RESUMEN

Ferroptosis is involved in the pathogenesis of osteoarthritis (OA) while suppression of chondrocyte ferroptosis has a beneficial effect on OA. However, the molecular mechanism of ferroptosis in OA remains to be elucidated. P21, an indicator of aging, has been reported to inhibit ferroptosis, but the relationship between P21 and ferroptosis in OA remains unclear. Here, we aimed to investigate the expression and function of P21 in OA chondrocytes, and the involvement of P21 in the regulation of ferroptosis in chondrocytes. First, we demonstrated that high P21 expression was observed in the cartilage from OA patients and destabilized medial meniscus (DMM) mice, and in osteoarthritic chondrocytes induced by IL-1ß, FAC and erastin. P21 knockdown exacerbated the reduction of Col2a1 and promoted the upregulation of MMP13 in osteoarthritic chondrocytes. Meanwhile, P21 knockdown exacerbated cartilage degradation in DMM-induced OA mouse models and decreased GPX4 expression in vivo. Furthermore, P21 knockdown sensitized chondrocytes to ferroptosis induced by erastin, which was closely associated with the accumulation of lipid peroxides. In mechanism, we demonstrated that P21 regulated the stability of GPX4 protein, and the regulation was independent of NRF2. Meanwhile, we found that P21 significantly affected the recruitment of GPX4 to linear ubiquitin chain assembly complex (LUBAC) and regulated the level of M1-linked ubiquitination of GPX4. Overall, our results suggest that P21 plays an essential anti-ferroptosis role in OA by regulating the stability of GPX4.

Asunto(s)

Ferroptosis , Osteoartritis , Humanos , Ratones , Animales , Condrocitos/metabolismo , Ferroptosis/genética , Cartílago/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Arriba , Osteoartritis/genética , Osteoartritis/metabolismo

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.

Asunto(s)

Bencilisoquinolinas , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Bencilisoquinolinas/farmacología , Bencilisoquinolinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores de Muerte Celular , Neoplasias Pancreáticas

RESUMEN

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4-2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.

Asunto(s)

Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/metabolismo , Antagonistas de Andrógenos , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Línea Celular Tumoral , Nitrilos/farmacología , Antagonistas de Receptores Androgénicos , Andrógenos/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Resistencia a Antineoplásicos , Ubiquitina-Proteína Ligasas

RESUMEN

Vinigrol is a natural diterpenoid with unprecedented chemical structure, driving great efforts into its total synthesis in the past decades. Despite anti-hypertension and anti-clot ever reported, comprehensive investigations on bioactions and molecular mechanisms of Vinigrol are entirely missing. Here we firstly carried out a complete functional prediction of Vinigrol using a transcriptome-based strategy coupled with multiple bioinformatic analyses and identified "anti-cancer" as the most prominent biofunction ahead of anti-hypertension and anti-depression/psychosis. Broad cytotoxicity was subsequently confirmed on multiple cancer types. Further mechanistic investigation on several breast cancer cells revealed that its anti-cancer effect was mainly through activating PERK/eIF2α arm of unfolded protein response (UPR) and subsequent non-apoptotic cell death independent of caspase activities. The other two branches of UPR, IRE1α and ATF6, were functionally irrelevant to Vinigrol-induced cell death. Using CRISPR/Cas9-based gene activation, repression, and knockout systems, we identified the essential contribution of ATF4 and DDIT3, not ATF6, to the death process. This study unraveled a broad anti-cancer function of Vinigrol and its underlying targets and regulatory mechanisms. It paved the way for further inspection on the structure-efficacy relationship of the whole compound family, making them a novel cluster of PERK-specific stress activators for experimental and clinical uses.

Asunto(s)

Factor de Transcripción Activador 4 , Neoplasias de la Mama , Diterpenos , Factor de Transcripción CHOP , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Diterpenos/farmacología , Estrés del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Femenino , Humanos , Proteínas Serina-Treonina Quinasas , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismo

