Changes in the Expression of Insulin Pathway, Neutrophil Elastase and Alpha 1 Antitrypsin Genes from Leukocytes of Young Individuals with Insulin Resistance.

Background: Chronic hyperinsulinemia is a hallmark of insulin resistance that affects a diversity of cells, including leukocytes modifying the expression of some genes involved in insulin signaling. Purpose: The aim of this study was to evaluate how hyperinsulinemia affects the expression of genes involved in the proximal insulin signaling pathway in leukocytes from 45 young individuals grouped: normal weight with not insulin resistance (NIR), with insulin resistance (IR) and with obesity (OB-IR). Methods: qPCR was performed to analyze the expression of insulin receptor (INSR), insulin receptor substrate 1 and 2 (IRS-1 and IRS-2), neutrophil elastase (NE), alpha 1 antitrypsin (A1AT), glucose transporters 1, 3 and 4 (GLUT-1, GLUT-3 and GLUT-4) by the 2-ΔCt method, and the correlation between the genes was determined by Spearman's test. Results: The mRNA expression analysis of all genes between NIR and IR individuals revealed no differences. However, when comparing NIR and IR individuals with OB-IR, an increase in NE and A1AT expression and a clear trend towards a decrease in IRS-2 expression was observed, whereas the comparison of IR and OB-IR showed a decrease in GLUT-3 expression. Overall, the correlation analysis showed that in the IR group there was a positive correlation only between NE with IRS-1 (r = 0.72, p = 0.003), while in the OB-IR group, there was a positive correlation between the NE and A1AT with INSR (r = 0.62, p = 0.01 and r = 0.74, p = 0.002, respectively) and with IRS-2 (r = 0.74, p = 0.002 and r = 0.76, p = 0.001, respectively). Conclusion: These results suggest that hyperinsulinemia and obesity are associated with changes in the expression of genes in leukocytes involved in the insulin pathway that are related to NE and A1AT.

The E6 Oncoprotein of HPV16 AA-c Variant Regulates Cell Migration through the MINCR/miR-28-5p/RAP1B Axis.

The E6 oncoprotein of HPV16 variants differentially alters the transcription of the genes involved in migration and non-coding RNAs such as lncRNAs. The role of the lncRNA MINCR in cervical cancer and its relationship with variants of oncogenic HPV remain unknown. Therefore, the objective of this study was to analyze the effect of the E6 oncoprotein of the AA-c variant of HPV16 in cell migration through the MINCR/miR-28-5p/RAP1B axis. To explore the functional role of MINCR in CC, we used an in vitro model of C33-A cells with exogenous expression of the E6 oncoprotein of the AA-c variant of HPV16. Interfering RNAs performed MINCR silencing, and the expression of miR-28-5p and RAP1B mRNA was analyzed by RT-qPCR. We found that C33-A/AA-c cells expressed MINCR 8-fold higher compared to the control cells. There is an inverse correlation between the expression of miR-28-5p and RAP1B in C33-A/AA-c cells. Our results suggest that MINCR might regulate the expression of RAP1B through the inhibition of miR-28-5p in CC cells expressing the E6 oncoprotein of HPV16 AA-c. We report, for the first time, that the MINCR/miR-28-5p/RAP1B axis positively regulates cell migration in CC-derived cells that express the E6 oncoprotein of the AA-c variant of HPV16.

Molecular insights into the interaction of HPV-16 E6 variants against MAGI-1 PDZ1 domain.

Oncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein's amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein-protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

The regulatory role of insulin in energy metabolism and leukocyte functions.

Insulin is the hormone responsible for maintaining glucose homeostasis in the body, in addition to participating in lipid metabolism, protein synthesis, and the inhibition of gluconeogenesis. These functions are well characterized in the classic organ target cells that are responsible for general energy regulation: the liver, skeletal muscle, and adipose tissue. However, these actions are not restricted to these tissues because insulin has been shown to affect most cells in the body. This review describes the role of insulin in leukocyte signaling pathways, metabolism and functions, and how insulin resistance could affect this signaling and deteriorate leukocyte metabolism and function, in addition to showing evidence that suggests leukocytes may substantially contribute to the development of systemic insulin resistance.

Effect of HPV 16 E6 Oncoprotein Variants on the Alterations of the Proteome of C33A Cells.

