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1.  Low Levels of CD36 in Peripheral Blood Monocytes in Subclinical Atherosclerosis in Rheumatoid Arthritis: A Cross-Sectional Study in a Mexican Population 
BioMed Research International  2014;2014:736786.
Patients with rheumatoid arthritis (RA) have a higher risk for atherosclerosis. There is no clinical information about scavenger receptor CD36 and the development of subclinical atherosclerosis in patients with RA. The aim of this study was to evaluate the association between membrane expression of CD36 in peripheral blood mononuclear cells (PBMC) and carotid intima-media thickness (cIMT) in patients with RA. Methods. We included 67 patients with RA from the Rheumatology Department of Hospital Civil “Dr. Juan I. Menchaca,” Guadalajara, Jalisco, Mexico. We evaluated the cIMT, considering subclinical atherosclerosis when >0.6 mm. Since our main objective was to associate the membrane expression of CD36 with subclinical atherosclerosis, other molecules related with cardiovascular risk such as ox-LDL, IL-6, and TNFα were tested. Results. We found low CD36 membrane expression in PBMC from RA patients with subclinical atherosclerosis (P < 0.001). CD36 mean fluorescence intensity had negative correlations with cIMT (r = −0.578, P < 0.001), ox-LDL (r = −0.427, P = 0.05), TNFα (r = −0.729, P < 0.001), and IL-6 (r = −0.822, P < 0.001). Conclusion. RA patients with subclinical atherosclerosis showed low membrane expression of CD36 in PBMC and increased serum proinflammatory cytokines. Further studies are needed to clarify the regulation of CD36 in RA.
doi:10.1155/2014/736786
PMCID: PMC4070538
2.  Anti-Cyclic Citrullinated Peptide (Anti-CCP) and Anti-Mutated Citrullinated Vimentin (Anti-MCV) Relation with Extra-Articular Manifestations in Rheumatoid Arthritis 
Journal of Immunology Research  2014;2014:536050.
We evaluated the association between anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-mutated citrullinated vimentin antibodies (anti-MCV) with the presence of extra-articular (ExRA) manifestations in 225 patients with rheumatoid arthritis (RA). Ninety-five patients had ExRA and 130 had no ExRA. There was no association of anti-CCP and anti-MCV levels with the presence of ExRA as total group (P = 0.40 and P = 0.91, resp.). Making an analysis of individual manifestations, rheumatoid nodules were associated with positivity for rheumatoid factor (RF); (P = 0.01), anti-CCP (P = 0.048), and anti-MCV (P = 0.02). Instead, RF, anti-CCP, or anti-MCV were not associated with SS, chronic anemia, or peripheral neuropathy. Levels of anti-CCP correlated with the score of the Health Assessment Questionnaire-Disability Index (HAQ-Di) (r = 0.154, P = 0.03), erythrocyte sedimentation rate (ESR); (r = 0.155, P = 0.03), and RF (P = 0.254, P < 0.001), whereas anti-MCV titres only correlated with RF (r = 0.169, P = 0.02). On adjusted analysis, ExRA was associated with longer age (P = 0.015), longer disease duration (P = 0.007), higher DAS-28 score (P = 0.002), and higher HAQ-DI score (P = 0.007), but serum levels of anti-CCP and anti-MCV were not associated. These findings show the need to strengthen the evaluation of the pathogenic mechanisms implied in each specific ExRA manifestation.
