Detectability of simulated apical lesions on mandibular premolars and molars between radiographic intraoral and cone-beam computed tomography images: an ex vivo study.

Adequate endodontic diagnostic is essential when making a therapy decision. Radiographic imagining acquisition methods (IAMs) are fundamental apical lesions of endodontic (ALE) origin diagnose tool. Thus, the aim of this research was to compare the simulated apical lesions (SALs) diagnose potential of digital intraoral radiography (DIR) and cone-beam computed tomography (CBCT), if there is a relationship between the IAMs, SALs-depth and their correct diagnose likelihood in human mandibular specimens' datasets. 1024 SALs were prepared in cancellous and cortical bone with different penetration depths. The SALs-stages were radiographed with CBCT and DIR. The IAMs were randomly evaluated by 16 observers in two trials. Possible SAL findings were analyzed according to a five-point scale. The null hypothesis established that SALs detection accuracy does not differ between CBCT and DIR. Significantly differences (first 0.935 and second trial 0.960) were found for the CBCT area under the curve when compared with the DIR (first 0.859 and second trial 0.862) findings. SALs of smaller size were earlier detected by CBCT. In SALs without cortical involvement the probability of detection increased from 90 to 100%. The SALs-depth had the highest detectability influence on cancellous bone lesions and CBCT SALs detectability was 84.9% higher than with DIR images. The CBCT diagnose reproducibility was higher than the one of DIR (Kappa CBCT 75.7-81.4%; DIR 53.4-57.1%). Our results showed that CBCT has a higher SALs IAM diagnosing accuracy and that SALs detection accuracy incremented as the SALs-size increased.

Receptor tyrosine kinase MET as potential target of multi-kinase inhibitor and radiosensitizer sorafenib in HNSCC.

BACKGROUND: The multi-kinase inhibitor sorafenib displays antitumoral effects in head and neck squamous cell carcinoma (HNSCC); however, the targeted kinases are unknown. Here we aimed to identify those kinases to determine the mechanism of sorafenib-mediated effects and establish candidate biomarkers for patient stratification. METHODS: The effects of sorafenib and MET inhibitors crizotinib and SU11274 were analyzed using a slide-based antibody array, Western blotting, proliferation, and survival assays. X-rays were used for irradiations. RESULTS: Sorafenib inhibited auto-phosphorylation of epidermal growth factor receptor and MET, which has not been described previously. MET expression in HNSCC cells was not always associated with activity/phosphorylation. Furthermore, sorafenib-dependent cell kill and radiosensitization was not associated with MET level. Although MET inhibitors blocked proliferation, they caused only mild cytotoxicity and no radiosensitization. CONCLUSION: We identified MET as a new potential target of sorafenib. However, MET inhibition is not the cause for sorafenib-mediated cytotoxicity or radiosensitization.

Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins.

The combination of radiotherapy and pharmacological inhibition of cellular signal transduction pathways offers promising strategies for enhanced cancer cell inactivation. However, the molecular effects of kinase inhibitors especially on DNA damage detection and repair after X-irradiation have to be understood to facilitate the development of efficient and personalized treatment regimens. Therefore, we applied differential proteomics for analyzing inhibitor-induced changes in either chromatin-bound or phosphorylated nuclear proteins. The effect of the multi kinase inhibitor sorafenib on DNA repair, chromatin binding and phosphorylation of nuclear proteins was analyzed in UT-SCC 42B head and neck cancer cells using metabolic labeling based differential proteomics (SILAC). Sorafenib significantly inhibited DNA repair but failed to significantly affect chromatin interactions of 90 quantified proteins. In contrast, analyzing nuclear phospho-proteins following sorafenib treatment, we detected quantitative changes in 9 out of 59 proteins, including DNA-repair proteins. In conclusion, the analysis of nuclear phospho-proteins by differential proteomics is an effective tool for determining the molecular effects of kinase inhibitors on X-irradiated cells. Analyzing chromatin binding might be less promising.

Effect of sorafenib on cisplatin-based chemoradiation in head and neck cancer cells.

