Erk Inhibition as a Promising Therapeutic Strategy for High IL-8-Secreting and Low SPTAN1-Expressing Colorectal Cancer.

Tumor recurrence and drug resistance are responsible for poor prognosis in colorectal cancer (CRC). DNA mismatch repair (MMR) deficiency or elevated interleukin-8 (IL-8) levels are characteristics of CRCs, which have been independently correlated with treatment resistance to common therapies. We recently demonstrated significantly impaired therapeutical response and increased IL-8 release of CRC cell lines with reduced expression of MMR protein MLH1 as well as cytoskeletal non-erythrocytic spectrin alpha II (SPTAN1). In the present study, decreased intratumoral MLH1 and SPTAN1 expression in CRCs could be significantly correlated with enhanced serum IL-8. Furthermore, using stably reduced SPTAN1-expressing SW480, SW620 or HT-29 cell lines, the RAS-mediated RAF/MEK/ERK pathway was analyzed. Here, a close connection between low SPTAN1 expression, increased IL-8 secretion, enhanced extracellular-signal-regulated kinase (ERK) phosphorylation and a mesenchymal phenotype were detected. The inhibition of ERK by U0126 led to a significant reduction in IL-8 secretion, and the combination therapy of U0126 with FOLFOX optimizes the response of corresponding cancer cell lines. Therefore, we hypothesize that the combination therapy of FOLFOX and U0126 may have great potential to improve drug efficacy on this subgroup of CRCs, showing decreased MLH1 and SPTAN1 accompanied with high serum IL-8 in affected patients.

Cytotoxic impact of nicotine products on periodontal ligament cells.

OBJECTIVES: The primary objective of this in vitro experiment was an assessment of proliferative capacity, metabolic activity, and potential cellular detriment of human periodontal ligament cells (hPDL) exposed to cigarette smoke (CS), electronic cigarette vapor (eCV), and heated tobacco product aerosol (HTP), or air (control). MATERIALS AND METHODS: Using a CAD/CAM-designed exposition chamber, hPDL were exposed to CS, eCV, HTP, or air (control) based on the Health Canada Intense Smoking Regime. Cell proliferation, metabolic activity, and cellular detriment were assessed at various time points. RESULTS: Compared to the control, hPDL exposed to CS exhibited significantly decreased cell numbers at all time points. HTP exposure led to reduced cell numbers 48 h and 72 h post-exposure, while eCV-exposed cells showed no significant decrease. The metabolic activity of eCV-treated hPDL was slightly reduced at 7 h but recovered at 24 h and 48 h. In contrast, CS-treated cells exhibited significantly decreased metabolic activity at 24 h and 48 h, and HTP-exposed cells showed a significant decrease after 48 h. Flow cytometry indicated both apoptotic and necrotic cell death following CS exposure, with necrotic cell death being more pronounced. CONCLUSIONS: eCV and HTP demonstrated comparatively reduced detrimental effects on hPDL compared to CS. CLINICAL RELEVANCE: The findings suggest that conventional cigarette smoke poses a substantial risk to periodontal health by significantly impairing cell proliferation and metabolic activity. However, alternatives such as eCV and HTP may offer a comparatively reduced risk.

Toxicological Analysis by Assessment of Vascularization and Cell Viability Using the Chicken's Chorioallantoic Membrane (CAM Assay).

The chorioallantoic membrane (CAM) assay is an increasingly popular method using a hen's egg as an experimental organism. Animal models have been established in scientific research for centuries. Yet, awareness of animal welfare in society rises, and the transferability of findings obtained in rodent models to human physiology is challenged. Thus, using fertilized eggs as an alternative platform for animal experimentation might be a promising alternative. The CAM assay is utilized for toxicological analysis by determination of CAM irritation as well as analysis of organ damage and ultimately death of the embryo. Furthermore the CAM provides a micromilieu suited for the implantation of xenografts. Xenogene tissues and tumors grow on the CAM due to a lack of rejection by the immune system and a dense vascular network providing oxygen and nutrients. Multiple analytical methods including in vivo microscopy and various imaging techniques are applicable to this model. Additionally, ethical aspects, a comparatively low financial burden, and low bureaucratic hurdles legitimize the CAM assay.We here describe an in ovo model utilized for xenotransplantation of a human tumor. The model can be used to evaluate the efficacy as well as the toxicity of different therapeutic agents after intravascular injection. Additionally, we present the evaluation of vascularization and viability by intravital microscopy, ultrasonography, and immunohistochemistry.

