The human-specific duplicated α7 gene inhibits the ancestral α7, negatively regulating nicotinic acetylcholine receptor-mediated transmitter release.

Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.

Organotin Dyes Bearing Anionic Boron Clusters as Cell-Staining Fluorescent Probes.

Within the cell nucleus, in the nucleoli, ribosomal RNAs are synthesized and participate in several biological processes. To better understand nucleoli-related processes, their visualization is often required, for which specific markers are needed. Herein, we report the design of novel fluorescent organotin compounds derived from 4-hydroxy-N'-((2-hydroxynaphthalen-1-yl)methylene)benzohydrazide and their cytoplasm and nucleoli staining of B16F10 cells in vitro. Tin compounds bearing an aliphatic carbon chain (-C12 H25 ) and an electron-donating group (-OH) were prepared, and the latter could be derivatized to bear the boron cluster anions [B12 H12 ]2- and [3,3'-Co(1,2-C2 B9 H11 )2 ]- (COSAN). All of the conjugates have been fully characterized and their luminescence properties have been assessed. In general, they show good quantum yields in solution (24-49 %), those for the COSAN derivatives being lower. Remarkably, the linking of [B12 H12 ]2- and COSAN to the complexes made them more soluble, without being detrimental to their luminescence properties. Living B16F10 cells were treated with all of the compounds to determine their fluorescence staining properties; the compounds bearing the aliphatic chain showed a reduced staining capacity due to the formation of aggregates. Notably, the complexes bearing different boron clusters showed different staining effects; those bearing [B12 H12 ]2- showed extraordinary staining of the nucleoli and cytoplasm, whereas those bearing COSAN were only detected in the cytoplasm. The remarkable fluorescence staining properties shown by these organotin compounds make them excellent candidates for fluorescence bioimaging in vitro.

Exosomes: From Garbage Bins to Promising Therapeutic Targets.

Intercellular communication via cell-released vesicles is a very important process for both normal and tumor cells. Cell communication may involve exosomes, small vesicles of endocytic origin that are released by all types of cells and are found in abundance in body fluids, including blood, saliva, urine, and breast milk. Exosomes have been shown to carry lipids, proteins, mRNAs, non-coding RNAs, and even DNA out of cells. They are more than simply molecular garbage bins, however, in that the molecules they carry can be taken up by other cells. Thus, exosomes transfer biological information to neighboring cells and through this cell-to-cell communication are involved not only in physiological functions such as cell-to-cell communication, but also in the pathogenesis of some diseases, including tumors and neurodegenerative conditions. Our increasing understanding of why cells release exosomes and their role in intercellular communication has revealed the very complex and sophisticated contribution of exosomes to health and disease. The aim of this review is to reveal the emerging roles of exosomes in normal and pathological conditions and describe the controversial biological role of exosomes, as it is now understood, in carcinogenesis. We also summarize what is known about exosome biogenesis, composition, functions, and pathways and discuss the potential clinical applications of exosomes, especially as biomarkers and novel therapeutic agents.

Therapeutic silencing of HPV 16 E7 by systemic administration of siRNA-neutral DOPC nanoliposome in a murine cervical cancer model with obesity.

PURPOSE: To evaluate the effectiveness of a neutral DOPC nanoliposome system for the delivery of siRNA to tumor cells in an obese murine cervical cancer model. METHODS: In vitro silencing of E6-E7 mRNA and E7 protein using siRNAE6 or siRNAE7 was analyzed in TC-1 cells by RT-PCR and Western blot. Silencing and antitumor capacities of siRNAE7-DOPC-nanoparticles (NP) were tested in vivo in both normal and obese mice using qPCR. These NPs were administered twice a week for 15 days and tumor volume and weight were recorded. RESULTS: Levels of in vitro E6-E7 silencing were 90% for mRNA and 60% for protein when siRNAE7 was used. On the other hand when siRNAE6 was used, the levels of silencing were 50% for E6-E7 mRNA and only 20% for protein. In vivo E7 mRNA silencing by siRNAE7-DOPC-NP was similar (60%) in both non-obese and obese mouse models. The therapeutic study showed a 65% decrease in tumor volume and a 57% reduction in tumor weight as compared to the control groups. CONCLUSION: There was no negative impact of obesity on the antitumor activity of siRNA-DOPC-NP in obese mice.

