Mechanisms of Action and Limitations of Monoclonal Antibodies and Single Chain Fragment Variable (scFv) in the Treatment of Cancer.

Monoclonal antibodies are among the most effective tools for detecting tumor-associated antigens. The U.S. Food and Drug Administration (FDA) has approved more than 36 therapeutic antibodies for developing novel alternative therapies that have significant success rates in fighting cancer. However, some functional limitations have been described, such as their access to solid tumors and low interaction with the immune system. Single-chain variable fragments (scFv) are versatile and easy to produce, and being an attractive tool for use in immunotherapy models. The small size of scFv can be advantageous for treatment due to its short half-life and other characteristics related to the structural and functional aspects of the antibodies. Therefore, the main objective of this review was to describe the current situation regarding the mechanisms of action, applications, and limitations of monoclonal antibodies and scFv in the treatment of cancer.

Biological Role and Aberrant Overexpression of Syntenin-1 in Cancer: Potential Role as a Biomarker and Therapeutic Target.

Syntenin-1 is a 298 amino acid protein codified by the melanoma differentiation-associated gene-9 (MDA-9). Structurally, it is composed of four domains: N-terminal, PDZ1, PDZ2, and C-terminal. The PDZ domains of syntenin-1 are involved in the stability and interaction with other molecules such as proteins, glycoproteins, and lipids. Domains are also associated with several biological functions such as the activation of signaling pathways related to cell-to-cell adhesion, signaling translation, and the traffic of intracellular lipids, among others. The overexpression of syntenin-1 has been reported in glioblastoma, colorectal, melanoma, lung, prostate, and breast cancer, which promotes tumorigenesis by regulating cell migration, invasion, proliferation, angiogenesis, apoptosis, and immune response evasion, and metastasis. The overexpression of syntenin-1 in samples has been associated with worst prognostic and recurrence, whereas the use of inhibitors such as shRNA, siRNA, and PDZli showed a diminution of the tumor size and reduction in metastasis and invasion. Syntenin-1 has been suggested as a potential biomarker and therapeutic target in cancer for developing more effective diagnostic/prognostic tests or passive/active immunotherapies.

The catE gene of Bacillus licheniformis M2-7 is essential for growth in benzopyrene, and its expression is regulated by the Csr system.

Benzopyrene is a high-molecular-weight polycyclic aromatic hydrocarbon that is highly recalcitrant and induces carcinogenic effects. CsrA is a conserved regulatory protein that controls the translation and stability of its target transcripts, having negative or positive effects depending on the target mRNAs. It is known that Bacillus licheniformis M2-7 has the ability to grow and survive in certain concentrations of hydrocarbons such as benzopyrene, prompted in part by CsrA, as is present in gasoline. However, there are a few studies that reveal the genes involved in that process. To identify the genes involved in the Bacillus licheniformis M2-7 degradation pathway, the plasmid pCAT-sp containing a mutation in the catE gene was constructed and used to transform B. licheniformis M2-7 and generate a CAT1 strain. We determined the capacity of the mutant B. licheniformis (CAT1) to grow in the presence of glucose or benzopyrene as a carbon source. We observed that the CAT1 strain presented increased growth in the presence of glucose but a statistically considerable decrease in the presence of benzopyrene compared with the wild-type parental strain. Additionally, we demonstrated that the Csr system positively regulates its expression since it was observed that the expression of the gene in the mutant strain LYA12 (M2-7 csrA:: Sp, SpR) was considerably lower than that in the wild-type strain. We were thus able to propose a putative regulation model for catE gene in B. licheniformis M2-7 strain by CsrA regulator in the presence of benzopyrene.

REST/NRSF Silencing Modifies Neuronal Gene Expression in siRNA-Treated HeLa Cells: A Preliminary Exploration in the Search for Neuronal Biomarkers of Cervical Cancer.