RESUMEN

Drug resistance in small cell lung cancer (SCLC) significantly affects the efficacy of chemotherapy treatment. However, due to the lack of tumor tissue samples, especially serial tumor samples during chemotherapy, the mechanism of chemotherapy resistance has not been fully studied. Circulating tumor DNA, which can be obtained in a noninvasive manner, can complement tumor sampling approaches for research in this field. We identified an SCLC patient with acquired drug resistance from 52 SCLC patients for whom follow-up data were available. By comparing somatic mutations in circulating tumor DNA before and after chemotherapy, for the first time, we found that the somatic mutation eIF3A R803K may be related to acquired chemotherapy resistance. Then, the association between the eIF3A R803K mutation and chemotherapy resistance was confirmed by samples from 254 lung cancer patients receiving chemotherapy. We found that the eIF3a R803K mutation weakened the proliferation ability of tumor cells but increased their resistance to chemotherapy. Further studies revealed that the eIF3A R803K mutation promotes cellular senescence. In addition, fisetin showed a synergistic effect with chemotherapy in eIF3A R803K mutant cells. These results suggest that the eIF3A R803K somatic mutation has the potential to predict chemotherapy resistance in SCLC. Moreover, the eIF3A R803K mutation promotes chemotherapy resistance by inducing senescence. Furthermore, a senolytic drug, fisetin, can reverse chemotherapy resistance mediated by the eIF3A R803K mutation.

Asunto(s)

Senescencia Celular/genética , Resistencia a Antineoplásicos/genética , Factor 3 de Iniciación Eucariótica/genética , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular , Movimiento Celular , Supervivencia Celular , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Mutación , Inhibidores de la Síntesis de la Proteína/farmacología , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/mortalidad

RESUMEN

Eukaryotic protein synthesis is the highly conserved, complex mechanism of translating genetic information into proteins. Although this process is essential for cellular homoeostasis, dysregulations are associated with cellular malfunctions and diseases including cancer and diabetes. In the challenging and ongoing search for adequate treatment possibilities, natural products represent excellent research tools and drug leads for new interactions with the translational machinery and for influencing mRNA translation. In this review, bacterial-, marine- and plant-derived natural compounds that interact with different steps of mRNA translation, comprising ribosomal assembly, translation initiation and elongation, are highlighted. Thereby, the exact binding and interacting partners are unveiled in order to accurately understand the mode of action of each natural product. The pharmacological relevance of these compounds is furthermore assessed by evaluating the observed biological activities in the light of translational inhibition and by enlightening potential obstacles and undesired side-effects, e.g. in clinical trials. As many of the natural products presented here possess the potential to serve as drug leads for synthetic derivatives, structural motifs, which are indispensable for both mode of action and biological activities, are discussed. Evaluating the natural products emphasises the strong diversity of their points of attack. Especially the fact that selected binding partners can be set in direct relation to different diseases emphasises the indispensability of natural products in the field of drug development. Discovery of new, unique and unusual interacting partners again renders them promising tools for future research in the field of eukaryotic mRNA translation.

Asunto(s)

Organismos Acuáticos , Bacterias , Productos Biológicos/farmacología , Extractos Vegetales/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Ribosomas/efectos de los fármacos , Animales , Organismos Acuáticos/química , Bacterias/química , Productos Biológicos/aislamiento & purificación , Desarrollo de Medicamentos , Humanos , Myxococcales/química , Extractos Vegetales/aislamiento & purificación , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , ARN Mensajero/genética , Ribosomas/genética , Ribosomas/metabolismo

RESUMEN

Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase ß (IKKß). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Benzopiranos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Selectina E/genética , Selectina E/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Ratones , FN-kappa B/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores de Factores de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

MEF2D-fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5 % of acute lymphoblastic leukemia (ALL) cases. Their introduction to cells has been reported to transform cell lines or increase the colony formation of bone marrow cells, suggesting their survival-supporting ability, which prompted us to examine M-fusion-targeting drugs. To identify compounds that reduce the protein expression level of MEF2D, we developed a high-throughput screening system using 293T cells stably expressing a fusion protein of MEF2D and luciferase, in which the protein expression level of MEF2D was easily measured by a luciferase assay. We screened 3766 compounds with known pharmaceutical activities using this system and selected staurosporine as a potential inducer of the proteolysis of MEF2D. Staurosporine induced the proteolysis of M-fusion proteins in M-fusion (+) ALL cell lines. Proteolysis was inhibited by caspase inhibitors, not proteasome inhibitors, suggesting caspase dependency. Consistent with this result, the growth inhibitory effects of staurosporine were stronger in M-fusion (+) ALL cell lines than in negative cell lines, and caspase inhibitors blocked apoptosis induced by staurosporine. We identified the cleavage site of MEF2D-HNRNPUL1 by caspases and confirmed that its caspase cleavage-resistant mutant was resistant to staurosporine-induced proteolysis. Based on these results, we investigated another Food and Drug Administration-approved caspase activator, venetoclax, and found that it exerted similar effects to staurosporine, namely, the proteolysis of M-fusion proteins and strong growth inhibitory effects in M-fusion (+) ALL cell lines. The present study provides novel insights into drug screening strategies and the clinical indications of venetoclax.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Caspasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Estaurosporina/farmacología , Sulfonamidas/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Fusión Génica , Células HEK293 , Humanos , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteolisis , Transducción de Señal