Determination of the LD50 with the chick embryo chorioallantoic membrane (CAM) assay as a promising alternative in nanotoxicological evaluation.

Toxicity tests in rodents are still considered a controversial topic concerning their ethical justifiability. The chick embryo chorioallantoic membrane (CAM) assay may offer a simple and inexpensive alternative. The CAM assay is easy to perform and has low bureaucratic hurdles. At the same time, the CAM assay allows the application of a broad variety of analytical methods in the field of nanotoxicological research. We evaluated the CAM assay as a methodology for the determination of nanotoxicity. Therefore we calculated the median lethal dose (LD50), performed in vivo microscopy and immunohistochemistry to identify organ-specific accumulation profiles, potential organ damage, and the kinetics of the in vivo circulation of the nanoparticles. Zinc oxide nanoparticles were intravascularly injected on day 10 of the egg development and showed an LD50 of 17.5 µM (1.4 µg/mLeggcontent). In comparison, the LD50 of equivalent amounts of Zn2+ was 4.6 µM (0.6 µg/mLeggcontent). Silica encapsulated ZnO@SiO2 nanoparticles conjugated with fluorescein circulated in the bloodstream for at least 24 h. Particles accumulated mostly in the liver and kidney. In immunohistochemical staining, organ damage was detected only in liver tissue after intravascular injection of zinc oxide nanoparticles in very high concentrations. Zinc oxide nanoparticles showed a different pharmacokinetic profile compared to Zn2+ ions. In conclusion, the CAM assay has proven to be a promising methodology for evaluating nanotoxicity and for the assessment of the in vivo accumulation profiles of nanoparticles. These findings may qualify the methodology for risk assessment of innovative nanotherapeutics in the future.

The Chorioallantoic Membrane Assay in Nanotoxicological Research-An Alternative for In Vivo Experimentation.

Nanomaterials unveil many applicational possibilities for technical and medical purposes, which range from imaging techniques to the use as drug carriers. Prior to any human application, analysis of undesired effects and characterization of their toxicological profile is mandatory. To address this topic, animal models, and rodent models in particular, are most frequently used. However, as the reproducibility and transferability to the human organism of animal experimental data is increasingly questioned and the awareness of animal welfare in society increases at the same time, methodological alternatives are urgently required. The chorioallantoic membrane (CAM) assay is an increasingly popular in ovo experimental organism suitable for replacement of rodent experimentation. In this review, we outline several application fields for the CAM assay in the field of nanotoxicology. Furthermore, analytical methods applicable with this model were evaluated in detail. We further discuss ethical, financial, and bureaucratic aspects and benchmark the assay with other established in vivo models such as rodents.

HSP27 regulates viability and migration of cancer cell lines following irradiation.

Despite improvements of radiotherapy and better outcomes of cancer patients resistances still limit the therapeutic success. The combined treatment of tumors by the use of irradiation as well as targeted therapies is a promising approach. By the use of a proteomic screening of lung and head and neck cancer cell lines we identified the heat shock protein HSP27 as a potential target protein for a combined treatment strategy. Overall expression of HSP27 was distinctly lower in HNSCCUM-02T cells which have a high HSP27 phosphorylation ratio, whereas A549 cells revealed the opposite. Irradiation and inhibition of HSP27 phosphorylation by MKII inhibition resulted in a significantly reduced viability in both cell lines. While irradiation impaired migration only in HNSCCUM-02T cells, MKII inhibition exerted that effect in both cell lines. In contrast, knockdown of HSP27 compromised the viability only in A549 cells. Additionally, MKII inhibition counteracts radiation-induced phosphorylation of HSP27 which causes an additive toxicity and reduced migratory capacity in HNSCCUM-02T when combined. Inhibition of HSP27 expression and phosphorylation in combination with radiotherapy may be an effective treatment option to overcome resistances.

Cigarette smoke modulates binding of the transcription factor MZF1 to the VEGF promoter and regulates VEGF expression in dependence of genetic variation SNP 405.

