RESUMEN

SPHK1 (sphingosine kinase type 1) is characterized as a rate-limiting enzyme in sphingolipid metabolism to phosphorylate sphingosine into sphingosine-1-phosphate (S1P) that can bind to S1P receptors (S1PRs) to initiate several signal transductions leading to cell proliferation and survival of normal cell. Many studies have indicated that SPHK1 is involved in several types of cancer development, however, a little is known in bladder cancer. The TCGA database analysis was utilized for analyzing the clinical relevance of SPHK1 in bladder cancer. Through CRISPR/Cas9 knockout (KO) and constitutive activation (CA) strategies on SPHK1 in the bladder cancer cells, we demonstrated the potential downstream target could be programmed cell death 1 ligand 2 (PD-L2). On the other hand, we demonstrated that FDA-approved SPHK1 inhibitor Gilenya® (FTY720) can successfully suppress bladder cancer metastasis by in vitro and in vivo approaches. This finding indicated that SPHK1 as a potent therapeutic target for metastatic bladder cancer by dissecting the mechanism of action, SPHK1/S1P-elicited Akt/ß-catenin activation promoted the induction of PD-L2 that is a downstream effector in facilitating bladder cancer invasion and migration. Notably, PD-L2 interacted with c-Src that further activates FAK. Here, we unveil the clinical relevance of SPHK1 in bladder cancer progression and the driver role in bladder cancer metastasis. Moreover, we demonstrated the inhibitory effect of FDA-approved SPHK1 inhibitor FTY720 on bladder cancer metastasis from both in vitro and in vivo models.

Asunto(s)

Fosfotransferasas (Aceptor de Grupo Alcohol) , Transducción de Señal , Neoplasias de la Vejiga Urinaria , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Línea Celular Tumoral , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Metástasis de la Neoplasia , Ratones , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Familia-src Quinasas/metabolismo , Movimiento Celular , Ratones Desnudos , Lisofosfolípidos/metabolismo , Proteína Tirosina Quinasa CSK/metabolismo , Clorhidrato de Fingolimod/farmacología , Proliferación Celular

RESUMEN

Many pathological conditions lead to defects in intestinal epithelial integrity and loss of barrier function; Sphingosine-1-phosphate (S1P) has been shown to augment intestinal barrier integrity, though the exact mechanisms are not completely understood. We have previously shown that overexpression of Sphingosine Kinase 1 (SphK1), the rate limiting enzyme for S1P synthesis, significantly increased S1P production and cell proliferation. Here we show that microRNA 495 (miR-495) upregulation led to decreased levels of SphK1 resultant from a direct effect at the SphK1 mRNA. Increasing expression of miR-495 in intestinal epithelial cells resulted in decreased proliferation and increased susceptibility to apoptosis. Transgenic expression of miR-495 inhibited mucosal growth, as well as decreased proliferation in the crypts. The intestinal villi also expressed decreased levels of barrier proteins and exaggerated damage upon exposure to cecal ligation-puncture. These results implicate miR-495 as a critical negative regulator of intestinal epithelial protection and proliferation through direct regulation of SphK1, the rate limiting enzyme critical for production of S1P.

Asunto(s)

Apoptosis , Mucosa Intestinal , Lisofosfolípidos , MicroARNs , Fosfotransferasas (Aceptor de Grupo Alcohol) , Esfingosina , MicroARNs/metabolismo , MicroARNs/genética , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Animales , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Mucosa Intestinal/metabolismo , Ratones , Proliferación Celular , Regulación hacia Abajo , Células Epiteliales/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

Mevalonate kinase deficiency is a rare autosomal recessive disease caused by mutations in the mevalonate kinase gene (MVK). Depending on the mutations, a patient with this deficiency can exhibit any one of a spectrum of rare autoinflammatory diseases, such as hypergammaglobulinemia D (hyper-IgD) with periodic fever syndrome and mevalonic aciduria. To date, approximately 300 cases with mutations in the MVK gene have been reported worldwide. Herein, we present a 3-year-old female from Puerto Rico with a history of fever, arthralgia, and skin lesions since her first month of age and who, upon genetic workup, was confirmed to have compound heterozygous mutations in the MVK gene. Given her medical history and the results of her genetic testing, she was diagnosed with hyper-IgD with periodic fever syndrome. She will be treated with canakinumab, an interleukin-1ß antagonist, after receiving the varicella and measles-mumps-rubella (MMR) vaccines.