doi:10.1155/2014/536050
PMCID: PMC3997885  PMID: 24804270
3.  Interaction of vitamin D with membrane-based signaling pathways 
Many studies in different biological systems have revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) modulates signaling pathways triggered at the plasma membrane by agents such as Wnt, transforming growth factor (TGF)-β, epidermal growth factor (EGF), and others. In addition, 1α,25(OH)2D3 may affect gene expression by paracrine mechanisms that involve the regulation of cytokine or growth factor secretion by neighboring cells. Moreover, post-transcriptional and post-translational effects of 1α,25(OH)2D3 add to or overlap with its classical modulation of gene transcription rate. Together, these findings show that vitamin D receptor (VDR) cannot be considered only as a nuclear-acting, ligand-modulated transcription factor that binds to and controls the transcription of target genes. Instead, available data support the view that much of the complex biological activity of 1α,25(OH)2D3 resides in its capacity to interact with membrane-based signaling pathways and to modulate the expression and secretion of paracrine factors. Therefore, we propose that future research in the vitamin D field should focus on the interplay between 1α,25(OH)2D3 and agents that act at the plasma membrane, and on the analysis of intercellular communication. Global analyses such as RNA-Seq, transcriptomic arrays, and genome-wide ChIP are expected to dissect the interactions at the gene and molecular levels.
doi:10.3389/fphys.2014.00060
PMCID: PMC3927071  PMID: 24600406
1α,25(OH)2D3; VDR; membrane-based signaling; Wnt; growth factors; cytokines; paracrine effects
4.  Plasma 25-Hydroxyvitamin D3 and Bladder Cancer Risk According to Tumor Stage and FGFR3 Status: A Mechanism-Based Epidemiological Study 
Background
Previous evidence suggests that 25-hydroxyvitamin D3 [25(OH)D3] protects against several cancers. However, little is known regarding urothelial bladder cancer (UBC). We analyzed the association between plasma 25(OH)D3 and overall risk of UBC, as well as according to stage and FGFR3 molecular subphenotypes.
Methods
Plasma concentrations of 25(OH)D3 in 1125 cases with UBC and 1028 control subjects were determined by a chemiluminescence immunoassay. FGFR3 mutational status and expression in tumor tissue were assessed. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression adjusting for potential confounders. Analyses were further stratified by tumor invasiveness and grade, FGFR3 expression, and smoking status. Cell proliferation was measured in human UBC cell lines cultured with 1α,25-dihydroxyvitamin D3.
Results
A statistically significantly increased risk of UBC was observed among subjects presenting the lowest concentrations of 25(OH)D3 (ORadj = 1.83; 95% CI = 1.19 to 2.82; P = .006), showing a dose–response effect (P trend = .004). The association was stronger for patients with muscle-invasive tumors, especially among low-FGFR3 expressers (ORadj = 5.94; 95% CI = 1.72 to 20.45; P = .005). The biological plausibility of these associations is supported by the fact that, in vitro, 1α,25-dihydroxyvitamin D3 upregulates FGFR3 expression in UBC cell lines with low levels of wild-type FGFR3.
Conclusion
These findings support a role of vitamin D in the pathogenesis of UBC and show that 25(OH)D3 levels are associated with FGFR3 expression in the tumor. Because FGFR3 mutation and overexpression are markers of better outcome, our findings suggest that individuals with low levels of plasma 25(OH)D3 may be at high risk of more aggressive forms of UBC.
doi:10.1093/jnci/djs444
PMCID: PMC3525815  PMID: 23108201
5.  Vitamin D Is a Multilevel Repressor of Wnt/β-Catenin Signaling in Cancer Cells 
Cancers  2013;5(4):1242-1260.
The Wnt/β-catenin signaling pathway is abnormally activated in most colorectal cancers and in a proportion of other neoplasias. This activation initiates or contributes to carcinogenesis by regulating the expression of a large number of genes in tumor cells. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits Wnt/β-catenin signaling by several mechanisms at different points along the pathway. Additionally, paracrine actions of 1,25(OH)2D3 on stromal cells may also repress this pathway in neighbouring tumor cells. Here we review the molecular basis for the various mechanisms by which 1,25(OH)2D3 antagonizes Wnt/β-catenin signaling, preferentially in human colon carcinoma cells, and the consequences of this inhibition for the phenotype and proliferation rate. The effect of the vitamin D system on Wnt/β-catenin signaling and tumor growth in animal models will also be commented in detail. Finally, we revise existing data on the relation between vitamin D receptor expression and vitamin D status and the expression of Wnt/β-catenin pathway genes and targets in cancer patients.
doi:10.3390/cancers5041242
PMCID: PMC3875938  PMID: 24202444
vitamin D; 1α,25-dihydroxyvitamin D3; Wnt; β-catenin; colon cancer; vitamin D receptor
6.  Thymidylate Synthase Expression Determines Pemetrexed Targets and Resistance Development in Tumour Cells 
PLoS ONE  2013;8(5):e63338.