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice.

The p53 protein integrates multiple upstream signals and functions as a tumor suppressor by activating distinct downstream genes. At the cellular level, p53 induces apoptosis, cell cycle arrest and senescence. A rare mutant form of p53 with the amino acid substitution R175P, found in human tumors, is completely defective in initiating apoptosis but still induces cell cycle arrest. To decipher the functional importance of these pathways in spontaneous tumorigenesis, we used homologous recombination to generate mice with mutant p53-R172P (the mouse equivalent of R175P in humans). Mice inheriting two copies of this mutation (Trp53(515C/515C)) escape the early onset of thymic lymphomas that characterize Trp53-null mice. At 7 months of age, 90% of Trp53-null mice had died, but 85% of Trp53(515C/515C) mice were alive and tumor-free, indicating that p53-dependent apoptosis was not required for suppression of early onset of spontaneous tumors. The lymphomas and sarcomas that eventually developed in Trp53(515C/515C) mice retained a diploid chromosome number, in sharp contrast to aneuploidy observed in tumors and cells from Trp53-null mice. The ability of mutant p53-R172P to induce a partial cell cycle arrest and retain chromosome stability are crucial for suppression of early onset tumorigenesis.

Therapeutic Effects of WT1 Silencing via Respiratory Administration of Neutral DOPC Liposomal-siRNA in a Lung Metastasis Melanoma Murine Model.

The lungs represent a frequent target for metastatic melanoma as they offer a high-oxygen environment for tumor development. The overexpression of the WT1 protein has been associated with the occurrence of melanoma. In this study, we evaluated the effects of silencing the WT1 protein by siRNA in both in vitro in the B16F10 melanoma cell line and in vivo in a murine model of lung metastatic melanoma. We did this by implementing a novel respiratory delivery strategy of a neutral DOPC liposomal-siRNA system (L-siRNA). In vitro studies showed an effective silencing of the WT1 protein in the siRNAs' WT1-treated cells when compared with controls, resulting in a loss of the cell's viability and proliferation by inducing G1 arrest, the inhibition of the migration and invasion capacities of the cells, as well as the induction of apoptosis. In vivo, the respiratory administration of L-WT1 siRNA showed an efficient biodistribution on the lungs. After two weeks of treatment, the silencing of the WT1 protein resulted in an important antitumor activity that reduced the tumor weight. In the survival study, L-WT1 treatment could significantly delay the death of the animals. This work demonstrates the efficacy of the L-siRNA respiratory administration as a novel therapy to reduce pulmonary tumors and to increase survivability by silencing specific cancer oncogenes as WT1.

Boron Schiff bases derived from α-amino acids as nucleoli/cytoplasm cell-staining fluorescent probes in vitro.

The size, shape, and number of nucleoli in a cell's nucleus might help to distinguish a malignant from a benign tumor. Cellular biology and histopathology often require better visualization to understand nucleoli-related processes, thus organelle-specific fluorescent markers are needed. Here, we report the design, synthesis, and fully chemo-photophysical characterization of fluorescent boron Schiff bases (BOSCHIBAs), derived from α-amino acids (i.e., phenylalanine, tyrosine and tryptophan), with nucleoli- and cytoplasm-specific staining in cells. It is the first time that Boron Schiff bases derived from α-amino acids act as notorious dual (nucleoli and cytoplasm) cell-staining fluorescent probes. The boron derivatives not only showed good photostability and acceptable quantum yields (∼5%) in solution, but also exhibited low cytotoxicity (>90% cell viability at 0.1 and 1 µg mL-1), which make them good candidates to be used in medical diagnosis.

