The effect of cilostazol on the platelet-derived growth factor-beta/beta isoform reduction on venous hyperplasia in an experimental balloon-induced injury model.

BACKGROUND: Intimal hyperplasia is the response to endothelial injury. Platelet-derived growth factor is released early and favors the formation of intimal hyperplasia. Although multiple treatments, from open surgery to endovascular techniques, have been used they remain controversial. There is currently interest in developing pharmacological strategies to address this pathology. Local vascular inflammation induced by vessel barotrauma generates intimal hyperplasia due to mechanical stress over the venous endothelium. Cilostazol is a selective phosphodiesterase type 3 (PDE3) selective inhibitor with a regulatory effect over intimal hyperplasia. The objective was to investigate cilostazol's role in inhibiting smooth muscle cell proliferation due to changes in the expression and release of PDGF-BB isoform and the effect on developing IH using an experimental model of vascular barotrauma (balloon-induced injury model). METHODS: We included 12 New Zealand rabbits. The balloon-induced injury model (BIIM) and experimental group cilostazol (20 mg/kg/day) included 6 rabbits each. Contralateral veins from 6 rabbits used in BIIM model has been taken as control group. We measured and compared the expression of PDGF-BB and the development of IH. A pathologist board chooses a PDGFRα antibody to localized its expression by immunohistochemistry analysis. Subsequently, using an automated immunohistochemical staining machine, the PDGFR expression was evaluated using a Zeiss Primo Star 4 light microscope. RESULTS: The measurement obtained in the intimal layer was: 126.12 µm2 in the CG, 232 µm2 in the BIIM group, and 178 µm2 in the EG. A statistically significant difference was observed. Baseline serum concentrations of PDGF-BB in the BIIM group were 0.22 pg/mL. At 12 h 0.42 pg/mL, and 0.17 pg/mL at seven days. In the experimental group, the basal levels were 0.33 pg/mL. With the use of cilostazol, a lower peak was obtained at 12 h (0.08 pg/mL). This difference was statistically significant. CONCLUSIONS: Cilostazol induced a significant reduction of IH caused by barotrauma in the venous endothelium, which correlates with decrease in the PDGF-BB in serum. This could be attributed to the pharmacologic effect on PDGFR expression.

Critical Role of the Transcription Factor AKNA in T-Cell Activation: An Integrative Bioinformatics Approach.

The human akna gene encodes an AT-hook transcription factor, the expression of which is involved in various cellular processes. The goal of this study was to identify potential AKNA binding sites in genes that participate in T-cell activation and validate selected genes. Here we analyzed ChIP-seq and microarray assays to determine AKNA-binding motifs and the cellular process altered by AKNA in T-cell lymphocytes. In addition, we performed a validation analysis by RT-qPCR to assess AKNA's role in promoting IL-2 and CD80 expression. We found five AT-rich motifs that are potential candidates as AKNA response elements. We identified these AT-rich motifs in promoter regions of more than a thousand genes in activated T-cells, and demonstrated that AKNA induces the expression of genes involved in helper T-cell activation, such as IL-2. The genomic enrichment and prediction of AT-rich motif analyses demonstrated that AKNA is a transcription factor that can potentially modulate gene expression by recognizing AT-rich motifs in a plethora of genes that are involved in different molecular pathways and processes. Among the cellular processes activated by AT-rich genes, we found inflammatory pathways potentially regulated by AKNA, suggesting AKNA is acting as a master regulator during T-cell activation.

HPV16 E6 and E7 Oncoproteins Stimulate the Glutamine Pathway Maintaining Cell Proliferation in a SNAT1-Dependent Fashion.