BACKGROUND/AIM: The E6 genotypic variants of HPV 16 identified in lesions of women with cervical cancer (CC) in Southern of Mexico include the E-G350, AAa, AAc, E-C188/G350, and E-A176/G350, transcriptomic analysis cells transfected with those variants showed to induce differential expression of the host genes involved in the development of CC, the aim of this work was to understand how the over-expression of the E6 oncoprotein and its variants can induce molecular mechanisms that lead to more aggressive HPV 16 phenotypes in cervical cancer and which proteins could be associated with the process. MATERIALS AND METHODS: Total extracts from C33A, C33A mock, C33A AAa, C33A E-C188/G350, C33A E-A176/G350, and C33A E-prototype cells were analyzed using 2D electrophoresis, PDQuest software and mass spectrometry, validation of results was performed through qPCR. RESULTS: Statistically significant differential expression of 122 spots was detected, 12 of the identified proteins were associated with metabolism and metabolic programming. Out of these CCT8, ENO and ALDH1A were further validated. CONCLUSION: CCT8 and ALDH1A were found to be over-expressed in C33A AAa and C33A E-A176/G350, compared to the E prototype. Both proteins could be associated with a most aggressive phenotype due to their relationship with metabolism, protein folding and stemness, mechanisms associated to E6 that could be useful in the design of new therapies.

Modeling and Molecular Dynamics of the 3D Structure of the HPV16 E7 Protein and Its Variants.

The oncogenic potential of high-risk human papillomavirus (HPV) is predicated on the production of the E6 and E7 oncoproteins, which are responsible for disrupting the control of the cell cycle. Epidemiological studies have proposed that the presence of the N29S and H51N variants of the HPV16 E7 protein is significantly associated with cervical cancer. It has been suggested that changes in the amino acid sequence of E7 variants may affect the oncoprotein 3D structure; however, this remains uncertain. An analysis of the structural differences of the HPV16 E7 protein and its variants (N29S and H51N) was performed through homology modeling and structural refinement by molecular dynamics simulation. We propose, for the first time, a 3D structure of the E7 reference protein and two of Its variants (N29S and H51N), and conclude that the mutations induced by the variants in N29S and H51N have a significant influence on the 3D structure of the E7 protein of HPV16, which could be related to the oncogenic capacity of this protein.

In silico prediction of structural changes in human papillomavirus type 16 (HPV16) E6 oncoprotein and its variants.

BACKGROUND: HPV16 infection is one of the main risk factors involved in the development of cervical cancer, mainly due to the high oncogenic potential of the viral proteins E6 and E7, which are involved in the different processes of malignant transformation. There is a broad spectrum of intratypical variation of E6, which is reflected in its high diversity, biological behavior, global distribution and risk of causing cervical cancer. Experimental studies have shown that the intratypical variants of the protein E6 from the European variants (E-G350, E-A176/G350, E-C188/G350) and Asian-American variants (AAa and AAc), are capable of inducing the differential expression of genes involved in the development of cervical cancer. RESULTS: An in silico analysis was performed to characterize the molecular effects of these variations using the structure of the HPV16 E6 oncoprotein (PDB: 4XR8; chain H) as a template. In particular, we evaluated the 3D structures of the intratypical variants by structural alignment, ERRAT, Ramachandran plots and prediction of protein disorder, which was further validated by molecular dynamics simulations. Our results, in general, showed no significant changes in the protein 3D structure. However, we observed subtle changes in protein physicochemical features and structural disorder in the N- and C-termini. CONCLUSIONS: Our results showed that mutations in the viral oncogene E6 of six high-risk HPV16 variants are effectively neutral and do not cause significant structural changes except slight variations of structural disorder. As structural disorder is involved in rewiring protein-protein interactions, these results suggest a differential pattern of interaction of E6 with the target protein P53 and possibly different patterns of tumor aggressiveness associated with certain types of variants of the E6 oncoprotein.

Cervical cancer stem cell-associated genes: Prognostic implications in cervical cancer.

Cervical cancer is the fourth most common type of gynecological malignancy to affect females, worldwide. Although high-risk human papillomavirus (HR-HPV) infection is the primary etiologic agent associated with the development of cervical cancer, cancer stem cells (CSCs) also serve a prominent role in the development, metastasis, recurrence and prognosis of the disease. CSCs are a small subpopulation of cells that have the ability to self-renew and are present in the majority of tumors, including cervical cancer. Studies describing the phenotype of cervical CSCs (CCSCs) vary in their definition of the expression pattern of principal biomarkers, including Musashi-1, aldehyde dehydrogenase 1, Oct3/4, Sox2 and CD49f. However, these markers are not observed in all cancers, although several may be present in multiple tumor types. The present review describes the potential biomarkers of CSCs in cervical cancer. These CCSC biomarkers may serve as molecular targets to enhance the efficacy and reduce the side effects associated with chemotherapeutic treatment in HR-HPV-positive cervical cancer.

