RESUMEN

Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity. Moreover, mispairing carries significant challenges for downstream purification, decreasing yields and increasing the cost of bioprocess development. In this study, quantitative transcriptomics and proteomics analyses were employed to investigate which signaling pathways correlated with low and high mispairing clone signatures. Gene and protein expression profiles of Chinese hamster ovary (CHO) clones producing an tsAb were analyzed in the exponential growth and stationary (tsAb production) phase of fed-batch culture. Functional analysis revealed activated endoplasmic reticulum stress in high mispairing clones in both culture phases, while low mispairing clones exhibited expression profiles indicative of activated protein translation, as well as higher endocytosis and target protein degradation, suggesting the clearance of unfolded proteins through ubiquitin-mediated mechanisms. In addition, through transcriptomic profiling, we identified a group of genes that have the potential to be used as a biomarker panel tool for identifying high mispairing levels in the early stages of bioprocess development.

RESUMEN

Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

Asunto(s)

Cromosomas Fúngicos , Replicación del ADN , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Telómero , Proteínas de Unión al ADN/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Complejo Shelterina , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Cromosomas Fúngicos/metabolismo

RESUMEN

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.

Asunto(s)

Dependovirus , Vectores Genéticos , Humanos , Dependovirus/genética , Células HEK293 , Células HeLa , Transfección

RESUMEN

Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS-polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.

Asunto(s)

Electroforesis Capilar , Virión , Electroforesis Capilar/métodos , Dodecil Sulfato de Sodio , Electroforesis en Gel de Poliacrilamida

RESUMEN

Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges. Discussed herein are key aspects related to R&D needs and Chemistry, Manufacturing and Control (CMC) efforts required to attend this growing demand. Authors provide their perspective on strategic topics for rAAV-based therapies success: scalability and productivity; improved safety; increased process understanding combined with development of orthogonal bioanalytics that are able to identify, monitor and control Critical Quality Attributes (CQAs) during bioprocessing.

Asunto(s)

Dependovirus , Vectores Genéticos , Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética

RESUMEN

The immigration of foreign retirees to Spain's southeast coast is a tradition that stretches back decades. This phenomenon has modified the demographic structure of many towns and also transformed their social and economic features, as well as the number and diversity of services available to the population. This study examines the effect of post-employment immigration on economic revitalisation, the increase in services and the arrival of new inhabitants. It demonstrates the transformative potential of the phenomenon for pre-coastal areas in the Spanish southeast and its influence on the configuration of friendly residential environments. The study analyses the evolution of basic demographic data and its spatial behaviour, establishing relationships between the quality of life of the local aging population and retired immigrants. The analysis focuses on the impact of residentialist areas on the configuration of friendly living environments. The results show how these communities, which were once somewhat stagnant and had a significantly aging population, either participated in the development or have improved their access to certain services and facilities, configuring new environments. The results reflect the improvement of the quality of life for the elderly in these settings, considering that they are often the majority age group.

Asunto(s)

Emigrantes e Inmigrantes , Calidad de Vida , Humanos , Anciano , Dinámica Poblacional , Emigración e Inmigración , Envejecimiento , Economía

RESUMEN

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA polymerases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72∆ mutants are defective in telomere replication and exhibit long 3'-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication.

Asunto(s)

Replicación del ADN , Evolución Molecular , Fosfoproteínas Fosfatasas/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Homeostasis del Telómero , Telómero/genética , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Secuencia Conservada , Humanos , Fosforilación , Schizosaccharomyces/enzimología , Homología de Secuencia

RESUMEN

Aneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison-Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors' cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes.