Although treatment options for cancer patients are increasing every year, the drug resistance problem remains very present. It is very difficult to find a drug that acts equally on tumours of the same histology as the individual's genetic characteristics often determine the response to treatment. Furthermore, tumours that initially respond to anti-tumour therapy are able to adapt and develop resistance to the drug, while others do not. In addition, this usually implies resistance development to agents to which the cells have not been exposed, a phenomenon called cross-resistance or multidrug resistance. Given this situation, it has been suggested that the most appropriate treatment would be able to act in parallel on multiple pathways constitutively altered in tumour cells. Pemetrexed is a multitargeted antifolate that exerts its activity against folate-dependent enzymes involved in de novo pyrimidine and purine synthesis. It is currently in use in combination with cisplatin against malignant pleural mesothelioma and non-squamous non-small cell lung cancer with favourable results. By real-time RT-PCR gene expression assays and restoration viability assays we demonstrated that Pemetrexed targets folate-dependent enzymes involved in de novo biosynthesis of purines differently depending on the intrinsic genetic characteristics of the tumour. These differences did not, however, interfere either with the initial response to the drug or with the activation of apoptotic pathways. In addition, these genetic fingerprints can differentiate two groups of tumours: those capable of developing resistance to antifolate, and not capable. These results may be useful to employ targets gene expression as resistance markers, a valuable tool for identifying patients likely to receive combination therapy to prevent the development of resistance.
doi:10.1371/journal.pone.0063338
PMCID: PMC3652812  PMID: 23675481
7.  c-Jun N-Terminal Kinase Phosphorylation Is a Biomarker of Plitidepsin Activity 
Marine Drugs  2013;11(5):1677-1692.
Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin.
doi:10.3390/md11051677
PMCID: PMC3707168  PMID: 23697951
plitidepsin; Aplidin; JNK; biomarker; xenograft
8.  Two Functional Motifs Define the Interaction, Internalization and Toxicity of the Cell-Penetrating Antifungal Peptide PAF26 on Fungal Cells 
PLoS ONE  2013;8(1):e54813.
The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW) is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW) and PAF96 (RKKAAA), were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i) its interaction with the cell envelope; (ii) its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii) its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the cytoplasm, which coincides with cell death. Peptide containment within vacuoles preserves fungal cells from peptide toxicity.
doi:10.1371/journal.pone.0054813
PMCID: PMC3549957  PMID: 23349973
9.  Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons 
PLoS ONE  2012;7(10):e48557.
Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation.
doi:10.1371/journal.pone.0048557
PMCID: PMC3485302  PMID: 23119056
10.  Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells 
Molecular Cancer  2012;11:25.
Background
Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA) is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for de novo synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC), and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet.
Results
In the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and –independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS) and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC) to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis.
Conclusions
We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and –independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.
doi:10.1186/1476-4598-11-25
PMCID: PMC3505171  PMID: 22537194
Pemetrexed; MTA; Antifolates; DNA damage; Apoptosis; Melanoma
12.  Vitamin D Receptor Deficiency Enhances Wnt/β-Catenin Signaling and Tumor Burden in Colon Cancer 
PLoS ONE  2011;6(8):e23524.