Murine interferon-gamma inducible protein-10 (IP-10) secreted by Lactococcus lactis chemo-attracts human CD3+ lymphocytes.

Chemokines are members of the super family of cytokines necessary for leukocyte recruitment in tissues and lymphoid organs. The interferon-gamma inducible protein-10 (IP-10) chemo-attracts CXCR3-expressing cells, such as activated T lymphocytes and monocytes. We have genetically engineered a strain of Lactococcus lactis to secrete a biologically active murine IP-10 that interacts with human CXCR3, its homolog receptor, and chemo-attracts human CD3+ T lymphocytes.

PTGER3 induces ovary tumorigenesis and confers resistance to cisplatin therapy through up-regulation Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis.

BACKGROUND: Inflammatory mediator prostaglandin E2-prostaglandin E2 receptor EP3 (PTGER3) signaling is critical for tumor-associated angiogenesis, tumor growth, and chemoresistance. However, the mechanism underlying these effects in ovarian cancer is not known. METHODS: An association between higher tumoral expression of PTGER3 and shorter patient survival in the ovarian cancer dataset of The Cancer Genome Atlas prompted investigation of the antitumor effects of PTGER3 downmodulation. PTGER3 mRNA and protein levels were higher in cisplatin-resistant ovarian cancer cells than in their cisplatin-sensitive counterparts. FINDINGS: Silencing of PTGER3 via siRNA in cancer cells was associated with decreased cell growth and less invasiveness, as well as cell-cycle arrest and increased apoptosis, mediated through the Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis. Furthermore, sustained PTGER3 silencing with multistage vector and liposomal 2'-F-phosphorodithioate-siRNA-mediated silencing of PTGER3 combined with cisplatin resulted in robust antitumor effects in cisplatin-resistant ovarian cancer models. INTERPRETATION: These findings identify PTGER3 as a potential therapeutic target in chemoresistant ovarian cancers expressing high levels of this oncogenic protein. FUND: National Institutes of Health/National Cancer Institute, USA.

The polyaromatic hydrocarbon ß-naphthoflavone alters binding of YY1, Sp1, and Sp3 transcription factors to the Dp71 promoter in hepatic cells.

The smallest product of the Duchenne muscular dystrophy gene, dystrophin (Dp)71, is ubiquitously expressed in nonmuscle tissues. We previously showed that Dp71 expression in hepatic cells is modulated in part by stimulating factor 1 (Sp1), stimulating protein 3 (Sp3), and yin yang 1 (YY1) transcription factors, and that the polyaromatic hydrocarbon, ß-naphthoflavone (ß­NF), downregulates Dp71 expression. The aim of the present study was to determine whether ß­NF represses Dp71 expression by altering mRNA stability or its promoter activity. Reverse transcription­quantitative polymerase chain reaction was used to measure half­life mRNA levels in ß­NF­treated cells exposed to actinomycin D, an inhibitor of transcription, for 0, 4, 8, 12 and 16 h. Transient transfections with a plasmid carrying the Dp71 basal promoter fused to luciferase reporter gene were carried out in control and ß­NF­treated cells. Electrophoretic mobility shift assays (EMSAs) were performed with labeled probes, corresponding to Dp71 promoter sequences, and nuclear extracts of control and ß­NF­treated cells. To the best of our knowledge, the results demonstrated for the first time that this negative regulation takes place at the promoter level rather than the mRNA stability level. Interestingly, using EMSAs, ß­NF reduced binding of YY1, Sp1, and Sp3 to the Dp71 promoter. It also suggests that ß­NF may modulate the expression of other genes regulated by these transcription factors. In conclusion, ß­NF represses Dp71 expression in hepatic cells by altering binding of YY1, Sp1, and Sp3 to the Dp71 promoter.

Focal adhesion kinase targeting using in vivo short interfering RNA delivery in neutral liposomes for ovarian carcinoma therapy.

