RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most devastating diseases; it has a considerably poor prognosis and may become the second most lethal malignancy in the next 10 years. Chemotherapeutic resistance is common in PDAC; thus, it is necessary to exploit effective alternative drugs. In recent years, traditional folk medicines and their extracts have shown great potential in cancer treatment. The seed of Lagenaria siceraria (Molina) Standl. is a traditional medicine in Asia. Because of its analgesic effects and ability to reduce swelling, it is often used as an adjuvant treatment for abdominal tumors. Cucurbitacin compounds are extracts abundant in Lagenaria siceraria (Molina) Standl. Here, we found that cucurbitacin C (CuC), a member of the cucurbitacin family, has apparent anti-PDAC therapeutic properties. CuC decreased the viability and suppressed the proliferation of PDAC cells in a time- and dose-dependent manner. Further studies revealed that CuC inhibited cell migration and invasion by inhibiting epithelial-mesenchymal transition (EMT). In addition, G2/M arrest was induced, and the apoptotic pathway was activated. Transcriptomic and bioinformatic analyses showed that CuC inhibited the cGMP-PKG-VASP axis, increasing the content of cGMP to restore tumor characteristics. The antitumor activity of CuC in vivo was verified through animal experiments, and no obvious side effects were observed. Overall, our study indicates a candidate therapeutic compound for PDAC that is worthy of further development.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Apoptosis , Cucurbitacinas/farmacología , Línea Celular Tumoral , Proliferación Celular , Puntos de Control de la Fase G2 del Ciclo Celular , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Transición Epitelial-Mesenquimal , Neoplasias PancreáticasRESUMEN
Potassium channels are involved in membrane hyperpolarization and ion homeostasis regulation during human sperm capacitation. However, the types of potassium channels in human sperm remain controversial. The voltage-gated ion channel KCNQ1 is ubiquitously expressed and regulates key physiological processes in the human body. In the present study, we investigated whether KCNQ1 is expressed in human sperm and what role it might have in sperm function. The expression and localization of KCNQ1 in human sperm were evaluated using Western blotting and indirect immunofluorescence. During capacitation incubation, human sperm were treated with KCNQ1- specific inhibitor chromanol 293B. Sperm motility was analyzed using a computer-assisted sperm analyzer. The acrosome reaction was studied using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Protein tyrosine phosphorylation levels and localization after capacitation were determined using Western blotting and immunofluorescence. Intracellular K+, Ca2+, Cl-, pH, and membrane potential were analyzed using fluorescent probes. The results demonstrate that KCNQ1 is expressed and localized in the head and tail regions of human sperm. KCNQ1 inhibition reduced sperm motility, acrosome reaction rates, and protein tyrosine phosphorylation but had no effect on hyperactivation. KCNQ1 inhibition also increased intracellular K+, membrane potential, and intracellular Cl-, while decreasing intracellular Ca2+ and pH. In conclusion, the KCNQ1 channel plays a crucial role during human sperm capacitation.
RESUMEN
BACKGROUND: Heat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s). As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37. Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility. Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways. METHODS: Human sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation. Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation. The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining. The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments. Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation. RESULTS: Human sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation. In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex. Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased. Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38. Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation. CONCLUSIONS: Taken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction. In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Adulto , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Masculino , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Heat shock protein 90 (Hsp90) signaling pathways participate in protein phosphorylation during sperm capacitation. However, the underlying mechanism is largely unknown. OBJECTIVE: The aim of this study was to explore the interaction between Hsp90 and its co-chaperone protein, cell division cycle protein Cdc37 (Cdc37), in human spermatozoa. MATERIALS AND METHODS: We examined the effects of H-89 (a protein kinase A [PKA] inhibitor) and Go6983 (a protein kinase C [PKC] inhibitor) on the phosphorylation of serine, threonine, and tyrosine residues in Hsp90; the effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG, Hsp90 inhibitor) on Y416-Src phosphorylation; and the effects of 17-AAG and geldanamycin on threonine phosphorylation during human sperm capacitation. RESULTS: Hsp90 co-localized and interacted with Cdc37. During human sperm capacitation, Hsp90 phosphorylation at serine, threonine, and tyrosine residues was inhibited by H-89 and Go6983. In addition, phosphorylation of residue Y416 in the tyrosine kinase Src (its active site) was inhibited by 17-AAG, and the threonine phosphorylation levels of some proteins were decreased by 17-AAG and geldanamycin. DISCUSSION AND CONCLUSION: Taken together, our data showed that the interaction of Hsp90 with Cdc37 regulates total protein threonine phosphorylation and Src phosphorylation via its serine, threonine, and tyrosine phosphorylation, which are controlled by PKA and PKC during human sperm capacitation. The results of this study help understand the mechanism underlying Hsp90 regulation of sperm function.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Capacitación Espermática , Espermatozoides/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Masculino , Fosforilación , Proteína Quinasa C/metabolismoRESUMEN
Extracellular polymeric substances (EPSs) extracted from fungal mycelium by four chemical methods (NaOH, H2SO4, formaldehyde-NaOH, glutaraldehyde-NaOH), three physical methods (heating, ultrasound, vibration), and a control method (centrifugation alone) were investigated. Results indicated formaldehyde-NaOH outperformed other methods with 186.6 ± 8.0 mg/g of polysaccharides and 23.2 ± 4.6 mg/g of protein extracted and ensured little contamination by intracellular substances. Thereafter, this method was applied in extracting EPS from a mixed fungal culture in the adaptation process with 0.5% (w/v) waste printed circuit boards (PCBs). With the four adaptation stages continuing, the culture tended to become more sensitive to respond to the external toxic environment characterized by secreting EPS more easily and quickly. The maximum amount of polysaccharides and protein could be achieved in only 3 days both at the 3rd and 4th adaptation stage. Three-dimensional excitation-emission matrix fluorescence spectrum indicated the peaks obtained for EPS were mainly associated to soluble microbial by-product-like and aromatic protein-like compounds. Transmission electron microscopic observation illustrated that although metal ions penetrated into hypha cells, parts of them could be absorbed by EPS, implying that EPS secretion may be a primary protective strategy adopted by the culture.