Background and Objectives: REST (RE1-silencing transcription factor) diminution is associated with transcriptional relaxation, neuropeptide overexpression, and phenotype redefinition in neuroendocrine cancers, but this effect has barely been studied in cervical cancer (CC). We previously reported reduced expressions of REST in samples with premalignant lesions and CC; however, the transcriptional consequences for neural genes associated with reduced REST expression in CC are unknown. Therefore, the objective of this work was to evaluate the expression of neuronal genes in cancerous cells with reduced expression levels of REST. Materials and Methods: Here, we monitored levels of REST by immunostaining along the premalignant lesions and in invasive cervical squamous cell carcinoma (SCC) and endocervical adenocarcinoma (ADC) in tissue samples from female patients from southern Mexico and the derivative cell lines SiHa and HeLa, respectively. Next, we selected REST target genes in silico and explored the effect of REST silencing by RT-PCR in siRNA-treated HeLa cells. Results: The results show a REST diminution in premalignant lesions, SCC, ADC, and cancerous cell lines. Further REST silencing in HeLa cells altered the expression of genes containing the RE1 (Restrictive Element 1) sequence, including CgA (chromogranin A), CHRNß2 (cholinergic receptor nicotinic ß 2 subunit), BDNF (brain-derived neurotrophic factor), CRF (corticotropin-releasing factor), and RASSF1A (Ras association domain family 1). Conclusions: This work provides preliminary evidence of the role of REST loss in the transcriptional regulation of its target genes in HeLa cells, which could have positive implications for the search for new biomarkers of cervical cancer.

Cooperative Binding of SRSF3 to Structured 3'ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA.

ZO-1α+ and ZO-1α- proteins are expressed in hermetic and leaky tight junctions, respectively. Two cis-acting distant exonic elements partly activate the 240 nucleotide-long α exon producing the ZO-1α+ isoform. However, the elements within and around the α exon and their respective factors involved in its splicing are unknown. To study the dynamic interaction between SRSF3 and its bioinformatically predicted target sites around the 3'ss upstream of the α exon during its activation, we performed EMSA, crosslinking, and in vivo splicing assays by ZO-1 minigene expression and siRNA-mediated silencing in transfected cells. Using V1 RNase, we probed the possible formation of a hairpin RNA structure between the intronic and proximal exonic SRSF3 binding sites. The hairpin sufficed for complex formations in the EMSA. The interaction of SRSF3 with the intronic site promoted the cooperative binding of SRSF3 to the exonic site. Finally, SRSF3 restored α exon activation in SRSF3 knockdown transfectants. Altogether, our results show that SRSF3-hairpin RNA interaction is crucial in the early recognition of 3'ss for α exon activation. It remains to be explored whether SRSF3 recruits or stabilizes the binding of other factors or brings separate splice sites into proximity.

Ficus crocata leaf extracts decrease the proliferation and invasiveness of breast cancer cells.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype due to its greater invasive capacity and non-response to hormone therapy. Several species of the Ficus genus have been used as an alternative to traditional medicine against malignant diseases. Previously, leaf extracts from Ficus crocata (Miq.) Mart. ex Miq. (F. crocata) showed antiproliferative activity in vitro against breast and cervical tumor cells without having a cytotoxic effect on non-tumor cell lines. The purpose of the study was to evaluate the effect of hexane (Hex-EFc), dichloromethane (Dic-EFc), and acetone (Ace-EFc) extracts from F. crocata on the proliferative and invasive capacity of breast cancer cells MCF-7 and MDA-MB-231. Materials and methods: The phytochemical profile was carried out by gas chromatography-mass spectrometry (GC-MS). Cell proliferation, migration, and invasion were determined by MTT, wound closure, and transwell assays, respectively. MMPs activity was analyzed using gelatin zymography, and fluorescence microscopy was used to visualize F-actin distribution. Results: Hex-EFc, Dic-EFc, and Ace-EFc showed cytotoxic activity on MDA-MB-231 tumor cells and, to a lesser extent, on MCF-7 cells, without presenting cytotoxicity at the same concentrations in MCF-10A non-tumor cells. Dic-EFc and Ace-EFc (5-10 µg/mL) reduced the migration capacity of MCF-7 and MDA-MB-231 cells. Interestingly, exposure to Dic-EFc and Ace-EFc (5-10 µg/mL) inhibited the invasive ability of MDA-MB-231 cells, reducing the secretion and activity of MMP-2 and MMP-9, as well as the F-actin distribution. Conclusions: Dic-EFc and Ace-EFc at low concentrations decreased breast cancer cell proliferation and invasiveness, mainly of MDA-MB-231 cells. The above supports the potential use of compounds from leaf extracts of F. crocata in neoadjuvant therapy to reduce the progression of breast cancer tumors, mainly triple-negative tumors.

