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Here, we report the design and synthesis of a Dâ¯πâ¯A-based fluorescent probe, (E)-4-(4-(dibutylamine)-2-hydroxystyryl)-1-methylquinolin-1-ium (DHMQ), which is nonfluorescent in â¼100% PBS buffer medium due to a twisted intra molecular charge transfer (TICT) phenomenon and it becomes highly fluorescent (â¼149 fold) in the presence of human serum albumin (HSA), owing to the restriction of its intramolecular free rotation inside the hydrophobic binding cavity of HSA. The site-selective fluorescence displacement assay and molecular docking studies clearly reveal that DHMQ selectively binds at subdomain IB of HSA. The 3σ/slope method was adopted to determine the limit of detection (LOD) value, which was as low as 2.39 nM in â¼100% PBS medium, indicating its high sensitivity towards HSA. The low dissociation constant value [Kd = (1.066 ± 0.017) µM] suggests a strong complexation between the DHMQ and HSA. Importantly, it has been demonstrated that DHMQ is capable of detecting HSA in real human serum and urine samples and was found to be suitable for live cell imaging of HSA.
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Human serum albumin (HSA) is regarded as a useful biomarker for rapid medical diagnosis of various disorders mainly related to the kidneys and liver. Hence, it is crucial to identify and monitor the HSA level in complex biofluids (urine and blood samples) using a simple approach. Herein, we have designed and synthesized an intramolecular charge transfer (ICT) based environment-sensitive fluorescent molecular probe, (E)-2-(3-(2-(5-methoxy-1H-indol-3-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (DCI-MIN), that can selectively interact with HSA in PBS buffer solution and exhibit a â¼78-fold enhancement in fluorescence intensity with a significant Stokes shift (â¼126 nm), which is important to avoid interference from the excitation light. The significant red fluorescence response can be attributed to the suppression of free intramolecular rotation of the DCI-MIN probe inside the hydrophobic binding cavity of HSA and the low polar microenvironment present within HSA. According to the 3σ/slope method, the detection limit was found to be 1.01 nM (0.0671 mg L-1) in aqueous solutions, which is significantly lower than the normal level of HSA in healthy urine and blood serum, indicating its high sensitivity. DCI-MIN has the ability to exhibit useful applications, including the detection and quantification of HSA concentration in complex biofluids (human urine and blood samples) as well as the imaging of serum albumin in living cells.
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Corantes Fluorescentes , Albumina Sérica Humana , Humanos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Albumina Sérica Humana/metabolismo , Albumina Sérica Humana/análise , Albumina Sérica Humana/química , Espectrometria de Fluorescência , Estrutura Molecular , Imagem ÓpticaRESUMO
Nitric oxide (NO) is a ubiquitous messenger molecule playing a key role in various physiological and pathological processes. However, producing a selective turn-on fluorescence response to NO is a challenging task due to (a) the very short half-life of NO (typically in the range of 0.1-10 s) in the biological milieu and (b) false positive responses to reactive carbonyl species (RCS) (e.g., dehydroascorbic acid and methylglyoxal etc.) and some other reactive oxygen/nitrogen species (ROS/RNS), especially with o-phenylenediamine (OPD) based fluorosensors. To avoid these limitations, NO sensors should be designed in such a way that they react spontaneously with NO to give turn-on response within the time frame of t1/2 (typically in the range of 0.1-10 s) of NO and λem in the visible wavelength along with good cell permeability to achieve biocompatibility. With these views in mind, a N-nitrosation based fluorescent sensor, NDAQ, has been developed that is highly selective to NO with â¼27-fold fluorescence enhancement at λem = 542 nm with high sensitivity (LOD = 7 ± 0.4 nM) and shorter response time, eliminating the interference of other reactive species (RCS/ROS/RNS). Furthermore, all the photophysical studies with NDAQ have been performed in 98% aqueous medium at physiological pH, indicating its good stability under physiological conditions. The kinetic assay illustrates the second-order dependency with respect to NO concentration and first-order dependency with respect to NDAQ concentration. The biological studies reveal the successful application of the probe to track both endogenous and exogenous NO in living organisms.