Asunto(s)
Matriz Extracelular de Sustancias Poliméricas/química , Formaldehído/química , Polisacáridos/química , Aclimatación , ProteínasRESUMEN
Purpureocillium lilacinum can promote the biomineralization of jarosite by secreting extracellular polymeric substances (EPS), but the detailed mechanism is not clear. In this study, the biosynthesis process of jarosite induced by P. lilacinum Y3 and hypha cell surface characterization were investigated. X-ray diffraction (XRD) analysis indicated that P. lilacinum Y3 could induce the formation of jarosite crystal and enhance mineralization kinetics. The kinetic and isotherm models confirmed that the metal ions transferring from the solution to the mycelium surface was controlled by diffusion process and the active interfacial sites on hypha cell surface played a pivotal role in the biomineralization process. Furthermore, transmission electron microscopy (TEM) pictures illustrated that the P. lilacinum Y3 mainly induced the generation of mineral precipitate extracellularly, but not intracellularly. Three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectrum results further revealed the extracellular compounds such as fulvic-acid-like and protein-like substances participated in the mineralization process.
Asunto(s)
Compuestos Férricos/metabolismo , Modelos Biológicos , Paecilomyces/metabolismo , Sulfatos/metabolismo , Biomineralización , Compuestos Férricos/química , Cinética , Paecilomyces/citología , Tamaño de la Partícula , Sulfatos/química , Propiedades de SuperficieRESUMEN
Glioblastoma (GBM) is the most common primary malignancy of the central nervous system (CNS) with <10% 5-year survival rate. The growth and invasion of GBM cells into normal brain make the resection and treatment difficult. A better understanding of the biology of GBM cells is crucial to the targeted therapies for the disease. In this study, we identified Septin9 (SEPT9) and Septin2 (SEPT2) as GBM-related genes through integrated multi-omics analysis across independent transcriptomic and proteomic studies. Further studies revealed that expression of SEPT9 and SEPT2 was elevated in glioma tissues and cell lines (A172, U87-MG). Knockdown of SEPT9 and SEPT2 in A172/U87-MG was able to inhibit GBM cell proliferation and arrest cell cycle progression in the S phase in a synergistic mechanism. Moreover, suppression of SEPT9 and SEPT2 decreased the GBM cell invasive capability and significantly impaired the growth of glioma xenografts in nude mice. Furthermore, the decrease in GBM cell growth caused by SEPT9 and SEPT2 RNAi appears to involve two parallel signaling pathway including the p53/p21 axis and MEK/ERK activation. Together, our integration of multi-omics analysis has revealed previously unrecognized synergistic role of SEPT9 and SEPT2 in GBM, and provided novel insights into the targeted therapy of GBM.
Asunto(s)
Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Septinas/genética , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Biología Computacional/métodos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Progresión de la Enfermedad , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Invasividad Neoplásica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/genética , Septinas/antagonistas & inhibidores , Septinas/metabolismo , Carga Tumoral , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cyanobacteria have been proven to be cheaper and more effective for the removal of metallic elements in aqueous solutions. In this study, the living cyanobacteria Synechocystis sp. PCC6803 was used to adsorb Cd(II) and its extracellular polymeric substances (EPS) were investigated in the adsorption process. The initial stage of adsorption of Cd(II) was a rapid process, and then increase slowly accompanied with the increases of biomass. The final adsorption percentage could achieve 86% when the Cd(II) concentration was 0.5 mg/L. It proved that Synechocystis sp. PCC6803 has a good adsorption capacity for heavy metal ions. EPS was extracted to investigate the secretion of which was dynamic and the maximum extracellular polysaccharides and proteins were 134.2 and 100.9 mg/g, respectively. Furthermore, the real-time PCR (RT-PCR) results of genes (slr0977 and exoD) involved in EPS synthesis and secretion indicated that the EPS production was firstly increased and then decreased slightly. Transmission electron microscope (TEM) observation revealed that heavy metal ions were absorbed into EPS layer. Fourier transform infrared spectrum (FT-IR) analysis showed that EPS was rich in functional groups which could combine with heavy metal ions, such as -OH and -NH groups. All the results obtained show that the secretion of EPS by cyanobacteria was one of the ways to resist heavy metal stress. And it shows a trend of rising first and then decreasing, the change regulation of which was consistent with adsorptive behavior.