BACKGROUND: Vascular endothelial growth factor (VEGF) affects carcinogenesis of the upper aerodigestive tract. Cigarette smoke (CSE) influences VEGF-gene regulation. The single nucleotide polymorphism +405 G/C (SNP +405 G/C) and the transcriptional factor (TF) myeloid zinc finger 1 (MZF1) are endogenic regulators of the VEGFpromoter as the polymorphism 405 potentially affects binding of the transcription factor MZF1. Therefore, this in vitro study analysed cancer cells of the upper aerodigestive tract after CSE incubation concerning MZF1-binding specificity and VEGF expression in dependency of VEGF polymorphism +405 G/C compared to wild type (wt). METHODS: In human alveolar epithelial-like type-II cells (A549) and oral squamous cell cancer cells (HNSCCUM-02T) SNP +405 G/C- and MZF1-dependent VEGF promoter activity and VEGF expression were analysed by qRT-PCR and Western blot after incubation with 10% CSE. Temporary knock-down of MZF1 was performed using siRNA. MZF1 binding was analysed by Co-Chromatin-Immunoprecipitation (Co-ChiP) (each test n = 3). RESULTS: We found a stronger MZF1 binding to VEGF polymorphism 405 in A549 cells (P < .05) compared to HNSCCUM-02T cells (P = .02), where MZF1 binding was reduced. MZF1 knock out reduced VEGF promoter activity in HNSCCUM-02T cells, showing the relevance of the factor for transcriptional activation of the VEGF promoter. Finally, we found that CSE increases promoter activity in both cell lines and no significant differences between the two analysed polymorphisms concerning their activating capacity. CONCLUSION: In summary, both VEGF promoter polymorphisms are similar effective in terms of transcriptional activity, and MZF1 is a transcriptional activator of VEGF promoter. Moreover, cigarette smoke increases MZF1 binding of VEGF-promoter and directly affects VEGF-gene regulation.

The impact of cigarette smoke on activity of single nucleotide polymorphisms of the vascular endothelial growth factor-promoter gene in cells of the upper aerodigestive tract.

BACKGROUND: The vascular endothelial growth factor (VEGF) is involved in tumorigenesis of the upper aerodigestive tract. Different single nucleotide polymorphisms (SNPs) turn the regulation of the VEGF gene into a highly complex process, particularly influenced by exogenic factors like cigarette smoke (CSE). Analysis of the SNP- and CSE-dependent VEGF-gene regulation can help to improve antiangiogenic therapies and prognosis. Therefore, the aim of this study was to analyse the influence of CSE on the SNP-dependent regulation of the VEGF-gene in vitro. METHODS: Human alveolar epithelial-like type-II cells (A549) were transfected with different SNPs and incubated with CSE. SNP- and CSE-dependent VEGF-promoter activity and mRNA stability was measured using qRT-PCR and Western blot analysis. RESULTS: Transfection with SNP -460 (ATC) and incubation with 10% CSE resulted in +19% elevated VEGF-promoter activity (P < 0.05). Transfection with SNP -2578/-460 (CTC) and 10% CSE incubation resulted in a 14% reduction of VEGF-promoter activity (P < 0.05). Regarding mRNA stability, transfection with SNP +936 T allele led to half-life of 1.11 hours, which decreased to 0.2 hours after incubation with CSE. In contrast, on protein level SNP +936 T transfection showed a not significant increase up to 176% (P > 0.05), while incubation with CSE led to a significant decrease to 61% (P = 0.002). CONCLUSION: Transcriptional regulation of the VEGF gene by SNP -460 (ATC) in combination with CSE represents a mechanism for elevated VEGF expression and may be associated with a worse prognosis. The influence of +SNP 936 on mRNA stability may be responsible for regulation of VEGF plasma levels.

Nanozymes in Nanofibrous Mats with Haloperoxidase-like Activity To Combat Biofouling.

