Integration of biochemical and topographic cues for the formation and spatial distribution of invadosomes in nasopharyngeal epithelial cells.

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-ß1, using engineered gratings with different width and depth. We first report that TGF-ß1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-ß1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells.

MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

IKKα-mediated biogenesis of miR-196a through interaction with Drosha regulates the sensitivity of cancer cells to radiotherapy.

Retraction to: Cell Death Differ 2016;23(9):1471­1482. doi:10.1038/cdd.2016.32

IKKα-mediated biogenesis of miR-196a through interaction with Drosha regulates the sensitivity of cancer cells to radiotherapy.

Radioresistance is a major obstacle in successful clinical cancer radiotherapy, and the underlying mechanisms are not clear. Here we show that IKKα-mediated miR-196a biogenesis via interaction with Drosha regulates the sensitivity of nasopharyngeal carcinoma (NPC) cells to radiotherapy. Phosphorylation of IKKα at T23 site (p-IKKαT23) promotes the binding of IKKα to Drosha that accelerates the processing of miR-196a primary transcripts, leading to increased expressions of both precursor and mature miR-196a. Dephosphorylation of p-IKKαT23 downregulates miR-196a expression and promotes the resistance of NPC cells to radiation treatment. The miR-196a mimic suppresses while its inhibitor promotes the resistance of NPC to radiation treatment. Importantly, the expression of p-IKKαT23 is positively related to the expression of miR-196a in human NPC tissues, and expression of p-IKKαT23 and miR-196a is inversely correlated with NPC clinical radioresistance. Thus, our studies establish a novel mechanistic link between the inactivation of IKKαT23-Drosha-miR-196a pathway and NPC radioresistance, and de-inactivation of IKKαT23-Drosha-miR-196a pathway would be an efficient way to restore the sensitivity of radioresistant NPC to radiotherapy.

Telomerase activity and its association with psychological stress, mental disorders, lifestyle factors and interventions: A systematic review.

OBJECTIVE: To summarise and discuss the association between telomerase activity and psychological stress, mental disorders and lifestyle factors. METHOD: A systematic review was carried out to identify prospective or retrospective studies and interventions published up to June 2015 that reported associations between telomerase activity and psychological stress, mental disorders and lifestyle factors. Electronic data bases of PubMed, ProQuest, CINAHL and Google Scholar were searched. RESULTS: Twenty six studies on humans measured telomerase activity in peripheral blood mononuclear cells (PBMCs) or leukocytes and examined its association with psychological stress, mental disorders and lifestyle factors. Of those studies, three reported significantly decreased telomerase activity in individuals under chronic psychological stress. Interestingly, one of the three studies found that acute laboratory psychological stress significantly increased telomerase activity. Nine studies reported mixed results on association between mental disorders and telomerase activity. Of the nine studies, five reported that major depressive disorder (MDD) was associated with significantly increased telomerase activity. In thirteen out of fourteen studies on lifestyle factors, it was reported that physical exercise, diet micronutrient supplementation, mindfulness meditation, Qigong practice or yoga mediation resulted in increase in telomerase activity. In addition, two studies on animal models showed that depression-like behaviour was associated with decreased hippocampus telomerase activity. Five animal studies showed that physical exercise increased telomerase activity by cell-type-specific and genotype-specific manners. CONCLUSION: Although multi-facet results were reported on the association between telomerase activity and psychological stress, mental disorders and lifestyle factors, there were some consistent findings in humans such as (1) decreased telomerase activity in individuals under chronic stress, (2) increased telomerase activity in individuals with MDD, and (3) increased telomerase activity in individuals under lifestyle interventions. Animal studies showed that physical exercise increased telomerase activity in specific cell-types. However, the exact mechanisms for the changes in telomerase activity have not been elucidated. We propose conglomerate models connecting chronic psychological stress, depression, mediation and physical exercise to telomerase activation. Several areas for future research are suggested.

Autophagy-induced RelB/p52 activation mediates tumour-associated macrophage repolarisation and suppression of hepatocellular carcinoma by natural compound baicalin.