Aberrant activation of the Wnt/β-catenin pathway is critical for the initiation and progression of most colon cancers. This activation provokes the accumulation of nuclear β-catenin and the induction of its target genes. Apcmin/+ mice are the most commonly used model for colon cancer. They harbor a mutated Apc allele and develop intestinal adenomas and carcinomas during the first months of life. This phenotype is caused by the mutation of the second Apc allele and the consequent accumulation of nuclear β-catenin in the affected cells. Here we describe that vitamin D receptor (VDR) is a crucial modulator of nuclear β-catenin levels in colon cancer in vivo. By appropriate breeding of Apcmin/+ mice and Vdr+/− mice we have generated animals expressing a mutated Apc allele and two, one, or none Vdr wild type alleles. Lack of Vdr increased the number of colonic Aberrant Crypt Foci (ACF) but not that of adenomas or carcinomas in either small intestine or colon. Importantly, colon ACF and tumors of Apcmin/+Vdr-/- mice had increased nuclear β-catenin and the tumors reached a larger size than those of Apcmin/+Vdr+/+. Both ACF and carcinomas in Apcmin/+Vdr-/- mice showed higher expression of β-catenin/TCF target genes. In line with this, VDR knock-down in cultured human colon cancer cells enhanced β-catenin nuclear content and target gene expression. Consistently, VDR depletion abrogated the capacity of 1,25(OH)2D3 to promote the relocation of β-catenin from the nucleus to the plasma membrane and to inhibit β-catenin/TCF target genes. In conclusion, VDR controls the level of nuclear β-catenin in colon cancer cells and can therefore attenuate the impact of oncogenic mutations that activate the Wnt/β-catenin pathway.
doi:10.1371/journal.pone.0023524
PMCID: PMC3156234  PMID: 21858154
13.  A genomic approach highlights common and diverse effects and determinants of susceptibility on the yeast Saccharomyces cerevisiae exposed to distinct antimicrobial peptides 
BMC Microbiology  2010;10:289.
Background
The mechanism of action of antimicrobial peptides (AMP) was initially correlated with peptide membrane permeation properties. However, recent evidences indicate that action of a number of AMP is more complex and involves specific interactions at cell envelopes or with intracellular targets. In this study, a genomic approach was undertaken on the model yeast Saccharomyces cerevisiae to characterize the antifungal effect of two unrelated AMP.
Results
Two differentiated peptides were used: the synthetic cell-penetrating PAF26 and the natural cytolytic melittin. Transcriptomic analyses demonstrated distinctive gene expression changes for each peptide. Quantitative RT-PCR confirmed differential expression of selected genes. Gene Ontology (GO) annotation of differential gene lists showed that the unique significant terms shared by treatment with both peptides were related to the cell wall (CW). Assays with mutants lacking CW-related genes including those of MAPK signaling pathways revealed genes having influence on sensitivity to peptides. Fluorescence microscopy and flow cytometry demonstrated PAF26 interaction with cells and internalization that correlated with cell killing in sensitive CW-defective mutants such as Δecm33 or Δssd1. GO annotation also showed differential responses between peptides, which included ribosomal biogenesis, ARG genes from the metabolism of amino groups (specifically induced by PAF26), or the reaction to unfolded protein stress. Susceptibility of deletion mutants confirmed the involvement of these processes. Specifically, mutants lacking ARG genes from the metabolism of arginine pathway were markedly more resistant to PAF26 and had a functional CW. In the deletant in the arginosuccinate synthetase (ARG1) gene, PAF26 interaction occurred normally, thus uncoupling peptide interaction from cell killing. The previously described involvement of the glycosphingolipid gene IPT1 was extended to the peptides studied here.
Conclusions
Reinforcement of CW is a general response common after exposure to distinct AMP, and likely contributes to shield cells from peptide interaction. However, a weakened CW is not necessarily indicative of a higher sensitivity to AMP. Additional processes modulate susceptibility to specific peptides, exemplified in the involvement of the metabolism of amino groups in the case of PAF26. The relevance of the response to unfolded protein stress or the sphingolipid biosynthesis, previously reported for other unrelated AMP, was also independently confirmed.
doi:10.1186/1471-2180-10-289
PMCID: PMC2996382  PMID: 21078184
14.  Epigenetic repression of ROR2 has a Wnt-mediated, pro-tumourigenic role in colon cancer 
Molecular Cancer  2010;9:170.
