant progression of this disease.


Subject(s)
Aspartic Acid Endopeptidases/metabolism , Colonic Neoplasms/pathology , Metalloendopeptidases/metabolism , Neoplasm Invasiveness/pathology , Blotting, Western , Casein Kinase II/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Endothelin-Converting Enzymes , Humans , Microscopy, Confocal , Protein Stability , RNA, Small Interfering , Transfection
11.
BMC Cancer ; 15: 463, 2015 Jun 10.
Article in English | MEDLINE | ID: mdl-26054531

ABSTRACT

BACKGROUND: Caveolin-1 (CAV1) has been implicated both in tumor suppression and progression, whereby the specific role appears to be context dependent. Endometrial cancer is one of the most common malignancies of the female genital tract; however, little is known about the role of CAV1 in this disease. METHODS: Here, we first determined by immunohistochemistry CAV1 protein levels in normal proliferative human endometrium and endometrial tumor samples. Then using two endometrial cancer cell lines (ECC: Ishikawa and Hec-1A) we evaluated mRNA and protein levels of CAV1 by real time qPCR and Western blot analysis, respectively. The role of CAV1 expression in ECC malignancy was further studied by either inducing its expression in endometrial cancer cells with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (4ß-TPA) or decreasing expression using short-hairpin RNA constructs, and then evaluating the effects of these changes on ECC proliferation, transmigration, matrigel invasion, and colony formation in soft agar. RESULTS: Immunohistochemical analysis of endometrial epithelia revealed that substantially higher levels of CAV1 were present in endometrial tumors than the normal proliferative epithelium. Also, in Ishikawa and Hec-1A endometrial cancer cells CAV1 expression was readily detectable. Upon treatment with 4ß-TPA CAV1 levels increased and coincided with augmented cell transmigration, matrigel invasion, as well as colony formation in soft agar. Reduction of CAV1 expression using short-hairpin RNA constructs ablated these effects in both cell types whether treated or not with 4ß-TPA. Alternatively, CAV1 expression appeared not to modulate significantly proliferation of these cells. CONCLUSION: Our study shows that elevated CAV1, observed in patients with endometrial cancer, is linked to enhanced malignancy of endometrial cancer cells, as evidenced by increased migration, invasion and anchorage-independent growth.


Subject(s)
Adenocarcinoma/genetics , Caveolin 1/biosynthesis , Endometrial Neoplasms/genetics , Neoplasm Invasiveness/genetics , Adenocarcinoma/pathology , Adult , Aged , Caveolin 1/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Endometrial Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , RNA, Messenger/biosynthesis
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