Andreas Neher

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation‐resistant prostate cancer cell lines, LNCaP‐IRR, PC3‐IRR, and Du145‐IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP‐IRR, PC3‐IRR, and Du145‐IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras‐mitogen‐activated protein kinase (MAPK) and phosphatidyl inositol 3‐kinase (PI3K)‐Akt and Jak‐STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2‐D DIGE followed by computational image analysis and MS. Twenty‐seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse

PURPOSE Radiation therapy cures malignant tumors of the head and neck region more effectively when it is combined with application of the anti-EGFR monoclonal antibody cetuximab. Despite the successes achieved, we still do not know how to select patients who will respond to this combination of anti-EGFR monoclonal antibody and radiation. This study was conducted to elucidate possible mechanisms which cause the combined treatment with cetuximab and irradiation to fail in some cases of squamous cell carcinomas. METHODS AND MATERIALS Mice bearing FaDu and A431 squamous cell carcinoma xenograft tumors were treated with cetuximab (total dose 3 mg, intraperitoneally), irradiation (10 Gy) or their combination at the same doses. Treatment was applied when tumors reached 8mm in size. To collect samples for further protein analysis (two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry MALDI-TOF/TOF, Western blot analysis, and ELISA), mice from each group were sacrificed on the 8th day after the first injection of cetuximab. Other mice were subjected to tumor growth delay assay. RESULTS In FaDu xenografts, treatment with cetuximab alone was nearly as effective as cetuximab combined with ionizing radiation, whereas A431 tumors responded to the combined treatment with significantly enhanced delay in tumor growth. Tumors extracted from the untreated FaDu and A431 xenografts were analysed for protein expression, and 34 proteins that were differently expressed in the two tumor types were identified. The majority of these proteins are closely related to intratumoral angiogenesis, cell adhesion, motility, differentiation, epithelial-to-mesenchymal transition (EMT), c-myc signaling and DNA repair. CONCLUSIONS The failure of cetuximab to enhance radiation response in FaDu xenografts was associated with the initiation of the program of EMT and with c-myc up-regulation in the carcinoma cells. For this reason, c-myc and EMT-related proteins (E-cadherin, vimentin) may be considered as potential biomarkers to predict squamous cell carcinoma response after treatment with cetuximab in combination with radiation.

Acute Otitis Externa: Efficacy and Tolerability of N-Chlorotaurine, a Novel Endogenous Antiseptic Agent†

Objective: The studys objective was to test the tolerability and efficacy of the endogenous antiseptic N‐chlorotaurine (NCT) in comparison with a standard clinical treatment according to a phase IIb clinical trial protocol.

[Otitis externa: etiology, diagnostic and therapy].

Otitis externa (OE) occurs during the lifetime in approximately 10% of the population, especially in warm and damp climates or from swimming (swimmers ear). Females are most often affected around the age of 50 years and males around 70 years of age. Both auditory canals are affected in approximately 10% of cases. Causes of infection are mostly bacteria, and more rarely fungi or viruses. OE can be accompanied not only by relatively slight pain and light swelling of the skin of the auditory canal, but also by severe pain, complete obstruction of the external meatus and retroauricular swelling. An uncomplicated infection can normally be treated by cleaning of the meatus by an ENT specialist and local application of a broad-spectrum antibiotic or an antiseptic supplemented with corticoids as well as antimycotics. Complicated infections, such as necrotizing OE, are rare and normally only occur in elderly patients with diabetes mellitus or in an immunosuppressed condition.

In vitro study on the influence of N-chlorotaurine on the ciliary beat frequency of nasal mucosa.

Background The aim of this study was to investigate the in vitro effects of N-chlorotaurine (NCT), a new endogenous antimicrobial agent, on the nasal ciliary beat frequency (CBF) in nasal mucosa samples of 10 adult patients who underwent conchotomy. Methods CBF was measured by a photometric technique, combining a light microscope, a photometer, a photograph multiplier, and a computed analyzing unit. Results CBF decreased ∼10% after 20 minutes of incubation in aqueous 1% NCT solution compared with 3% in 0.9% saline, and 0.1 and 0.01% NCT had no effect. However, a solution of 7% cocaine, which is used routinely for shrinking the nasal mucosa before rhinoscopy lowered the CBF to 50% after the same incubation time. Conclusion These results confirm the very low toxic potential of NCT, and they are fundamental for clinical investigations regarding its topical application in the nasal and paranasal sinuses.

Influence of essential and fatty oils on ciliary beat frequency of human nasal epithelial cells.