RESUMEN

Cardiac injury is followed by fibrosis, characterized by myofibroblast activation. Excessive deposition of extracellular matrix (ECM) impairs the plasticity of myocardium and results in myocardial systolic and diastolic dysfunction. Mangiferin is a xanthonoid derivative rich in plants mangoes and iris unguicularis, exhibiting the ability to ameliorate metabolic disorders. This study aims to investigate whether mangiferin attenuates cardiac fibrosis via redox regulation. The transverse aortic constriction (TAC) in mice induced cardiac fibrosis with impaired heart function. Oral administration of mangiferin (50 mg/kg, 4 weeks) inhibited myofibroblast activation with reduced formation of ECM. The impaired left ventricular contractive function was also improved by mangiferin. TGF-ß1 stimulation increased glutaminolysis to fuel intracellular glutamate pool for the increased demands of nutrients to support cardiac myofibroblast activation. Mangiferin degraded Keap1 to promote Nrf2 protein accumulation by improving its stability, leading to Nrf2 activation. Nrf2 transcriptionally promotes the synthesis of antioxidant proteins. By activating Nrf2, mangiferin promoted the synthesis of glutathione (GSH) in cardiac fibroblasts, likely due to the consumption of glutaminolysis-derived glutamate as a source. Meanwhile, mangiferin promoted the exchange of intracellular glutamate for the import of extracellular cystine to support GSH generation. As a result of redistribution, the reduced glutamate availability failed to support myofibroblast activation. In support of this, the addition of extracellular glutamate or α-ketoglutarate diminished the inhibitory effects of mangiferin on cardiac myofibroblast proliferation and activation. Moreover, cardiac knockdown of Nrf2 attenuated the cardioprotective effects of mangiferin in mice subjected to TAC. In conclusion, we demonstrated that activated myofibroblasts were sensitive to glutamate availability. Mangiferin activated Nrf2 and redistributed intracellular glutamate for the synthesis of GSH, consequently impairing cardiac myofibroblast activation due to decreased glutamate availability. These results address that pharmacological activation of Nrf2 could restrain cardiac fibrosis via metabolic regulation.

Asunto(s)

Cardiomiopatías/prevención & control , Ácido Glutámico/metabolismo , Glutatión/metabolismo , Miocardio/metabolismo , Miofibroblastos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/agonistas , Xantonas/farmacología , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Fibrosis , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Células 3T3 NIH , Ratas Sprague-Dawley , Transducción de Señal

RESUMEN

Excessive activation of osteoclast activity is responsible for many bone diseases, such as osteoporosis, rheumatoid arthritis, periprosthetic osteolysis, and periodontitis. Natural compounds that inhibit osteoclast formation and/or function have therapeutic potential for treating these diseases. Catalpol, a bioactive iridoid extracted from a traditional herbal medicine Rehmannia glutinosa, exhibits various pharmacological properties, including anti-inflammatory, antioxidant, antidiabetic, and antitumor effects. However, its effects on osteoclast formation and function remain unknown. In the present study, we showed that catalpol inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation and bone resorption, as well as the expression of osteoclast-related marker genes. The investigation of molecular mechanisms showed that catalpol upregulated phosphatase and tensin homolog (PTEN) activity by reducing its ubiquitination and degradation, subsequently suppressing RANKL-induced NF-κB and AKT signaling pathways, leading to an inhibition on NFATc1 induction. Furthermore, catalpol protected mice against inflammation- and ovariectomy-induced bone loss by inhibiting osteoclast activity in vivo. These results suggest that catalpol might be developed as a promising candidate for treating osteoclast-related bone diseases.