PURPOSE: Focal adhesion kinase (FAK) plays a critical role in ovarian cancer cell survival and in various steps in the metastatic cascade. Based on encouraging in vitro results with FAK silencing, we examined the in vivo therapeutic potential of this approach using short interfering RNA (siRNA) in the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). EXPERIMENTAL DESIGN: Therapy experiments of FAK siRNA with or without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8MDR in nude mice. Additional experiments with a cisplatin-resistant cell line (A2780-CP20) were also done. Assessments of angiogenesis (CD31), cell proliferation (proliferating cell nuclear antigen), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) were done using immunohistochemical analysis. RESULTS: A single dose of FAK siRNA-DOPC was highly effective in reducing in vivo FAK expression for up to 4 days as assayed by Western blot and immunohistochemical analysis. Therapy experiments were started 1 week after injection of the ovarian cancer cells. Treatment with FAK siRNA-DOPC (150 mug/kg twice weekly) reduced mean tumor weight by 44% to 72% in the three cell lines compared with the control group (Ps < 0.05 for HeyA8, A2780-CP20, and SKOV3ip1). When FAK siRNA-DOPC was combined with docetaxel, there was even greater reduction in mean tumor weight in all models (all Ps < 0.05). Similar results were observed in combination with cisplatin. Treatment with FAK siRNA-DOPC plus docetaxel resulted in decreased microvessel density, decreased expression of vascular endothelial growth factor and matrix metalloproteinase-9, and increased apoptosis of tumor-associated endothelial cells and tumor cells. CONCLUSIONS: Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.

Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery.

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 microg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.

Regulation of hnRNPA1 by microRNAs controls the miR-18a-K-RAS axis in chemotherapy-resistant ovarian cancer.

The regulation of microRNA (miRNA) biogenesis, function and degradation involves a range of mechanisms, including interactions with RNA-binding proteins. The potential contribution of regulatory miRNAs to the expression of these RNA interactor proteins that could control other miRNAs expression is still unclear. Here we demonstrate a regulatory circuit involving oncogenic and tumor-suppressor miRNAs and an RNA-binding protein in a chemotherapy-resistant ovarian cancer model. We identified and characterized miR-15a-5p and miR-25-3p as negative regulators of hnRNPA1 expression, which is required for the processing of miR-18a-3p, an inhibitor of the K-RAS oncogene. The inhibition of miR-25-3p and miR-15a-5p decreased the proliferation, motility, invasiveness and angiogenic potential and increased apoptosis when combined with docetaxel. Alteration of this regulatory circuit causes poor overall survival outcome in ovarian cancer patients. These results highlight miR-15a-5p and miR-25-3p as key regulators of miR-18a-3p expression and its downstream target K-RAS, through direct modulation of hnRNPA1 expression. Our results demonstrate the therapeutic potential of inhibiting miR-25-3p and miR-15a-5p and the use of miR-18a-3p/KRAS ratio as a prominent outcome prognostic factor.

Synergistic Antimicrobial Effects of Silver/Transition-metal Combinatorial Treatments.

Due to the emergence of multi-drug resistant strains, development of novel antibiotics has become a critical issue. One promising approach is the use of transition metals, since they exhibit rapid and significant toxicity, at low concentrations, in prokaryotic cells. Nevertheless, one main drawback of transition metals is their toxicity in eukaryotic cells. Here, we show that the barriers to use them as therapeutic agents could be mitigated by combining them with silver. We demonstrate that synergism of combinatorial treatments (Silver/transition metals, including Zn, Co, Cd, Ni, and Cu) increases up to 8-fold their antimicrobial effect, when compared to their individual effects, against E. coli and B. subtilis. We find that most combinatorial treatments exhibit synergistic antimicrobial effects at low/non-toxic concentrations to human keratinocyte cells, blast and melanoma rat cell lines. Moreover, we show that silver/(Cu, Ni, and Zn) increase prokaryotic cell permeability at sub-inhibitory concentrations, demonstrating this to be a possible mechanism of the synergistic behavior. Together, these results suggest that these combinatorial treatments will play an important role in the future development of antimicrobial agents and treatments against infections. In specific, the cytotoxicity experiments show that the combinations have great potential in the treatment of topical infections.