Peptide-Based Vaccines in Clinical Phases and New Potential Therapeutic Targets as a New Approach for Breast Cancer: A Review.

Breast cancer is the leading cause of death in women from 20 to 59 years old. The conventional treatment includes surgery, chemotherapy, hormonal therapy, and immunotherapy. This immunotherapy is based on administering monoclonal therapeutic antibodies (passive) or vaccines (active) with therapeutic purposes. Several types of vaccines could be used as potential treatments for cancer, including whole-cell, DNA, RNA, and peptide-based vaccines. Peptides used to develop vaccines are derived from tumor-associated antigens or tumor-specific antigens, such as HER-2, MUC1, ErbB2, CEA, FRα, MAGE A1, A3, and A10, NY-ESO-1, among others. Peptide-based vaccines provide some advantages, such as low cost, purity of the antigen, and the induction of humoral and cellular immune response. In this review, we explore the different types of vaccines against breast cancer with a specific focus on the description of peptide-based vaccines, their composition, immune response induction, and the description of new potential therapeutic targets.

Chronic Leptin Treatment Induces Epithelial-Mesenchymal Transition in MCF10A Mammary Epithelial Cells.

Leptin is a cytokine-like hormone that functions as a link between obesity and breast cancer (BC). Leptin treatment induces Epithelial to Mesenchymal Transition (EMT) in BC cell lines. In non-tumoral breast epithelial MCF10A cells, acute leptin treatment induces partial EMT. However, the effect of chronic leptin treatment on EMT in non-tumorigenic breast cells has not been fully explored. This study aimed to evaluate the effect of chronic leptin treatment on the induction of EMT in MCF10A cells. We found that chronic leptin treatment induces a switch from an epithelial to a mesenchymal morphology, partial loss of E-cadherin and gain of vimentin expression. Immunolocalization experiments showed a partial loss of E-cadherin at cell junctions and increased cytoplasmic localization of vimentin in leptin-treated cells. Moreover, chronic leptin treatment increased collective cell migration and invasion. Furthermore, when cultured in non-adherent conditions leptin treated cells exhibited reduced cell aggregation, increased survival, and decreased apoptosis, which correlates with increased FAK and AKT phosphorylation. Finally, bioinformatic analysis in two publicly available RNAseq datasets from normal breast tissue shows that high levels of leptin mRNA correlate positively with the expression of mesenchymal markers, and negatively with epithelial markers. Thus, our results demonstrate that chronic leptin treatment induces EMT in non-tumorigenic MCF10A cells and suggest that high leptin expression in normal breast tissue may induce EMT and contribute to increased risk of breast cancer.

Natural isoflavonoids in invasive cancer therapy: From bench to bedside.

Cancer is a public health problem worldwide, and one of the crucial steps within tumor progression is the invasion and metastasis of cancer cells, which are directly related to cancer-associated deaths in patients. Recognizing the molecular markers involved in invasion and metastasis is essential to find targeted therapies in cancer. Interestingly, about 50% of the discovered drugs used in chemotherapy have been obtained from natural sources such as plants, including isoflavonoids. Until now, most drugs are used in chemotherapy targeting proliferation and apoptosis-related molecules. Here, we review recent studies about the effect of isoflavonoids on molecular targets and signaling pathways related to invasion and metastasis in cancer cell cultures, in vivo assays, and clinical trials. This review also reports that glycitein, daidzein, and genistein are the isoflavonoids most studied in preclinical and clinical trials and displayed the most anticancer activity targeting invasion-related proteins such as MMP-2 and MMP-9 and also EMT-associated proteins. Therefore, the diversity of isoflavonoids is promising molecules to be used as chemotherapeutic in invasive cancer. In the future, more clinical trials are needed to validate the effectiveness of the various natural isoflavonoids in the treatment of invasive cancer.

TOP2A/MCM2, p16INK4a, and cyclin E1 expression in liquid-based cytology: a biomarkers panel for progression risk of cervical premalignant lesions.

