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BACKGROUND: Epigenome, genetic variants, and other environmental factors involved in gene regulation are highly inter-dependent in several chronic diseases, including obesity, cardiovascular disease, and diabetes. The present study aimed at testing the associations and the mechanism involved in silencing of CYP2R1 gene in normal and obese Saudi women patients. Height, weight, BMI, 25-hydroxy vitamin D, parathyroid hormone, glycemic status, and lipid profile (TG, LDL, HDL, and TC) of CYP2R1 were measured in 100 women (31 normal and 69 obese patients). RESULTS: Our result shows that hypermethylation in site 2 of the CYP2R1 gene with body weight (p < 0.004), BMI (p < 0.002), waist circumference (p < 0.002), total-LDL (p < 0.027), total cholesterol (p < 0.022), and vitamin D (VD) (close to borderline significance p < 0.06) and site 4 of CYP2R1 with LDL (p < 0.041) in the four tested sites among normal and obese women was significantly associated. Moreover, we tested five different CpG sites in the CYP27B1 gene where site 5 correlated significantly with VD levels. CONCLUSION: Our present study clearly indicates that hypermethylation of specific sites in the CYP2R1 and CYP27B1 genes might regulate gene expression with special reference to the risk of obesity and vitamin D metabolism.
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The identification of lipolytic bioactive compounds via the functional stimulation of carbohydrate response element-binding protein-1 (CREBp-1) and AMP-activated protein kinase (AMPK) is most warranted. Nano lipid carriers (NLCs) are currently being considered within drug delivery development as they facilitate controlled drug release and have intracellular bioavailability after encapsulating the active principles with lipid matrix. The present study has been designed to synthesize punicalagin, and ketogenic amino acids (KAA) loaded with organic lipid carriers to optimize the liposome-assisted intracellular delivery's bioavailability. Punicalagin (PUNI) and KAA (tryptophan, methionine, threonine, lysine, and leucine) were encapsulated with chia seed phospholipids by homogenization, emulsification, and cold ultra-sonication method to obtain nano lipid carriers (NLC). The physicochemical characterization of NLCs has been carried out using Zetasizer, FT-IR, and TEM analysis. Punicalagin and ketogenic amino acid-loaded NLCs (NLC-PUNI-KAA) were identified with an average diameter of 240 to 800 nm. The biosafety of NLC-PUNI-KAA has been evaluated in human mesenchymal stem cells. PI staining confirmed that a 0.4, 0.8 or 1.6µg/dL dose of NLC-PUNI-KAA potentially maintains nuclear integration. NLC-PUNI-KAA treated with maturing adipocytes decreased lipid accumulation and significantly increased the gene expression levels of fatty acid beta-oxidation (PPARγC1α, UCP-1 and PRDM-16) pathways when compared to free PUNI (5 µg/dL) treatment. The lipolytic potential has been confirmed by the functional activation of AMPK and CREBp-1 protein levels. In conclusion, NLC-PUNI-KAA treatment effectively increased mitochondrial efficiency more than free punicalagin or orlistat treated maturing adipocyte. Enhanced lipolysis and decreased hypertrophic adipocyte resulted in decreased adipokine secretion, which has been associated with the suppression of obesity-associated comorbidities and vascular cell inflammation. The bioefficacy and lipolytic potential of water-soluble punicalagin have been improved after functional modification into NLCs.
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Excessive storage of lipids in visceral or ectopic sites stimulates adipokine production, which attracts macrophages. This process determines the pro- and anti-inflammatory response regulation in adipose tissue during obesity-associated systemic inflammation. The present study aimed to identify the composition of Ocimum basilicum L. (basil) seed extract and to determine its bio-efficacy on adipocyte thermogenesis or fatty acid oxidation and inhibition of lipid accumulation and adipokine secretion. Ocimum basilicum L. seed methanol extract (BSME) was utilized to analyze the cytotoxicity vs. control; lipid accumulation assay (oil red O and Nile red staining), adipogenesis and mitochondrial-thermogenesis-related gene expression vs. vehicle control were analyzed by PCR assay. In addition, vehicle control and BSME-treated adipocytes condition media were collected and treated with lipopolysaccharide (LPS)-induced macrophage to identify the macrophage polarization. The results shown that the active components present in BSME did not produce significant cytotoxicity in preadipocytes or macrophages in the MTT assay. Furthermore, oil red O and Nile red staining assay confirmed that 80 and 160 µg/dL concentrations of BSME effectively arrested lipid accumulation and inhibited adipocyte maturation, when compared with tea polyphenols. Gene expression level of adipocyte hyperplasia (CEBPα, PPARγ) and lipogenesis (LPL)-related genes have been significantly (p ≤ 0.05) downregulated, and mitochondrial-thermogenesis-associated genes (PPARγc1α, UCP-1, prdm16) have been significantly (p ≤ 0.001) upregulated. The BSME-treated, maturing, adipocyte-secreted proteins were detected with a decreased protein level of leptin, TNF-α, IL-6 and STAT-6, which are associated with insulin resistance and macrophage recruitment. The "LPS-stimulated macrophage" treated with "BSME-treated adipocytes condition media", shown with significant (p ≤ 0.001) decrease in metabolic-inflammation-related proteins-such as PGE-2, MCP-1, TNF-α and NF-κB-were majorly associated with the development of foam cell formation and progression of atherosclerotic lesion. The present findings concluded that the availability of active principles in basil seed effectively inhibit adipocyte hypertrophy, macrophage polarization, and the inflammation associated with insulin resistance and thrombosis development. Ocimum basilicum L. seed may be useful as a dietary supplement to enhance fatty acid oxidation, which aids in overcoming metabolic complications.