Persistent high-risk human papillomavirus infection is the main risk factor for cervical cancer establishment, where the viral oncogenes E6 and E7 promote a cancerous phenotype. Metabolic reprogramming in cancer involves alterations in glutamine metabolism, also named glutaminolysis, to provide energy for supporting cancer processes including migration, proliferation, and production of reactive oxygen species, among others. The aim of this work was to analyze the effect of HPV16 E6 and E7 oncoproteins on the regulation of glutaminolysis and its contribution to cell proliferation. We found that the E6 and E7 oncoproteins exacerbate cell proliferation in a glutamine-dependent manner. Both oncoproteins increased the levels of transporter SNAT1, as well as GLS2 and GS enzymes; E6 also increased LAT1 transporter protein levels, while E7 increased ASCT2 and xCT. Some of these alterations are also regulated at a transcriptional level. Consistently, the amount of SNAT1 protein decreased in Ca Ski cells when E6 and E7 expression was knocked down. In addition, we demonstrated that cell proliferation was partially dependent on SNAT1 in the presence of glutamine. Interestingly, SNAT1 expression was higher in cervical cancer compared with normal cervical cells. The high expression of SNAT1 was associated with poor overall survival of cervical cancer patients. Our results indicate that HPV oncoproteins exacerbate glutaminolysis supporting the malignant phenotype.

Synthesis and optimization of photothermal properties of NIR emitting LiGa5O8: Cr3+ and gold nanorods as hybrid materials for theranostic applications.

The increase in incidence of degenerative diseases has fueled the development of novel materials, mostly focused on reducing adverse effects caused by current medical therapies. Theranostic materials represent an alternative to treat degenerative diseases, since they combine diagnostic properties and localized therapy within the same material. This work presents the synthesis and characterization of hybrid materials designed for theranostic purposes. The hybrid materials were composed of LiGa5O8:Cr3+ (LGO) with emission lines in the near infrared (NIR), hence providing an excellent diagnostic ability. As for the therapy part, the hybrid nanomaterials contained gold nanorods (AuR) with localized surface plasmon resonance (LSPR). Once AuR are excited, plasmonic processes are triggered at their surface resulting in increased localized temperature capable of inducing irreversible damage to the cells. A detailed characterization of the hybrid materials confirmed proper assembly of LGO and AuR. Moreover, these nanocomposites preserved their luminescent properties and LSPR. Finally, the cytotoxic potential of the hybrid material was evaluated in different cell lines by cell viability colorimetric assays to determine its possible use as theranostic agent. The success in the synthesis of hybrid materials based on LGO with emission in the NIR coupled with AuR, provides a new perspective for the design of hybrid materials with improved properties to be used in biomedical fields.

HPV Prevalence and Predictive Biomarkers for Oropharyngeal Squamous Cell Carcinoma in Mexican Patients.

BACKGROUND: Worldwide prevalence of Oropharyngeal Squamous Cell Carcinoma (OPSCC) has increased, affecting mostly young males. OPSCC associated with Human Papillomavirus (HPV) infection exhibits particular characteristics in terms of response to treatment, hence HPV has been proposed as a prognostic factor. The impact of HPV positivity and associated biomarkers on OPSCC in the Mexican population has not been addressed. Therefore, the analysis of OPSCC prognostic markers in the Mexican population is necessary. METHODS: Retrolective study in Mexican OPSCC patients, where HPV prevalence, p16 and EGFR levels were assessed using INNO-LiPA and immunohistochemistry. RESULTS: We found an HPV prevalence of 57.6% in OPSCC cases treated at a reference center in Mexico. HPV and p16 positivity, as well as EGFR, associate with better outcomes in OPSCC patients, and they also promote reduced death risk. Notably, HPV presence and p16 positivity showed a significant association with disease-free survival (DFS), with a HR of 0.15 (p = 0.006) and a HR of 0.17 (p = 0.012), respectively, indicating a possible role as predictive biomarkers in Mexican OPSCC patients. CONCLUSIONS: Our results reflect the clinical utility of p16 analysis to improve overall survival (OS) and to predict recurrence in oropharyngeal cancer. These results position p16 and HPV as predictive biomarkers for OPSCC.

Alteration of the IFN-Pathway by Human Papillomavirus Proteins: Antiviral Immune Response Evasion Mechanism.