Asunto(s)

Resorción Ósea/prevención & control , Glucósidos Iridoides/farmacología , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Animales , Resorción Ósea/genética , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Medicamentos Herbarios Chinos/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoclastos/metabolismo , Osteogénesis/genética , Fosfohidrolasa PTEN/genética , Ligando RANK/farmacología , Células RAW 264.7

RESUMEN

Lactobacillus sanfranciscensis is the predominant lactic acid bacteria (LAB) species in Chinese traditional sourdoughs and conduces to the flavor and rheology properties of Chinese steamed bread, a staple food originated in China over 1500years ago. The aim of this study is to describe the intraspecific diversity of 98 L. sanfranciscensis isolates from 11 Chinese traditional sourdoughs in different regions by multilocus sequence typing (MLST) and multiplex random amplified polymorphic DNA-polymerase chain reaction (multiplex RAPD-PCR). MLST scheme was reduced from six gene fragments (gdh, gyrA, mapA, nox, pgmA and pta) to five (gdh, gyrA, mapA, nox and pta) since the fragment of pgmA displayed only one allele. 10 different sequence types (STs) were revealed by MLST and 6 of them containing 79.8% of the isolates were classified into one clonal complex, demonstrating a close relationship among them. The multiplex-RAPD analysis was performed by employing the combined primers OPL-05+RD1 and divided the 98 L. sanfranciscensis isolates into 6 types with the similarity level of 70%. According to the result, it seems that the genotypic variations of L. sanfranciscensis strains showed by MLST have no relations to geographical origin. MLST seems to have a higher discriminatory power compared with multiplex-RAPD since it produced more groups, but multiplex-RAPD could help to distinguish some strains in the same ST. Hence, an optimal genotypic characterization of L. sanfranciscensis was obtained under the combination of MLST and multiplex-RAPD analysis, targeting different genetic variations.

Asunto(s)

Pan/microbiología , Microbiología de Alimentos/métodos , Genes Esenciales/genética , Lactobacillus/genética , Tipificación de Secuencias Multilocus , Técnica del ADN Polimorfo Amplificado Aleatorio , China , Cartilla de ADN/genética , Variación Genética/genética , Genotipo , Lactobacillus/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex

RESUMEN

Dihydropyrimidinase (DHP, EC 3.5.2.2), encoded by the gene DPYS, is the second enzyme in the catabolic pathway of pyrimidine and of fluoropyrimidine drugs such as 5-fluorouracil, which are commonly used in anticancer treatment; DHP catalyzes the hydrolytic ring opening of dihydrouracil and dihydro-5-fluorouracil. DPYS mutations are known to contribute to interindividual variations in the toxicity of fluoropyrimidine drugs, but the functional characterization of DHP allelic variants remains inadequate. In this study, in vitro analysis was performed on 22 allelic variants of DHP by transiently expressing wild-type DHP and 21 DHP variants in 293FT cells and characterizing their enzymatic activities by using dihydrouracil and dihydro-5-fluorouracil as substrates. DHP expression levels and oligomeric forms were determined using immunoblotting and blue native PAGE, respectively, and the stability of the DHP variants was assessed by examining the proteins in variant-transfected cells treated with cycloheximide or bortezomib. Moreover, three kinetic parameters, Km, Vmax, and intrinsic clearance (Vmax/Km), for the hydrolysis of dihydrouracil and dihydro-5-fluorouracil were determined. We found that 5/21 variants showed significantly decreased intrinsic clearance as compared to wild-type DHP, and that 9/21 variants were expressed at low levels and were inactive due to proteasome-mediated degradation. The band patterns observed in the immunoblotting of blue native gels corresponded to DHP activity, and, notably, 18/21 DHP variants exhibited decreased or null enzymatic activity and these variants also showed a drastically reduced ability to form large oligomers. Thus, detection of DPYS genetic polymorphisms might facilitate the prediction severe adverse effects of fluoropyrimidine-based treatments.

Asunto(s)

Amidohidrolasas , Antineoplásicos/metabolismo , Fluorouracilo/análogos & derivados , Variación Genética , Uracilo/análogos & derivados , Alelos , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Fluorouracilo/metabolismo , Humanos , Modelos Moleculares , Estabilidad Proteica , Especificidad por Sustrato , Transfección , Uracilo/metabolismo