Asunto(s)

Deficiencia de Mevalonato Quinasa , Mutación , Humanos , Puerto Rico , Femenino , Preescolar , Deficiencia de Mevalonato Quinasa/diagnóstico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fiebre/etiología , Anticuerpos Monoclonales Humanizados , Inmunoglobulina D

RESUMEN

Herpesviral protein kinases, such as the therapy-relevant pUL97 of human cytomegalovirus (HCMV), are important for viral replication efficiency as well as pathogenesis, and represent key antiviral drug targets. HCMV pUL97 is a viral cyclin-dependent kinase (CDK) ortholog, as it shares functional and structural properties with human CDKs. Recently, the formation of vCDK/pUL97-cyclin complexes and the phosphorylation of a variety of viral and cellular substrate proteins has been demonstrated. Genetic mapping and structural modeling approaches helped to define two pUL97 interfaces, IF1 and IF2, responsible for cyclin binding. In particular, the regulatory importance of interactions between vCDK/pUL97 and host cyclins as well as CDKs has been highlighted, both as determinants of virus replication and as a novel drug-targeting option. This aspect was substantiated by the finding that virus replication was impaired upon cyclin type H knock-down, and that such host-directed interference also affected viruses resistant to existing therapies. Beyond the formation of binary interactive complexes, a ternary pUL97-cyclin H-CDK7 complex has also been described, and in light of this, an experimental trans-stimulation of CDK7 activity by pUL97 appeared crucial for virus-host coregulation. In accordance with this understanding, several novel antiviral targeting options have emerged. These include kinase inhibitors directed to pUL97, to host CDKs, and to the pUL97-cyclin H interactive complexes. Importantly, a statistically significant drug synergy has recently been reported for antiviral treatment schemes using combinations of pharmacologically relevant CDK7 and vCDK/pUL97 inhibitors, including maribavir. Combined, such findings provide increased options for anti-HCMV control. This review focuses on regulatory interactions of vCDK/pUL97 with the host cyclin-CDK apparatus, and it addresses the functional relevance of these key effector complexes for viral replication and pathogenesis. On this basis, novel strategies of antiviral drug targeting are defined.

Asunto(s)

Antivirales , Quinasas Ciclina-Dependientes , Citomegalovirus , Proteínas Virales , Humanos , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/fisiología , Proteínas Virales/metabolismo , Proteínas Virales/química , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/virología , Animales , Ciclinas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)

RESUMEN

Leishmania donovani relies on specific vitamins and cofactors crucial for its survival and pathogenesis. Tailoring therapies to disrupt these pathways offers a promising strategy for the treatment of Visceral Leishmaniasis. Current treatment regimens are limited due to drug resistance and high costs. The dependency of Leishmania parasites on Vitamin B2 and its metabolic products is not known. In this study, we have biochemically and biophysically characterized a Vitamin B2 metabolism enzyme, riboflavin kinase from L. donovani (LdRFK) which converts riboflavin (vitamin B2) into flavin mononucleotide (FMN). Sequence comparison with human counterpart reflects 31.58 % identity only, thus opening up the possibility of exploring it as drug target. The rfk gene was cloned, expressed and the recombinant protein was purified. Kinetic parameters of LdRFK were evaluated with riboflavin and ATP as substrates which showed differential binding affinity when compared with the human RFK enzyme. Thermal and denaturant stability of the enzyme was evaluated. The rfk gene was overexpressed in the parasites and its role in growth and cell cycle was evaluated. In the absence of crystal structure, homology modelling and molecular dynamic simulation studies were performed to predict LdRFK structure. The data shows differences in substrate binding between human and parasite enzyme. This raises the possibility of exploring LdRFK for specific designing of antileishmanial molecules. Gene disruption studies can further validate its candidature as antileishmanial target.