A persistent infection with the so-called high-risk Human Papillomaviruses (hr-HPVs) plays a fundamental role in the development of different neoplasms. The expression of the HPV proteins throughout the different steps of the viral life cycle produce a disruption of several cellular processes, including immune response, which can lead to cell transformation. The interferon-mediated response plays an important role in eliminating HPV-infected and -transformed cells. The ability of HPV to disrupt the proper function of the interferon response is based on a series of molecular mechanisms coordinated by HPV proteins intended to prevent clearance of infection, ultimately producing an immunotolerant environment that facilitates the establishment of persistence and cancer. In this review, we focus on the molecular actions performed by HPV E1, E2, E5, E6 and E7 proteins on IFN signaling elements and their contribution to the establishment of infection, viral persistence and the progression to cancer.

Fas and Fas ligand are highly expressed in lymphocytes from cervical intraepithelial neoplasia and cervical cancer patients: A possible role for immune escaping.

Objectives: Infection with high-risk human papillomavirus is required to develop cervical cancer. Some viruses modulate the Fas/FasL signaling to evade the immune response; the role of these molecules in cervical cancer is not clear. In this study, we measured the expression levels of Fas and FasL mRNA, soluble proteins, and cell surface proteins in peripheral blood mononuclear cells from patients with low- and high-grade squamous intraepithelial lesions and cervical cancer in relation to healthy women, to gain new insights into the role of Fas/FasL in cervical cancer development. Materials and Methods: Fas/FasL mRNA expression was measured in cervical tissues and peripheral blood mononuclear cells from patients and healthy subjects; serum soluble proteins Fas/FasL were measured by ELISA, and cell-surface protein expression was detected by flow cytometry. Results: Varying expression levels were found for both molecules. Cervical Fas and FasL mRNA expression was decreased in low- and high-grade lesions, but it was increased in cervical cancer cases. While, systemic Fas mRNA expression increased as malignity progressed; systemic FasL mRNA expression was increased in low- and high-grade lesions, but it was decreased in cancer patients. Soluble FasL levels decreased as lesions progressed, while soluble Fas levels increased. Finally, overexpression of Fas/FasL on the surface of peripheral blood mononuclear cells was found in patients with low-grade lesion with respect to healthy donors. Conclusion: Fas and FasL act as negative modulators of the immune response, probably by removing specific cytotoxic T lymphocytes against papillomavirus -infected cells and tumor cells.

Extracellular vesicles from blood of breast cancer women induce angiogenic processes in HUVECs.

Breast cancer is the most frequent malignancy among women in developed countries and the main cause of death related to cancer in women worldwide. Extracellular vesicles (EVs) are vesicles with a variable size enclosed within a phospholipid bilayer that contain a variety of molecules with biological activity. Cancer cells release EVs that induce proliferation, escape from apoptosis, reprogramming energy metabolism, invasion and metastasis. In this study we studied whether EV fractions deprived of platelet EVs from breast cancer women (BC EVs) can mediate cell processes related with angiogenesis in human umbilical vein endothelial cells (HUVECs). Our findings demonstrate that BC EVs enhance migration, invasion and formation of new tubules in HUVECs, compared with EV fractions deprived of platelet EVs from healthy women (Ctrl EVs). In summary, we demonstrate, for the first time, that BC EVs induce cellular processes in HUVECs that participate in angiogenesis.

Functional Role of AKNA: A Scoping Review.

Human akna encodes an AT-hook transcription factor whose expression participates in various cellular processes. We conducted a scoping review on the literature regarding the functional role of AKNA according to the evidence found in human and in vivo and in vitro models, stringently following the "PRISMA-ScR" statement recommendations. METHODS: We undertook an independent PubMed literature search using the following search terms, AKNA OR AKNA ADJ gene OR AKNA protein, human OR AKNA ADJ functions. Observational and experimental articles were considered. The selected studies were categorized using a pre-determined data extraction form. A narrative summary of the evidence was produced. RESULTS: AKNA modulates the expression of CD40 and CD40L genes in immune system cells. It is a negative regulator of inflammatory processes as evidenced by knockout mouse models and observational studies for several autoimmune and inflammatory diseases. Furthermore, AKNA contributes to the de-regulation of the immune system in cancer, and it has been proposed as a susceptibility genetic factor and biomarker in CC, GC, and HNSCC. Finally, AKNA regulates neurogenesis by destabilizing the microtubules dynamics. CONCLUSION: Our results provide evidence for the role of AKNA in various cellular processes, including immune response, inflammation, development, cancer, autoimmunity, and neurogenesis.