BACKGROUND: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer. METHODS: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit. RESULTS: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%). CONCLUSIONS: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample.

Significant decrease of a master regulator of genes (REST/NRSF) in high-grade squamous intraepithelial lesion and cervical cancer.

BACKGROUND: The repressor element 1-silencing transcription factor (REST) is a regulator of gene expression, and the Ras association domain family member 1 A (RASSF1A) is an important tumor suppressor gene involved in cancer development. Although extensive characterization of the roles of REST and RASSF1A in cancer development have been reported in cellular models, the link between them and their possible role in the development of squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) of the cervix have not been explored. The aim of this study was to evaluate the expression of REST and RASSF1A in cervical cytological and histological samples from patients diagnosed with SIL or SCC and in CC-derived cell lines. METHODS: We analyzed the expression of REST and RASSF1A by immunocyto/histochemistry in cervical samples from patients (n = 271) and in cancer cell lines. Data analyses were performed using the Kruskal-Wallis test and generalized linear models. RESULTS: We identified binding sites for REST in RASSF1A and observed a significant reduction in REST and RASSF1A nuclear expression in samples from patients with high-grade SIL (HSIL) and SCC. For REST, we observed an average decrease of 334 and 423 r.u.d. for HSIL (n = 21) and SCC (n = 18) compared with non-LSIL (n = 72), whereas for RASSF1A, this decrease was 126 and 217 r.u.d., respectively (p < 0.001). CONCLUSION: Our results provide evidence of the altered expression of REST and RASSF1A in SIL and SCC, with a significant decrease in HSIL, SCC, and SCC-derived cell lines; findings that can contribute to the diagnosis, prognosis, and post-treatment follow-up of patients diagnosed with SIL or SCC.

New Actors Driving the Epithelial-Mesenchymal Transition in Cancer: The Role of Leptin.

Leptin is a hormone secreted mainly by adipocytes; physiologically, it participates in the control of appetite and energy expenditure. However, it has also been linked to tumor progression in different epithelial cancers. In this review, we describe the effect of leptin on epithelial-mesenchymal transition (EMT) markers in different study models, including in vitro, in vivo, and patient studies and in various types of cancer, including breast, prostate, lung, and ovarian cancer. The different studies report that leptin promotes the expression of mesenchymal markers and a decrease in epithelial markers, in addition to promoting EMT-related processes such as cell migration and invasion and poor prognosis in patients with cancer. Finally, we report that leptin has the greatest biological relevance in EMT and tumor progression in breast, lung, prostate, esophageal, and ovarian cancer. This relationship could be due to the key role played by the enriched tumor microenvironment in adipose tissue. Together, these findings demonstrate that leptin is a key biomolecule that drives EMT and metastasis in cancer.

Biofilm Production by Enterotoxigenic Strains of Bacillus cereus in Different Materials and under Different Environmental Conditions.

Foodborne illnesses, such as infections or food poisoning, can be caused by bacterial biofilms present in food matrices or machinery. The production of biofilms by several strains of Bacillus cereus on different materials under different culture conditions was determined, as well as the relationship of biofilms with motility, in addition to the enterotoxigenic profile and candidate genes that participate in the production of biofilms. Biofilm production of B. cereus strains was determined on five materials: glass, polystyrene, polyethylene, polyvinylchloride (PVC), PVC/glass; in three culture media: Phenol red broth, tryptic soy broth, and brain heart infusion broth; in two different temperatures (37 °C and 25 °C), and in two different oxygen conditions (oxygen and CO2 tension). Furthermore, the strains were molecularly characterized by end-point polymerase chain reaction. Motility was determined on semi-solid agar. The B. cereus strains in this study were mainly characterized as enterotoxigenic strains; statistically significant differences were found in the PVC material and biofilm production. Motility was positively associated with the production of biofilm in glass/PVC. The sipW and tasA genes were found in two strains. The results of this study are important in the food industry because the strains carry at least one enterotoxin gene and produce biofilms on different materials.

Phytochemical profile and antiproliferative effect of Ficus crocata extracts on triple-negative breast cancer cells.