Background
Wnt factors control cell differentiation through semi-independent molecular cascades known as the β-catenin-dependent (canonical) and -independent (non-canonical) Wnt signalling pathways. Genetic and epigenetic alteration of components of the canonical Wnt signalling pathway is one of the primary mechanisms underlying colon cancer. Despite increasing evidence of the role of the non-canonical pathways in tumourigenesis, however, the underlying molecular mechanisms are poorly understood.
Results
Here we report that the receptor tyrosine kinase-like orphan receptor 2 (ROR2), a transmembrane receptor for Wnt factors that activates non-canonical pathways, is frequently repressed by aberrant promoter hypermethylation in human colon cancer cell lines and primary tumours. By restoring ROR2 activity in colon cancer cells harbouring ROR2 promoter hypermethylation, we show that the role of ROR2 in colon cancer cells is mediated, at least in part, by canonical Wnt and that its epigenetic-dependent loss can be pro-tumourigenic.
Conclusions
Our data show the importance of epigenetic alterations of ROR2 in colon cancer, highlighting the close interconnection between canonical and non-canonical Wnt signalling pathways in this type of tumour.
doi:10.1186/1476-4598-9-170
PMCID: PMC2903502  PMID: 20591152
15.  Novel Snail1 Target Proteins in Human Colon Cancer Identified by Proteomic Analysis 
PLoS ONE  2010;5(4):e10221.
Background
The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT), a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT.
Methodology/Principal Findings
We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4) that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and the Splicing factor proline/glutamine-rich (SFPQ), was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3ε, 14-3-3τ, 14-3-3ζ and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3σ and Snail1 expression in human colorectal tumors.
Conclusions/Significance
We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.
doi:10.1371/journal.pone.0010221
PMCID: PMC2857666  PMID: 20421926
16.  Imaging and Clinical Findings in Patients with Aberrant Course of the Cervical Internal Carotid Arteries 
Background and Purpose:
Aberrrant course of the cervical internal carotid arteries (ICAs) may result in submucosal masses in the posterior pharyngeal wall, may cause confusion at physical examination, may be symptomatic, and can be at risk of surgical injury. The aim of this report is to present the clinical and imaging characteristics associated with aberrant course of the cervical portion of the ICAs.
Methods:
Imaging studies of 5.500 patients were prospectively selected from CT studies of the head and neck performed in a five years period, in which the course of the one or both ICAs at the level of the hypopharynx and oropharynx was assessed as aberrant by means of a proposed classification. We then reviewed the medical records to establish which symptoms were present and if these symptoms could be caused by these variations in the course of the ICAs. In selected cases, further studies including magnetic resonance (MR) imaging, MR angiography (MRA), or selective catheter angiography were obtained.
Results:
In our restricted classification, we found 14 (0.2%) patients who met the cervical ICA aberrancy criteria. In all patients contrast enhanced CT or CT angiography was performed, 4 also have had MRI and MRA, and in two additional catheter angiograms were performed. Mean age was 62 years. Eight patients were male and seven were female. Four patients (28%) were considered to have clinical symptoms related to aberrant course of the ICAs. In most of the symptomatic patients both ICAs had aberrant courses. Overall, the course of the right ICA was aberrant in 43%, the left ICA in 14%, and both in 43%. In 50% of the cases the aberrancy of the artery was focal (localized to the oropharynx or laryngopharynx) and in the other 50% it involved the entire cervical course of the ICA.
Conclusions:
In most of our patients variations in the course of the cervical portion of the ICA involved the right side and were asymptomatic, except with regard to potential surgical risks. However, in about 25% of our patients these variations were thought to be the culprit of patient complaints, particularly oropharyngeal pulsatile sensation. Furthermore, extreme degrees of medialization of the ICAs resulted in progressive symptoms including hoarseness and upper respiratory distress.
doi:10.2174/1874440001004010174
PMCID: PMC3026334  PMID: 21283647
Internal carotid artery; anomalies; internal carotid artery; aberrancy; head and neck; carotid artery.