HPV oral and oropharynx infection dynamics in young population.

The incidence of human papillomavirus (HPV)-associated cancers, especially those from the head and neck region, has increased. The relatively early age of presentation of HPV-positive head and neck cancer (HNC) indicates that viral infection might be acquired early in life. Persistent HPV infection has been recognized as the main risk factor for cancer development, but most studies have focused on evaluating HPV persistence in the genital region. Thus, in this work, we aimed to evaluate the prevalence of HPV in oral cavity and oropharynx in a young population, as well as the possible persistence of the infection after 12 months. Our results indicate that almost half (46.8%) of the analyzed population harbors an HPV infection either in the oral cavity or in the oropharynx. Furthermore, after 1 year of initial identification, half of them eliminated the infection, and only one person (5.26%) exhibited persistence. Interestingly, 50% of the individuals who successfully eliminated the infection acquired a new viral type, indicating that even when the primary infection is effectively eliminated by the immune system, there is a dynamic circulation of HR-HPV types that produce reinfection. This dynamic HPV infection among young individuals could influence the future establishment of cancer in some proportion of the cases.

The role of extracellular DNA (exDNA) in cellular processes.

Nowadays, extracellular DNA or circulating cell-free DNA is considered to be a molecule with clinical applications (diagnosis, prognosis, monitoring of treatment responses, or patient follow-up) in diverse pathologies, especially in cancer. Nevertheless, because of its molecular characteristics, it can have many other functions. This review focuses on the participation of extracellular DNA (exDNA) in fundamental processes such as cell signaling, coagulation, immunity, evolution through horizontal transfer of genetic information, and adaptive response to inflammatory processes. A deeper understanding of its role in each of these processes will allow development of better tools to monitor and control pathologies, as well as helping to generate new therapeutic options, beyond the applicability of DNA in liquid biopsy.

Comprehensive bioinformatic analysis reveals oncogenic role of H2A.Z isoforms in cervical cancer progression.

Objectives: Cervical cancer ranks as the fourth most common neoplasia in women worldwide in which epigenetic alterations play an important role. Several studies have reported pro-oncogenic role of the histone variant H2A.Z in different types of cancer; however, the role of H2A.Z in cervical cancer remains poorly studied. This study aimed to determine the potential role of H2A.Z in cervical cancer through a bioinformatic approach. Materials and Methods: H2A.Z expression was analyzed in The Human Protein Atlas, The Cancer Genome Atlas, and Gene Expression Omnibus datasets. The promoter regions of H2AZ1 and H2AZ2 genes were downloaded from Expasy, and the prediction of transcription factor binding motifs was performed using CONSITE, Alibaba, and ALGGEN. ChIP-seq and RNA-seq data from HeLa-S3 cells were downloaded from ENCODE. The discovery motif was investigated using MEME-ChIP. The functional annotation was examined in Enrich. Results: The expression of H2A.Z is elevated in cervical cancer. Interestingly, DNA methylation, copy number, and transcription factors AP2α and ELK1 are involved in H2A.Z overexpression. Additionally, H2A.Z is enriched on promoter and enhancer regions of genes involved in pathways associated with cancer development. In these regions, H2A.Z enables the recruitment of transcription factors such as NRF1, NFYA, and RNA Pol II. Finally, H2A.Z allows the expression of genes associated with proliferation in patients with cervical cancer. Conclusion: Our findings suggest that H2A.Z overexpression and its presence in promoters and enhancers could be regulating the transcription of genes involved in cervical carcinogenesis.

New insights in Hippo signalling alteration in human papillomavirus-related cancers.