Asunto(s)

Leishmania donovani , Fosfotransferasas (Aceptor de Grupo Alcohol) , Riboflavina , Leishmania donovani/enzimología , Leishmania donovani/genética , Riboflavina/metabolismo , Riboflavina/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Humanos , Cinética , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Especificidad por Sustrato , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Clonación Molecular , Estabilidad de Enzimas , Mononucleótido de Flavina/metabolismo

RESUMEN

PURPOSE: Colorectal cancer (CRC) is one of the top five cancer-related causes of mortality globally. Acquired resistance has hindered the effectiveness of 5-fluorouracil (5-FU), the main chemotherapeutic drug used to treat CRC. Sphingosine kinase 2 (SphK2) may be a cancer treatment target and involved in 5-FU resistance. METHODS: Cell growth was examined using MTT and clone formation assays for SphK2 expression. To identify immune cells in mice, flow cytometry was performed. West blotting demonstrated alterations in cell division and inflammation-related proteins. SphK2 levels and inflammation-related variables were studied using Elisa. RESULTS: Due to SphK2 overexpression, immunosuppression, and 5-FU resistance are caused by the development of myeloid-derived suppressor cells (MDSCs) subsequent to IL-6/STAT3 activation and alterations in the arginase (ARG-1) protein. After therapy, the combination of SphK2 inhibitors and 5-FU can effectively suppress MDSCs while increasing CD4+ and CD8+ T cell infiltration into the tumor microenvironment, lowering tumor burden, and exhibiting a therapeutic impact on CRC. CONCLUSIONS: Our findings suggest that 5-FU treatment combined with simultaneous Spkh2 inhibition by ABC294640 has anti-tumor synergistic effects by influencing multiple effects on tumor cells, T cells, and MDSCs, potentially improving the poor prognosis of colorectal cancer patients.

Asunto(s)

Neoplasias Colorrectales , Resistencia a Antineoplásicos , Fluorouracilo , Células Supresoras de Origen Mieloide , Fosfotransferasas (Aceptor de Grupo Alcohol) , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Animales , Ratones , Humanos , Línea Celular Tumoral , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

BACKGROUND: Approximately 25-30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML. METHODS: We performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, ß-catenin, PP2A, and GSK3ß. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results. RESULTS: We demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates ß-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit ß-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3ß (GSK3ß) pathway. CONCLUSIONS: These findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.

Asunto(s)

Glucógeno Sintasa Quinasa 3 beta , Leucemia Mieloide Aguda , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteína Fosfatasa 2 , beta Catenina , Tirosina Quinasa 3 Similar a fms , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , beta Catenina/metabolismo , beta Catenina/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Animales , Ratones , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/antagonistas & inhibidores , Línea Celular Tumoral , Sorafenib/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética

RESUMEN

BACKGROUND: Sphingosine kinase 1 (SphK1) is a lipid enzyme whose role in the etiology of cancer has been well explored. Here, a systematic review and meta-analysis were conducted to evaluate the association of SphK1 expression with hematological malignancy. MATERIALS AND METHODS: Relevant studies were identified through electronic databases (PubMed, Scopus, Embase, and OVID) and evaluated based on predefined inclusion and exclusion criteria. Quality assessment using the Newcastle-Ottawa Scale (NOS) was conducted, and pooled odds ratio (OR) was calculated to assess the association between SphK1 expression and hematological malignancy. RESULTS: Nine studies meeting the inclusion criteria were included in the systematic review. These studies utilized various techniques to assess SphK1 expression in hematological malignancies. The quality assessment reported that the included studies were of moderate quality. Meta-analysis of eligible studies revealed a positive association between SphK1 expression and hematological malignancies at the protein level (OR = 52.37, 95% CI = 10.10 to 271.47, and P = 0.00001). The funnel plot indicated no publication bias among the included studies. However, the certainty of the evidence was low according to the GRADE assessment. CONCLUSION: Our study's findings support the link between SphK1 expression and hematological malignancies. SphK1 gene dysregulation may contribute to various malignancies, suggesting it could be a therapeutic target to improve patient outcomes. Further research is needed to understand SphK1's mechanistic role in hematological malignancies and its therapeutic potential.