RESUMEN

A large number of correctors have been identified that can partially repair defects in folding, stability and trafficking of CFTR processing mutants that cause cystic fibrosis (CF). The best corrector, VX-809 (Lumacaftor), has shown some promise when used in combination with a potentiator (Ivacaftor). Understanding the mechanism of VX-809 is essential for development of better correctors. Here, we tested our prediction that VX-809 repairs folding and processing defects of CFTR by promoting interactions between the first cytoplasmic loop (CL1) of transmembrane domain 1 (TMD1) and the first nucleotide-binding domain (NBD1). To investigate whether VX-809 promoted CL1/NBD1 interactions, we performed cysteine mutagenesis and disulfide cross-linking analysis of Cys-less TMD1 (residues 1-436) and ΔTMD1 (residues 437-1480; NBD1-R-TMD2-NBD2) truncation mutants. It was found that VX-809, but not bithiazole correctors, promoted maturation (exited endoplasmic reticulum for addition of complex carbohydrate in the Golgi) of the ΔTMD1 truncation mutant only when it was co-expressed in the presence of TMD1. Expression in the presence of VX-809 also promoted cross-linking between R170C (in CL1 of TMD1 protein) and L475C (in NBD1 of the ΔTMD1 truncation protein). Expression of the ΔTMD1 truncation mutant in the presence of TMD1 and VX-809 also increased the half-life of the mature protein in cells. The results suggest that the mechanism by which VX-809 promotes maturation and stability of CFTR is by promoting CL1/NBD1 interactions.

Asunto(s)

Aminopiridinas/metabolismo , Benzodioxoles/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Secuencia de Aminoácidos/genética , Sitios de Unión/fisiología , Células HEK293 , Humanos , Unión Proteica/fisiología

RESUMEN

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2µg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piranos/farmacología , Timidilato Sintasa/metabolismo , Antibacterianos/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Regulación hacia Abajo , Quimioterapia Combinada , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Timidilato Sintasa/genética

RESUMEN

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 µM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Mitomicina/toxicidad , Proteína Oncogénica v-akt/metabolismo , Recombinasa Rad51/biosíntesis , Antibióticos Antineoplásicos/toxicidad , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Mitomicina/uso terapéutico , Proteína Oncogénica v-akt/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , Xantófilas/toxicidad

RESUMEN

The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gßγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gßγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gßγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gßγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gßγ. The Tctex-1-Gßγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gßγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway.

Asunto(s)

Dineínas/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Receptor Cannabinoide CB2/biosíntesis , Agonismo Inverso de Drogas , Regulación de la Expresión Génica , Células HEK293 , Humanos , Indoles/farmacología , Unión Proteica/fisiología , Receptor Cannabinoide CB2/antagonistas & inhibidores

RESUMEN

Accumulating evidence suggests that prolyl-isomerase (Pin1) is an important regulator of apoptosis. In the present study, Pin1 was shown to negatively regulate the stabilization of glycogen synthase kinase (GSK) 3αß serine phosphorylation by inhibiting ubiquitin (Ub)-proteasome degradation, and to induce autophagy following cadmium (Cd) exposure. Cd-induced autophagy in human hepatoma (HepG2) cells has been demonstrated by green fluorescent protein (GFP)-LC3B plasmid DNA transfection, and LC3-II conversion by autophagy inhibitors. Atg5 silencing inhibited Cd-induced apoptosis, indicating that autophagy is involved in cell death. Exposing HepG2 cells to Cd (≤6 µM) initially increased p-Ser GSK3αß, but then resulted in a gradual decrease, which correlated with polyubiquitinated protein levels, which is indicative of protein degradation. However, high Cd concentrations lead to p-Ser GSK3αß and polyubiquitinated protein accumulation, indicating proteasome impairment. Cd-induced p-Ser GSK3αß was enhanced by proteasome inhibition (MG132) and ubiquitin deficiency (cyclohexamide), but no ubiquitination of p-Ser GSK3αß or GSK3αß was detected. Cd exposure resulted in p-Ser GSK3αß redistribution from the nucleus into the cytosol, and this along with autophagy, was inhibited by leptomycin B. Cd concentrations <6 µM did not alter Pin1; however, Cd≥6 µM decreased Pin1. Pin1 overexpression decreased Cd-induced p-Ser GSK3αß, autophagy, and polyubiquitinated protein levels, while its inhibition reversed the effects. Silencing and overexpression of GSK3αß had no effect on Cd-induced Pin1 levels. Immunoprecipitation studies showed that Pin1 and p-Ser GSK3αß interact. Collectively, Pin1 protects cells by inhibiting p-Ser GSK3αß, and Pin1 decrease upregulates p-Ser GSK3αß, through the inhibition of Ub-mediated proteasome degradation, and stimulates cell death via autophagy.