17.  Cystatin D is a candidate tumor suppressor gene induced by vitamin D in human colon cancer cells 
The Journal of Clinical Investigation  2009;119(8):2343-2358.
The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] has wide but not fully understood antitumor activity. A previous transcriptomic analysis of 1α,25(OH)2D3 action on human colon cancer cells revealed cystatin D (CST5), which encodes an inhibitor of several cysteine proteases of the cathepsin family, as a candidate target gene. Here we report that 1α,25(OH)2D3 induced vitamin D receptor (VDR) binding to, and activation of, the CST5 promoter and increased CST5 RNA and protein levels in human colon cancer cells. In cells lacking endogenous cystatin D, ectopic cystatin D expression inhibited both proliferation in vitro and xenograft tumor growth in vivo. Furthermore, cystatin D inhibited migration and anchorage-independent growth, antagonized the Wnt/β-catenin signaling pathway, and repressed c-MYC expression. Cystatin D repressed expression of the epithelial-mesenchymal transition inducers SNAI1, SNAI2, ZEB1, and ZEB2 and, conversely, induced E-cadherin and other adhesion proteins. CST5 knockdown using shRNA abrogated the antiproliferative effect of 1α,25(OH)2D3, attenuated E-cadherin expression, and increased c-MYC expression. In human colorectal tumors, expression of cystatin D correlated with expression of VDR and E-cadherin, and loss of cystatin D correlated with poor tumor differentiation. Based on these data, we propose that CST5 has tumor suppressor activity that may contribute to the antitumoral action of 1α,25(OH)2D3 in colon cancer.
doi:10.1172/JCI37205
PMCID: PMC2719930  PMID: 19662683
18.  RhoA–ROCK and p38MAPK-MSK1 mediate vitamin D effects on gene expression, phenotype, and Wnt pathway in colon cancer cells 
The Journal of Cell Biology  2008;183(4):697-710.
The active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation and promotes differentiation of colon cancer cells through the activation of vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Additionally, 1,25(OH)2D3 has several nongenomic effects of uncertain relevance. We show that 1,25(OH)2D3 induces a transcription-independent Ca2+ influx and activation of RhoA–Rho-associated coiled kinase (ROCK). This requires VDR and is followed by activation of the p38 mitogen-activated protein kinase (p38MAPK) and mitogen- and stress-activated kinase 1 (MSK1). As shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, RhoA–ROCK, and p38MAPK-MSK1 activation is necessary for the induction of CDH1/E-cadherin, CYP24, and other genes and of an adhesive phenotype by 1,25(OH)2D3. RhoA–ROCK and MSK1 are also required for the inhibition of Wnt–β-catenin pathway and cell proliferation. Thus, the action of 1,25(OH)2D3 on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA–ROCK and p38MAPK-MSK1.
doi:10.1083/jcb.200803020
PMCID: PMC2582889  PMID: 19015318
19.  Studies on the Mode of Action of the Antifungal Hexapeptide PAF26 
Antimicrobial Agents and Chemotherapy  2006;50(11):3847-3855.
The small antimicrobial peptide PAF26 (Ac-RKKWFW-NH2) has been identified by a combinatorial approach and shows preferential activity toward filamentous fungi. In this work, we investigated the mode of action and inhibitory effects of PAF26 on the fungus Penicillium digitatum. The dye Sytox Green was used to demonstrate that PAF26 induced cell permeation. However, microscopic observations showed that sub-MIC concentrations of PAF26 produced both alterations of hyphal morphology (such as altered polar growth and branching) and chitin deposition in areas of no detectable permeation. Analysis of dose-response curves of inhibition and permeation suggested that growth inhibition is not solely a consequence of permeation. In order to shed light on the mode of PAF26 action, its antifungal properties were compared with those of melittin, a well-known pore-forming peptide that kills through cytolysis. While the 50% inhibitory concentrations and MICs of the two peptides against P. digitatum mycelium were comparable, they differed markedly in their fungicidal activities toward conidia and their hemolytic activities toward human red blood cells. Kinetic studies showed that melittin quickly induced Penicillium cell permeation, while PAF26-induced Sytox Green uptake was significantly slower and less efficient. Therefore, the ultimate growth inhibition and morphological alterations induced by PAF26 for P. digitatum are not likely a result of conventional pore formation. Fluorescently labeled PAF26 was used to demonstrate its specific in vivo interaction and translocation inside germ tubes and hyphal cells, at concentrations as low as 0.3 μM (20 times below the MIC), at which no inhibitory, morphological, or permeation effects were observed. Interestingly, internalized PAF26 could bind to cellular RNAs, since in vitro nonspecific RNA binding activity of PAF26 was demonstrated by electrophoretic mobility shift assays. We propose that PAF26 is a short, de novo-designed penetratin-type peptide that has multiple detrimental effects on target fungi, which ultimately result in permeation and killing.