Asunto(s)

Neoplasias Hematológicas , Fosfotransferasas (Aceptor de Grupo Alcohol) , Humanos , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Neoplasias Hematológicas/epidemiología , Neoplasias Hematológicas/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Pronóstico

RESUMEN

DNA damage has been implicated in the stimulation of the type 1 interferon (T1IFN) response. Here, we show that downregulation of the DNA repair protein, polynucleotide kinase/phosphatase (PNKP), in a variety of cell lines causes robust phosphorylation of STAT1, upregulation of interferon-stimulated genes and persistent accumulation of cytosolic DNA, all of which are indicators for the activation of the T1IFN response. Furthermore, this did not require damage induction by ionizing radiation. Instead, our data revealed that production of reactive oxygen species (ROS) synergises with PNKP loss to potentiate the T1IFN response, and that loss of PNKP significantly compromises mitochondrial DNA (mtDNA) integrity. Depletion of mtDNA or treatment of PNKP-depleted cells with ROS scavengers abrogated the T1IFN response, implicating mtDNA as a significant source of the cytosolic DNA required to potentiate the T1IFN response. The STING signalling pathway is responsible for the observed increase in the pro-inflammatory gene signature in PNKP-depleted cells. While the response was dependent on ZBP1, cGAS only contributed to the response in some cell lines. Our data have implications for cancer therapy, since PNKP inhibitors would have the potential to stimulate the immune response, and also to the neurological disorders associated with PNKP mutation.

Asunto(s)

Enzimas Reparadoras del ADN , ADN Mitocondrial , Interferón Tipo I , Fosfotransferasas (Aceptor de Grupo Alcohol) , Radiación Ionizante , Especies Reactivas de Oxígeno , Humanos , Interferón Tipo I/metabolismo , Interferón Tipo I/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Reparación del ADN , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Daño del ADN , Línea Celular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Fosforilación , Citosol/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética

RESUMEN

Conversion of pantothenate to phosphopantothenate in humans is the first dedicated step in the coenzyme A (CoA) biosynthesis pathway and is mediated by four isoforms of pantothenate kinase. These enzymes are allosterically regulated by acyl-CoA levels, which control the rate of CoA biosynthesis. Small molecule activators of the PANK enzymes that overcome feedback suppression increase CoA levels in cultured cells and animals and have shown great potential for the treatment of pantothenate kinase-associated neurodegeneration and propionic acidemias. In this study, we detail the further optimization of PANK pyridazine activators using structure-guided design and focus on the cellular CoA activation potential, metabolic stability, and solubility as the primary drivers of the structure-activity relationship. These studies led to the prioritization of three late-stage preclinical lead PANK modulators with improved pharmacokinetic profiles and the ability to substantially increase brain CoA levels. Compound 22 (BBP-671) eventually advanced into clinical testing for the treatment of PKAN and propionic acidemia.

Asunto(s)

Encéfalo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Piridazinas , Humanos , Animales , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Piridazinas/farmacocinética , Piridazinas/farmacología , Piridazinas/química , Piridazinas/síntesis química , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Relación Estructura-Actividad , Ratas , Activadores de Enzimas/farmacología , Activadores de Enzimas/química , Activadores de Enzimas/farmacocinética , Activadores de Enzimas/síntesis química , Coenzima A/metabolismo , Ratones

RESUMEN

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) catalyzes the final step in phytic acid (InsP6) synthesis. In this study, the effects of OsIPK1 mutations on InsP6 synthesis, grain filling and their underlying mechanisms were investigated. Seven gRNAs were designed to disrupt the OsIPK1 gene via CRISPR/CAS9 system. Only 4 of them generated 29 individual insertion or deletion T0 plants, in which nine biallelic or heterozygous genotypes were identified. Segregation analysis revealed that OsIPK1 frameshift mutants are homozygous lethality. The biallelic and heterozygous frameshift mutants exhibited significant reduction in yield-related traits, particularly in the seed-setting rate and yield per plant. Despite a notable decline in pollen viability, the male and female gametes had comparable transmission rates to their progenies in the mutants. A significant number of the filling-aborted (FA) grains was observed in mature grains of these heterozygous frameshift mutants. These grains exhibited a nearly complete blockage of InsP6 synthesis, resulting in a pronounced increase in Pi content. In contrast, a slight decline in InsP6 content was observed in the plump grains. During the filling stage, owing to the excessive accumulation of Pi, starch synthesis was significantly impaired, and the endosperm development-specific gene expression was nearly abolished. Consistently, the activity of whereas AGPase, a key enzyme in starch synthesis, was significantly decreased and Pi transporter gene expression was upregulated in the FA grains. Taken together, these results demonstrate that OsIPK1 frameshift mutations result in excessive Pi accumulation, decreased starch synthesis, and ultimately leading to lower yields in rice.