The plasticity of tumour-associated macrophages (TAMs) has implicated an influential role in hepatocellular carcinoma (HCC). Repolarisation of TAM towards M1 phenotype characterises an immune-competent microenvironment that favours tumour regression. To investigate the role and mechanism of TAM repolarisation in suppression of HCC by a natural compound baicalin, Orthotopic HCC implantation model was used to investigate the effect of baicalin on HCC; liposome-clodronate was introduced to suppress macrophage populations in mice; bone marrow-derived monocytes (BMDMs) were induced to unpolarised, M1-like, M2-like macrophages and TAM using different conditioned medium. We observed that oral administration of baicalin (50 mg/kg) completely blocked orthotopic growth of implanted HCC. Suppression of HCC by baicalin was diminished when mice macrophage was removed by clodronate treatment. Baicalin induced repolarisation of TAM to M1-like phenotype without specific toxicity to either phenotype of macrophages. Baicalin initiated TAM reprogramming to M1-like macrophage, and promoted pro-inflammatory cytokines production. Co-culturing of HCC cells with baicalin-treated TAMs resulted in reduced proliferation and motility in HCC. Baicalin had minimal effect on derivation of macrophage polarisation factors by HCC cells, while directly induced repolarisation of TAM and M2-like macrophage. This effect was associated with elevated autophagy, and transcriptional activation of RelB/p52 pathway. Suppression of autophagy or RelB abolished skewing of baicalin-treated TAM. Autophagic degradation of TRAF2 in baicalin-treated TAM might be responsible for RelB/p52 activation. Our findings unveil the essential role of TAM repolarisation in suppressive effect of baicalin on HCC, which requires autophagy-associated activation of RelB/p52.

High prevalence of human herpesvirus type 8 infection in patients with pulmonary tuberculosis in Taiwan.

Blood samples were collected from 101 untreated pulmonary tuberculosis (TB) patients and 101 age- and sex-matched healthy control subjects. TB patients had lower lymphocyte and a higher monocyte counts than control subjects (p <0.0001 for both). The seropositive rate of human herpesvirus (HHV) type 8 antibody was higher in patients (30/101) than in control subjects (15/101) (p = 0.01). Antibody titres in patients also exceeded those in control subjects (p 0.006). Lymphocyte and monocyte counts between seronegative and seropositive subjects were not different. Four patients were positive for HHV-8 DNA. The study revealed a significantly higher HHV-8 seroprevalence in untreated pulmonary TB patients than in general population.

Insulin sensitivity and brain reward activation in overweight Hispanic girls: a pilot study.

BACKGROUND: Insulin resistance is a link between obesity and the associated disease risk. In addition to its role as an energy regulatory signal to the hypothalamus, insulin also modulates food reward. OBJECTIVE: To examine the relationship of insulin sensitivity (SI) and fasting insulin with cerebral activation in response to food and non-food cues in children. METHODS: Twelve overweight Hispanic girls (age: 8-11) participated in two study visits, a frequently sampled intravenous glucose tolerance test and a functional neuroimaging session (GE HDxt 3.0Tesla) with visual stimulation tasks. Blocks of images (high calorie [HC], low calorie [LC] and non-food [NF]) were presented in randomized order. RESULTS: Comparing HC with NF, SI was inversely associated with activation in the anterior cingulate (r(2) = 0.65; P < 0.05), the insula (r(2) = 0.69; P < 0.05), the orbitofrontal cortex (r(2) = 0.74; P < 0.05), and the frontal and rolandic operculum (r(2) = 0.76; P < 0.001). Associations remained significant after adjustment for body mass index. Association of fasting insulin and cerebral activation disappeared after adjustment for waist circumference. CONCLUSION: In addition to weight loss, insulin sensitivity may pose an important target to regulate neural responses to food cues in the prevention of excessive weight gain.

Differential expression of Toll-like receptor 4 in healthy and diseased human gingiva.