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Óxido Nítrico , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio , Nitrosação , Fluorescência , OxigênioRESUMO
BACKGROUND: Oral submucous fibrosis (OSF) is a debilitating collagen-metabolic disorder leading to submucosal fibrosis and trismus. Lysyl oxidase (LOX), a critical collagen biosynthetic enzyme, is up-regulated in OSF. Polymorphisms in the Lysyl oxidase gene have been associated with increased risk of OSF and might affect normal collagen synthesis, accumulation, or degradation, crucial in determining fibrosis severity. METHODS: One hundred OSF cases and 100 controls were genotyped for LOX G473A(Arg158Gln) polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The expression of LOX was estimated both by quantitative mRNA analysis and western blot. Total soluble collagen was evaluated from mucosal tissue obtained from OSF cases. Immunohistochemical (IHC) localization of type 1 collagen was performed in mucosal tissue obtained from patients carrying various genotypes. RESULTS: Heterozygous G473A genotype was significantly higher in OSF cases [2.063(95% CI =1.059-4.016)], among 26-40 years age-group [4.375(95% CI=1.323-14.267),p=0.029] and in male patients [2.38 (95% CI= 1.107-5.121), p= 0.042]. LOX expression was significantly higher in cases of the heterozygous or homozygous carrier (p <0.001). We found the total soluble collagen level significantly (p <0.001) higher among patients carrying GA or AA genotype. IHC revealed focal deposition of type1 collagen in the submucosal tissue; comparatively higher deposition was evident in mucosal tissue of OSF patients carrying AA genotype. CONCLUSIONS: These findings suggest LOX G473A polymorphism confers an increased risk of OSF and may affect collagen accumulation in OSF cases.
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Colágeno/metabolismo , Predisposição Genética para Doença , Fibrose Oral Submucosa/patologia , Polimorfismo de Nucleotídeo Único , Proteína-Lisina 6-Oxidase/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Índia/epidemiologia , Lactente , Recém-Nascido , Masculino , Fibrose Oral Submucosa/epidemiologia , Fibrose Oral Submucosa/genética , Fibrose Oral Submucosa/metabolismo , Prognóstico , Fatores de Risco , Adulto JovemRESUMO
Human populations of Black African ancestry have a relatively high risk of aggressive cancer types, including keratinocyte-derived squamous cell carcinomas (SCCs). We show that primary keratinocytes (HKCs) from Black African (Black) versus White Caucasian (White) individuals have on average higher oncogenic and self-renewal potential, which are inversely related to mitochondrial electron transfer chain activity and ATP and ROS production. HSD17B7 is the top-ranked differentially expressed gene in HKCs and Head/Neck SCCs from individuals of Black African versus Caucasian ancestries, with several ancestry-specific eQTLs linked to its expression. Mirroring the differences between Black and White HKCs, modulation of the gene, coding for an enzyme involved in sex steroid and cholesterol biosynthesis, determines HKC and SCC cell proliferation and oncogenicity as well as mitochondrial OXPHOS activity. Overall, the findings point to a targetable determinant of cancer susceptibility among different human populations, amenable to prevention and management of the disease.
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Carcinoma de Células Escamosas , Proliferação de Células , Humanos , Queratinócitos , OncogenesRESUMO
Melanoma susceptibility differs significantly in male versus female populations. Low levels of androgen receptor (AR) in melanocytes of the two sexes are accompanied by heterogeneous expression at various stages of the disease. Irrespective of expression levels, genetic and pharmacological suppression of AR activity in melanoma cells blunts proliferation and induces senescence, while increased AR expression or activation exert opposite effects. AR down-modulation elicits a shared gene expression signature associated with better patient survival, related to interferon and cytokine signaling and DNA damage/repair. AR loss leads to dsDNA breakage, cytoplasmic leakage, and STING activation, with AR anchoring the DNA repair proteins Ku70/Ku80 to RNA Pol II and preventing RNA Pol II-associated DNA damage. AR down-modulation or pharmacological inhibition suppresses melanomagenesis, with increased intratumoral infiltration of macrophages and, in an immune-competent mouse model, cytotoxic T cells. AR provides an attractive target for improved management of melanoma independent of patient sex.