Asunto(s)

Mutación del Sistema de Lectura , Regulación de la Expresión Génica de las Plantas , Homeostasis , Oryza , Fósforo , Proteínas de Plantas , Almidón , Oryza/genética , Oryza/metabolismo , Oryza/crecimiento & desarrollo , Almidón/biosíntesis , Almidón/metabolismo , Fósforo/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/genética , Semillas/metabolismo , Semillas/crecimiento & desarrollo , Sistemas CRISPR-Cas , Grano Comestible/genética , Grano Comestible/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Plantas Modificadas Genéticamente , Ácido Fítico/metabolismo , Ácido Fítico/biosíntesis

RESUMEN

Despite advances in therapies, glioblastoma (GBM) recurrence is almost inevitable due to the aggressive growth behavior of GBM cells and drug resistance. Temozolomide (TMZ) is the preferred drug for GBM chemotherapy, however, development of TMZ resistance is over 50% cases in GBM patients. To investigate the mechanism of TMZ resistance and invasive characteristics of GBM, analysis of combined RNA-seq and ChIP-seq was performed in GBM cells in response to TMZ treatment. We found that the PERK/eIF2α/ATF4 signaling was significantly upregulated in the GBM cells with TMZ treatment, while blockage of ATF4 effectively inhibited cell migration and invasion. SPHK1 expression was transcriptionally upregulated by ATF4 in GBM cells in response to TMZ treatment. Blockage of ATF4-SPHK1 signaling attenuated the cellular and molecular events in terms of invasive characteristics and TMZ resistance. In conclusion, GBM cells acquired chemoresistance in response to TMZ treatment via constant ER stress. ATF4 transcriptionally upregulated SPHK1 expression to promote GBM cell aggression and TMZ resistance. The ATF4-SPHK1 signaling in the regulation of the transcription factors of EMT-related genes could be the underlying mechanism contributing to the invasion ability of GBM cells and TMZ resistance. ATF4-SPHK1-targeted therapy could be a potential strategy against TMZ resistance in GBM patients.

Asunto(s)

Movimiento Celular , Resistencia a Antineoplásicos , Estrés del Retículo Endoplásmico , Glioblastoma , Invasividad Neoplásica , Transducción de Señal , Temozolomida , Animales , Humanos , Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 4/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/patología , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Ratones Desnudos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Transducción de Señal/efectos de los fármacos , Temozolomida/farmacología , Temozolomida/uso terapéutico

RESUMEN

Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli. Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1-/-) or SphK2 (SphK2-/-) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2-/- mice, but exacerbated in the SphK1-/- mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1-/- and SphK2-/- in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research.

Asunto(s)

Lesión Renal Aguda , Síndrome Hemolítico-Urémico , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol) , Animales , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/genética , Ratones , Síndrome Hemolítico-Urémico/patología , Síndrome Hemolítico-Urémico/genética , Modelos Animales de Enfermedad , Esfingolípidos/metabolismo , Riñón/patología , Riñón/metabolismo , Ratones Endogámicos C57BL , Toxina Shiga II , Eliminación de Gen , Masculino

RESUMEN

The infectivity of Leishmania is determined by its ability to invade and evade host and its thriving capacity within the macrophage. Our study revealed the role of Leishmania donovani mevalonate kinase (MVK), an enzyme of mevalonate pathway in visceral leishmaniasis pathogenesis. Peritoneal exudate cells (PEC)-derived macrophages from BALB/c mice were infected with wild type (WT), MVK over expressing (MVK OE) and knockdown (KD) parasites and MVK OE parasites were found to be more infective than WT and MVK KD parasites. Incubation of macrophages with MVK OE parasites declined inducible nitric oxide synthase (iNOS) expression as well as nitric oxide (NO) production, both by 2 times in comparison to WT parasites. Moreover, ∼3 fold increase in Arginase1 expression indicated that MVK might induce polarization of macrophage towards M2, favouring the survival of parasite within the macrophages. Post 24 h infection of the macrophages with mutant strains, the levels of different cytokines (TNF-α, IL-12, IL-10 and IFN-γ) were measured. Infection of macrophages with MVK OE parasites showed an increase in the level of anti-inflammatory cytokine: IL-10 while infection with MVK KD parasites exhibited an increase in the level of pro-inflammatory cytokines: TNF-α, IL-12, and IFN-γ. Hence, Leishmania donovani mevalonate kinase (LdMVK) modulates macrophage functions and has a significant role in pathogenesis.