HER2/neu increases the expression of Wilms' Tumor 1 (WT1) protein to stimulate S-phase proliferation and inhibit apoptosis in breast cancer cells.

High levels of the Wilms' Tumor 1 (WT1) protein and mRNA had been associated with aggressive phenotypes of breast tumors. Here we report that the HER2/neu oncogene increases WT1 expression. Approximately threefold higher levels of WT1 protein were observed in MCF-7 breast cancer cells transfected with the HER2/neu oncogene than in parental MCF-7 cells. Conversely, inhibition of HER2/neu with the anti-HER2/neu trastuzumab (Herceptintrade mark) antibody decreased WT1 protein levels in HER2/neu-overexpressing BT-474 and SKBr3 cells. We also found that HER2/neu engages Akt to regulate WT1 levels since inhibition of Akt reduced WT1 levels. Decreased expression of WT1 protein led to cell cycle arrest at the G1 phase and increased apoptosis in HER2/neu-overexpressing cells, which is correlated with decreased cyclin D1 and Bcl-2 levels. Our data indicate that HER2/neu engages Akt to increase WT1 expression, and that WT1 protein plays a vital role in regulating cell cycle progression and apoptosis in HER2/neu-overexpressing breast cancer cells.

Switching mechanisms of cell death in mdm2- and mdm4-null mice by deletion of p53 downstream targets.

The p53 tumor suppressor ensures maintenance of genome integrity by initiating either apoptosis or cell cycle arrest in response to DNA damage. Deletion of either mdm2 or mdm4 genes, which encode p53 inhibitors, results in embryonic lethality. The lethal phenotypes are rescued in the absence of p53, which indicates that increased activity of p53 is the cause of lethality in the mdm2- and mdm4-null embryos. Here we show that mdm2-null embryos die because of apoptosis initiated at 3.5 days postcoitum (dpc). Partial rescue of mdm2-null embryos by deletion of bax allows survival to 6.5 dpc and alters the mechanism of death from apoptosis to cell cycle arrest, indicating that bax is a critical component of the p53 pathway in early embryogenesis. The death of mdm4-null embryos is due to p53-initiated cell cycle arrest at 7.5 dpc. Deletion of p21(p21(waf1/cip1)), a p53 downstream target partially responsible for cell cycle arrest, does not rescue this phenotype; however, deletion of p21 alters the mechanism of cell death from lack of proliferation to apoptosis. Thus, in both examples, deletion of a p53 downstream target gene allows p53 to redirect its efforts, highlighting a high degree of plasticity in p53 function.

New application of fluorescent organotin compounds derived from Schiff bases: synthesis, X-ray structures, photophysical properties, cytotoxicity and fluorescent bioimaging.

A series of eight new organotin compounds derived from Schiff bases has been prepared by a multicomponent reaction from 2-hydroxy-1-naphthaldehyde or 4-substituted-2-hydroxybenzalhedyde, benzhydrazine, and the corresponding diorganotin oxide (R2SnO, R = nBu or Ph). All of the compounds were fully characterized by NMR (1H, 13C, and 119Sn), IR, UV/vis, elemental analyses and fluorescence spectroscopy. The crystal structures for some organotin compounds were determined by single crystal X-ray diffraction analysis. All of the compounds display fluorescence at room temperature with quantum yields of about 2 × 10-4 to 0.56. The cytotoxic activity and cellular imaging studies were carried out with the newly synthesized compounds. To the best of our knowledge, this is the first report of organotin compounds with Schiff base ligands investigated for fluorescence bioimaging (FBI).