Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

Surgical rescue for persistent head and neck cancer after first-line treatment.

PURPOSE: Surgical rescue is a treatment option for persistent disease after first-line treatment treatment of head and neck cancer (HNC). METHODS: Patients with persistent HNC treated with rescue surgery between 2008 and 2016 were included. Patients who received a rescue neck dissection (ND only) and who received primary site surgery ± ND were analysed separately (primary site surgery ± ND). RESULTS: During the observation period, 35 patients received ND only and 17 primary site surgery ± ND. No perioperative mortality was observed. In nine patients with ND only and 12 patients with primary site surgery ± ND at least one complication was encountered. 41/52 (79%) patients had a complete response. Median overall survival of patients receiving rescue surgery was 56 months (95% CI 44-69 months). Median overall survival was best for patients with initial laryngeal and oropharyngeal cancer and worst for patients with hypopharyngeal cancer (p = 0.02). Functional deficits following rescue surgery were mainly observed in the domains speech, nutrition, and shoulder/arm mobility. The risk of functional impairment was higher for patients with rescue surgery at the primary tumor site (OR 2.5 ± 2; p = 0.07). CONCLUSION: Rescue surgery offers patients with resectable, persistent disease a realistic chance to achieve long-term survival. Especially patients with laryngeal and oropharyngeal cancer profited from rescue surgery. Rescue neck dissection is an effective and safe procedure. Patients with rescue surgery at the primary tumor site ± ND should expect complications and permanent functional impairment.

New treatment strategies for HPV-positive head and neck cancer.

Epidemiological studies show an increasing incidence of human papilloma virus-associated oropharyngeal cancer. HPV-positive head and neck squamous cell carcinoma (HNSCC) is recognized as a special subgroup of HNSCC. Because HPV-positive patients are often younger and have an outstanding prognosis, long-term toxicities of therapy have become an important issue. Current clinical trials focus on a reduction of treatment-related toxicity and the development of HPV-specific therapies. New treatment strategies include a dose reduction of radiotherapy, the use of cetuximab instead of cisplatin for chemoradiation and transoral robotic surgery (TORS). Increasing comprehension of the molecular background of HPV-associated HNSCC has also lead to more specific treatment attempts including immunotherapeutic strategies. Whereas recently published data shed light on immune mechanisms resulting in a tolerogenic niche for HPV and HPV-associated HNSCC, other studies focus on specific vaccination of HPV-positive HNSCC. This study will summarize current therapy approaches and illustrate ongoing clinical trials in the field of HPV-positive HNSCC.

The neuropeptide galanin is a novel inhibitor of human hair growth.

BACKGROUND: Galanin is a trophic factor of the central and peripheral nervous system that shows widespread distribution in human skin. However, the exact localization and the role of galanin in the hair follicle (HF) remain to be clarified. OBJECTIVES: To characterize galanin expression in human scalp HFs and to examine the effects of galanin on normal human scalp HF growth in organ culture. METHODS: Immunohistochemistry was performed on cryosections of human female scalp skin. Anagen HFs were microdissected and cultured up to 9 days and treated with 100 nmol L(-1) galanin. Staining for Ki-67, TUNEL and Masson-Fontana were used to analyse proliferation, apoptosis and hair cycle staging of the HFs. Functional effects of galanin were tested in serum-free HF organ culture. RESULTS: Galanin-like immunoreactivity was detected in the outer root sheath (ORS) and inner root sheath. Additionally, galanin mRNA was detected in ORS keratinocytes and all HF samples tested. Galanin receptor transcripts (GalR2, GalR3) were also detected in selected samples. Galanin reduced proliferation of hair matrix keratinocytes in situ compared with vehicle-treated controls, shortened the hair growth phase (anagen) in vitro and reduced hair shaft elongation. This was accompanied by the premature development of a catagen-like morphology of galanin-treated HFs. CONCLUSIONS: We present the first evidence that human HFs are both a source and a functionally relevant target of galanin. Due to its hair growth-inhibitory properties in vitro, galanin application deserves further exploration as a potential new treatment strategy for unwanted hair growth (hirsutism, hypertrichosis).