Electrospun polymer mats are widely used in tissue engineering, wearable electronics, and water purification. However, in many environments, the polymer nanofibers prepared by electrospinning suffer from biofouling during long-term usage, resulting in persistent infections and device damage. Herein, we describe the fabrication of polymer mats with CeO2- x nanorods that can prevent biofouling in an aqueous environment. The embedded CeO2- x nanorods are functional mimics of natural haloperoxidases that catalyze the oxidative bromination of Br- and H2O2 to HOBr. The generated HOBr, a natural signaling molecule, disrupted the bacterial quorum sensing, a critical step in biofilm formation. The polymer fibers provide porous structures with high water wettability, and the embedded cerium oxide nanozymes act as a catalyst that can efficiently trigger oxidative bromination, as shown by a haloperoxidase assay. Additionally, the embedded nanozymes enhance the mechanical property of polymer mats, as shown by a single-fiber bending test using atomic force microscopy. We envision that the fabricated polymer mats with CeO2- x nanorods may be used to provide mechanically robust coatings with antibiofouling properties.

An acute exposure to ozone impairs human olfactory functioning.

INTRODUCTION: Ozone is a ubiquitous and irritant gas. We questioned whether an acute exposure to 0.2 ppm ozone impaired olfactory functioning. METHODS: Healthy, normosmic subjects were exposed according to a parallel group design either to 0.2 ppm ozone (n = 15) or to sham (n = 13) in an exposure chamber for two hours. Possible irritating effects were assessed by questionnaire (range 0-5). The detection threshold of n-butanol was measured with the Sniffin' Sticks test before and after exposure. Olfactory thresholds were logarithmized and a two-way analysis of variance (ANOVA) with repeated measurements was carried out to test the effects of exposure (ozone vs. sham) and time (before vs. after exposure). Additionally, nasal secretions were taken at a preliminary examination and after exposure to determine interleukins 1ß and 8. RESULTS: No irritating effects to the upper airways were observed. In the ozone group, the median score for cough increased from 0 to 2 at the end of exposure (sham group 0 and 0, respectively, p < 0.001). The ANOVA showed a main effect for ozone exposure (F (1, 26) = 27.6, p = 0.0002), indicating higher olfactory thresholds in the ozone group. Concentrations of interleukins in nasal secretions did not increase following ozone exposure. CONCLUSIONS: This study shows a clear impairment of olfactory functioning following an acute exposure to 0.2 ppm ozone.

Knockdown of hnRNPK leads to increased DNA damage after irradiation and reduces survival of tumor cells.

Radiotherapy is an important treatment option in the therapy of multiple tumor entities among them head and neck squamous cell carcinoma (HNSCC). However, the success of radiotherapy is limited by the development of radiation resistances. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a cofactor of p53 and represents a potential target for radio sensitization of tumor cells. In this study, we analyzed the impact of hnRNPK on the DNA damage response after gamma irradiation. By yH2AX foci analysis, we found that hnRNPK knockdown increases DNA damage levels in irradiated cells. Tumor cells bearing a p53 mutation showed increased damage levels and delayed repair. Knockdown of hnRNPK applied simultaneously with irradiation reduced colony-forming ability and survival of tumor cells. Taken together, our data shows that hnRNPK is a relevant modifier of DNA damage repair and tumor cell survival. We therefore recommend further studies to evaluate the potential of hnRNPK as a drug target for improvement of radiotherapy success.

Glycine-functionalized copper(ii) hydroxide nanoparticles with high intrinsic superoxide dismutase activity.

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide (O2-) radicals into molecular oxygen (O2) and H2O2 as a first line of defense against oxidative stress. Here, we show that glycine-functionalized copper(ii) hydroxide nanoparticles (Gly-Cu(OH)2 NPs) are functional SOD mimics, whereas bulk Cu(OH)2 is insoluble in water and catalytically inactive. In contrast, Gly-Cu(OH)2 NPs form water-dispersible mesocrystals with a SOD-like activity that is larger than that of their natural CuZn enzyme counterpart. Based on this finding, we devised an application where Gly-Cu(OH)2 NPs were incorporated into cigarette filters. Cigarette smoke contains high concentrations of toxic reactive oxygen species (ROS, >1016 molecules per puff) including superoxide and reactive nitrogen species which lead to the development of chronic and degenerative diseases via oxidative damage and subsequent cell death. Embedded in cigarette filters Gly-Cu(OH)2 NPs efficiently removed ROS from smoke, thereby protecting lung cancer cell lines from cytotoxic effects. Their stability, ease of production and versatility make them a powerful tool for a wide range of applications in environmental chemistry, biotechnology and medicine.