BACKGROUND AND OBJECTIVE: Lipopolysaccharide (LPS)-mediated signaling in host cells involves Toll-like receptor 4 (TLR4) accessory molecules, including LPS-binding protein (LBP), cluster of differentiation 14 (CD14) and lymphocyte antigen 96 (MD-2). However, expression of these innate defense molecules in various compartments of the human periodontium is unclear. The aim of this study was to investigate the expression profile of TLR4 in human gingiva. MATERIAL AND METHODS: Human gingival biopsies were collected from healthy gingival or chronic periodontitis tissue. Primary gingival keratinocytes and fibroblasts were cultured. Immunohistochemical analysis for TLR4 was performed. Transcripts of TLR4, MD-2, CD14 and LBP, and their protein products, were examined using RT-PCR, immunoprecipitation and immunoblotting. The interactions between these molecules in keratinocytes and fibroblasts were investigated by co-immunoprecipitation. RESULTS: TLR4 immunoreactivity was found in healthy gingival epithelium and periodontitis tissue, and appeared to be lower in junctional epithelium ( p ≤ 0.01). Fibroblasts and inflammatory cells stained more strongly for TLR4 in diseased periodontal tissues (p < 0.001). Three TLR4 splicing variants, two MD-2 splicing variants and one CD14 mRNA were expressed by gingival keratinocytes and fibroblasts. Expression of TLR4, CD14 and MD-2 proteins was detected in keratinocytes and fibroblasts in vitro. TLR4 protein from gingival keratinocytes and fibroblasts could be co-immunoprecipitated with CD14 or MD-2, suggesting an association between the related molecules in vivo. LBP transcript was detected in gingival biopsies, but not in primary cultures of gingival keratinocytes or fibroblasts. CONCLUSION: TLR4, CD14 and MD-2, but not LBP, are expressed in human gingival keratinocytes and fibroblasts. The TLR4 expression level in the junctional epithelium appeared to be lowest within the periodontal epithelial barrier.

Trends in the susceptibility of methicillin-resistant Staphylococcus aureus to nine antimicrobial agents, including ceftobiprole, nemonoxacin, and tyrothricin: results from the Tigecycline In Vitro Surveillance in Taiwan (TIST) study, 2006-2010.

This study investigated the in vitro susceptibilities of methicillin-resistant Staphylococcus aureus (MRSA) to nine antimicrobial agents in Taiwan. A total of 1,725 isolates were obtained from 20 hospitals throughout Taiwan from 2006 to 2010. The minimum inhibitory concentrations (MICs) of the nine agents were determined by the agar dilution method. The MICs of mupirocin and tyrothricin were determined for 223 MRSA isolates collected from 2009 to 2010. For vancomycin, 99.7 % were susceptible; however, 30.0 % (n = 517) exhibited MICs of 2 µg/ml and 0.3 % (n = 6) demonstrated intermediate susceptibility (MICs of 4 µg/ml). Nearly all isolates (≥ 99.9 %) were susceptible to teicoplanin, linezolid, and daptomycin. The MIC90 values were 2 µg/ml for ceftobiprole and 1 µg/ml for nemonoxacin. The MIC90 values of mupirocin and tyrothricin were 0.12 and 4 µg/ml, respectively. MIC creep was noted for daptomycin during this period, but not for vancomycin, teicoplanin, linezolid, or tigecycline. For isolates with vancomycin MICs of 2 µg/ml, the MIC90 values were 2 µg/ml for teicoplanin, 0.5 µg/ml for daptomycin, and 0.5 µg/ml for tigecycline. Those values were four- to eight-fold higher than those among isolates with vancomycin MICs of 0.5 µg/ml (2, 0.06, and 0.12 µg/ml, respectively). Of the nine MRSA isolates exhibiting non-susceptibility to vancomycin (n = 6), teicoplanin (n = 1), daptomycin (n = 2), or tigecycline (n = 1), all had different pulsotypes, indicating the absence of intra-hospital or inter-hospital spread. The presence of a high proportion of MRSA isolates with elevated MICs (2 µg/ml) and MIC creep of daptomycin might alert clinicians on the therapy for serious MRSA infections in Taiwan.

Clinical outcomes of tigecycline alone or in combination with other antimicrobial agents for the treatment of patients with healthcare-associated multidrug-resistant Acinetobacter baumannii infections.