Despite aggressive chemoradiation (CRT) protocols in the treatment of patients with head and neck squamous cell carcinomas (HNSCC), the outcome is still unfavorable. To improve therapy efficacy we had already successfully tested the multikinase inhibitor sorafenib in combination with irradiation (IR) in previous studies on HNSCC cell lines. In this study we investigated its effect on combined CRT treatment using cisplatin.Radio- and chemosensitivity with and without sorafenib was measured in four HNSCC cell lines and normal fibroblasts (NF) by colony formation assay. Apoptosis and cell cycle analysis were performed by flow cytometry.In HNSCC cells, sorafenib enhanced the antiproliferative effect of cisplatin without affecting apoptosis induction and with only minor effects on cell inactivation. Sorafenib added prior to irradiation enhanced cellular radiosensitivity in three of the tested HNSCC cell lines and caused massive overall cell inactivation when combined with CRT. In contrast, sorafenib did not radiosensitize NF and reduced cisplatin-induced cell inactivation. Cell inactivation by IR and cisplatin is further increased by the addition of sorafenib in HNSCC, but not in NF cells. Therefore, sorafenib is a promising candidate to improve therapy efficacy for HNSCC.

Quantitative proteomics unveiled: Regulation of DNA double strand break repair by EGFR involves PARP1.

BACKGROUND: EGFR inhibition blocks DNA double strand break (DSB) repair but the detailed mechanisms are still unclear. We asked whether EGFR inhibition blocks DSB repair by reducing the X-ray-induced phosphorylation of repair proteins using a phosphoproteomic approach. MATERIALS AND METHODS: Using UT-SCC5 and SAS head and neck cancer cells we established a differential phosphoproteomic approach for quantitative analysis of DNA repair proteins by stable isotope labeling with amino acids. Nuclear phosphoproteins were isolated and analyzed by liquid chromatography/tandem mass spectrometry. Erlotinib, PD98059 and olaparib were used to inhibit EGFR, MEK1/2 and PARP1, respectively. PARP1 was knocked down by siRNA. DSB repair was measured by quantifying residual 53BP1 foci. RESULTS: Over 150 nuclear phosphoproteins were quantified after irradiation, including 24 DNA repair proteins. Two of these, including PARP1, were consistently reduced in both cell lines upon erlotinib treatment. PARP1 inhibition or knock-down and EGFR inhibition resulted in an analog number of residual foci which was not further increased by combination of both strategies. MEK1/2 inhibition with or without blockage of EGFR or PARP1 caused similar effects. CONCLUSION: We have established a powerful, quantitative phosphoproteomic approach to investigate regulatory mechanisms in DSB repair, dependent on protein phosphorylation after irradiation. Using this approach we have identified PARP1 as a mediator of EGFR/MEK-dependent regulation of DSB repair.

Sorafenib sensitizes head and neck squamous cell carcinoma cells to ionizing radiation.

BACKGROUND AND PURPOSE: There is a great need to improve the outcome of locoregionally advanced squamous cell carcinomas of the head and neck (HNSCC). Standard treatment includes a combination of surgery, radio- and chemotherapy. The addition of molecular targeting agents to conventional treatment may improve outcomes. In this study the Raf inhibitor sorafenib was used to increase the radiosensitivity of HNSCC cell lines. MATERIAL AND METHODS: In a panel of six cell lines (A549, FaDu, UTSCC 60A, UTSCC 42A, UTSCC 42B, UTSCC 29) radiosensitivity was measured by colony formation assay and apoptosis and cell cycle analysis were performed by flow cytometry. DNA repair was analyzed by 53BP1 immunohistochemistry. RESULTS: Sorafenib added prior to irradiation resulted in an increased cellular radiosensitivity (DEF0.5=1.11-1.84). Radiosensitization was not caused by an enhanced rate of apoptosis or cell cycle effects. In contrast, sorafenib was shown for the first time to block the repair of DNA double-strand breaks (DSB). CONCLUSION: Our data suggest that sorafenib may be used to overcome the radioresistance of HNSCC through the inhibition of DSB repair.