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BACKGROUND: Kidney diseases are a major global health problem affecting millions of people. Despite this, there is as yet no effective drug therapy improving outcome in patients with renal disease. The aim of this study was to examine the nephroprotective effect of α-lipoic acid (ALA) in vitro and to examine the effect of ALA administered in vivo on the production of reactive sulfur species (RSS), including hydrogen sulfide (H2S) and compounds containing sulfane sulfur. METHODS: The effect of ALA was studied in vitro by determining the viability of human embryonic kidney cells (HEK293) in normoxic and hypoxic conditions as well as in vivo in two groups of chronic kidney disease (CKD) patients: non-dialyzed (ND) and undergoing continuous ambulatory peritoneal dialysis (PD) after 30 days of ALA supplementation. RESULTS: The results revealed that the viability of HEK293 cells was significantly decreased by hypoxic conditions, while ALA administered during hypoxia increased the viability to the level observed in normoxic conditions. Studies performed in plasma of CKD patients after ALA supplementation suggested that ALA did not affect the parameters of oxidative stress, while significantly increased the level of reactive sulfane sulfur in both ND and PD patients suffering from CKD. The results suggest that ALA can exert nephroprotective effects which are related to sulfane sulfur production.
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Insuficiencia Renal Crónica , Ácido Tióctico , Humanos , Ácido Tióctico/farmacología , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/tratamiento farmacológico , Células HEK293 , Masculino , Femenino , Supervivencia Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Persona de Mediana Edad , Antioxidantes/farmacología , Sulfuro de Hidrógeno/farmacologíaRESUMEN
The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.
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Cápside , Parvovirinae , Humanos , Células HEK293 , Vectores Genéticos/genética , Dependovirus/genética , Plásmidos/genética , Proteínas de la Cápside/genética , Parvovirinae/genéticaRESUMEN
INTRODUCTION AND OBJECTIVE: Mobile phones and Wi-Fi are the most commonly used forms of telecommunications. Initiated with the first generation, the mobile telephony is currently in its fifth generation without being screened extensively for any biological effects that it may have on humans or on animals. Some studies indicate that high frequency electromagnetic radiation emitted by mobile phone and Wi-Fi connection can have a negative effect upon human health, and can cause cancer, including brain tumour. OBJECTIVE: The aim of the study was to investigate the influence of 2.4 GHz radiofrequency electromagnetic field (RF-EMF) on the proliferation and morphology of normal (human embryonic kidney cell line Hek-293) and cancer cells (glioblastoma cell line U-118 MG). MATERIAL AND METHODS: The cell cultures were incubated in RF-EMF at the frequency of 2.4 GHz, with or without dielectric screen, for 24, 48 and 72h. In order to analyse the influence of the electromagnetic field on cell lines, Cytotoxicity test Cell Counting Kit-8 was performed. To protect cells against emission of the electromagnetic field, a dielectric screen was used. RESULTS: It was found that 2.4 GHz RF electromagnetic field exposure caused a significant decrease in viability of U-118 MG and Hek-293 cells. The impact of the electromagnetic field was strongest in the case of cancer cells, and the decrease in their survival was much greater compared to the healthy (normal) cells of the Hek-293 line. CONCLUSIONS: Results of the study indicate that using a radio frequency electromagnetic field (2.4 GHz) has a clearly negative effect on the metabolic activity of glioblastoma cells. RF-EMF has much less impact on reducing the viability of normal cells (Hek -293) than cancer cells.