Asunto(s)

Envejecimiento/genética , Aneuploidia , Fibroblastos/metabolismo , Proteína Forkhead Box M1/genética , Mitosis , Progeria/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Senescencia Celular/genética , Niño , Preescolar , Fibroblastos/citología , Proteína Forkhead Box M1/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Lactante , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Progeria/etnología , Progeria/metabolismo , Progeria/patología , Población Blanca

RESUMEN

Mammalian CST (CTC1-STN1-TEN1) complex fulfills numerous functions including rescue of the stalled replication forks and termination of telomerase action. In fission yeast lacking the CTC1 ortholog, the Stn1-Ten1 complex restricts telomerase action via its sumoylation-mediated interaction with Tpz1TPP1. We identify a small ubiquitin-like modifier (SUMO)-interacting motif (SIM) in the carboxyl-terminal part of Stn1 and show that this domain is crucial for SUMO and Tpz1-SUMO interactions. Point mutations in the SIM (Stn1-226) lead to telomere elongation, impair Stn1-Ten1 recruitment to telomeres, and enhance telomerase binding, revealing that Stn1 SIM domain contributes to the inhibition of telomerase activity at chromosome ends. Our results suggest that Stn1-Ten1 promotes DNA synthesis at telomeres to limit single-strand DNA accumulation. We further demonstrate that Stn1 functions in the replication of telomeric and subtelomeric regions in a Taz1-independent manner. Genetic analysis reveals that misregulation of origin firing and/or telomerase inhibition circumvents the replication defects of the stn1-226 mutant. Together, our results show that the Stn1-Ten1 complex has a dual function at telomeres by limiting telomerase action and promoting chromosome end replication.

Asunto(s)

Chaperonas Moleculares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/genética , Telómero/metabolismo , Replicación del ADN , ADN de Cadena Simple , Expresión Génica , Modelos Biológicos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutación , Unión Proteica , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genética

RESUMEN

ARF is a tumour suppressor activated by oncogenic stress, which stabilizes p53. Although p53 is a key component of the response to DNA damage, a similar function for ARF has not been ascribed. Here we show that primary mouse and human cells lacking the tumour suppressor BRCA2 accumulate DNA damage, which triggers checkpoint signalling and ARF activation. Furthermore, senescence induced by Brca2 deletion in primary mouse and human cells is reversed by the loss of ARF, a phenotype recapitulated in cells lacking RAD51. Surprisingly, ARF is not necessary for p53 accumulation per se but for altering the spectrum of genes activated by this transcription factor. Specifically, ARF enables p53 transcription of Dusp4 and Dusp7, which encode a pair of phosphatases known to inactivate the MAP kinases ERK1/2. Our results ascribe a previously unanticipated function to the ARF tumour suppressor in genome integrity, controlled by replicative stress and ATM/ATR-dependent checkpoint responses.

Asunto(s)

Proteína BRCA2/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Cricetinae , Daño del ADN , Epigénesis Genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/metabolismo , Eliminación de Gen , Células HeLa , Humanos , Ratones , Fenotipo , Monoéster Fosfórico Hidrolasas/metabolismo , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismo , Transducción de Señal , Factores de Transcripción

RESUMEN

Shelterin component TRF2 prevents ATM activation, while POT1 represses ATR signalling at telomeres. Here, we investigate the mechanism of G2/M arrest triggered by telomeres uncapped through TRF2 or POT1 inhibition in human cells. We find that telomere damage-activated ATR and ATM phosphorylate p53, as well as CHK1 and CHK2, thus activating two independent pathways to prevent progression into mitosis with uncapped telomeres. Surprisingly, telomere damage targets the CDC25C phosphatase for proteasome degradation in G2/M. CHK1/CHK2-dependent phosphorylation of CDC25C at Ser 216 is required for CDC25C nuclear export and destruction, which in turn acts to sustain the G2/M arrest elicited by TRF2- or POT1-depleted telomeres. In addition, CDC25C is transcriptionally downregulated by p53 in response to telomere damage. These mechanisms are distinct from the canonical DNA damage response to ionizing radiation, which triggers cell-cycle arrest through CDC25A destruction. Thus, dysfunctional telomeres promote ATM/ATR-dependent degradation of CDC25C phosphatase to block mitotic entry, thereby preventing telomere dysfunction-driven genomic instability.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Citocinesis , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Serina-Treonina Quinasas/metabolismo , Telómero/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Fosfatasas cdc25/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular , Perfilación de la Expresión Génica , Humanos , Modelos Biológicos