Background In alternative and complementary medicine, the use of essential and fatty oils has become more and more popular. In addition to conventional medical therapies, self-medication is showing increasing popularity, using agents with unclear compounds and poorly controlled dosages. Among other disorders, these alternative treatments are used in bronchitis and rhinitis, including some topical applications. Thus, the influence on ciliated epithelia should be evaluated, because a disturbance of the ciliary function can lead to recurrent sinusitis and chronic rhinosinusitis. The aim of this study was to test the influence of fatty and essential oils on the ciliary beat frequency (CBF) of nasal mucosa in vivo. Methods The influence of sesame oil, soy oil, peanut oil, Miglyol 840, thyme oil, lavender oil, eucalyptus oil, and menthol on the ciliary activity of nasal brushings was evaluated by digital high-speed imaging. Results The presence of most fatty oils resulted in an increase in CBF, the effect being highest for peanut oil. Miglyol 840 had no significant influence on CBF. The essential oils were tested at a concentration of 0.2 and 2%. Thyme oil did not affect CBF, whereas the presence of all other essentials oils resulted in an increase in CBF; the effect was higher at 0.2% than at 2%. Conclusion Except thyme oil and Miglyol 840, all tested oils caused an increase in CBF. Interestingly, the 0.2% concentrations of essential oils resulted in stronger effects when compared with the 2% concentrations.

Tolerability of N-Chlorotaurine in the Guinea Pig Middle Ear: A Pilot Study Using an Improved Application System

The tissue tolerance of N-chlorotaurine (NCT), a mild endogenous antimicrobial oxidant, has been investigated by application to the guinea pig middle ear. The animals were implanted with a novel cannula system that allows chronic external drug delivery to the round window niche. In the first part of the study, 3 animals each received 100 μL of 0.1% NCT (5.5 mmol/L) and 1% NCT, respectively, in aqueous solution twice daily for 8 days. In the second part, NCT was dissolved in phosphate-buffered saline solution to 300 milliosmolar (isotonic), and 27 μL was injected in 3 additional animals twice daily for 7 days. The guinea pigs injected with 100 μL of NCT developed immediate dizziness and nystagmus and did not thrive. Other reactions included mucosal thickening in the middle ear, rupture of the tympanic membrane, and blood and gelatinous material in the cochlea accompanied by hair cell loss and a 10- to 90-dB elevation of the hearing threshold as determined by auditory brain stem responses. The effects seemed to be dose-dependent, but the rate of variability was high across animals. In contrast, the guinea pigs treated with 27 μL of isotonic NCT showed no signs of discomfort, no or only moderate thickening of the middle ear mucosa, no shift of the hearing threshold, and no hair cell loss. Positive control animals injected with 10% neomycin sulfate developed extensive hair cell loss. Provided that the membranes of the inner ear are intact and that low single-dose volumes are used to avoid increased middle ear pressure, isotonic NCT seems to be well tolerated in the tympanic cavity. The new drug delivery system proved to be advantageous for ototoxicity studies.

Antimicrobial Activity of Dexamethasone and Its Combination With N-Chlorotaurine

OBJECTIVE To investigate the antimicrobial effect of dexamethasone phosphate, the endogenous antiseptic N-chlorotaurine (NCT), and their combination on ear, nose, and throat microorganisms. DESIGN In vitro study. SUBJECTS Strains of Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus milleri, Aspergillus flavus, and Aspergillus fumigatus. INTERVENTIONS Bacterial and fungal strains were cultured with 0.1% dexamethasone with and without a low (0.1%) or high (1%) concentration of NCT. The killing effects of dexamethasone, NCT, and the combination were monitored. RESULTS Dexamethasone killed S. milleri and A. flavus after incubation times of 24 to 48 hours. The low concentration of NCT caused a 90% reduction of S. aureus and P. aeruginosa within 30 minutes and 99.9% reduction within 50 minutes. The high concentration of NCT reduced viable counts of S. aureus and P. aeruginosa to the detection limit within 10 minutes. The low-concentration combination (0.1% dexamethasone and 0.1% NCT) showed significant (P < .01) synergistic killing of S. aureus with 2- to 3-fold shorter killing times. The high-concentration combination (0.1% dexamethasone and 1% NCT) demonstrated more rapid killing than NCT alone in both S. aureus and P. aeruginosa. CONCLUSIONS With short and intermediate exposure times, the combination of dexamethasone and NCT showed significantly stronger antimicrobial effects than treatment with NCT alone. Significant killing of S. milleri, A. flavus, and A. fumigatus was observed after extended exposure to dexamethasone. The combined application of dexamethasone and NCT might be a promising therapeutic option, producing high efficacy with low side effects.

Antitumor activity of CTFB, a novel anticancer agent, is associated with the down-regulation of nuclear factor-κB expression and proteasome activation in head and neck squamous carcinoma cell lines

This study aimed to characterize the antitumor activity of 5-Chloro-N-{2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl}-2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 μmol/L with 17.16 ± 2.08%, 10.92 ± 1.22%, and 27.03 ± 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G0-G1 phase that was associated with the up-regulation of p21/WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-κB (NF-κB) and NF-κB p65 phosphorylated at Ser536. The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-κB is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer. [Mol Cancer Ther 2007;6(6):1898–908]

Influence of amphotericin B on the ciliary beat frequency of nasal mucosa.

Objective: A new therapeutic approach in eosinophilic fungal rhinosinusitis is topical application of antifungals such as amphotericin B. The tolerability of a recently applied concentration of this drug by human nasal mucosa was tested in vitro.