[New aspects of HPV-positive head and neck cancer. Highlights from the 2011 ASCO Congress]. / Neue Aspekte HPV-positiver Kopf-Hals-Karzinome. Highlights vom ASCO-Kongress 2011.

HPV-positive head and neck carcinoma is significantly different than tobacco- and alcohol-induced cancer. Between 30% and 50% of oropharyngeal cancers are associated with human papillomavirus (HPV). Studies still show an increasing incidence. HPV-positive head and neck cancer patients have a better prognosis due to a better response to therapy. Especially patients with gene overexpression of immunological proteins in the antigen presentation are suggested to benefit from radiotherapy. A current retrospective study shows better outcomes for patients treated with radiotherapy in combination with biological targets compared to radiochemotherapy.

Postoperative course and clinical significance of biochemical blood tests following hepatic resection.

BACKGROUND: Hepatic resection continues to be associated with substantial morbidity. Although biochemical tests are important for the early diagnosis of complications, there is limited information on their postoperative changes in relation to outcome in patients with surgery-related morbidity. METHODS: A total of 835 consecutive patients underwent hepatic resection between January 2002 and January 2008. Biochemical blood tests were assessed before, and 1, 3, 5 and 7 days after surgery. Analyses were stratified according to the extent of resection (3 or fewer versus more than 3 segments). RESULTS: A total of 451 patients (54·0 per cent) underwent resection of three or fewer anatomical segments; resection of more than three segments was performed in 384 (46·0 per cent). Surgery-related morbidity was documented in 258 patients (30·9 per cent) and occurred more frequently in patients who had a major resection (P = 0·001). Serum bilirubin and international normalized ratio as measures of serial hepatic function differed significantly depending on the extent of resection. Furthermore, they were significantly affected in patients with complications, irrespective of the extent of resection. The extent of resection had, however, little impact on renal function and haemoglobin levels. Surgery-related morbidity caused an increase in C-reactive protein levels only after a minor resection. CONCLUSION: Biochemical data may help to recognize surgery-related complications early during the postoperative course, and serve as the basis for the definition of complications after hepatic resection.

Slug Is A Surrogate Marker of Epithelial to Mesenchymal Transition (EMT) in Head and Neck Cancer.

BACKGROUND: Epithelial to mesenchymal transition (EMT) promotes therapy resistance in head and neck cancer (HNC) cells. In this study, EMT was quantified in HNC tumor samples by the cellular co-localization of cytokeratin/vimentin, E­cadherin/ß­catenin and by Slug expression. METHODS: Tissue samples from HNC patients were stained with antibody pairs against cytokeratin/vimentin and E-cadherin/ß-catenin. Epithelial-mesenchymal co-localization was quantified using immunofluorescence multichannel image cytometry. Double positivity was confirmed using confocal microscopy. Slug was semi-quantified by 2 specialists and quantified by bright field image cytometry. RESULTS: Tumor samples of 102 patients were investigated. A loss of E-cadherin positive cells (56.9 ± 2.6% vs. 97.9 ± 1.0%; p < 0.0001) and E-cadherin/ß-catenin double positive cells (15.4 ± 5.7% vs. 85.4 ± 1.2%; p < 0.0001) was observed in tumor samples. The percentage of Slug positive cells was increased in tumor samples (12.1 ± 3.6% vs. 3.2 ± 2.6%; p = 0.001). Ordinal Slug scores judged by two specialists closely correlated with percentage of Slug-positive cells (Spearman's rho = 0.81; p < 0.001). Slug score correlated negatively with the percentage of E-cadherin positive cells (r = 0.4; p = 0.006), the percentage of E-cadherin/ß-catenin positive cells (r = 0.5; p = 0.001) and positively with cytokeratin/vimentin positive cells (r = 0.4, p = 0.003). CONCLUSION: EMT can be assessed in HNC tumor probes by cytokeratin/vimentin co-expression and loss of E-cadherin/ß-catenin co-expression. Slug score provides a convenient surrogate marker for EMT.

Lack of complex I is associated with oncocytic thyroid tumours.