Tigecycline (TG) has been shown to be active in vitro against Acinetobacter baumannii, although data on the clinical efficacy of TG alone or in combination for the treatment of infections due to multidrug-resistant A. baumannii (MDRAB) remain limited. The purpose of this study was to investigate the clinical outcomes of patients with healthcare-associated infections (HAIs) caused by MDRAB who were treated with imipenem/cilastatin and sulbactam, and TG alone or in combination with other antibiotics. A total of 386 patients with HAIs caused by MDRAB were retrospectively analyzed and grouped into TG and non-TG groups, depending on whether they received TG treatment. Of the 266 patients in the TG group, 108 were treated with TG alone and 158 were treated with TG in combination with ceftazidime, ceftriaxone, piperacillin/tazobactam, or a carbapenem. All 120 patients in the non-TG group were treated with imipenem/cilastatin and sulbactam. The primary outcome measure was 30-day mortality after TG treatment and the secondary outcome was clinical outcome. There were no significant differences in survival rates between the two groups. However, the rate of unfavorable outcome was significantly lower (p < 0.05) among patients in the TG group than among patients in the non-TG group. The most significant predictor of unfavorable outcome was sepsis, whereas TG treatment and microbial eradication were the most significant predictors of favorable outcomes. Our study represents the largest study of patients with MDRAB infection treated with TG and expands our understanding of the role of TG therapy alone or in combination with other agents for the treatment of HAI caused by MDRAB.

Micronucleus scoring in liver fine needle aspiration cytology.

OBJECTIVE: This study evaluated the role of the micronucleus (MN) in liver fine needle aspiration (FNA) cytology. METHODS: Histological features of 75 cases of hepatocellular carcinoma (HCC), of which 25 were well differentiated, 37 moderately differentiated and 13 poorly differentiated, and 58 benign hepatic lesions (total, 133 cases) were correlated with MN expression observed in FNA smears reported as benign (n =40), atypical (n = 14), suspicious (n = 30) and malignant (n =49). RESULTS: Stepwise increases in the MN score (0.4 ± 0.6, 1.2 ± 1.3, 6.3 ± 4.2 and 14.8 ± 8.8) correlated with the degree of cytological abnormality: benign, atypia, suspicious and malignant, respectively. The mean MN scores for well-, moderately and poorly differentiated HCC were 5.4 ± 2.2, 11.5 ± 4.5 and 24.9 ± 9.1, respectively, which was significantly different between malignant and suspicious (P < 0.0001), between suspicious and atypical (P= 0.008) but not between atypical and benign. The MN scores differed significantly between all degrees of differentiation of HCC and between the HCC and benign hepatic lesions (P < 0.0001). High sensitivity, specificity and accuracy of liver FNA for diagnosing HCC (96%, 98%, and 96%, respectively) were obtained at a cutoff of three for the MN score. CONCLUSIONS: The MN score is an effective HCC biomarker and has a good potential use as an ancillary tool for diagnosing HCC using FNA cytology.

Preclinical evaluation of the AKT inhibitor MK-2206 in nasopharyngeal carcinoma cell lines.

Nasopharyngeal carcinoma (NPC) is endemic to Asia and over 40 % of NPC tissues harbor PIK3CA amplifications. This study characterized the preclinical activity of MK-2206, an oral allosteric inhibitor of AKT in 6 NPC cell lines: C666-1, HK1, HONE-1-EBV, HONE-1, CNE-2 and HNE-1. Exposure to increasing concentrations of MK-2206 resulted in over 95 % of growth inhibition in all NPC cell lines with IC50 values in the low micromolar range. Further experiments were performed in 3 representative NPC cell lines: CNE-2 (harbor PIK3CA mutation and most sensitive to MK-2206), C666-1 (carries PIK3CA amplification), and HONE-1-EBV (least sensitive to MK-2206). MK-2206 induced G0/G1 cycle arrest in all 3 cell lines, but could induce apoptosis only in CNE-2 cells. MK-2206 significantly abrogated AKT signaling in all 3 cell lines by inhibiting the activation of AKT and its downstream effectors (FKHR, GSK3ß and BAD). MK-2206 also reduced mTOR signaling by reducing activation of mTOR and its downstream 4E-BP1 and p70S6 kinase. MAPK activation was observed in HONE-1 and C666-1 cells, but not in CNE-2 cells following exposure to MK-2206. The addition of MK-2206 to cisplatin (but not with paclitaxel) has a supra-additive inhibitory effect on growth in vitro. In summary, MK-2206 can inhibit growth and abrogate AKT and mTOR signaling in NPC cell lines. This agent is currently being evaluated in a phase II study in metastatic NPC.