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Campos Electromagnéticos , Glioblastoma , Animales , Humanos , Campos Electromagnéticos/efectos adversos , Exposición a Riesgos Ambientales/análisis , Células HEK293 , Ondas de Radio/efectos adversosRESUMEN
Nanoparticle uptake by cells has been studied for applications both in nanomedicine and in nanosafety. While the majority of studies have focused on the biological mechanisms underlying particle internalization, less attention has been given to questions of a more quantitative nature, such as how many nanoparticles enter cells and how rapidly they do so. To address this, we exposed human embryonic kidney cells to 40-200 nm carboxylated polystyrene nanoparticles and the particles were observed by live-cell confocal and super-resolution stimulated emission depletion fluorescence microscopy. How long a particle remained at the cell membrane after adsorbing onto it was monitored, distinguishing whether the particle ultimately desorbed again or was internalized by the cell. We found that the majority of particles desorb, but interestingly, most of the particles that are internalized do so within seconds, independently of particle size. As this is faster than typical endocytic mechanisms, we interpret this observation as the particles entering via an endocytic event that is already taking place (as opposed to directly triggering their own uptake) or possibly via an as yet uncharacterized endocytic route. Aside from the rapidly internalizing particles, a minority of particles remain at the membrane for tens of seconds to minutes before desorbing or being internalized. We also followed particles after cell internalization, observing particles that appeared to exit the cell, sometimes as rapidly as within tens of seconds. Overall, our results provide quantitative information about nanoparticle cell internalization times and early trafficking.
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Nanopartículas , Tetranitrato de Pentaeritritol , Humanos , Transporte Biológico , Ácidos Carboxílicos , Membrana Celular , RiñónRESUMEN
BACKGROUND AND PURPOSE: GABAA receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate. EXPERIMENTAL APPROACH: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C-21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABAA receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations. KEY RESULTS: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six- (compound 5) or five-membered heterocyclic ring (compound 6) on C-21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABAA receptor, are the most likely cause of the distinctive functional profiles. CONCLUSIONS AND IMPLICATIONS: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next-generation GABAA receptor drug design and development.
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Pregnenolona , Receptores de GABA-A , Receptores de GABA-A/metabolismo , Pregnenolona/farmacología , Pregnenolona/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
BACKGROUND: Zearalenone (ZEA) is a mycotoxin produced by fungi and induces cytotoxicity by the generation of reactive oxygen species. The aim of this study was to evaluate and compare the nephroprotective effects of crocin and nano-crocin against ZEA-induced toxicity in HEK293 cell line via modulation of oxidative stress and special formulation to make nano-crocin. METHOD: Nano-crocin physicochemical properties, such as size, load, appearance, and drug release profile were determined. Also, the viability of intoxicated HEK293 cells was evaluated by MTT assay. Furthermore, lactate dehydrogenase lipid Peroxidation (LPO), and oxidative stress biomarkers were measured. RESULT: The best nano-crocin formulation with superior entrapment effectiveness (54.66 ± 6.02), more significant drug loading (1.89 ± 0.01), better zeta potential (-23.4 ± 2.844), and smaller particle size (140.3 ± 18.0 nm) was chosen. This study showed that treatment with crocin and nano-crocin in ZEA-induced cells, significantly decreased LDH and LPO levels and increased superoxide dismutase (SOD), catalase (CAT) activities, and total antioxidant capacity (TAC) levels compared to the control group. Moreover, nano-crocin had a more curative effect against oxidative stress than crocin. CONCLUSION: Niosomal structure of crocin, when administered with the special formulation, may be more beneficial in reducing ZEA-induced in vitro toxicity than conventional crocin.
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Zearalenona , Humanos , Células HEK293 , Zearalenona/toxicidad , Antioxidantes/uso terapéutico , Carotenoides/farmacología , Carotenoides/uso terapéutico , Estrés OxidativoRESUMEN
Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.
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Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iß (SET/TAF-Iß), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iß is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iß is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iß:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iß (a.k.a. SET/TAF-Iß ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.
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Human embryonic kidney cells are the host of adenovirus type-5 (Ad5) amplification. An Ad5-vector-based COVID-19 vaccine has been proven to be tolerated and immunogenic in healthy adults. Therefore, a rationally designed scaffold for culturing human embryonic kidney cells is useful for further studying its mechanism of action. Herein, a three-dimensional layered reticulated polycaprolactone (PCL) scaffold coated with poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) was developed to proliferate human embryonic kidney cells and to be used to amplify the Ad5 vector. The results indicate that PCEC improves the hydrophilicity and the cell culture ability of PCL cell culture scaffolds, resulting in a three times higher cell proliferation ratio of human embryonic kidney cells compared with those grown on bare PCL cell culture scaffolds. Meanwhile, the cytotoxicity test results showed that the scaffold material is noncytotoxic. This work provides an effective and scalable method for the in-depth study of adenoviruses.