Oncocytic tumours are characterised by hyperproliferation of mitochondria. We immunohistochemically analysed all enzymes of the oxidative phosphorylation system in 19 oncocytic thyroid tumours. A specific lack of complex I was detected, which was expressed at <5% of the level determined in surrounding non-cancerous tissue.

Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond.

Epithelial mesenchymal crosstalk (EMC) describes the interaction of the tumor stroma and associated fibroblasts with epithelial cancer cells. In this study we analysed the effects of EMC on head and neck cancer cells. In tumor cell lines EMC was induced using media conditioned from a mix-culture of cancer cells and fibroblasts. Cell proliferation and chemotherapy response were assessed using direct cell counting. Flow cytometry, immunohistochemistry of markers of epithelial-mesenchymal transition (EMT) and subsequent TissueFaxs™ acquisition and quantification and western blot analysis were performed. Holotomographic microscopy imaging was used to visualize the effects of EMC on Cisplatin response of SCC-25 cells. EMC induced a hybrid epithelial-mesenchymal phenotype in SCC-25 cells with co-expression of vimentin and cytokeratin. This hybrid phenotype was associated with chemotherapy resistance and increased proliferation of the cells. The EMC conditioned medium led to an activation of the IL-6/STAT3 pathway with subsequent phosphorylation of STAT3. EMC induced a hybrid epithelial-mesenchymal phenotype in HNSCC cells accompanied by increased therapy resistance and cell proliferation. The IL-6/STAT3 pathway might be one of the major pathways involved in these EMC-related effects.

Galanin modulates human and murine neutrophil activation in vitro.

AIMS: Polymorphonuclear neutrophils are key players in innate immunity. The innate immune system needs to be tightly controlled to ensure proper activation but also no overactivation. Galanin has been shown to regulate inflammatory reactions, and therefore, we aimed to elucidate the expression of galanin and its three receptors (GAL1 -GAL3 ) in polymorphonuclear neutrophils and to evaluate whether galanin exerts direct or indirect effects on human and murine polymorphonuclear neutrophils. METHODS: Human peripheral polymorphonuclear neutrophils were isolated from fresh blood of healthy donors, and murine polymorphonuclear neutrophils were isolated from bone marrow of C57BL/6N mice. Gene expression was evaluated by qRT-PCR. As a marker for polymorphonuclear neutrophil activation, CD11b integrin surface expression was measured by FACS analysis. Furthermore, a label-free technology measuring ligand-induced dynamic mass redistribution was used to evaluate the response of polymorphonuclear neutrophils to galanin. RESULTS: GAL2 receptor expression was found in both human and murine polymorphonuclear neutrophils, galanin and GAL3 receptor were exclusively expressed in murine bone marrow polymorphonuclear neutrophils, and GAL1 receptor was not detectable in polymorphonuclear neutrophils of either species. Galanin treatment was not able to induce CD11b integrin surface expression or dynamic mass redistribution in human polymorphonuclear neutrophils and murine bone marrow polymorphonuclear neutrophils. However, galanin treatment significantly enhanced the response of polymorphonuclear neutrophils of both species to interleukin-8. CONCLUSION: Galanin can be regarded as an immunomodulatory peptide as it can sensitize polymorphonuclear neutrophils towards pro-inflammatory cytokines in humans and mice.

Molecular cloning and characterisation of the mouse preprogalanin gene.

Using a probe obtained by PCR amplification from mouse genomic DNA, a genomic clone was isolated covering the entire mouse preprogalanin gene. The mouse gene has an exon:intron organisation very similar to that of the rat and human genes. The first exon is noncoding while exons 2-5 carry the coding region. Exon 6 also encodes the stop codon and a polyadenylation signal. The deduced amino-acid sequence of mouse preprogalanin is 94% and 68% identical to the rat and human peptide, respectively. The amino-acid sequence of mouse galanin was confirmed by RT-PCR amplification of mouse brain RNA. The cloning of the mouse galanin gene should allow elucidation of the regulatory characteristics of its promoter and facilitate transgenic approaches to the analysis of galanin gene function in this species.

On the biological role of the nuclear polymerizing NAD+: protein(ADP-ribosyl) transferase (ADPRT): ADPRT from Dictyostelium discoideum and inactivation of the ADPRT gene in the mouse.