doi:10.1128/AAC.00650-06
PMCID: PMC1635192  PMID: 17065623
20.  Gender Susceptibility to Chronic Hepatitis C Virus Infection Associated with Interleukin 10 Promoter Polymorphism 
Journal of Virology  2006;80(18):9144-9150.
Elevated levels of interleukin 10 (IL-10) were previously described for chronically hepatitis C virus (HCV)-infected patients. We determined by a sequence-specific oligonucleotide probing technique the IL-10 promoter genotypes in 286 Argentinean HCV patients grouped according to disease outcome. The GG genotype (position −1082) is known to be associated with high IL-10 production, GA is considered an intermediate producer, and AA is associated with low IL-10 production. We found an increase in frequency of the GG genotype in female patients who do not eliminate the virus (RNA+). In these patients, the GG frequency was 0.19, versus 0.10 in controls (P = 0.03). This association became more significant in those RNA+ female patients with elevated hepatic transaminases (GG frequency of 0.25; P = 0.0013). Additionally, this genotype frequency was higher in noncirrhotic female patients than in controls (GG frequency for noncirrhotic female patients was 0.31; P = 0.009). In RNA− patients, the GA frequency was elevated compared with that in controls (GA frequency of 0.76 in RNA− patients versus 0.48 in controls; P = 0.01), that in all HCV patients (GA frequency of 0.43; P = 0.001), and that in RNA+ patients (GA frequency of 0.40; P = 0.0005). We conclude that a gender effect is observed with women carrying the GG high IL-10 producer genotype. The higher levels of IL-10 present in those individuals are associated with a higher risk of an inefficient clearance of the HCV and the development of a chronic HCV infection together with a lower risk of progression to cirrhosis in female patients.
doi:10.1128/JVI.00339-06
PMCID: PMC1563933  PMID: 16940525
21.  Snail1 transcriptional repressor binds to its own promoter and controls its expression 
Nucleic Acids Research  2006;34(7):2077-2084.
The product of Snail1 gene is a transcriptional repressor of E-cadherin expression and an inductor of the epithelial–mesenchymal transition in several epithelial tumour cell lines. Transcription of Snail1 is induced when epithelial cells are forced to acquire a mesenchymal phenotype. In this work we demonstrate that Snail1 protein limits its own expression: Snail1 binds to an E-box present in its promoter (at −146 with respect to the transcription start) and represses its activity. Therefore, mutation of the E-box increases Snail1 transcription in epithelial and mesenchymal cells. Evidence of binding of ectopic or endogenous Snail1 to its own promoter was obtained by chromatin immunoprecipitation (ChIP) experiments. Studies performed expressing different forms of Snail1 under the control of its own promoter demonstrate that disruption of the regulatory loop increases the cellular levels of Snail protein. These results indicate that expression of Snail1 gene can be regulated by its product and evidence the existence of a fine-tuning feed-back mechanism of regulation of Snail1 transcription.
doi:10.1093/nar/gkl141
PMCID: PMC1440880  PMID: 16617148
22.  Role of HuR in Skeletal Myogenesis through Coordinate Regulation of Muscle Differentiation Genes 
Molecular and Cellular Biology  2003;23(14):4991-5004.