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Adipocitos/efectos de los fármacos , Adipoquinas/metabolismo , Macrófagos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ocimum basilicum/química , Extractos Vegetales/farmacología , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Inmunohistoquímica , Inflamación , Metabolismo de los Lípidos/efectos de los fármacos , Macrófagos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metanol/química , Oxidación-Reducción , Extractos Vegetales/química , Termogénesis/efectos de los fármacosRESUMEN
This research was aimed at finding the cytotoxic potential of the mixed ligand copper(II) complex [Cu(tdp)(phen)](ClO4)-where H(tdp) is the tetradentate ligand 2-[(2-(2-hydroxyethylamino)-ethylimino)methyl]phenol, and phen is 1,10-phenanthroline-to two genotypically different breast cancer cells, MCF-7 (p53+ and ER+) and MDA-MB-231 (p53- and ER-). The complex has been already shown to be cytotoxic to ME180 cervical carcinoma cells. The special focus in this study was the induction of cell death by apoptosis and necrosis, and its link with ROS. The treatment brought about nuclear fragmentation, phosphatidylserine externalization, disruption of mitochondrial trans-membrane potential, DNA damage, cell cycle arrest at sub-G1 phase, and increase of ROS generation, followed by apoptotic death of cells during early hours and a late onset of necrosis in the cells surviving the apoptosis. The efficacy of the complex against genotypically different breast cancer cells is attributed to a strong association through p53-mitochondrial redox-cell cycle junction. The ADMET properties and docking of the complex at the active site of Top1 are desirable attributes of a lead molecule for development into a therapeutic. Thus, it is shown that the copper(II)-phenolate complex[Cu(tdp)(phen)]+ offers potential to be developed into a therapeutic for breast cancers in general and ER-negative ones in particular.
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Neoplasias de la Mama/patología , Simulación por Computador , Cobre/farmacología , Hidroxibenzoatos/farmacología , Receptores de Estrógenos/metabolismo , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayo Cometa , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN , ADN de Neoplasias/metabolismo , Femenino , Fluorescencia , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Coloración y EtiquetadoRESUMEN
Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications.