Asunto(s)

Citocinas , Leishmania donovani , Leishmaniasis Visceral , Macrófagos Peritoneales , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico , Fosfotransferasas (Aceptor de Grupo Alcohol) , Leishmania donovani/enzimología , Leishmania donovani/patogenicidad , Leishmania donovani/genética , Leishmania donovani/fisiología , Animales , Ratones , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/inmunología , Óxido Nítrico/metabolismo , Macrófagos Peritoneales/parasitología , Macrófagos Peritoneales/enzimología , Citocinas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Arginasa/metabolismo , Arginasa/genética , Femenino , Técnicas de Silenciamiento del Gen

RESUMEN

Sphingosine-1-phosphate (S1P) formed via catalytic actions of sphingosine kinase 1 (SphK1) behaves as a pro-survival substance and activates downstream target molecules associated with various pathologies, including initiation, inflammation, and progression of cancer. Here, we aimed to investigate the SphK1 inhibitory potentials of thymoquinone (TQ), Artemisinin (AR), and Thymol (TM) for the therapeutic management of lung cancer. We implemented docking, molecular dynamics (MD) simulations, enzyme inhibition assay, and fluorescence measurement studies to estimate binding affinity and SphK1 inhibitory potential of TQ, AR, and TM. We further investigated the anti-cancer potential of these compounds on non-small cell lung cancer (NSCLC) cell lines (H1299 and A549), followed by estimation of mitochondrial ROS, mitochondrial membrane potential depolarization, and cleavage of DNA by comet assay. Enzyme activity and fluorescence binding studies suggest that TQ, AR, and TM significantly inhibit the activity of SphK1 with IC50 values of 35.52 µM, 42.81 µM, and 53.68 µM, respectively, and have an excellent binding affinity. TQ shows cytotoxic effect and anti-proliferative potentials on H1299 and A549 with an IC50 value of 27.96 µM and 54.43 µM, respectively. Detection of mitochondrial ROS and mitochondrial membrane potential depolarization shows promising TQ-induced oxidative stress on H1299 and A549 cell lines. Comet assay shows promising TQ-induced oxidative DNA damage. In conclusion, TQ, AR, and TM act as potential inhibitors for SphK1, with a strong binding affinity. In addition, the cytotoxicity of TQ is linked to oxidative stress due to mitochondrial ROS generation. Overall, our study suggests that TQ is a promising inhibitor of SphK1 targeting lung cancer therapy.

Asunto(s)

Artemisininas , Benzoquinonas , Proliferación Celular , Neoplasias Pulmonares , Fosfotransferasas (Aceptor de Grupo Alcohol) , Timol , Humanos , Células A549 , Antineoplásicos/farmacología , Artemisininas/farmacología , Benzoquinonas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Timol/farmacología

RESUMEN

Small molecules that exhibit broad-spectrum enteroviral inhibitory activity by targeting viral replication proteins are highly desired in antiviral drug discovery studies. To discover new human rhinovirus (hRV) inhibitors, we performed a high-throughput screening of 100,000 compounds from the Korea Chemical Bank library. This search led to identification of two phosphatidylinositol-4-kinase IIIß (PI4KIIIß) inhibitors having the pyrazolo-pyrimidine core structure, which display moderate anti-rhinoviral activity along with mild cytotoxicity. The results of a study aimed at optimizing the activity of the hit compounds showed that the pyrazolo-pyrimidine derivative 6f exhibits the highest activity (EC50 = 0.044, 0.066, and 0.083 µM for hRV-B14, hRV-A16, and hRV-A21, respectively) and moderate toxicity (CC50 = 31.38 µM). Furthermore, 6f has broad-spectrum activities against various hRVs, coxsackieviruses and other enteroviruses, such as EV-A71, EV-D68. An assessment of kinase inhibition potencies demonstrated that 6f possesses a high and selective kinase inhibition activity against PI4KIIIß (IC50 value of 0.057 µM) and not against PI4KIIIα (>10 µM). Moreover, 6f exhibits modest hepatic stability (46.9 and 55.3 % remaining after 30 min in mouse and human liver microsomes, respectively). Finally, an in vivo study demonstrated that 6f possesses a desirable pharmacokinetic profile reflected in low systemic clearance (0.48 L∙h-1 kg-1) and modest oral bioavailability (52.4 %). Hence, 6f (KR-26549) appears to be an ideal lead for the development of new antiviral drugs.