The human cadherin 11 is a pro-apoptotic tumor suppressor modulating cell stemness through Wnt/ß-catenin signaling and silenced in common carcinomas.

Genetic alterations of 16q21-q22, the locus of a 6-cadherin cluster, are frequently involved in multiple tumors, suggesting the presence of critical tumor suppressor genes (TSGs). Using 1 Mb array comparative genomic hybridization (aCGH), we refined a small hemizygous deletion (~1 Mb) at 16q21-22.1, which contains a single gene Cadherin-11 (CDH11, OB-cadherin). CDH11 was broadly expressed in human normal adult and fetal tissues, while its silencing and promoter CpG methylation were frequently detected in tumor cell lines, but not in immortalized normal epithelial cells. Aberrant methylation was also frequently detected in multiple primary tumors. CDH11 silencing could be reversed by pharmacologic or genetic demethylation, indicating an epigenetic mechanism. Ectopic expression of CDH11 strongly suppressed tumorigenecity and induced tumor cell apoptosis. Moreover, CDH11 was found to inhibit Wnt/ß-catenin and AKT/Rho A signaling, as well as actin stress fiber formation, thus further inhibiting tumor cell migration and invasion. CDH11 also inhibited epithelial-to-mesenchymal transition and downregulated stem cell markers. Thus, our work identifies CDH11 as a functional tumor suppressor and an important antagonist of Wnt/ß-catenin and AKT/Rho A signaling, with frequent epigenetic inactivation in common carcinomas.

Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma.

Fibulin-2 (FBLN2) has been identified as a candidate tumor-suppressor gene in nasopharyngeal carcinoma (NPC). Originally identified through a chromosome 3 NotI genomic microarray screen, it shows frequent deletion or methylation in NPC. FBLN2 is located on chromosome 3p25.1 and is associated with tumor development through its important interactions with the extracellular matrix (ECM) proteins. FBLN2 encodes two isoforms. The short isoform (FBLN2S) is expressed abundantly in normal tissues, but is dramatically downregulated in NPC, while the long isoform (FBLN2L) is either not detectable or is expressed only at low levels in both normal and tumor tissues. Reintroduction of this FBLN2S inhibited cell proliferation, migration, invasion and angiogenesis in vitro. Furthermore, in vivo studies in nude mice show its expression is associated with tumor and angiogenesis suppression. FBLN2-associated angiogenesis occurs via concomitant downregulation of vascular endothelial growth factor and matrix metalloproteinase 2. This study provides compelling evidence that FBLN2S has an important tumor-suppressive and anti-angiogenic role in NPC.

Light-activated tissue bonding for excisional wound closure: a split-lesion clinical trial.

BACKGROUND: Apposition of wound edges by sutures provides a temporary scaffold and tension support for healing. We have developed a novel tissue-sealing technology, photoactivated tissue bonding (PTB), which immediately crosslinks proteins between tissue planes, thereby sealing on a molecular scale. OBJECTIVES: To determine the effectiveness of PTB for superficial closure of skin excisions and to compare the results with standard epidermal suturing. METHODS: A split-lesion, paired comparison study of 31 skin excisions was performed. Following deep closure with absorbable sutures, one-half of each wound was superficially closed with nonabsorbable nylon sutures while the other half was stained with Rose Bengal dye and treated with green light. Overall appearance and scar characteristics were rated at 2weeks and 6months in a blinded manner by three dermatologists viewing photographs, by two onsite physicians and by patients. RESULTS: At 2weeks, neither sutured nor PTB-treated segments showed dehiscence; however, PTB-sealed segments showed less erythema than sutured segments as determined by photographic (P=0·001) and onsite evaluations (P=0·005). Overall appearance after PTB was judged better than after sutures (P=0·002). At 6months, scars produced by PTB were deemed superior to scars resulting from sutures in terms of appearance (P<0·001), width (P=0·002) and healing (P=0·003). Patients were more satisfied with the appearance of the PTB-sealed wound half after 2weeks and 6months (P=0·013 and P=0·003, respectively). CONCLUSIONS: A novel molecular suturing technique produces effective wound sealing and less scarring than closure with nylon interrupted epidermal sutures. Comparisons with better suturing techniques are warranted.