Asunto(s)

Autofagia/efectos de los fármacos , Cadmio/toxicidad , Glucógeno Sintasa Quinasa 3/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Procesamiento Proteico-Postraduccional , Serina/metabolismo , Ubiquitina/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Estabilidad de Enzimas , Glucógeno Sintasa Quinasa 3 beta , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA , Fosforilación

RESUMEN

Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Minociclina/farmacología , Mitomicina/farmacología , Recombinasa Rad51/genética , Antibióticos Antineoplásicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Minociclina/administración & dosificación , Mitomicina/administración & dosificación

RESUMEN

Transforming growth factor-ß (TGF-ß) has both tumor suppressive and oncogenic activities. Autocrine TGF-ß signaling supports tumor survival and growth in certain types of cancer, and the TGF-ß signaling pathway is a potential therapeutic target for these types of cancer. TGF-ß induces p21 expression, and p21 is considered as an oncogene as well as a tumor suppressor, due to its anti-apoptotic activity. Thus, we hypothesized that autocrine TGF-ß signaling maintains the expression of p21 at levels that can support cell growth. To verify this hypothesis, we sought to examine p21 expression and cell growth in various cancer cells following the inhibition of autocrine TGF-ß signaling using siRNAs targeting TGF-ß signaling components and SB431542, a TGF-ß receptor inhibitor. Results from the present study show that p21 expression and cell growth were reduced by knockdown of TGF-ß signaling components using siRNA in MDA-MB231 and A549 cells. Cell growth was also reduced in p21 siRNA-transfected cells. Downregulation of p21 expression induced cellular senescence in MDA-MB231 cells but did not induce apoptosis in both cells. These data suggest that autocrine TGF-ß signaling is required to sustain p21 levels for positive regulation of cell cycle. On the other hand, treatment with SB431542 up-regulated p21 expression while inhibiting cell growth. The TGF-ß signaling pathway was not associated with the SB431542-mediated induction of p21 expression. Specificity protein 1 (Sp1) was downregulated by treatment with SB431542, and p21 expression was increased by Sp1 knockdown. These findings suggest that downregulation of Sp1 expression is responsible for SB43154-induced p21 expression.

Asunto(s)

Benzamidas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Dioxoles/farmacología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Receptores de Factores de Crecimiento Transformadores beta/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Comunicación Autocrina/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células COS , Células CACO-2 , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Chlorocebus aethiops , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Células MCF-7 , Masculino , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factores de Tiempo , Transfección

RESUMEN

Inhibition of hypoxia inducible factor-prolyl hydroxylase-2 (HPH), leading to activation of hypoxia inducible factor (HIF)-1 is a potential therapeutic strategy for the treatment of colitis. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid is a naturally occurring polyphenolic compound with two catechols, a or inhibition of HPH. To improve accessibility of highly hydrophilic RA to HPH, an intracellular target, RA was chemically modified to decrease hydrophilicity. Of the less-hydrophilic derivatives, rosmarinic acid methyl ester (RAME) most potently inhibited HPH. Accordingly, RAME prevented hydroxylation of HIF-1α and consequently stabilized HIF-1α protein in cells. RAME inhibition of HPH and induction of HIF-1α were diminished by elevated doses of the required factors of HPH, 2-ketoglutarate and ascorbate. RAME induction of HIF-1α led to activation of an ulcer healing pathway, HIF-1-vascular endothelial growth factor (VEGF), in human colon carcinoma cells. RAME administered rectally ameliorated TNBS-induced rat colitis and substantially decreased the levels of pro-inflammatory mediators in the inflamed colonic tissue. In parallel with the cellular effects of RAME, RAME up-regulated HIF-1α and VEGF in the inflamed colonic tissue. Thus, lipophilic modification of RA improves its ability to inhibit HPH, leading to activation of the HIF-1-VEGF pathway. RAME, a lipophilic RA derivative, may exert anti-colitic effects via activation of the ulcer healing pathway.

Asunto(s)

Cinamatos/química , Cinamatos/farmacología , Colitis/tratamiento farmacológico , Depsidos/química , Depsidos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Animales , Ácido Ascórbico/farmacología , Ácidos Carboxílicos/química , Línea Celular Tumoral , Cinamatos/uso terapéutico , Coenzimas/fisiología , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Depsidos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Ésteres , Humanos , Hidroxilación/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ácidos Cetoglutáricos/farmacología , Estabilidad Proteica/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Ácido Trinitrobencenosulfónico/efectos adversos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ácido Rosmarínico