The persistent infection with high-risk human papillomavirus (HPV) is an etiologic factor for the development of different types of cancers, mainly attributed to the continuous expression of E6 and E7 HPV oncoproteins, which regulate several cell signalling pathways including the Hippo pathway. It has been demonstrated that E6 proteins promote the increase of the Hippo elements YAP, TAZ and TEAD, at protein level, as well as their transcriptional targets. Also, E6 and E7 oncoproteins promote nuclear YAP localization and a decrease in YAP negative regulators such as MST1, PTPN14 or SOCS6. Interestingly, Hippo signalling components modulate HPV activity, such as TEAD1 and the transcriptional co-factor VGLL1, induce the activation of HPV early and late promoters, while hyperactivation of YAP in specific cells facilitates virus infection by increasing putative HPV receptors and by evading innate immunity. Additionally, alterations in Hippo signalling elements have been found in HPV-related cancers and particularly, the involvement of HPV oncoproteins on the regulation of some of these Hippo components has been also proposed, although the precise mechanisms remain unclear. The present review addresses the recent findings describing the interplay between HPV and Hippo signalling in HPV-related cancers, a fact that highlights the importance of developing more in-depth studies in this field to establish key therapeutic targets.

IMMUNOLOGY OF CERVICAL CANCER.

Optimal function of the immune system allows the recognition and elimination of infected and tumor cells. However, these cells can develop mechanisms to evade the cellular immune response. In human papillomavirus (HPV) infection, dysregulation of major histocompatibility complex Class I molecules and other components of the innate immune system promote the survival of infected cells by allowing the infection to persist which, in turn, favors the development of cancer. Further, tumor cells possess inherent mechanisms designed to block the recognition and activation of cytotoxic lymphocytes: particularly, HPV proteins such as E1 and E2 and oncoproteins E5, E6, and E7 that inhibit immune mechanisms and/or stimulate the expression of immunosuppressive cytokines. These mechanisms include a decrease in receptor activation and costimulating molecules on the surface of immune cells, as well as the constitutive expression of molecules that inhibit their function, which allow HPV persistence and tumor progression. Immunotherapy-based therapeutic options are positioned as excellent candidates for the treatment of cervical cancer.

MOLECULAR MARKERS FOR THE DIAGNOSIS OF HIGH-RISK HUMAN PAPILLOMAVIRUS INFECTION AND TRIAGE OF HUMAN PAPILLOMAVIRUS-POSITIVE WOMEN.

Infection with high-risk human papillomavirus (HPV) increases the likelihood of developing cervical cancer (CC). A plethora of cellular processes is required to produce pre-malignant lesions, which in turn may become malignant if left untreated. Those changes are induced by viral oncoproteins, which represent an ideal target to identify the viral presence, or by some particularities of the host that ultimately promote the establishment of CC. This article describes the different methods used for HPV detection and quantification, as well as the current trend of secondary screening approaches to detect premalignant lesions and CC. In addition, we analyzed validated biomarkers and those under clinical investigation for the classification (triage) of women at risk of developing CC after an initial positive HPV test and that could be used as prognostic biomarkers for CC. The use of molecular biomarkers, together with the detection of HPV DNA sequences, provides a high impact diagnostic and prognostic tool in the detection of patients at increased risk of developing CC and also may guide their clinical management. In addition, some of those biomarkers could represent pharmacological targets for the future design of therapeutic approaches to CC treatment.

Immunology of cervical cancer

ABSTRACT Optimal function of the immune system allows the recognition and elimination of infected and tumor cells. However, these cells can develop mechanisms to evade the cellular immune response. In human papillomavirus (HPV) infection, dysregulation of major histocompatibility complex Class I molecules and other components of the innate immune system promote the survival of infected cells by allowing the infection to persist which, in turn, favors the development of cancer. Further, tumor cells possess inherent mechanisms designed to block the recognition and activation of cytotoxic lymphocytes: particularly, HPV proteins such as E1 and E2 and oncoproteins E5, E6, and E7 that inhibit immune mechanisms and/or stimulate the expression of immunosuppressive cytokines. These mechanisms include a decrease in receptor activation and costimulating molecules on the surface of immune cells, as well as the constitutive expression of molecules that inhibit their function, which allow HPV persistence and tumor progression. Immunotherapy-based therapeutic options are positioned as excellent candidates for the treatment of cervical cancer.