BACKGROUND: Some species of the Ficus genus show pharmacological activity, including antiproliferative activity, in cell lines of several cancer Types. ficus crocata is distributed in Mexico and used in traditional medicine, as it is believed to possess anti-inflammatory, analgesic, and antioxidant properties. However, as of yet, there are no scientific reports on its biological activity. This study aims to evaluate the phytochemical profile of F. crocata leaf extracts and their effects on breast cancer MDA-MB-231 cells proliferation. Moreover, the study aims to unearth possible mechanisms involved in the decrease of cell proliferation. METHODS: The extracts were obtained by the maceration of leaves with the solvents hexane, dichloromethane, and acetone. The phytochemical profile of the extracts was determined using gas chromatography coupled with mass analysis. Cell proliferation, apoptosis, and cell cycle analysis in MDA-MB-231 cells were determined using a Crystal violet assay, MTT assay, and Annexin-V/PI assay using flow cytometry. The data were analyzed using ANOVA and Dunnett's test. RESULTS: The hexane (Hex-EFc), dichloromethane (Dic-EFc), and acetone (Ace-EFc) extracts of F. crocata decreased the proliferation of MDA-MB-231 cells, with Dic-EFc having the strongest effect. Dic-EFc was fractioned and its antiproliferative activity was potentiated, which enhanced its ability to induce apoptosis in MDA-MB-231 cells, as well as increased p53, procaspase-8, and procaspase-3 expression. CONCLUSIONS: This study provides information on the biological activity of F. crocata extracts and suggests their potential use against triple-negative breast cancer.

Leptin induces cell migration and invasion in a FAK-Src-dependent manner in breast cancer cells.

Breast cancer is the most common invasive neoplasia, and the second leading cause of the cancer deaths in women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloprotease secretion, and invasion. We found that leptin activates FAK and Src and induces the localization of FAK to the focal adhesions. Interestingly, leptin promotes the activation of FAK through a Src- and STAT3-dependent canonical pathway. Specific inhibitors of FAK, Src and STAT3 showed that the effect exerted by leptin in cell migration in breast cancer cells is dependent on these proteins. Moreover, we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9 and invasion in a FAK and Src-dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.

Extracellular-Signal Regulated Kinase: A Central Molecule Driving Epithelial-Mesenchymal Transition in Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible cellular process, characterized by changes in gene expression and activation of proteins, favoring the trans-differentiation of the epithelial phenotype to a mesenchymal phenotype. This process increases cell migration and invasion of tumor cells, progression of the cell cycle, and resistance to apoptosis and chemotherapy, all of which support tumor progression. One of the signaling pathways involved in tumor progression is the MAPK pathway. Within this family, the ERK subfamily of proteins is known for its contributions to EMT. The ERK subfamily is divided into typical (ERK 1/2/5), and atypical (ERK 3/4/7/8) members. These kinases are overexpressed and hyperactive in various types of cancer. They regulate diverse cellular processes such as proliferation, migration, metastasis, resistance to chemotherapy, and EMT. In this context, in vitro and in vivo assays, as well as studies in human patients, have shown that ERK favors the expression, function, and subcellular relocalization of various proteins that regulate EMT, thus promoting tumor progression. In this review, we discuss the mechanistic roles of the ERK subfamily members in EMT and tumor progression in diverse biological systems.

Production and characterization of monoclonal antibodies against the DNA binding domain of the RE1-silencing transcription factor.

The use of monoclonal antibodies for the detection of cellular biomarkers during carcinogenesis provides new strategies for cancer diagnosis or prognosis in patients. Loss of the Restrictive Element 1-Silencing Transcription (REST) factor has been observed in previous molecular and immunological approaches in aggressive breast cancer, small cell lung cancer, liver carcinoma, and colo-rectal cancer; however, for clinic diagnosis, monoclonal antibodies for REST recognition are unavailable. The goal of this work was to design, produce and characterize monoclonal antibodies against the REST DNA binding damain (DBD) that would be suitable for immunoassays. We searched for conserved domains, and immunogenic and antigenic sites in the REST structure via in silico analysis. For mice immunization, we used a recombinant REST DBD purified by affinity chromatography, and then Hybridomas were generated by mouse spleen fusion with myeloma cells. Finally, for monoclonal antibody characterization, we performed enzyme-linked immunosorbent (ELISA), western blot, dot blot, immunocytochemistry (ICC) and immunoprecipitation assays. Results showed that the DBD is conserved in REST isoforms and contains immunogenic and antigenic sites. We generated three clones producing monoclonal antibodies against REST DBD, one of them specifically recognized native REST and was suitable for ICC in samples from patients.