In this report, we investigate the role of the RNA-binding protein HuR during skeletal myogenesis. At the onset of myogenesis in differentiating C2C12 myocytes and in vivo in regenerating mouse muscle, HuR cytoplasmic abundance increased dramatically, returning to a predominantly nuclear presence upon completion of myogenesis. mRNAs encoding key regulators of myogenesis-specific transcription (myogenin and MyoD) and cell cycle withdrawal (p21), bearing AU-rich regions, were found to be targets of HuR in a differentiation-dependent manner. Accordingly, mRNA half-lives were highest during differentiation, declining when differentiation was completed. Importantly, HuR-overexpressing C2C12 cells displayed increased target mRNA expression and half-life and underwent precocious differentiation. Our findings underscore a critical function for HuR during skeletal myogenesis linked to HuR's coordinate regulation of muscle differentiation genes.
doi:10.1128/MCB.23.14.4991-5004.2003
PMCID: PMC162217  PMID: 12832484
23.  HuD binds to three AU-rich sequences in the 3′-UTR of neuroserpin mRNA and promotes the accumulation of neuroserpin mRNA and protein 
Nucleic Acids Research  2002;30(10):2202-2211.
Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3′-untranslated region (3′-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3′-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3′-UTR, which prolongs the mRNA lifetime and increases protein level.
PMCID: PMC115279  PMID: 12000840
24.  Vitamin D3 promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of β-catenin signaling 
The Journal of Cell Biology  2001;154(2):369-388.
The β-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1α,25-dehydroxyvitamin D3) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1α,25(OH)2D3 induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of β-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for β-catenin binding. Accordingly, 1α,25(OH)2D3 repressed β-catenin–TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic β-catenin and reduced by TCF-4. Also, 1α,25(OH)2D3 inhibited expression of β-catenin–TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor δ, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1α,25(OH)2D3 induces E-cadherin and modulates β-catenin–TCF-4 target genes in a manner opposite to that of β-catenin, promoting the differentiation of colon carcinoma cells.
doi:10.1083/jcb.200102028
PMCID: PMC2150773  PMID: 11470825
vitamin D; vitamin D receptor; β-catenin; E-cadherin; colon cancer
25.  Glucocorticoids Antagonize Ap-1 by Inhibiting the Activation/Phosphorylation of Jnk without Affecting Its Subcellular Distribution 
The Journal of Cell Biology  2000;150(5):1199-1208.
The immunosuppressive and antiinflammatory actions of glucocorticoid hormones are mediated by their transrepression of activating protein-1 (AP-1) and nuclear factor-kappa B (NFκB) transcription factors. Inhibition of the c-Jun NH2-terminal kinase (JNK) signaling pathway, the main mediator of AP-1 activation, has been described in extracts of hormone-treated cells. Here, we show by confocal laser microscopy, enzymatic assays, and immunoblotting that the synthetic glucocorticoid dexamethasone inhibited tumor necrosis factor α (TNF-α)–induced phosphorylation and activation of JNK in the cytoplasm and nucleus of intact HeLa cells. As a result, c-Jun NH2-terminal domain phosphorylation and induction were impaired. Dexamethasone did not block the TNF-α–induced JNK nuclear translocation, but rather induced, per se, nuclear accumulation of the enzyme. Consistently with previous findings, a glucocorticoid receptor mutant (GRdim), which is deficient in dimerization, DNA binding, and transactivation, but retains AP-1 transrepressing activity, was as efficient as wild-type GR in mediating the same effects of dexamethasone on JNK in transfected Cos-7 cells. Our results show that glucocorticoids antagonize the TNF-α–induced activation of AP-1 by causing the accumulation of inactive JNK without affecting its subcellular distribution.
PMCID: PMC2175250  PMID: 10974006
dexamethasone; activating protein-1; tumor necrosis factor α; c-Jun NH2-terminal kinase; nuclear translocation

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