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Carcinogênese/genética , Proliferação de Células/genética , Melanoma/genética , Receptores Androgênicos/genética , Transdução de Sinais/genética , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Dano ao DNA/genética , Reparo do DNA/genética , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Polimerase II/genéticaRESUMO
Cancer associated fibroblasts (CAFs) are a key component of the tumor microenvironment. Genomic alterations in these cells remain a point of contention. We report that CAFs from skin squamous cell carcinomas (SCCs) display chromosomal alterations, with heterogeneous NOTCH1 gene amplification and overexpression that also occur, to a lesser extent, in dermal fibroblasts of apparently unaffected skin. The fraction of the latter cells harboring NOTCH1 amplification is expanded by chronic UVA exposure, to which CAFs are resistant. The advantage conferred by NOTCH1 amplification and overexpression can be explained by NOTCH1 ability to block the DNA damage response (DDR) and ensuing growth arrest through suppression of ATM-FOXO3a association and downstream signaling cascade. In an orthotopic model of skin SCC, genetic or pharmacological inhibition of NOTCH1 activity suppresses cancer/stromal cells expansion. Here we show that NOTCH1 gene amplification and increased expression in CAFs are an attractive target for stroma-focused anti-cancer intervention.
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Fibroblastos Associados a Câncer/metabolismo , Amplificação de Genes , Receptor Notch1/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Dano ao DNA , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Humanos , Camundongos , Camundongos SCID , Receptor Notch1/genética , Pele/metabolismo , Neoplasias Cutâneas/genéticaRESUMO
Fluorescence spectroscopy is a significant bio-analytical technique for specific detection of nitric oxide (NO) and for broadcasting the in vitro and in vivo biological activities of this gasotransmitter. Herein, a benzo-coumarin embedded smart molecular probe (BCM) is employed for NO sensing through detailed fluorescence studies in purely aqueous medium. All the spectroscopic analysis and literature reports clearly validate the mechanistic insight of this sensing strategy i.e., the initial formation of 1,2,3,4-oxatriazole on treatment of the probe with NO which finally converted to its carboxylic acid derivative. This oxatriazole formation results in a drastic enhancement in fluoroscence intensity due to the photoinduced electron transfer (PET) effect. The kinetic investigation unveils the second and first-order dependency on [NO] and [BCM] respectively. The very low detection limit (16 nM), high fluorescence enhancement (123 fold) in aqueous medium and good formation constant (Kf = (4.33 ± 0.48) × 104 M-1) along with pH invariability, non-cytotoxicity, biocompatibility and cell permeability make this probe a very effective one for tracking NO intracellularly.
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CumarínicosRESUMO
Genomic instability is a hallmark of cancer. Whether it also occurs in Cancer Associated Fibroblasts (CAFs) remains to be carefully investigated. Loss of CSL/RBP-Jκ, the effector of canonical NOTCH signaling with intrinsic transcription repressive function, causes conversion of dermal fibroblasts into CAFs. Here, we find that CSL down-modulation triggers DNA damage, telomere loss and chromosome end fusions that also occur in skin Squamous Cell Carcinoma (SCC)-associated CAFs, in which CSL is decreased. Separately from its role in transcription, we show that CSL is part of a multiprotein telomere protective complex, binding directly and with high affinity to telomeric DNA as well as to UPF1 and Ku70/Ku80 proteins and being required for their telomere association. Taken together, the findings point to a central role of CSL in telomere homeostasis with important implications for genomic instability of cancer stromal cells and beyond.
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Fibroblastos Associados a Câncer/metabolismo , Carcinoma de Células Escamosas/metabolismo , Fibroblastos/metabolismo , Instabilidade Genômica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Telômero/metabolismo , Carcinoma de Células Escamosas/genética , Dano ao DNA , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Homeostase , Humanos , Autoantígeno Ku/metabolismo , Proteínas de Membrana , Simulação de Acoplamento Molecular , Mutagênese , RNA Helicases/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Pele/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Transativadores/metabolismoRESUMO
A new rhodamine 6G-benzylamine-based sensor (L1), having only hydrocarbon skeletons in the extended part, was synthesized and characterized by single-crystal X-ray crystallographic study. It exhibited excellent selective and sensitive recognition of trivalent metal ions M3+ (M = Fe, Al and Cr) over mono- and di-valent and other trivalent metal ions. A large enhancement of the fluorescence intensity for Fe3+ (41-fold), Al3+ (31-fold) and Cr3+ (26-fold) was observed upon the addition of 3.0 equivalent of these metal ions into the probe in H2O/CH3CN (4 : 1, v/v, pH 7.2) with naked eye detection. The corresponding Kf values were evaluated to be 9.4 × 103 M-1 (Fe3+), 1.34 × 104 M-1 (Al3+) and 8.7 × 103 M-1 (Cr3+). Quantum yields of the L1, [L1-Fe3+], [L1-Al3+] and [L1-Cr3+] complexes in H2O/CH3CN (4 : 1, v/v, pH 7.2) were found to be 0.012, 0.489, 0.376 and 0.310, respectively, using rhodamine-6G as standard. LODs for Fe3+, Al3+ and Cr3+ were determined by 3σ methods and found to be 1.28, 1.34 and 2.28 µM, respectively. Cyanide ion scavenged Fe3+ from the [Fe3+-L1] complex and quenched its fluorescence via its ring-closed spirolactam form. Advanced level molecular logic devices using different inputs (2 and 4 inputs) as advanced level logic gates and memory devices were constructed. The large enhancement in fluorescence emission of L1 upon complexation with M3+ metal ions makes the probe suitable for the bio-imaging of M3+ (M = Fe, Al and Cr) in living cells.