Asunto(s)
Cadmio/análisis , Contaminantes Ambientales/análisis , Polisacáridos/metabolismo , Synechocystis/metabolismo , Adsorción , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomasa , Cadmio/toxicidad , Contaminantes Ambientales/toxicidad , Polisacáridos/genética , Synechocystis/efectos de los fármacos , Synechocystis/crecimiento & desarrolloRESUMEN
Biohydrometallurgy is generally considered as a green technology for the recycling of industrial solid waste. In this study, an indigenous fungal strain named Y5 with the ability of high-yielding organic acids was isolated and applied in bioleaching of waste printed circuit boards (PCBs). The strain Y5 was identified as Penicillium chrysogenum by morphological and molecular identification. Meanwhile, we investigated that an optimal set of culturing conditions for the fungal growth and acids secretion was 15 g/L glucose with initial pH 5.0, temperature 25°C and shaking speed 120 rpm in shaken flasks culture. Moreover, three bioleaching processes such as one-step, two-step and spent medium processes were conducted to extract copper from waste PCBs. Spent medium bioleaching showed higher copper extraction percentage and it was 47% under 5%(w/v) pulp density. Transmission electron microscope (TEM) observation combining with energy dispersive analysis of X-rays (EDAX) showed that the leached metal ions did not obviously damage the hypha cells. All above results indicated that P.chrysogenum strain Y5 has the tolerance to metal ions, suggesting its potential in recycling of metals from waste PCBs in industry.
Asunto(s)
Cobre/farmacocinética , Residuos Electrónicos , Residuos Industriales , Penicillium chrysogenum/aislamiento & purificación , Penicillium chrysogenum/metabolismo , Reciclaje/métodos , Biodegradación Ambiental , Cobre/análisis , Cobre/aislamiento & purificación , Tecnología Química Verde/métodos , Metalurgia/métodos , Metales Pesados/química , Metales Pesados/aislamiento & purificación , Metales Pesados/farmacocinética , Microscopía Electrónica de Transmisión , Penicillium chrysogenum/citología , Contaminantes del Suelo/química , Contaminantes del Suelo/aislamiento & purificación , Contaminantes del Suelo/farmacocinéticaRESUMEN
Accurate quantification of gene expression is fundamental for understanding the molecular, genetic and functional bases of tissue development and diseases. Quantitative real-time PCR (qPCR) is now the most widely used method of quantifying gene expression due to its simplicity, specificity, sensitivity, and wide quantification range. The use of appropriate reference genes to ensure accurate normalization is crucial for the correct quantification of gene expression from the early development, maturation, aging to injury processes in the central nervous system (CNS). In this study, we have determined the expression profiles of 12 candidate housekeeping genes (ACTB, CYC1, HMBS, GAPDH, HPRT1, RPL13A, YWHAZ, PPIA, RPLP0, TFRC, GUS, and 18S rRNA) in developing mouse brain and spinal cord. Throughout development, there was a significant degree of fluctuations in their expression levels, indicating the importance and complexity of finding appropriate reference genes. Three software including BestKeeper, geNorm and NormFinder were used to evaluate the stability of potential reference genes. GUS was the most stable gene and GUS/YWHAZ were the most stable reference gene pair across different developmental stages in different CNS regions, whereas HPRT1 and GAPDH were the most variable genes and thus inappropriate to use as reference genes. Therefore, our results identified GUS and YWHAZ as the best combination of two reference genes for expression data normalization in CNS developmental studies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 39-50, 2018.
Asunto(s)
Lesiones Traumáticas del Encéfalo/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
In this study, the biosynthesis of jarosite by Purpureocillium lilacinum was investigated. Firstly, we found when the pH value was lower than 2.50 at 30 °C, the concentration of Fe3+ in the solution significantly dropped about 72% after inoculation and a yellow-ocher precipitate was observed on the mycelium surface. X-ray diffraction analysis revealed the precipitate was jarosite. Thereafter, the characterization of the biomineralization process by scanning electron microscopy showed that mineral precipitates started on the cell surface, and then thoroughly covered it. Furthermore the effect of extracellular polymeric substances (EPS) on the biosynthesis of jarosite was investigated. The results suggested Fe3+ only dropped 5.2% in 2 days when EPS were stripped. Finally, through monitoring the changes of mycelium surface groups by Fourier transform infrared spectroscopy, we found the biomineralization process originated from the existence of free P[double bond, length as m-dash]O groups in EPS which acted as crystallization nuclei to promote Fe(OH)3 transformation into jarosite by the formation of P-O-Fe bonds.