Two approaches have been used to elucidate the role of the nuclear polymerizing NAD+:protein(ADP-ribosyl)-transferase (ADPRT): i) comparison of the primary structure of Dictyostelium discoideum ADPRT derived from a 2 kb, partial cDNA sequence with the mammalian, fish, amphibian and insect counterparts revealed an overall homology of 25%. Whereas the automodification domain was not conserved at all, the NAD+ binding domain (aa 859-908) showed more than 70% identical amino acids in all species. Together with the similar enzymatic properties of the ADPRTs the genetic conservation underlined the notion that ADPRT plays a major role in various cellular processes; and ii) inactivation of the ADPRT gene in murine embryonic stem cells by homologous recombination led to mouse strains with a complete lack of nuclear poly(ADP-ribosyl)ation. These ADPRT mutant mice were viable and fertile indicating that ADPRT is dispensable in mouse development. Moreover, repair of UV and MNNG induced DNA damage was not affected in ADPRT/3T3 like fibroblasts, as measured by reactivation of in vitro damaged reporter plasmids and unscheduled DNA synthesis. However, about 30% of the ADPRT mutant mice developed pathological skin aberrations on a mixed 129/Sv x C57B1/6 genetic background. These mice will be extremely useful to define the precise biological role of poly(ADP-ribosyl)ation.

Characterization of the 5'-flanking region of the human preprogalanin gene.

Expression of the human galanin gene was analysed using a 3.5-kb DNA fragment comprising the 5'-flanking sequence of the gene. This sequence contains a TATA box (ATATATA) preceded by numerous potential binding sites for transcription factors such as SP1, AP2, and NF kappa B. Three half-palindromic estrogen response elements (EREs, GGTCA) are also found at positions -1,162, -361, and -122 bp relative to the transcription start site. To localize functionally important portions of the promoter region, several shorter fragments of the galanin 5'-flanking region were placed upstream from the chloramphenicol acetyltransferase (CAT) reporter gene. In transient transfection assays, all constructs demonstrated substantial transcriptional activity in both rat glioma/mouse neuroblastoma hybrid cells (NG108-15) and Chinese hamster ovary (CHO-K1) cells. Comparison of the basal expression levels of the different constructs suggests the presence of a negative modulator between positions -1,891 and -207. When cotransfected into NG108-15 cells with the human estrogen receptor cDNA, estrogen did not induce transcription of the human galanin gene at physiological levels of estrogen receptor, although transcription was induced up to 30-fold in the presence of high levels of receptor.

Simplified PCR set-up using a frozen preformulated mix for the detection of cytomegalovirus.

A simple PCR set-up for the detection of cytomegalovirus in clinical specimen was developed. All components of the PCR master mix including Taq DNA polymerase, uracil N-glycosilase, and primers were preformulated and stored frozen in aliquots. After thawing the master mix aliquots, the PCR was immediately started after the addition of sample DNA. This method gave excellent reproducible PCR-results without loss of enzyme activities following storage at -20 degrees C for at least 4 months.

Poor storage and handling of tissue mimics mitochondrial DNA depletion.

Analysis of the mitochondrial DNA (mtDNA) is an important part in the diagnosis of mitochondrial disorders. Besides point mutations and deletions in the mitochondrial genome a reduction in the amount of mtDNA molecules (mtDNA depletion) can also be the reason for mitochondrial defects. The DNA stability in clinical samples is essential for proper performance and interpretation of DNA based diagnosis. The stability of mtDNA was compared with that of nuclear DNA under poor handling and storage conditions. Fresh and thawed muscle tissue specimens were kept at different temperatures for a certain period of time before DNA isolation. Quantitative Southern blot analysis revealed a time-dependent decrease in the amount of mtDNA compared with nuclear DNA in thawed tissue specimens. Therefore, the current study demonstrates that proper specimen storage is a critical issue in quantitative mtDNA analysis and that poor handling and storage of tissue may mimic a severe mtDNA depletion.

Brain-stem involvement in multiple sclerosis: a comparison between brain-stem auditory evoked potentials and the acoustic stapedius reflex.