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Alumínio/análise , Técnicas Biossensoriais , Cromo/análise , Compostos Férricos/análise , Corantes Fluorescentes/farmacologia , Alumínio/química , Benzilaminas/química , Benzilaminas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cromo/química , Cristalografia por Raios X , Compostos Férricos/química , Corantes Fluorescentes/química , Células Hep G2 , Humanos , Íons , Limite de Detecção , Imagem Óptica , Rodaminas/química , Rodaminas/farmacologiaRESUMO
We report a novel phenazine-embedded fluorescent probe (2-[2-(pyridin-2-ylmethoxy)-phenyl]-1H-imidazo[4,5-b]phenazine, PIP), which upon complexation with Cu(II)-ion-forming [(PIP)CuII(Cl)] becomes nonfluorescent but regenerates fluorescence in a selective reaction with NO and HNO over different biologically reactive oxygen and nitrogen (ROS/RNS) species under physiological conditions. The fluorescence intensity of PIP gets quenched due to the formation of the [(PIP)CuII(Cl)] complex, which regenerates the fluorescence by 67 and 84% upon reaction either with NO or HNO, respectively, in the presence of other biological reducing species. Details of photophysical properties of PIP, [(PIP)CuII(Cl)], and [(PIP)CuI] have been studied by density functional theory (DFT) calculations. The recognition efficacy of [(PIP)CuII(Cl)] for exogenous and endogenous NO and HNO in A549 and RAW 264.7 cells with the flow cytometry application has also been demonstrated successfully.
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The aging-associated increase of cancer risk is linked with stromal fibroblast senescence and concomitant cancer-associated fibroblast (CAF) activation. Surprisingly little is known about the role of androgen receptor (AR) signaling in this context. We have found downmodulated AR expression in dermal fibroblasts underlying premalignant skin cancer lesions (actinic keratoses and dysplastic nevi) as well as in CAFs from the 3 major skin cancer types, squamous cell carcinomas (SCCs), basal cell carcinomas, and melanomas. Functionally, decreased AR expression in primary human dermal fibroblasts (HDFs) from multiple individuals induced early steps of CAF activation, and in an orthotopic skin cancer model, AR loss in HDFs enhanced tumorigenicity of SCC and melanoma cells. Forming a complex, AR converged with CSL/RBP-Jκ in transcriptional repression of key CAF effector genes. AR and CSL were positive determinants of each other's expression, with BET inhibitors, which counteract the effects of decreased CSL, restoring AR expression and activity in CAFs. Increased AR expression in these cells overcame the consequences of CSL loss and was by itself sufficient to block the growth and tumor-enhancing effects of CAFs on neighboring cancer cells. As such, the findings establish AR as a target for stroma-focused cancer chemoprevention and treatment.
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Fibroblastos Associados a Câncer/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/metabolismo , Ativação Transcricional , Animais , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas de Neoplasias/genética , Receptores Androgênicos/genética , Proteínas Repressoras/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
A simple molecular probe displays highly selective turn-on response toward NO by the unprecedented NO-induced formation of a 1,2,3,4-oxatriazole ring exhibiting no interference from various endogenous biomolecules including DHA, AA, etc. Kinetics of the reactions between NO and the probe provide a mechanistic insight into the formation of 1,2,3,4-oxatriazole which showed that, though initially 1,2,3,4-oxatriazole is formed and extractable in solid form, it exists in equilibrium with the ring opened azide form which ultimately hydrolyzed and converted to carboxylic acid and nitrate. The reaction displays second-order dependence on [NO] and first-order on [Probe]. The probe is water-soluble, cell permeable, and noncytotoxic and appropriates for live cell imaging. This constitutes the first report where there is a direct evidence of NO-induced ring closing reaction of an acyl hydrazide moiety leading to the formation of 1,2,3,4-oxatriazole.