A possible association between the -2518 A>G MCP-1 polymorphism and insulin resistance in school children.

Objective Monocyte chemoattractant protein 1 (MCP-1) has been suggested to be involved in the pathophysiology of insulin resistance (IR); therefore, variants in the MCP-1 gene may contribute to the development of this disease. The aim of this study was to analyze the relationship of the -2518 A>G MCP-1 (rs1024611) gene polymorphism with insulin resistance in Mexican children. Subjects and methods A cross-sectional study was performed in 174 children, including 117 children without insulin resistance and 57 children with IR, with an age range of 6-11 years. Levels for serum insulin and high-sensitivity C-reactive protein were determined. The -2518 A>G MCP-1 polymorphism was identified by the polymerase chain reaction-restriction fragment length polymorphism method. Insulin resistance was defined as a HOMA-IR in the upper 75th percentile, which was ≥ 2.4 for all children. Results Genotype frequencies of the rs1024611 polymorphism for the insulin-sensitive group were 17% AA, 48% AG and 35% GG, and the frequency of G allele was 59%, whereas frequencies for the insulin-resistant group were 12% AA, 37% AG and 51% GG, and the frequency of G allele was 69%. The genotype and allele frequencies between groups did not show significant differences. However, the GG genotype was the most frequent in children with IR. The GG genotype was associated with insulin resistance (OR = 2.2, P = 0.03) in a genetic model. Conclusion The -2518 A>G MCP-1 gene polymorphism may be related to the development of insulin resistance in Mexican children.

A possible association between the -2518 A>G MCP-1 polymorphism and insulin resistance in school children

ABSTRACT Objective Monocyte chemoattractant protein 1 (MCP-1) has been suggested to be involved in the pathophysiology of insulin resistance (IR); therefore, variants in the MCP-1 gene may contribute to the development of this disease. The aim of this study was to analyze the relationship of the -2518 A>G MCP-1 (rs1024611) gene polymorphism with insulin resistance in Mexican children. Subjects and methods A cross-sectional study was performed in 174 children, including 117 children without insulin resistance and 57 children with IR, with an age range of 6-11 years. Levels for serum insulin and high-sensitivity C-reactive protein were determined. The -2518 A>G MCP-1 polymorphism was identified by the polymerase chain reaction-restriction fragment length polymorphism method. Insulin resistance was defined as a HOMA-IR in the upper 75th percentile, which was ≥ 2.4 for all children. Results Genotype frequencies of the rs1024611 polymorphism for the insulin-sensitive group were 17% AA, 48% AG and 35% GG, and the frequency of G allele was 59%, whereas frequencies for the insulin-resistant group were 12% AA, 37% AG and 51% GG, and the frequency of G allele was 69%. The genotype and allele frequencies between groups did not show significant differences. However, the GG genotype was the most frequent in children with IR. The GG genotype was associated with insulin resistance (OR = 2.2, P = 0.03) in a genetic model. Conclusion The -2518 A>G MCP-1 gene polymorphism may be related to the development of insulin resistance in Mexican children.

Obesidad y análisis proteómico: su utilidad en la búsqueda de biomarcadores y blancos terapéuticos de enfermedades metabólicas / Utility of proteomic analysis in the search for biomarkers and therapeutic targets in metabolic alterations associated with obesity

La obesidad es una enfermedad compleja y multifactorial, caracterizada por un aumento de grasa corporal que puede ser ocasionado por un desequilibrio entre la ingesta de alimentos y el gasto energético. En el proceso de aumento de peso intervienen factores como la susceptibilidad genética, factores ambientales y el estilo de vida. Está bien documentado que la obesidad aumenta el riesgo de padecer numerosas enfermedades y trastornos metabólicos como la resistencia a la insulina, diabetes tipo 2, hipercolesterolemia, enfermedades cardiovasculares, hígado graso, inflamación de bajo grado, algunos tipos de cáncer y trastornos sicológicos. Debido al incremento de la obesidad y sus comorbilidades en los últimos años en la población mundial, los gastos médicos erogados para su tratamiento representan un problema grave para los sistemas de salud pública. El análisis proteómico a gran escala, es una herramienta potente y prometedora para el descubrimiento de biomarcadores tempranos y para la comprensión de los mecanismos moleculares que subyacen a las alteraciones metabólicas asociadas con la obesidad. No obstante, es imprescindible considerar las limitaciones técnicas y el análisis e interpretación de los hallazgos proteómicos, para avanzar en la comprensión funcional integral de la dinámica del proteoma ligado a la obesidad. Adicionalmente, los abordajes con un enfoque proteómico, permitirán el desarrollo de nuevas terapias preventivas, así como el descubrimiento de agentes terapéuticos potenciales en el tratamiento de disfunciones metabólicas. El objetivo de esta revisión es analizar las contribuciones más recientes de la proteómica en la búsqueda de biomarcadores relacionados con la obesidad.