Multikinase inhibitors sorafenib and sunitinib as radiosensitizers in head and neck cancer cell lines.

BACKGROUND: Radioresistance is a common feature of head and neck squamous cell carcinoma (HNSCC). We previously showed that the irradiation- activated vascular endothelial growth factor (VEGF)-extracellular signal-regulated kinase (ERK)-axis is fundamental for the survival of resistant tumors. In this study, we examined if treatment with potent multikinase (MK) inhibitors, sorafenib and sunitinib, could radiosensitize tumor cells. METHODS: Cultured HNSCC cell lines were treated with inhibitors and subsequently irradiated. Radiosensitizing effects were functionally assessed by annexin-V apoptosis and clonogenic assays and confirmed by Western blot. Additionally, we surveyed human HNSCC tissue microarrays (TMAs) for activated ERK expression. RESULTS: Based on combination indexes, we found that combining irradiation with both inhibitors exerted strong and supra-additive antitumor effects on clonogenic survival. Kinase inhibition enhanced irradiation-induced apoptotic rates and inhibited postradiogenic phospho-ERK-expression. Patients with recurrent HNSCC displayed significantly lower extracellular signal-regulated kinase phosphorylation (pERK) levels than relapse-free patients. CONCLUSION: We propose further evaluation of sorafenib and sunitinib as potential radiosensitizing agents in HNSCC treatment. © 2017 Wiley Periodicals, Inc. Head Neck 39: 623-632, 2017.

Semiquantifiable angiogenesis parameters in association with the malignant transformation of oral leukoplakia.

BACKGROUND: Aim of the study was to assess the role of angiogenesis in the process of malignant transformation of clinical diagnosed oral leucoplakia (OL). MATERIALS AND METHODS: A total of 131 histological preparations [oral leukoplakia/hyperkeratosis without dysplasia (OL; n = 49), oral leukoplakia/hyperkeratosis with mild dysplasia (OL-SIN1; n = 33), with moderate dysplasia (OL-SIN2; n = 13) and leukoplakia-derived oral squamous cell carcinoma (OL-OSCC; n = 36)] were evaluated for microvessel density (MVD), vessel diameter as well as for vascular endothelial growth factor (VEGF-A) expression. Data were compared within the groups. RESULTS: For MVD, there were significant differences between OL and OL-SIN 2/OL-OSCC (P < 0.05) and between OL-SIN 1 and OL-OSCC (P < 0.05). For OL-OSCC, vessel diameters were significantly increased compared with OL (P < 0.05). Expression of VEGF-A increased significantly gradually from OL-SIN 1 to OSCC (each P < 0.05). This was especially evident for lesions of the tongue when compared to the others. CONCLUSION: Angiogenesis increases during the transition from OL through dysplasia to OL-OSCC. In particular, OL-OSCCs of the tongue, VEGF-A expression may be used for estimation of malignant progression of OL.

Downregulation of EGFR in hypoxic, diffusion-limited areas of squamous cell carcinomas of the head and neck.

BACKGROUND: The receptor for the epidermal growth factor (EGFR) is widely considered to be one of the central drivers of oncogenesis in squamous cell carcinomas of the head and neck region (HNSCC). Inhibition of EGFR using monoclonal antibodies is established both in the curative and the palliative setting in this disease. HNSCCs are known to contain abundant hypoxic tissue areas and hypoxia has been shown to be involved in the (down)regulation of EGFR membrane expression. METHODS: A novel method for multiplex immunofluorescence and single-cell segmentation (via DAPI-stained nuclei) was established, to study the expression of EGFR, the endogenous hypoxia marker CA IX, and intratumoural diffusion distances from microvessels (using CD34 staining) in 58 human HNSCCs and 9 normal/cancer adjacent tissues. RESULTS: EGFR was found to be significantly downregulated with increasing distance from tumour microvessels, whereas the opposite was true for CA IX. Larger diffusion-limited areas were correlated with higher expression of CA IX. CONCLUSIONS: The hypoxic tumour microenvironment may have a major role in mediating resistance against anti-EGFR strategies by downregulating EGFR molecules on tumour cells.