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Methylglyoxal (MGO) is a reactive carbonyl species that can cause cellular damage and is closely related to kidney disease, especially diabetic nephropathy. The toxic effect of MGO (0.5, 1, and 2â mM) on human embryonic kidney (HEK293) cells and its underlying mechanisms were explored in this study. Cell viability, apoptosis and the signaling pathways were measured with MTT, fluorescent staining and western blot experiments, the results showed that MGO could induce oxidative stress and cell inflammation, the level of reactive oxygen species (ROS) increased, and p38MAPK, JNK and NF-κB signaling pathways were activated. Meanwhile, MGO also induced DNA damage. The expression of DNA oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) increased, the expression of double-strand break marker γH2AX increased significantly, and ATM/Chk2/p53 DNA damage response signaling pathway was activated. Furthermore, the expression of the receptor for advanced glycation end products (RAGE) also increased. Finally, mitochondrial membrane potential (MMP) decreased, fluorescence intensity of Hoechst33258 increased, and the protein expression ratio of Bax/Bcl-2 increased significantly after the treatment of MGO. These results demonstrated that MGO might induce HEK293 cells damage by regulating oxidative stress, inflammation, DNA damage, and cell apoptosis, which revealed the specific mechanisms of MGO-induced damage to HEK293 cells.
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Estrés Oxidativo , Piruvaldehído , Apoptosis , Daño del ADN , Células HEK293 , Humanos , Riñón , Piruvaldehído/metabolismo , Piruvaldehído/toxicidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
By using only half of the total evolution time for dephasing pulses, C{N} rotational-echo double resonance (REDOR) for clusters of 13C spins (RDX) results in the same universal REDOR behavior as observed for isolated 13C-15N pairs. RDX combines Hahn echoes with solid echoes to suppress interference from scalar J couplings. This is crucial for long evolution times. The modified version (which we call RDX24) makes RDX quantitative for 13C clusters. We apply this scheme to human embryonic kidney cells labeled in culture by L-[13C5 -15N2]-glutamine. We quantitatively characterize three separate nitrogen isotopic enrichments for: (i) the alpha nitrogens of glutamine residues in proteins (including the residues of the five amino acids synthesized from glutamine); (ii) the alpha nitrogens of the five amino-acid residues synthesized from glucose, together with those of the nine essential amino acids added to the growth medium; and (iii) the side-chain nitrogens of glutamine (and of asparagine derived from glutamine).
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Espectroscopía de Resonancia Magnética , Isótopos de Carbono , Humanos , Isótopos de NitrógenoRESUMEN
Several plants have traditionally been used since antiquity to treat various gastroenteritis and respiratory symptoms similar to COVID-19 outcomes. The common symptoms of COVID-19 include fever or chills, cold, cough, flu, headache, diarrhoea, tiredness/fatigue, sore throat, loss of taste or smell, asthma, shortness of breath, or difficulty breathing, etc. This study aims to find out the plants and plant-derived products which are being used by the COVID-19 infected patients in Bangladesh and how those plants are being used for the management of COVID-19 symptoms. In this study, online and partially in-person survey interviews were carried out among Bangladeshi respondents. We selected Bangladeshi COVID-19 patients who were detected Coronavirus positive (+) by RT-PCR nucleic acid test and later recovered. Furthermore, identified plant species from the surveys were thoroughly investigated for safety and efficacy based on the previous ethnomedicinal usage reports. Based on the published data, they were also reviewed for their significant potentialities as antiviral, anti-inflammatory, and immunomodulatory agents. We explored comprehensive information about a total of 26 plant species, belonging to 23 genera and 17 different botanical families, used in COVID-19 treatment as home remedies by the respondents. Most of the plants and plant-derived products were collected directly from the local marketplace. According to our survey results, greatly top 5 cited plant species measured as per the highest RFC value are Camellia sinensis (1.0) > Allium sativum (0.984) > Azadirachta indica (0.966) > Zingiber officinale (0.966) > Syzygium aromaticum (0.943). Previously published ethnomedicinal usage reports, antiviral, anti-inflammatory, and immunomodulatory activity of the concerned plant species also support our results. Thus, the survey and review analysis simultaneously reveals that these reported plants and plant-derived products might be promising candidates for the treatment of COVID-19. Moreover, this study clarifies the reported plants for their safety during COVID-19 management and thereby supporting them to include in any future pre-clinical and clinical investigation for developing herbal COVID-19 therapeutics.