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A new sensor (L3) based on Rhodamine-B-en (2) and 2-(pyridin-2-ylmethoxy)benzaldehyde (1) has been developed for highly sensitive and selective recognition of NO in purely aqueous medium where the reaction of NO with the fluorophore leads to an unusual formation of nitrosohydroxylamine with the selective opening of the spirolactam ring over different cations, anions, amino-acids and other biological species with prominent enhancement in absorption and emission intensities. A large enhancement of fluorescence intensity for NO (11 fold) was observed upon addition of 3 equivalents of NO into the sensor in aqueous HEPES buffer (20 mM) at pH 7.20, µ = 0.05 M NaCl with naked eye detection. The corresponding Kf value was evaluated to be (7.55 ± 2.04) × 104 M-1 from the fluorescence titration plot. Quantum yields of L3 and the [L3 + NO] compound are found to be 0.07 and 0.77, respectively, using Rhodamine-6G as the standard. The LOD for NO was determined by the 3σ method and found to be 83.4 nM. The L3 sensor has low cytotoxicity, and is cell permeable and suitable for in vitro NO sensing. The in vivo compatibility of the sensor was also checked on zebrafish.
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Benzaldeídos/química , Imagem Molecular/métodos , Óxido Nítrico/análise , Rodaminas/química , Animais , Morte Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Fluorescência , Hidroxilaminas/química , Limite de Detecção , Óxido Nítrico/química , Água , Peixe-ZebraRESUMO
A smart molecule, QT490, containing thiosemicarbazide moiety acts as a highly selective turn-on in vitro NO sensor through the unprecedented NO-induced transformation of thiosemicarbazide moiety to 1,3,4-oxadiazole heterocycle with the concomitant release of HSNO, thereby eliminating any interference from various endogenous biomolecules including dehydroascorbic acid, ascorbic acid, etc. The kinetic studies of the reactions between QT490 and NO provide a mechanistic insight into formation of HSNO/RSNO from the reaction between H2S/RSH and NO in the biological system. This novel probe is non-cytotoxic, cell permeable, water-soluble, and appropriate for intracellular cytoplasmic NO sensing with the possibilities of in vivo applications.
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Óxido Nítrico/química , S-Nitrosotióis/síntese química , Semicarbazidas/química , Células HeLa , Humanos , Estrutura Molecular , S-Nitrosotióis/químicaRESUMO
BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the common malignancies in Southeast Asia. Epigenetic changes, mainly the altered DNA methylation, have been implicated in many cancers. Considering the varied environmental and genotoxic exposures among the Indian population, we conducted a genome-wide DNA methylation study on paired tumor and adjacent normal tissues of ten well-differentiated OSCC patients and validated in an additional 53 well-differentiated OSCC and adjacent normal samples. RESULTS: Genome-wide DNA methylation analysis identified several novel differentially methylated regions associated with OSCC. Hypermethylation is primarily enriched in the CpG-rich regions, while hypomethylation is mainly in the open sea. Distinct epigenetic drifts for hypo- and hypermethylation across CpG islands suggested independent mechanisms of hypo- and hypermethylation in OSCC development. Aberrant DNA methylation in the promoter regions are concomitant with gene expression. Hypomethylation of immune genes reflect the lymphocyte infiltration into the tumor microenvironment. Comparison of methylome data with 312 TCGA HNSCC samples identified a unique set of hypomethylated promoters among the OSCC patients in India. Pathway analysis of unique hypomethylated promoters indicated that the OSCC patients in India induce an anti-tumor T cell response, with mobilization of T lymphocytes in the neoplastic environment. Survival analysis of these epigenetically regulated immune genes suggested their prominent role in OSCC progression. CONCLUSIONS: Our study identified a unique set of hypomethylated regions, enriched in the promoters of immune response genes, and indicated the presence of a strong immune component in the tumor microenvironment. These methylation changes may serve as potential molecular markers to define risk and to monitor the prognosis of OSCC patients in India.