Molecular markers for the diagnosis of high-risk human papillomavirus infection and triage of human papillomavirus-positive women

ABSTRACT Infection with high-risk human papillomavirus (HPV) increases the likelihood of developing cervical cancer (CC). A plethora of cellular processes is required to produce pre-malignant lesions, which in turn may become malignant if left untreated. Those changes are induced by viral oncoproteins, which represent an ideal target to identify the viral presence, or by some particularities of the host that ultimately promote the establishment of CC. This article describes the different methods used for HPV detection and quantification, as well as the current trend of secondary screening approaches to detect premalignant lesions and CC. In addition, we analyzed validated biomarkers and those under clinical investigation for the classification (triage) of women at risk of developing CC after an initial positive HPV test and that could be used as prognostic biomarkers for CC. The use of molecular biomarkers, together with the detection of HPV DNA sequences, provides a high impact diagnostic and prognostic tool in the detection of patients at increased risk of developing CC and also may guide their clinical management. In addition, some of those biomarkers could represent pharmacological targets for the future design of therapeutic approaches to CC treatment.

High expression of Helicobacter pylori VapD in both the intracellular environment and biopsies from gastric patients with severity.

Helicobacter pylori is a Gram-negative bacterium that causes chronic atrophic gastritis and peptic ulcers and it has been associated with the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT). One of the more remarkable characteristics of H. pylori is its ability to survive in the hostile environment of the stomach. H. pylori regulates the expression of specific sets of genes allowing it to survive high acidity levels and nutrient scarcity. In the present study, we determined the expression of virulence associated protein D (VapD) of H. pylori inside adenocarcinoma gastric (AGS) cells and in gastric biopsies. Using qRT-PCR, VapD expression was quantified in intracellular H. pylori-AGS cell cultures at different time points and in gastric mucosa biopsies from patients suffering from chronic atrophic gastritis, follicular gastritis, peptic ulcers, gastritis precancerous intestinal metaplasia and adenocarcinoma. Our results show that vapD of H. pylori presented high transcription levels inside AGS cells, which increased up to two-fold above basal values across all assays over time. Inside AGS cells, H. pylori acquired a coccoid form that is metabolically active in expressing VapD as a protection mechanism, thereby maintaining its permanence in a viable non-cultivable state. VapD of H. pylori was expressed in all gastric biopsies, however, higher expression levels (p = 0.029) were observed in gastric antrum biopsies from patients with follicular gastritis. The highest VapD expression levels were found in both antrum and corpus gastric biopsies from older patients (>57 years old). We observed that VapD in H. pylori is a protein that is only produced in response to interactions with eukaryotic cells. Our results suggest that VapD contributes to the persistence of H. pylori inside the gastric epithelial cells, protecting the microorganism from the intracellular environment, reducing its growth rate, enabling long-term infection and treatment resistance.

Lactate in the Regulation of Tumor Microenvironment and Therapeutic Approaches.

Tumor cells must generate sufficient ATP and biosynthetic precursors in order to maintain cell proliferation requirements. Otto Warburg showed that tumor cells uptake high amounts of glucose producing large volumes of lactate even in the presence of oxygen, this process is known as "Warburg effect or aerobic glycolysis." As a consequence of such amounts of lactate there is an acidification of the extracellular pH in tumor microenvironment, ranging between 6.0 and 6.5. This acidosis favors processes such as metastasis, angiogenesis and more importantly, immunosuppression, which has been associated to a worse clinical prognosis. Thus, lactate should be thought as an important oncometabolite in the metabolic reprogramming of cancer. In this review, we summarized the role of lactate in regulating metabolic microenvironment of cancer and discuss its relevance in the up-regulation of the enzymes lactate dehydrogenase (LDH) and monocarboxilate transporters (MCTs) in tumors. The goal of this review is to expose that lactate is not only a secondary product of cellular metabolic waste of tumor cells, but also a key molecule involved in carcinogenesis as well as in tumor immune evasion. Finally, the possible targeting of lactate production in cancer treatment is discussed.