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The formation of ß-glucuronides is a major route by which mammals detoxify and remove breakdown products, such as l-tyrosine, as well as many xenobiotics, from their systems. In humans, dietary l-tyrosine is broken down largely by the action of the anaerobic gut bacterium C. difficile to p-cresol, providing a competitive advantage in the gut microbiota. Ortho- (o-) and meta- (m-), cresols, also present in the environment, may share a common degradative pathway. Relatively little work has been done on cresyl glucuronides. Here, a direct synthesis of o-, m-, and p-cresyl ß-D-glucuronides from methyl 1,2,3,4 tetra-O-acetyl-ß-d-glucuronate and the respective cresol employing trimethylsilyltriflate as promoter is presented. The protected intermediates were hydrolysed using aqueous sodium carbonate to yield the cresyl ß-glucuronides. The toxicities of the o-, m- and p-cresyl ß-D-glucuronides were compared. All three were less toxic to HEK293 cells than their respective cresol precursors: toxicity followed the order o < m < p for Na+ salts and o < p < m for Ca2+ salts. The m-cresyl-glucuronide Ca2+ salt and p-cresyl-glucuronide Na+ salt reduced colony formation by 11% and 9% (v. 30% reduction from the aglycone) respectively, whereas o-cresyl-glucuronide (both Na+ and Ca2+ salts), mildly stimulated HEK293 cell growth.
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Cresoles/farmacología , Glucurónidos/farmacología , Supervivencia Celular/efectos de los fármacos , Cresoles/síntesis química , Cresoles/química , Relación Dosis-Respuesta a Droga , Glucurónidos/síntesis química , Glucurónidos/química , Células HEK293 , Humanos , Estructura Molecular , EstereoisomerismoRESUMEN
Curdlan or laminarin, a ß-1,3-glucan was hydrolysed by ß-1,3-endoglucanase (CtLam81A) from Clostridium thermocellum to produce laminari-oligosaccharides. TLC analysis of hydrolysed curdlan showed the presence of laminari-oligosaccharides of the degree of polymerization, DP2-DP7. This mixture of laminari-oligosaccharides displayed prebiotic properties. Laminari-oligosaccharides showed an increase in the growth of probiotic bacteria such as Lactobacillus plantarum DM5 and Lactobacillus acidophilus, while they did not promote the growth of non-probiotic bacteria (Escherichia coli and Enterobacter aerogenes). Laminari-oligosaccharides showed higher prebiotic activity score of 0.92 ± 0.01 and 0.64 ± 0.08 for L. plantarum DM5 and L. acidophilus NRRL B-4496, respectively, similar to those shown by inulin. Laminari-oligosaccharides showed higher resistance or low digestibility against α-amylase, artificial gastric juice and intestinal fluid than inulin indicating their bioavailability to the probiotic bacteria present in the gastrointestinal tract of human. The probiotic bacteria consumed laminaribiose and laminariotriose more readily than higher laminari-oligosaccharides as carbon source for their growth. The in vitro cytotoxicity assay of laminari-oligosaccharides (1 mg/ml) on human embryonic kidney (HEK 293) cells showed that the cell viability was not affected even after 72 h indicating their biocompatible nature. All the results amply indicated that laminari-oligosaccharides can serve as potential prebiotic additives for functional food products.