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Células Madre Pluripotentes Inducidas/patología , Mutación Puntual , Neoplasias Cutáneas/genética , Xerodermia Pigmentosa/genética , Línea Celular Tumoral , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de la radiación , Modelos Biológicos , Análisis de Secuencia de ADN , Neoplasias Cutáneas/etiología , Xerodermia Pigmentosa/complicaciones , Xerodermia Pigmentosa/patología , Proteína de la Xerodermia Pigmentosa del Grupo A/genéticaRESUMEN
Incidence of breast cancer shows geographical variation, even within areas of ethnic homogeneity. Saudi Arabia has witnessed an increase in occurrence of breast cancer in its unexplored ethnic populations over the past few years. We aimed at determining whether any association exists between single nucleotide polymorphisms in breast cancer associated gene 1 (BRCA1) and breast cancer associated gene 2 (BRCA2) and the risk of breast cancer. TaqMan based Real Time Polymerase chain reaction genotyping assays were used to determine the frequency of single nucleotide polymorphisms in BRCA1 (rs799917) and BRCA2 (rs144848) in a group of 100 breast cancer patients and unaffected age matched controls of Saudi Arabian origin. The present data revealed that neither BRCA1 nor the BRCA2 studied variant show any significant association with the disease. This study failed to find any role of the concerned variants in breast cancer either as risk or as prognostic factors. The small number of patients registered was one of the limitations of this study. In summary, comparison of mutation profile with other ethnic populations and regions reflected both differences and similarities indicating co-exposure to a unique set of risk factors. The differences could be due to exposure to particular environmental carcinogens; different lifestyle, reproductive pattern; dietary or cultural practices of Saudi Arabian women that need further investigations.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Neoplasias de la Mama/epidemiología , Etnicidad/genética , Femenino , Humanos , Incidencia , Persona de Mediana Edad , Riesgo , Factores de Riesgo , Arabia SauditaRESUMEN
Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes, high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties, although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extract from Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF- 7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion, TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in the presence of FME at 65 µg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptor pathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However, such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by a time-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FME induced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, as well as p53 interdependently.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Extractos Vegetales/farmacología , Trigonella/química , Receptor fas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Humanos , Células MCF-7 , Extractos Vegetales/química , Plantas Medicinales/química , Proteína p53 Supresora de Tumor/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Anisomelic acid (AA), one of the major compounds in Anisomeles malabarica, was tested for its cytotoxicity and apoptosis-inducing potential in breast and cervical cancer cells. The MTT assay for cell viability indicated that AA is cytotoxic to all of the four cell lines tested in a dose- and duration-dependent manner. Acridine Orange & Ethidium Bromide (AO & EB) and Hoechst 33258 staining of AA-treated cells revealed typical apoptotic morphology such as condensed chromatin and formation of apoptotic bodies. The comet assay revealed DNA strand break(s), indicating that AA induces DNA damage which culminates in apoptosis. Thus, the study revealed the anti-proliferative and apoptosis-inducing properties of AA in both breast and cervical cancer cells. Therefore, anisomelic acid offers potential for application in breast and cervical cancer therapy.
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OBJECTIVES: To find the efficacy of serial extracts of Anisomeles malabarica in inhibiting proliferation of and inducing apoptosis in human cervical cancer cells, SiHa and ME 180, that are HPV 16-positive. MATERIALS AND METHODS: The whole plant was extracted in n-hexane, chloroform, ethyl acetate, n-butanol, methanol, and water. The cells were treated with the extracts at increasing concentrations to find the IC(50), adopting MTT ([3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. Acridine orange (AO) and ethidium bromide (EB) and Hoechst 33258 staining were adopted to assess the mode of cell death, Annexin V-Cy3 staining to evaluate one of the early apoptotic features, JC-1 staining to assess the mitochondrial membrane depolarization, comet assay for DNA fragmentation, and cell cycle analysis for the distribution of cells after treatment. RESULTS: n-Hexane and chloroform extracts were cytotoxic to the cervical cancer cells in dose- and duration-dependent manner. The cells that responded to the treatments revealed typical apoptotic features. Early features of apoptosis, phosphatidyl serine translocation and loss of mitochondrial trans-membrane potential, were observed in the treated cells, and comet assay revealed DNA damage. In the FACS analysis, the cells accumulated in the sub-G0/G1 phase of the cell cycle, except in n-hexane- and chloroform extract-treated SiHa cells at 24 h, which showed arrest in S- and G2/M phases. CONCLUSIONS: n-Hexane and chloroform extracts of A. malabarica inhibit proliferation of and induce death in HPV16-positive cervical cancer cells, mostly by apoptosis and to some extent by necrosis.
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UNLABELLED: Aristolochia manshuriensis has been used for centuries in Chinese medicinal system for their versatile medicinal uses. Recent studies have revealed two new aristolactames (compound A and B) with γ-lactame ring fused with the phenentherene ring as potent inhibitors of human Cycline Dependent Kinase2 (CDK2). Studies on aristolactames and related compounds claim for their CDK2 inhibition without delineating the involved mechanism and structural basis of interaction. Molecular structural model was used to we propose a structural basis of CDK2 inhibition. We showed that these compounds (A and B) can successfully dock into the inhibitor binding pockets of human CDK2. Predicted binding affinities are comparable to known inhibitors of CDK2. Results were in agreement with the earlier biochemical studies. Hence, suggest that studied compounds A and B can be a promising scaffold for rational design of novel and potential drugs against cancer. ABBREVIATIONS: CDK2 - cyclin-dependent kinase 2, OLO - Olomoucine, NW1 - Cyclohexylmethyloxy-5-Nitroso-Pyrimidine- 2, 4-Diamine, CMG - 6-O-Cyclohexylmethyl Guanine.