Obesity is a complex and multifactorial disease characterized by an increase in body fat that can be caused by an imbalance between food intake and energy expenditure. In the process of weight gain, factors such as genetic susceptibility, environmental factors and lifestyle are involved. It is well documented that obesity increases the risk of numerous diseases and metabolic disorders such as insulin resistance, type 2 diabetes, hypercholesterolemia, cardiovascular disease, fatty liver, low grade inflammation, some types of cancer and psychological disorders. Due to the increase in obesity and its comorbidities in recent years at the global level, medical expenses incurred for its treatment represent a serious problem for public health systems. Large-scale proteomic analysis is a powerful and promising tool for the discovery of early biomarkers and for the understanding of the molecular mechanisms underlying the metabolic alterations associated with obesity. Nevertheless, it is essential to consider the technical limitations and the analysis and interpretation of the proteomic findings, to advance in the integral functional understanding of the dynamics of the proteome linked to obesity. In addition, approaches with a proteomic viewpoint will allow the development of new preventive therapies, as well as the discovery of potential therapeutic agents in the treatment of metabolic dysfunctions. The aim of this review is to analyze the most recent contributions of proteomics in the search for biomarkers related to obesity.

Differential proteins among normal cervix cells and cervical cancer cells with HPV-16 infection, through mass spectrometry-based Proteomics (2D-DIGE) in women from Southern México.

BACKGROUND: Cervical cancer (CC) is the fourth most common cancer in women worldwide with an estimated 528,000 new cases in 2012. The same year México had an incidence of 13,960 and a mortality of 4769 cases. There are several diagnosis methods of CC; among the most frequents are the conventional Pap cytology (Pap), colposcopy, and visual inspection with acetic acid (VIA), histopathological examination, tests of imaging and detection of high-risk papilloma virus (HR-HPV) with molecular tests (PCR, hybridization, sequencing). Proteomics is a tool for the detection of new biomarkers that can be associated with clinical stage, histological type, prognosis, and/or response to treatment. In this study we performed a comparative analysis of CC cells with normal cervical cells. The proteomic analysis was carried out with the fluorescent two-dimensional electrophoresis (2D-DIGE) technique to subsequently identify differential protein profiles using Decyder Software, and the selected proteins were identified by Mass Spectrometry (MALDI-TOF). RESULTS: The proteins that showed an increased expression in cervical cancer in comparison with normal cervix cells were: Mimecan, Actin from aortic smooth muscle and Lumican. While Keratin, type II cytoskeletal 5, Peroxiredoxin-1 and 14-3-3 protein sigma showed a decrease in their protein expression level in cervical cancer in comparison with normal cervix cells. CONCLUSIONS: Thus, this study was successful in identifying biomarker signatures for cervical cancer, and might provide new insights into the mechanism of CC progression.

Integrins and haptoglobin: Molecules overexpressed in ovarian cancer.

Integrins are adhesion molecules whose expression is upregulated during different cellular processes such as adhesion, growth, proliferation, migration, survival and differentiation, all of which are involved in neoplastic development. Several reports have linked the overexpression of integrins with epithelial ovarian cancer (EOC). Furthermore, fucosylated haptoglobin (Hp) isoforms with antioxidant activity and synthesized primarily in the liver have also been associated with various types of cancer, including ovarian cancer. Here, we determined the level of expression of three integrin heterodimers (α5ß1, α6ß4, and αVß3) and fucosyltated Hp in two different settings: cell cultures and biopsies from ovarian cancer patients. On the one hand, integrin heterodimers were analyzed in the ovarian cancer cell line (SKOV-3), two primary cultures (INCan017 and INCan019) and a tumor derived from INCan017 (T-017) by Western blot. Statistical analysis was performed using one-way ANOVA. The SKOV-3 cell line, INCan017 and INCan019 primary cultures, and the T-017 tumor showed increased expression patterns of the α5, αV, ß1, ß3, and ß4 integrin subunits when compared with healthy ovary tissue. We then analyzed the expression pattern of the integrin subunits as well as the fucosylated Hp in biopsies from patients with different histotypes of EOC by immunofluorescence. α5ß1 and α6ß4 integrins were expressed by 90% of the samples, whereas 80% expressed the αVß3 integrin. Furthermore, Hp, fucosylated or not, was present at high levels in most biopsies. In fact, there was a statistical correlation between the expression of integrins or Hp and the presence of the disease given that α5ß1, α6ß4, and αVß3 integrins, Hp, fucosylated Hp and additional fucosylation state of proteins were highly expressed in biopsies of EOC histotypes when compared with healthy ovarian tissue. However, the statistical analysis showed no association of the presence of integrins, Hp or fucosylation with clinical or pathological characteristics of EOC patients. These results suggest that increased expression of these molecules and of the fucosylation modification are characteristics of the malignant process itself. Therefore, these molecules may be promising therapeutic targets in patients with this type of neoplasia.