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INTRODUCTION: Basic fibroblast growth factor (bFGF) is a promising cytokine in regenerative therapy for spinal cord injury. In this study, recombinant canine bFGF (rc-bFGF) was synthesized for clinical use in dogs, and the ability of rc-bFGF to differentiate canine bone marrow mesenchymal stem cells (BMSCs) into functional neurons was investigated. METHODS: The rc-bFGF was synthesized using a wheat germ cell-free protein synthesis system. The expression of rc-bFGF mRNA in the purification process was confirmed using a reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to confirm the antigenic property of the purified protein. To verify function of the purified protein, phosphorylation of extracellular signal-regulated kinase (ERK) was examined by in vitro assay using HEK293 cells. To compare the neuronal differentiation capacity of canine BMSCs in response to treatment with rc-bFGF, the cells were divided into the following four groups: control, undifferentiated, rh-bFGF, and rc-bFGF groups. After neuronal induction, the percentage of cells that had changed to a neuron-like morphology and the mRNA expression of neuronal markers were evaluated. Furthermore, to assess the function of the canine BMSCs after neuronal induction, changes in the intracellular Ca2+ concentrations after stimulation with KCl and l-glutamate were examined. RESULTS: The protein synthesized in this study was rc-bFGF and functioned as bFGF, from the results of RT-PCR, western blotting, and the expression of pERK in HEK293 cells. Canine BMSCs acquired a neuron-like morphology and expressed mRNAs of neuronal markers after neuronal induction in the rh-bFGF and the rc-bFGF groups. These results were more marked in the rc-bFGF group than in the other groups. Furthermore, an increase in intracellular Ca2+ concentrations was observed after the stimulation of KCl and l-glutamate in the rc-bFGF group, same as in the rh-bFGF group. CONCLUSIONS: A functional rc-bFGF was successfully synthesized, and rc-bFGF induced the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells. Our purified rc-bFGF may contribute, on its own, or in combination with canine BMSCs, to regenerative therapy for spinal cord injury in dogs.
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Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and ß isoforms to study their interaction with amyloid ß peptide (Aß42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/ß and immobilized Aß42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.
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Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Motivos EF Hand , Glucógeno Sintasa Quinasas/aislamiento & purificación , Calcio/química , Expresión Génica , Glucógeno Sintasa Quinasa 3/aislamiento & purificación , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta/aislamiento & purificación , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasas/química , Glucógeno Sintasa Quinasas/genética , Glucógeno Sintasa Quinasas/metabolismo , Células HEK293 , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Graphene, a two-dimensional carbon sheet with single-atom thickness, shows immense promise in several nanoscientific and nanotechnological applications, including in sensors, catalysis, and biomedicine. Although several studies have shown the cytotoxicity of graphene oxide in different cell types, there are no comprehensive studies on human embryonic kidney (HEK293) cells that include transcriptomic analysis and an in vitro investigation into the mechanisms of cytotoxicity following exposure to graphene oxide. Therefore, we exposed HEK293 cells to different concentrations of graphene oxide for 24 h and performed several cellular assays. Cell viability and proliferation assays revealed a significant dose-dependent cytotoxic effect on HEK293 cells. Cytotoxicity assays showed increased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) generation, and decreased levels of reduced glutathione (GSH) and increased level of oxidized glutathione indicative of oxidative stress. This detailed mechanistic approach showed that graphene oxide exposure elicits significant decreases in mitochondrial membrane potential and ATP synthesis, as well as in DNA damage and caspase 3 activity. Furthermore, our RNA-Seq analysis revealed that HEK293 cells exposed to graphene oxide significantly altered the expression of genes involved in multiple apoptosis-related biological pathways. Moreover, graphene oxide exposure perturbed the expression of key transcription factors, promoting these apoptosis-related pathways by regulating their downstream genes. Our analysis provides mechanistic insights into how exposure to graphene oxide induces changes in cellular responses and massive cell death in HEK293 cells. To our knowledge, this is the first study describing a combination of cellular responses and transcriptome in HEK293 cells exposed to graphene oxide nanoparticles, providing a foundation for understanding the molecular mechanisms of graphene oxide-induced cytotoxicity and for the development of new therapeutic strategies.