Hypoxia-Inducible Factor 2α Mutation-Related Paragangliomas Classify as Discrete Pseudohypoxic Subcluster.

Recently, activating mutations of the hypoxia-inducible factor 2α gene (HIF2A/EPAS1) have been recognized to predispose to multiple paragangliomas (PGLs) and duodenal somatostatinomas associated with polycythemia, and ocular abnormalities. Previously, mutations in the SDHA/B/C/D, SDHAF2, VHL, FH, PHD1, and PHD2 genes have been associated with HIF activation and the development of pseudohypoxic (cluster-1) PGLs. These tumors overlap in terms of tumor location, syndromic presentation, and noradrenergic phenotype to a certain extent. However, they also differ especially by clinical outcome and by presence of other tumors or abnormalities. In the present study, we aimed to establish additional molecular differences between HIF2A and non-HIF2A pseudohypoxic PGLs. RNA expression patterns of HIF2A PGLs (n=6) from 2 patients were compared with normal adrenal medullas (n=8) and other hereditary pseudohypoxic PGLs (VHL: n=13, SDHB: n=15, and SDHD: n=14). Unsupervised hierarchical clustering showed that HIF2A PGLs made up a separate cluster from other pseudohypoxic PGLs. Significance analysis of microarray yielded 875 differentially expressed genes between HIF2A and other pseudohypoxic PGLs after normalization to adrenal medulla (false discovery rate 0.01). Prediction analysis of microarray allowed correct classification of all HIF2A samples based on as little as three genes (TRHDE, LRRC63, IGSF10; error rate: 0.02). Genes with the highest expression difference between normal medulla and HIF2A PGLs were selected for confirmatory quantitative reverse transcriptase polymerase chain reaction. In conclusion, HIF2A PGLs show a characteristic expression signature that separates them from non-HIF2A pseudohypoxic PGLs. Unexpectedly, the most significantly differentially expressed genes have not been previously described as HIF target genes.

Growth differentiation factor 15 as a radiation-induced marker in oral carcinoma increasing radiation resistance.

BACKGROUND: Growth differentiation factor 15 (GDF15) is involved in tumor pathogenesis of oral squamous cell carcinoma (OSCC). The aim of this study was an investigation of the potential influence of GDF15 on radioresistance of OSCC cells in vitro. METHODS: Oral squamous cell carcinoma cell lines were irradiated with 0, 2, or 6 Gy, and GDF15 expression in the supernatant per survived cell colony was examined with ELISA. Non-irradiated and OSCC cell lines irradiated with 6 Gy were evaluated for GDF15 expression using immunofluorescent staining. For further investigation of GDF15 effects on radioresistance, a GDF15 knockdown model in a human OSCC cell line was established, and apoptotic activity after radiation was measured using the Caspase-Glo 3/7 system. RESULTS: ELISA and immunofluorescent staining indicated an increased GDF15 expression in 5 OSCC cell lines compared with human gingival epithelial cells. Irradiation with two and six gray resulted in a significant elevation of GDF15 expression per survived cell colony in the irradiated OSCC cell lines (P < 0.001). Furthermore, a dose-dependent expression of GDF15 was seen. Immunofluorescent staining confirmed an elevated GDF15 expression in irradiated OSCC cell lines (n = 10; P ≤ 0.001). Apoptotic activity was significantly increased after irradiation in the GDF15 knockdown group compared with control cells (n = 24; P < 0.001). CONCLUSION: This study describes for the first time the vital role of GDF15 both in tumorigenesis and in radioresistance of OSCC cells. With its anti-apoptotic effects, GDF15 possibly promotes tumor progression and might protect carcinoma cells against irradiation effects. Consequently, GDF15 may be a promising therapeutic target in oral cancer.

Silica-coated Au@ZnO Janus particles and their stability in epithelial cells.