Asunto(s)

Antivirales , Pirimidinas , Rhinovirus , Replicación Viral , Humanos , Rhinovirus/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Replicación Viral/efectos de los fármacos , Pirimidinas/farmacología , Pirimidinas/química , Pirimidinas/síntesis química , Animales , Relación Estructura-Actividad , Estructura Molecular , Pirazoles/farmacología , Pirazoles/química , Pirazoles/síntesis química , Ratones , Relación Dosis-Respuesta a Droga , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Pruebas de Sensibilidad Microbiana , Fosfotransferasas (Aceptor de Grupo Alcohol)

RESUMEN

AIMS: Depression is a potentially fatal illness affecting millions of individuals worldwide, across all age groups. Neuroinflammation is a key factor in depression development. Paclitaxel (PXL), a well-known chemotherapeutic agent has been used as therapy for several types of cancer. This study aims to evaluate the ameliorative effect of low-dose PXL against lipopolysaccharide (LPS)-induced depression in rats. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were administrated a single dose of LPS (5 mg/kg, i.p.); 2 h later, rats received PXL (0.3 mg/kg, i.p. three times/week) for one week. KEY FINDINGS: Low-dose PXL alleviated LPS-induced depressive-like behavior in rats as evidenced by significantly improving behavioral changes in both forced swim test (FST) and open field test (OFT), successfully mitigated depletion of monoamines (serotonin, norepinephrine, and dopamine), in addition to markedly decreasing lipid peroxidation with antioxidant levels elevation in brain tissues. Low-dose PXL substantially decreased inflammation triggered by LPS in brain tissue via repressing the expression of NLRP3 and its downstream markers level, caspase-1 and IL-1ß jointly with a corresponding decrease in proinflammatory cytokine levels (TNF-α). Furthermore, low-dose PXL remarkably down-regulated Sphk1/S1P signaling pathway. Concurrent with these biochemical findings, there was a noticeable improvement in the brain tissue's histological changes. SIGNIFICANCE: These findings prove the role of low-dose PXL in treatment of LPS-induced neuroinflammation and depressive-like behavior through their anti-depressant, antioxidant and anti-inflammatory actions. The suggested molecular mechanism may entail focusing the interconnection among Sphk1/S1P, and NLRP3/caspase-1/IL-1ß signaling pathways. Hence PXL could be used as a novel treatment against LPS-induced depression.

Asunto(s)

Caspasa 1 , Depresión , Interleucina-1beta , Lipopolisacáridos , FN-kappa B , Proteína con Dominio Pirina 3 de la Familia NLR , Paclitaxel , Ratas Sprague-Dawley , Transducción de Señal , Animales , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Lipopolisacáridos/toxicidad , Transducción de Señal/efectos de los fármacos , Depresión/tratamiento farmacológico , Depresión/inducido químicamente , Depresión/metabolismo , Caspasa 1/metabolismo , Interleucina-1beta/metabolismo , Ratas , FN-kappa B/metabolismo , Paclitaxel/toxicidad , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/metabolismo , Conducta Animal/efectos de los fármacos , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Antiinflamatorios/farmacología

RESUMEN

Flavins and their associated proteins have recently emerged as compelling players in the landscape of cancer biology. Flavins, encompassing flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), serve as coenzymes in a multitude of cellular processes, such as metabolism, apoptosis, and cell proliferation. Their involvement in oxidative phosphorylation, redox homeostasis, and enzymatic reactions has long been recognized. However, recent research has unveiled an extended role for flavins in the context of cancer. In parallel, riboflavin transporters (RFVTs), FAD synthase (FADS), and riboflavin kinase (RFK) have gained prominence in cancer research. These proteins, responsible for riboflavin uptake, FAD biosynthesis, and FMN generation, are integral components of the cellular machinery that governs flavin homeostasis. Dysregulation in the expression/function of these proteins has been associated with various cancers, underscoring their potential as diagnostic markers, therapeutic targets, and key determinants of cancer cell behavior. This review embarks on a comprehensive exploration of the multifaceted role of flavins and of the flavoproteins involved in nucleus-mitochondria crosstalk in cancer. We journey through the influence of flavins on cancer cell energetics, the modulation of RFVTs in malignant transformation, the diagnostic and prognostic significance of FADS, and the implications of RFK in drug resistance and apoptosis. This review also underscores the potential of these molecules and processes as targets for novel diagnostic and therapeutic strategies, offering new avenues for the battle against this relentless disease.