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In our continued efforts to develop metal based therapeutic agents, we have synthesized a novel copper(II) complex, [{Cu(hpdbal-sbdt)}2] (2) tethered with a biocompatible ONS2- donor backbone [H2hpdbal-sbdt] (1) [H2hpdbal-sbdt is a tridentate ligand derived from S-benzyldithiocarbazate (Hsbdt) and 2-hydroxy-5-(phenyldiazenyl)benzaldehyde (Hhpdbal)]. The metal complex (2) was characterized using attenuated total reflection-infrared (ATR-IR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, thermogravimetry and differential scanning calorimetric (TG-DSC) analysis, field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS) and elemental (CHNS) analysis. The antineoplastic ability of copper complex was evaluated in vitro against human cervical cancer (HeLa) cells. MTT assay results showed that the copper complex exhibited significant growth inhibition of HeLa cells with an IC50 value of 4.46⯵M and this value was compared with reported standards. Cytotoxicity of the copper complex towards human embryonic kidney cells (HEK-293) was also evaluated. The potentially active copper complex was studied for its solution state stability at a pH range of 3-9. Following this, the interactive behaviour of the bioactive copper complex with a drug transporter protein (BSA) was deciphered through multi-spectrosopic investigations like steady-state fluorescence, three-dimensional fluorescence, deconvoluted-IR and UV-Visible techniques.
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Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Evaluación Preclínica de Medicamentos , Albúmina Sérica Bovina/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Bovinos , Muerte Celular/efectos de los fármacos , Cobre , Espectroscopía de Resonancia por Spin del Electrón , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/química , Soluciones , Espectrometría por Rayos X , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Termodinámica , TermogravimetríaRESUMEN
Schiff bases are versatile organic compounds which are widely used and synthesized by condensation reaction of different amino compound with aldehydes or ketones known as imine. Schiff base ligands are considered as privileged ligands as they are simply synthesized by condensation. They show broad range of application in medicine, pharmacy, coordination chemistry, biological activities, industries, food packages, dyes, and polymer and also used as an O2 detector. Semicarbazone is an imine derivative which is derived from condensation of semicarbazide and suitable aldehyde and ketone. Imine ligand-containing transition metal complexes such as copper, zinc, and cadmium have shown to be excellent precursors for synthesis of metal or metal chalcogenide nanoparticles. In recent years, the researchers have attracted enormous attention toward Schiff bases, semicarbazones, thiosemicarbazones, and their metal complexes owing to numerous applications in pharmacology such as antiviral, antifungal, antimicrobial, antimalarial, antituberculosis, anticancer, anti-HIV, catalytic application in oxidation of organic compounds, and nanotechnology. In this review, we summarize the synthesis, structural, biological, and catalytic application of Schiff bases as well as their metal complexes.
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Renal failures treatment has been faced with several problems during the last decades. Kidney tissue engineering has been created many hopes to improve treatment procedures with scaffold fabrication that can modulate kidney cells/stem cells migration to the lesion site and increase the survival of these cells at that site with imitating the role of the kidney extracellular matrix. In this study, bone morphogenetic protein-7 (BMP7) as a vital factor for kidney development and regeneration was incorporated in the polycaprolactone (PCL) nanofibers and after morphological, mechanical, and biocompatible characterization, proliferation, and survival of the human embryonic kidney cells (HEK) were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and gene expression while cultured on scaffolds. Mechanical properties of the PCL nanofibers modulated after combining with BMP7 and hydration degree, protein adsorption and cell adhesion were enhanced in PCL-BMP7 compared to the pure PCL. Proliferation rate and growth increased significantly in HEK cells cultured on PCL-BMP7 when compared with that of PCL and tissue culture plate, whereas these data were also confirmed via significant decrease in apoptotic genes expression level in HEK cell cultured on PCL-BMP7. According to the results, PCL-BMP7 demonstrated positive effects on the survival and proliferation rate of the kidney cells and showed has also a great potential to use as a bioimplant for kidney tissue engineering applications.