Brain-stem auditory evoked potentials (BAEPs) and the acoustic stapedius reflex (ASR) were recorded in 68 patients with definite, probable and possible multiple sclerosis (using the definitions of McAlpine). The high incidence of abnormal results, 68% and 60%, respectively, pointed to the diagnostic value of these two measures in detecting brain-stem dysfunction. Combination of the methods increased the diagnostic yield to 85%. Since in part the same brain-stem generator sites underlie BAEPs and the ASR, it was considered that a study of their correlation might serve to increase the reliability and validity of these techniques. There was 71% agreement overall between results from the two measures. Furthermore, 72% of the joint BAEP and ASR abnormalities corresponded in detection of the brain-stem lesion site. It was concluded that the combined approach may supply powerful, complementary information on brain-stem dysfunction, which may aid in establishing the diagnosis of multiple sclerosis.

Biphasic response to human galanin of extracellular acidification in human Bowes melanoma cells.

The metabolic response of galanin GAL1 receptor subtype, endogenously expressed in human Bowes melanoma (HBM) cells, was investigated. Cytosensor microphysiometry was used to determine the extracellular acidification rate. A biphasic response, consisting of a rapid increase in the extracellular acidification rate followed by a decrease below the basal level, was observed after perfusion with human galanin. The magnitude and the rate of onset of both phases were dependent on the galanin concentration. The increase in the extracellular acidification rate (maximum of 25% of basal level; -log(EC(50))=7.23+/-0.14) was transient, whereas the following decrease (maximum of 40% of basal level; -log(EC(50))=7.77+/-0.23) was sustained. The EC(50) values for the increase and decrease were in a similar range. After consecutive galanin administration, the magnitude of the response was the same as for the unexposed cells, indicating the absence of galanin receptor desensitization or internalization in HBM cells. Responses were blocked by pretreatment with pertussis toxin and phorbol-12-myristate-13-acetate (PMA), indicating a G-protein/protein kinase C signalling pathway. Our microphysiometry results show a biphasic response of the extracellular acidification rate mediated by the galanin receptor expressed in HBM cells which has not been described previously for any other endogenously expressed neuropeptide receptor.

Compatibility study between acetylcysteine and some commonly used tablet excipients.

Differential scanning calorimetry (DSC), Fourier transform infra-red spectroscopy (FT-IR), HPLC and TLC were used to investigate the interactions between the mucolytic drug acetylcysteine and a number of commonly used tablet and capsule excipients. Acetylcysteine was found to be compatible with microcrystalline cellulose (Avicel PH 101), sodium carboxymethylcellulose, amorphous silicon dioxide (Aerosil), PVP, cross-linked PVP (Polyplasdone XL), corn starch, saccharose and magnesium stearate. Acetylcysteine thermal stability (onset degradation temperature) was decreased in mixtures with corn starch, magnesium stearate, saccharose and lactose. Interactions of acetylcysteine with lactose, PEG 4000 and 6000, glycine, adipic acid and saccharin sodium were found using DSC and studied in detail with FT-IR, HPLC and TLC. The results suggest that acetylcysteine in mixtures with PEG 4000, glycine or saccharin sodium is degraded during storage at conditions of high temperature and humidity.

Contingent negative variation (CNV) differences between cerebrovascular patients with and without dementia.

This study investigated the clinical usefulness of the contingent negative variation (CNV) as a neurophysiological index of cognitive dysfunction associated with cerebrovascular disease. A total of 31 patients ranging in age from 45 to 88 years with the diagnosis of a stroke were included. Nineteen patients were classified as demented according to clinical assessment and the Mental Deterioration Index (MDI) based on the WAIS, the other 12 patients as non-demented. In a discriminative CNV-paradigm two different tones served as warning stimuli for a flash of light presented 1.5 s later that could be turned off by a press on a button only after the relevant tone. According to our hypothesis, CNV amplitudes of demented patients were significantly smaller than those of the non-demented group. Moreover, a significant relationship was observed between severity of dementia (MDI) and degree of amplitude reduction. Thus, it was concluded that the CNV method could make an important clinical contribution to the assessment and course of dementia.