Multicomponent particles have emerged in recent years as new compartmentalized colloids with two sides of different chemistry or polarity that have opened up a wide field of unique applications in medicine, biochemistry, optics, physics and chemistry. A drawback of particles containing a ZnO hemisphere is their low stability in biological environment due to the amphoteric properties of Zn2+. Therefore we have synthesized monodisperse Au@ZnO Janus particles by seed-mediated nucleation and growth whose ZnO domain was coated selectively with a thin SiO2 layer as a protection from the surrounding environment that imparts stability in aqueous media while the Au domain remained untouched. The thickness of the SiO2 layer could be precisely controlled. The SiO2 coating of the oxide domain allows biomolecule conjugation (e.g. antibodies, proteins) in a single step for converting the photoluminescent and photocatalytic active Janus nanoparticles into multifunctional efficient vehicles for cell targeting. The SiO2-coated functionalized nanoparticles were stable in buffer solutions and other aqueous systems. Biocompatibility and potential biomedical applications of the Au@ZnO@SiO2 Janus particles were assayed by a cell viability analysis by co-incubating the Au@ZnO@SiO2 Janus particles with epithelia cells and compared to those of uncoated ZnO.

Proteomic identification of the heterogeneous nuclear ribonucleoprotein K as irradiation responsive protein related to migration.

Irradiation resistance is a major obstacle of head and neck squamous cell carcinoma (HNSCC) therapy, limiting treatment success and patient survival. The aim of our experiments was to identify irradiation-regulated proteins as potential drug targets. Two established HNSCC cell lines (HNSCCUM-01T and HNSCCUM-02T) were treated with a single 8Gy (Gray) fraction of irradiation. Changes in cellular protein expression were studied after 24h by means of 2D-electrophoresis and MALDI-TOF-mass spectrometry. Ninety-four differentially expressed proteins were identified. The expression levels of four proteins were regulated similarly in both cell lines after irradiation treatment, i.e., GRP78, PRDX, ACTC, and the heterogeneous nuclear ribonucleoprotein K (hnRNPK), suggesting a relevant role during irradiation response. hnRNPK as a p53 interacting protein was verified by Western blotting and immunocytochemical staining as well as functionally analyzed. Knock-down by the use of siRNA resulted in only slightly reduced viability, however, migratory activity was strongly reduced. Combined application of siRNA against hnRNPK and irradiation reduced migration almost completely. We conclude that hnRNPK is potentially implicated in the radiogenic response of HNSCC. The inhibition of hnRNPK might reduce the metastasizing potential of HNSCC especially in combination with irradiation and suggest that this molecule should be further evaluated in this context. BIOLOGICAL SIGNIFICANCE: We showed completely impaired migration of irradiated hnRNPK-knock-out HNSCC cells, suggesting this molecule as a potential drug target in combined treatment schedules.

The effect of extracellular matrix proteins on the cellular response of HUVECS and HOBS after covalent immobilization onto titanium.

Biomimetic surface modifications are regarded as promising approach to stimulate cellular behavior at the interface of implant materials. Aim of the study was an evaluation of the cellular response of human umbilical cord cells (HUVECS) and human osteoblasts (HOBS) on titanium covalently coated with the extracellular matrix (ECM) proteins fibrinogen, collagen, laminin, and osteopontin. For the surface modification, titanium discs were first amino-functionalized by plasma polymerization of allylamine. The ECM protein conjugation was performed using the linker molecule α, ω-bis-N-hydroxysuccinimide polyethylene glycol (Di-NHS linker). For surface characterization, infrared spectroscopy and fluorescein isothiocyanate staining (FITC) were used to evaluate the presence and distribution of primary amines in the plasma polymer film. Real-time analyses of the respective protein conjugation processes were performed via surface plasmon resonance kinetic measurements. All ECM proteins were immobilized successfully. Furthermore, the biological functionality of the conjugated factors fibronectin and collagen could be proven as they led to a distinct stimulation of cell adhesion of HUVECS and HOBS when compared to the control group. The highest cell coverage of HUVECS was observed on fibronectin-modified surfaces with approximately 35% and on collagen with 33% after 24 h (PT: 9.4%). For laminin, no additional effect was observed, and for osteopontin, only a slight enhancement of cell adhesion was found. A similar, cell-stimulating tendency of fibronectin and collagen was seen as well after 3 and 7 days. Biomimetic surface modification via plasma polymerization is a powerful method for biomolecule conjugation with a high retention of biological functionality and offer promising clinical perspectives.