RESUMEN

The tumor suppressor protein BRCA2 is a key component of the homologous recombination pathway of DNA repair, acting as the loader of RAD51 recombinase at sites of double-strand breaks. Here we show that BRCA2 associates with telomeres during the S and G2 phases of the cell cycle and facilitates the loading of RAD51 onto telomeres. Conditional deletion of Brca2 and inhibition of Rad51 in mouse embryonic fibroblasts (MEFs), but not inactivation of Brca1, led to shortening of telomeres and accumulation of fragmented telomeric signals--a hallmark of telomere fragility that is associated with replication defects. These findings suggest that BRCA2-mediated homologous recombination reactions contribute to the maintenance of telomere length by facilitating telomere replication and imply that BRCA2 has an essential role in maintaining telomere integrity during unchallenged cell proliferation. Mouse mammary tumors that lacked Brca2 accumulated telomere dysfunction-induced foci. Human breast tumors in which BRCA2 was mutated had shorter telomeres than those in which BRCA1 was mutated, suggesting that the genomic instability in BRCA2-deficient tumors was due in part to telomere dysfunction.

Asunto(s)

Proteína BRCA2/fisiología , Recombinasa Rad51/metabolismo , Telómero/metabolismo , Animales , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Reparación del ADN , Fase G2 , Eliminación de Gen , Inestabilidad Genómica , Ratones , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Fase S , Telómero/química

RESUMEN

Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

Asunto(s)

Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Genes BRCA1 , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Mutación , Animales , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/deficiencia , Proteína BRCA2/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Ciclo Celular , Proliferación Celular , Proteínas Cromosómicas no Histona , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Eliminación de Gen , Genes BRCA2 , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Mutagénesis Insercional , Células Tumorales Cultivadas , Proteína 1 de Unión al Supresor Tumoral P53

RESUMEN

Telomeres are protected by capping structures consisting of core protein complexes that bind with sequence specificity to telomeric DNA. In their absence, telomeres trigger a DNA damage response, materialized in accumulation at the telomere of damage response proteins, e.g., phosphorylated histone H2AX (gammaH2AX), into telomere-dysfunction-induced foci. Telomere uncapping occurs transiently in every cell cycle in G2, following DNA replication, but little is known about how protective structures are reassembled or whether this process is controlled by the cell-cycle surveillance machinery. Here, we report that telomere capping is monitored at the G2/M transition by the p53/p21 damage response pathway. Unlike their wild-type counterparts, human and mouse cells lacking p53 or p21 progress into mitosis prematurely with persisting uncapped telomeres. Furthermore, artificially uncapped telomeres delay mitotic entry in a p53- and p21-dependent manner. Uncapped telomeres that persist in mitotic p53-deficient cells are shorter than average and religate to generate end-to-end fusions. These results suggest that a p53-dependent pathway monitors telomere capping after DNA replication and delays G2/M progression in the presence of unprotected telomeres. This mechanism maintains a cell-cycle stage conducive for capping reactions and prevents progression into stages during which uncapped telomeres are prone to deleterious end fusions.

Asunto(s)

Mitosis/fisiología , Telómero/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Proteínas de Ciclo Celular/fisiología , Línea Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Daño del ADN , Proteínas de Unión al ADN/fisiología , Células HeLa , Histonas/metabolismo , Humanos , Ratones , Ratones Noqueados , Mitosis/genética , Proteínas Serina-Treonina Quinasas/fisiología , ARN Interferente Pequeño/genética , Telómero/genética , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/fisiología