Proteomic identification of fucosylated haptoglobin alpha isoforms in ascitic fluids and its localization in ovarian carcinoma tissues from Mexican patients.

BACKGROUND: Ovarian cancer is the most lethal gynecologic disease due to delayed diagnosis, and ascites production is a characteristic of patients in advanced stages. The aim of this study was to perform the proteomic analysis of ascitic fluids of Mexican patients with ovarian carcinoma, in order to detect proteins with a differential expression pattern in the continuing search to identify biomarkers for this disease. METHODS: Samples were collected from 50 patients from the Instituto Nacional de Cancerología of México under informed consent and with approval of the bioethics and scientific committees. After elimination of abundant proteins (Albumin/IgGs) samples were processed for 2D electrophoresis and further protein identification by Mass Spectrometry (MALDI-TOF). Molecules of interest were followed by western blot and lectin binding assays, and their tissue location by histo-immunofluorescence and confocal analysis. RESULTS AND DISCUSSION: An area with a differential expression pattern among samples was located in the 2D gels. Identified proteins were 6 alpha 1 isoforms and 1 alpha 2 isoform of Haptoglobin, and 2 isoforms of Transthyretin. While Transthyretin isoforms were constitutively expressed in all samples, clear differences in the expression pattern of Haptoglobin alpha isoforms were found. Moreover, increased levels of fucosylation of Haptoglobin alpha isoforms analyzed in 40 samples by Aleuria aurantia lectin binding by 1D overlay assay showed a positive correlation with advanced stages of the disease. Tissue detection of Haptoglobin and its fucosylated form, by histo-immunofluorescence in biopsies of ovarian cancer, also showed a correlation with ovarian cancer progression. Moreover, results show that fucosylated Haptoglobin is produced by tumor cells. CONCLUSIONS: Increased numbers of highly fucosylated Haptoglobin alpha isoforms in ascitic fluids and the presence of fucosylated Haptoglobin in tumor tissues of ovarian cancer Mexican patients associated with advanced stages of the disease, reinforce the potential of fucosylated Haptoglobin alpha isoforms to be characterized as biomarkers for disease progression.

Physical, structural, and functional properties of the beta1 integrin-like fibronectin receptor (beta1EhFNR) in Entamoeba histolytica.

The presence in Entamoeba histolytica of a fibronectin (FN) receptor, which is antigenically related to beta1 integrin-like molecules and shows 99% homology with the intermediate subunit-2 of the Gal/GalNAc-specific lectin has been described. The E. histolytica genome has been sequenced, and its analysis shows no integrin sequences. Here we provide further evidence to demonstrate that this molecule behaves as integrin-like in its physical, structural and functional properties. The purified beta1EhFNR complex is resolved into three polypeptides of 150, 140, and 130 kDa. Transmission electron microscopy showed individual complexes consisting of oblong heads of 3 nm x 4 nm and two projecting arms 6-7 nm in length. In the absence of detergent, these complexes formed aggregates that were composed of clusters or "rosettes" of between two and six or more beta1EhFNR complexes. The physical properties of the purified beta1EhFNR complexes were: R(S)=5.8 nm, S(20)W=8.3, f/f(0)=1.4. This complex was seen in close physical association with adhesion plates and phagocytic invaginations, using confocal microscopy and the 3C10 mAb that recognizes these three subunits complex. Regulation of its surface expression is not dependent on protein synthesis; rather it is regulated by inward and outward mobilization of the molecules. The presence and antigenic similarity of putative beta1EhFNRs in different strains and species of Entamoeba was analyzed using the 3C10 mAb; this mAb recognized the complex in all E. histolytica species, however there was no recognition in E. dispar, E. invadens, and Laredo strains. Finally, evidence is provided about post-translational modifications such as tyrosine phosphorylation and glycosylation suffered by the beta1EhFNR complex.