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Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Metilação de DNA/imunologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias Bucais/genética , Neoplasias Bucais/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/patologia , Ilhas de CpG , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/imunologia , Estudo de Associação Genômica Ampla/métodos , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Prognóstico , Regiões Promotoras Genéticas , Carcinoma de Células Escamosas de Cabeça e Pescoço , Análise de Sobrevida , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Neo-tanshinlactone (NTL) a natural product is known for its specificity and selectivity towards the breast cancer cells. By NTL D-ring modification approach, 13 new analogues were synthesized (1A-1M). Among them 1J showed the best anticancer activity in MCF-7 (ER+, PR+/-, HER2-), SKBR3 (ER-, PR-, HER2+) and MDA-MB-231 (ER-, PR-, HER2-) cells lines with IC50 value 11.98nM, 23.71nM, and 62.91nM respectively. 1J showed minor grove binding interaction with DNA at AT-rich region and induced DNA double strand breaks (DDSBs). This had triggered several key molecular events involving, activation of ATM, Chk2 and p53, reduction in mitochondrial potential (Δψm) leading to caspase-3 and PARP cleavage mediated apoptosis. These results along with other biochemical studies strongly suggest that novel NTL analogue 1J caused DNA cleavage mediated apoptosis in the breast cancer cells and this may serve as potential lead for future breast cancer treatment.
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Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Furanos/farmacologia , Pironas/farmacologia , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Fator de Transcrição E2F1/metabolismo , Furanos/síntese química , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Pironas/síntese química , Proteína do Retinoblastoma/metabolismo , Relação Estrutura-AtividadeRESUMO
Cholera, an acute infection of the small intestine, is caused by Vibrio cholerae. The present study identified a hypothetical protein in V. cholerae O395, which was predicted to be acquired through horizontal gene transfer the origin of which was found to be from a phage. Its expression was further confirmed by RT-PCR. Homology based 3D model of the hypothetical protein indicated DksA like homologue. Protein binding site of 3D-model revealed a deep cleft which may influence the dimer formation and interaction with ds-DNA molecule. Also, canonical function of direct interaction with RNA polymerase (RNAP) holoenzyme in complex with ppGpp suggests its dual role in the pathogenesis of cholera.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Vibrio cholerae/genética , Vibrio cholerae/fisiologia , Sítios de Ligação , Simulação por Computador , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Perfilação da Expressão Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A newly designed oxime based probe, 2,4-dihydroxyacetophenone-oxime (DHAO), was found to recognize H2AsO4- selectively with â¼3 fold "turn-on" fluorescence enhancement and LOD of 29 µM, K = (2.10 ± 0.54) × 104 M-1 in purely aqueous medium. The structures of the DHAO and its adduct with H2AsO4- were optimized by DFT calculations. Intracellular imaging of As(V) in HepG2 cells demonstrate the possibilities of in vitro/in vivo applications for selective monitoring of such species.
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Arseniatos/análise , Corantes Fluorescentes/síntese química , Imagem Óptica/métodos , Oximas/química , Corantes Fluorescentes/química , Células Hep G2 , HumanosRESUMO
Context The effect of 6-gingerol (6G), the bioactive component of Zingiber officinale Roscoe (Zingiberaceae), in the reduction of Vibrio cholerae (Vibrionaceae)-induced inflammation has not yet been reported. Materials and methods Cell viability assay was performed to determine the working concentration of 6G. Elisa and RT-PCR were performed with Int 407 cells treated with 50 µM 6G and 100 multiplicity of infection (MOI) V. cholerae for 0, 2, 3, 3.5, 6 and 8 h to determine the concentration of IL-8, IL-6, IL-1α and IL-1ß in both protein and RNA levels. Furthermore, the effect of 50 µM 6G on upstream MAP-kinases and NF-κB signalling pathways was evaluated at 0, 10, 15, 30, 60 and 90 min. Results The effective dose (ED50) value of 6G was found to be 50 µM as determined by cell viability assay. Pre-treatment with 50 µM 6G reduced V. cholerae infection-triggered levels of IL-8, IL-6, IL-1α and IL-1ß by 3.2-fold in the protein level and two-fold in the RNA level at 3.5 h. The levels of MAP-kinases signalling molecules like p38 and ERK1/2 were also reduced by two- and three-fold, respectively, after 30 min of treatment. Additionally, there was an increase in phosphorylated IκBα and down-regulation of p65 resulting in down-regulation of NF-κB pathway. Conclusion Our results showed that 6G could modulate the anti-inflammatory responses triggered by V. cholerae-induced infection in intestinal epithelial cells by modulating NF-κB pathway.