RESUMEN

Cucurbitacin R is known to exhibit an anti-inflammatory effect in different experimental models of inflammation. In this article, we outline the effect of cucurbitacin R on T lymphocyte proliferation, cytokine production, and nuclear factor activation, as well as its influence on various experimental models of delayed-type hypersensitivity (DTH) in mice. Cucurbitacin R reduced the proliferation of phytohemagglutinin A-stimulated human T lymphocytes (IC(50), 18 microM), modifying the cell cycle, as well as the production of cytokines [interleukin (IL)-2, IL-4, IL-10, and especially interferon-gamma] and the induction of the principal cyclins implicated in the cell cycle (A(1), B(1), D(2), and E). These effects are brought on by a novel, selective inhibition of nuclear factor AT (NFAT) by cucurbitacin R, with no concomitant effect on other transcription factors such as activator protein-1. In addition, we tested the in vivo effects of cucurbitacin R in three experimental models of DTH, as well as its effects on T lymphocyte proliferation, the cell cycle, cytokines, and cyclins. Although cucurbitacin R was found to reduce the inflammatory response brought on by both oxazolone and dinitrofluorobenzene, its activity was even more pronounced against sheep red blood cell-induced edema in mouse paws, with a clear reduction in the production of IL-1beta, IL-4, and tumor necrosis factor alpha in the inflamed paw. In conclusion, cucurbitacin R has the potential to be a new immunosuppressive agent with antiproliferative effects through the inhibition of the NFAT with anti-inflammatory activity in DTH reactions.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Hipersensibilidad a las Drogas/tratamiento farmacológico , Hipersensibilidad Tardía/prevención & control , Factores de Transcripción NFATC/antagonistas & inhibidores , Triterpenos/farmacología , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Ciclo Celular/efectos de los fármacos , Ciclinas/metabolismo , Dinitrofluorobenceno/farmacología , Oído/patología , Femenino , Pie/patología , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/patología , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Células Jurkat , Ratones , Factores de Transcripción NFATC/metabolismo , Oxazolona/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Triterpenos/uso terapéutico

RESUMEN

Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Proteínas Proto-Oncogénicas/metabolismo , Triterpenos/farmacología , Proteínas ras/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Proteínas Proto-Oncogénicas p21(ras) , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

We have studied the effects of dihydrocucurbitacin B, a triterpene isolated from Cayaponia tayuya roots, on different models of delayed type hypersensitivity (DTH) in mice, as well as on T-lymphocyte proliferation and the mediators involved. In experiments with mice, dihydrocucurbitacin B inhibited the inflammatory reactions induced by oxazolone, dinitrofluorobenzene, and sheep red blood cells, reducing both the edema and cell infiltration. Moreover, the analysis of inflamed tissues showed that dihydrocucurbitacin B reduced the presence of the most relevant cytokines implicated in these processes, including interleukin-1 beta, interleukin-4, and tumor necrosis factor-alpha. Dihydrocucurbitacin B was also found to inhibit the proliferation of phytohemagglutinin-stimulated human T lymphocytes (IC(50) = 1.48 microM), halting the cell cycle in the G(0) phase. In addition, the triterpene reduced the production of interleukin-2, interleukin-4, interleukin-10, and interferon-gamma in human T lymphocytes, and it hampered the induction of the principal cyclins involved in the cell cycle, including A(1), B(1), D(2), and E(1). Finally, dihydrocucurbitacin B was found to exert a selective inhibition on the nuclear factor of activated T cells (NFAT) in human lymphocytes without affecting the calcium influx. Taken together, these results suggest that dihydrocucurbitacin B curbs DTH reactions by inhibiting NFAT, which in turn suppresses the proliferation of the most relevant cells involved in DTH reactions, namely the T cells.

Asunto(s)

Hipersensibilidad Tardía/tratamiento farmacológico , Activación de Linfocitos/efectos de los fármacos , Triterpenos/farmacología , Animales , Ciclinas/efectos de los fármacos , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Modelos Animales de Enfermedad , Inflamación/prevención & control , Ratones , Factores de Transcripción NFATC/antagonistas & inhibidores , Fase de Descanso del Ciclo Celular

RESUMEN

Scropolioside A, an iridoid isolated from Scrophularia auriculata ssp. pseudoauriculata, showed anti-inflammatory properties against different experimental models of delayed-type hypersensitivity. This iridoid reduced the oedema induced by oxazolone by 79% (72 h) at 0.5 mg/ear while reducing that induced by sheep red blood cells by 47% (18 h), 45% (24 h) and 36% (48 h) at 10 mg/kg. In vivo it reduced both oedema formation and cell infiltration whereas in vitro it reduced the proliferation of activated T-lymphocytes (IC50 of 67.74 microM). Treatment with scropolioside A (100 microM) 18 and 24 h after phytohemagglutinin stimulation increased the number of cells arrested in the subG(0) phase whereas treatment 3 h after stimulation clearly increased the number of cells that passed to the S phase. Scropolioside A also inhibited the production of prostaglandin E2, leukotriene B4, nitric oxide, interleukin-1beta, interleukin-2, interleukin-4, tumour necrosis factor-alpha and interferon-gamma, but had no effect on the production of interleukin-10. Moreover, it modified the expression of both nitric oxide synthase-2 and cyclooxygenase-2, as well as the activation of nuclear factor-kappaB in RAW 264.7 macrophages.