Ouabain Modulates the Adherens Junction in Renal Epithelial Cells.

BACKGROUND/AIMS: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. METHODS: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, ß-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²âº removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. RESULTS: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, ß-catenin and γ-catenin and displaced ß-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and ß-catenin in the plasma membrane after Ca²âº replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of ß-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase. CONCLUSION: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.

Unexplored Molecular Features of the Entamoeba histolytica RNA Lariat Debranching Enzyme Dbr1 Expression Profile.

The RNA lariat debranching enzyme (Dbr1) has different functions in RNA metabolism, such as hydrolyzing the 2'-5' linkage in intron lariats, positively influencing Ty1 and HIV-1 retrotransposition, and modulating snRNP recycling during splicing reactions. It seems that Dbr1 is one of the major players in RNA turnover. It is remarkable that of all the studies carried out to date with Dbr1, to our knowledge, none of them have evaluated the expression profile of the endogenous Dbr1 gene. In this work, we describe, for the first time, that Entamoeba histolytica EhDbr1 mRNA has a very short half-life (less than 30 min) and encodes a very stable protein that is present until trophozoite cultures die. We also show that the EhDbr1 protein is present in the nuclear periphery on the cytoplasmic basal side, contrary to the localization of human Dbr1. Comparing these results with previous hypotheses and with results from different organisms suggests that Dbr1 gene expression is finely tuned and conserved across eukaryotes. Experiments describing the aspects of Dbr1 gene expression and Dbr1 mRNA turnover as well as other functions of the protein need to be performed. Particularly, a special emphasis is needed on the protozoan parasite E. histolytica, the causative agent of amoebiasis, since even though it is a unicellular organism, it is an intron-rich eukaryote whose intron lariats seem to be open to avoid intron lariat accumulation and to process them in non-coding RNAs that might be involved in its virulence.

Ezrin and E-cadherin expression profile in cervical cytology: a prognostic marker for tumor progression in cervical cancer.

BACKGROUND: Cervical cancer (CC) is the fourth cause of mortality by neoplasia in women worldwide. The use of immunomarkers is an alternative tool to complement currently used algorithms for detection of cancer, and to improve selection of therapeutic schemes. Aberrant expression of Ezrin and E-cadherin play an important role in tumor invasion. In this study we analyzed Ezrin and E-cadherin expression in liquid-based cervical cytology samples, and evaluated their potential use as prognostic immunomarkers. METHODS: Immunocytochemical staining of Ezrin and E-cadherin was performed in cervical samples of 125 patients. The cytological or histological diagnostic was performed by Papanicolaou staining or H&E staining, respectively. HPV genotyping was determined using INNO-LIPA Genotyping Extra kit and the HPV physical status by in situ hybridization. Ezrin expression in HaCaT, HeLa and SiHa cell lines was determined by immunocytochemistry, immunofluorescence and Western blot. RESULTS: High Ezrin expression was observed in cervical cancer samples (70%), samples with multiple infection by HR-HPV (43%), and samples with integrated viral genome (47%). High Ezrin expression was associated with degree of SIL, viral genotype and physical status. In contrast, low E-cadherin expression was found in cervical cancer samples (95%), samples with multiple infection by HR-HPV/LR-HPV (87%) and integrated viral genome (72%). Low E-cadherin expression was associated with degree of SIL and viral genotype. Interestingly, Ezrin nuclear staining was associated with degree of SIL and viral genotype. High Ezrin expression, high percent of nuclear Ezrin and low E-cadherin expression behaved as risk factors for progression to HSIL and cervical cancer. CONCLUSIONS: Ezrin and E-cadherin expression profile in cervical cytology samples could be a potential prognostic marker, useful for identifying cervical lesions with a high-risk of progression to cervical cancer.