Asunto(s)

Flavinas , Homeostasis , Neoplasias , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Flavinas/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Animales , Apoptosis , Fosfotransferasas (Aceptor de Grupo Alcohol)

RESUMEN

Beauveria bassiana is a popular and eco-friendly biopesticide. During its pathogen-pest interaction, both N-acetylglucosamine (GlcNAc) catabolism and anabolism are crucial for nutrient supply and cell-wall construction. The initiation of GlcNAc metabolism relies on the catalysis of GlcNAc kinase, which has been extensively studied in the human pathogen Candida albicans. However, the physiological function of GlcNAc kinase remains poorly understood in entomopathogenic fungi. In the present study, a GlcNAc kinase homolog was identified and designated as BbHxk1 in B. bassiana. Deletion of BbHxk1 resulted in viable but reduced vegetative growth on various carbon sources. ΔBbHxk1 mutants displayed severe defects in cell wall integrity, making them more susceptible to cell wall stress cues. Furthermore, the absence of BbHxk1 resulted in an increase in conidial yield and blastospore production, and a faster rate of germination and filamentation, potentially attributed to higher intracellular ATP levels. BbHxk1 deficiency led to a reduction in the activities of cuticle-degrading enzymes, which might contribute to the attenuated pathogenicity specifically through cuticle penetration rather than hemocoel infection towards Galleria mellonella larvae. Being different from C. albicans Hxk1, which facultatively acts as a catalyzing enzyme and transcriptional regulator, BbHxk1 primarily acts as a catalyzing enzyme and metabolic regulator. The altered metabolomic profiling correlated with the phenotypic defects in ΔBbHxk1 mutants, further implicating a potential metabolism-dependent mechanism of BbHxk1 in mediating physiologies of B. bassiana. These findings not only unveil a novel role for GlcNAc kinase in B. bassiana, but also provide a solid theoretical basis to guide metabolic reprogramming in order to maintain or even enhance the efficiency of fungi for practical applications.

Asunto(s)

Beauveria , Pared Celular , Fosfotransferasas (Aceptor de Grupo Alcohol) , Beauveria/patogenicidad , Beauveria/genética , Pared Celular/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Esporas Fúngicas , Mariposas Nocturnas/microbiología , Agentes de Control Biológico

RESUMEN

The SARS-CoV-2 pandemic has had an unprecedented impact on global public health and the economy. Although vaccines and antivirals have provided effective protection and treatment, the development of new small molecule-based antiviral candidates is imperative to improve clinical outcomes against SARS-CoV-2. In this study, we identified UNI418, a dual PIKfyve and PIP5K1C inhibitor, as a new chemical agent that inhibits SARS-CoV-2 entry into host cells. UNI418 inhibited the proteolytic activation of cathepsins, which is regulated by PIKfyve, resulting in the inhibition of cathepsin L-dependent proteolytic cleavage of the SARS-CoV-2 spike protein into its mature form, a critical step for viral endosomal escape. We also demonstrated that UNI418 prevented ACE2-mediated endocytosis of the virus via PIP5K1C inhibition. Our results identified PIKfyve and PIP5K1C as potential antiviral targets and UNI418 as a putative therapeutic compound against SARS-CoV-2.

Asunto(s)

Antivirales , COVID-19 , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol) , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Internalización del Virus/efectos de los fármacos , Antivirales/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , COVID-19/virología , COVID-19/metabolismo , Tratamiento Farmacológico de COVID-19 , Animales , Catepsina L/metabolismo , Catepsina L/antagonistas & inhibidores , Chlorocebus aethiops , Endocitosis/efectos de los fármacos , Células Vero , Enzima Convertidora de Angiotensina 2/metabolismo , Células HEK293