Asunto(s)

Antiinflamatorios/farmacología , Glucósidos/farmacología , Piranos/farmacología , Alérgenos , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Citocinas/biosíntesis , Dinoprostona/inmunología , Oído/patología , Edema/inducido químicamente , Eritrocitos/inmunología , Femenino , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/tratamiento farmacológico , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/patología , Leucotrieno B4/inmunología , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Oxazolona , Elastasa Pancreática/inmunología , Ovinos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología

RESUMEN

The aim of this study was to investigate the effects of cucurbitacin R on an experimental model of adjuvant-induced arthritis in rats. The treatment of arthritic rats with cucurbitacin R (1 mg/kg p.o. daily) modified the evolution of the clinical symptoms, whereas the histopathology of paws demonstrated a reduction in the signs of arthritis. Compared with the control group, radiography of the tibiotarsal joints of cucurbitacin R-treated rats showed a decrease in joint damage and soft tissue swelling of the footpad. The in vivo study of the expression of proinflammatory enzymes (nitric-oxide synthase-2 and cyclooxygenase-2) with the aid of the Western blot technique, and that of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) by means of enzyme-linked immunosorbent assays demonstrated a clear decrease in both the enzymes and the mediators in paw homogenates. The analysis for prostaglandin E(2), nitric oxide, and TNF-alpha production in RAW 264.7 macrophages, as well as that for TNF-alpha in human lymphocytes, indicated a reduction of all mediators. The expression of cyclooxygenase-2 was not modified in RAW 264.7 macrophages, whereas the expression of nitric-oxide synthase-2 was clearly diminished. Moreover, cucurbitacin R was found to inhibit signal transducer and activator of transcription 3 activation in the lymphocytes of both healthy and arthritic men. These experimental data on the chronic model, together with previously reported activity on acute and subchronic experimental models, justify the anti-inflammatory activity of cucurbitacin R and provide further evidence for the therapeutic potential of a group of natural products as anti-inflammatory agents.

Asunto(s)

Antiinflamatorios/farmacología , Artritis Experimental/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Triterpenos/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Línea Celular , Cucurbitacinas , Dinoprostona/biosíntesis , Femenino , Humanos , Ratones , Elastasa Pancreática/metabolismo , Ratas , Ratas Endogámicas Lew , Factor de Transcripción STAT3/metabolismo , Superóxidos/metabolismo , Triterpenos/uso terapéutico

RESUMEN

23,24-Dihydrocucurbitacin B, from the anti-rheumatic plant Cayaponia tayuya, was tested on arthritis induced by adjuvant to corroborate the anti-inflammatory properties of this plant. Arthritis was induced in Lewis rats; the resulting arthritic rats were then treated with dihydrocucurbitacin B (1 mg/kg orally, daily, 1 week). The effect of dihydrocucurbitacin B on the synthesis, release, and activity of pro-inflammatory enzymes (elastase, cyclooxygenase-2, and nitric oxide synthase-2) as well as its effect on different mediators (tumor necrosis factor-alpha and interleukin-1beta) were determined. Dihydrocucurbitacin B modified the evolution of the clinical symptoms, reducing the swelling and bone and tissue damage along with the development of the disease, modifying the cell infiltration and the expression of both nitric oxide synthase-2 and cyclooxygenase-2. In addition, it decreased the tumor necrosis factor-alpha and interleukin-1beta production in lymphocytes, but did not modify it in macrophages.

Asunto(s)

Artritis Experimental/prevención & control , Cucurbitaceae/química , Triterpenos/uso terapéutico , Administración Oral , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Antirreumáticos/administración & dosificación , Antirreumáticos/aislamiento & purificación , Antirreumáticos/uso terapéutico , Artritis Experimental/metabolismo , Artritis Experimental/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Dolor/prevención & control , Elastasa Pancreática/metabolismo , Fitoterapia , Extractos Vegetales/administración & dosificación , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Raíces de Plantas/química , Ratas , Ratas Endogámicas Lew , Superóxidos/metabolismo , Triterpenos/administración & dosificación