Melittin Induced G1 Cell Cycle Arrest and Apoptosis in Chago-K1 Human Bronchogenic Carcinoma Cells and Inhibited the Differentiation of THP-1 Cells into Tumour- Associated Macrophages

Background: Bronchogenic carcinoma (lung cancer) is one of the leading causes of death. Although many compounds isolated from natural products have been used to treat it, drug resistance is a serious problem, and alternative anti-cancer drugs are required. Here, melittin from Apis mellifera venom was used, and its effects on bronchogenic carcinoma cell proliferation and tumour-associated macrophage differentiation were evaluated. Methods: The half maximal inhibitory concentration (IC50) of melittin was measured by MTT. Cell death was observed by annexin V and propidium iodide (PI) co-staining followed by flow cytometry. Cell cycle arrest was revealed by PI staining and flow cytometry. To investigate the tumour microenvironment, differentiation of circulating monocytes (THP-1) into tumour-associated macrophages (TAMs) was assayed by sandwich-ELISA and interleukin (IL)-10 levels were determined. Cell proliferation and migration was observed by flat plate colony formation. Secretion of vascular endothelial growth factor (VEGF) was detected by ELISA. The change in expression levels of CatS, Bcl-2, and MADD was measured by quantitative RT-PCR. Results: Melittin was significantly more cytotoxic (p < 0.01) to human bronchogenic carcinoma cells (ChaGo-K1) than to the control human lung fibroblasts (Wi-38) cells. At 2.5 µM, melittin caused ChaGo-K1 cells to undergo apoptosis and cell cycle arrest at the G1 phase. The IL-10 levels showed that melittin significantly inhibited the differentiation of THP-1 cells into TAMs (p < 0.05) and reduced the number of colonies formed in the treated ChaGo-K1 cells compared to the untreated cells. However, melittin did not affect angiogenesis in ChaGo-K1 cells. Unlike MADD, Bcl-2 was up-regulated significantly (p < 0.05) in melittin-treated ChaGo-K1 cells. Conclusion: Melittin can be used as an alternative agent for lung cancer treatment because of its cytotoxicity against ChaGo-K1 cells and the inhibition of differentiation of THP-1 cells into TAMs.

Soluble Triggering Receptor Expressed on Myeloid Cells 1 in lung cancer.

Soluble Triggering Receptor Expressed on Myeloid Cells 1 (sTREM-1) can be found in the sera of patients with infectious, autoimmune and malignant diseases. The primary objective of this study was to investigate the prognostic significance of sTREM-1 in lung cancer patients. We analyzed the sera of 164 patients with lung cancer of all histologies and all stages at the time of diagnosis. We employed an ELISA using the anti-TREM-1 clone 6B1.1G12 mAb and recombinant human TREM-1. Patient data was collected retrospectively by chart review. In ROC-analysis, a sTREM-1 serum level of 163.1 pg/ml showed the highest Youden-Index. At this cut-off value sTREM-1 was a marker of short survival in patients with NSCLC (median survival 8.5 vs. 13.3 months, p = 0.04). A Cox regression model showed stage (p < 0.001) and sTREM-1 (p = 0.011) to indicate short survival. There were no differences in sTREM-1 serum values among patients with or without infection, pleural effusion or COPD. sTREM-1 was not associated with metastasis at the time of diagnosis and was not a predictor of subsequent metastasis. In SCLC patients sTREM-1 levels were lower than in NSCLC patients (p = 0.001) and did not predict survival. sTREM-1 did not correlate with CRP or the number of neutrophils. In non-small cell lung cancer patients, sTREM-1 in serum has prognostic significance.

miRSNPs of miR1274 and miR3202 Genes that Target MeCP2 and DNMT3b Are Associated with Lung Cancer Risk: A Study Conducted on MassARRAY Genotyping.

Genetic variants of miRNAs that target DNMTs and MBDs involved in DNA methylation were scanned with current databases, and 35 miRSNPs in 22 miRNA genes were identified. The aim of the study was to determine the association between these variants of miRNA genes and lung cancer (LC). DNA samples were isolated from blood samples and genotyped using a Sequenom MassARRAY System. An association between the rs188912830 gene variant of miR3202 that targets the MeCP2 protein and LC was indicated in both subtypes. The presence of the C-allele in patients with LC and its subtypes was significantly lower, and the absence of the C-allele was determined to increase the risk of LC by 7,429-times compared to the presence (p=0,010). The rs318039 gene variant of miR1274 that targets DNMT3b was found to be associated with LC subtypes. When allele distributions were compared, the numbers of individuals with the C-allele were significantly lower in the NSCLC and SCLC groups. No significant associations were found for the rs72563729 variant of the miR200b gene that targets DNMT3a or for the rs145416750 variant of the miR513c gene that targets TRDMT1. The other 33 variants were found to be ancestral genotypes. Consequently, rs188912830 and rs318039 variations were associated with LC subtypes. Importantly, this study is the first to indicate the functional characterisation of miRSNPs of genes that target DNA methylation.

Expression of Rab1A is upregulated in human lung cancer and associated with tumor size and T stage.

Rab1A expression is associated with malignant phenotypes in several human tumors; however, the role of Rab1A in lung cancer is still unclear. In this study, we attempted to establish the role of Rab1A in major human lung cancer subtypes. Rab1A expression in different histological types of human lung cancer was analyzed in lung cancer tissues with paired adjacent noncancerous tissues and a large panel of lung cancer cell lines. The effect of Rab1A expression on multiple cancer-associated signaling pathways was also examined. The results demonstrated that Rab1A was significantly overexpressed in the different histological types of lung cancer as compared to non-cancerous tissues, and Rab1A expression was correlated with tumor volume and stage. In a large panel of lung cancer cell lines, high Rab1A expression was observed as compared to a normal lung/bronchus epithelial cell line. However, Rab1A protein levels were not correlated with mTORC1 (P-S6K1), mTORC2 (P-AKT), MEK (P-ERK), JNK (P-c-Jun) or p38MAPK (P-MK2) signaling. Rab1A knockdown had no effect on mTOR signaling or cell growth. These data suggested that Rab1A may be involved in the pathogenesis of human lung cancer in an mTOR- and MAPK-independent manner.

Lactoferrin-appended solid lipid nanoparticles of paclitaxel for effective management of bronchogenic carcinoma.

Lung cancer is a dreadful disease which claims to be more life threatening as compared to total sum up of colon, prostate and breast cancers. Thus, there is an urgent need to develop an effective delivery approach for its management. Paclitaxel (PTX) is one of the well-known choice as antineoplasitic agent used for the treatment of different types of human cancers such as non-small-cell lung, head and neck cancers, leukemia, breast, ovarian and melanoma. Lactoferrin (Lf), a "multifunctional protein" is crucial for natural immunity which is secreted by exocrine glands. Lf receptors are expressed on the apical surface on bronchial epithelial cells. These over-expressed LF receptors can be utilized for the transportation of Lf-conjugated drug or nanocarrier devices. The present study was aimed to develop PTX-loaded Lf-coupled solid lipid nanoparticles (SLNs) for the treatment of lung cancer. PTX-loaded SLNs were prepared, characterized and then coupled with Lf using carbodiimide chemistry. The formulations were characterized by transmission electron microscopy, particle size, polydispersity index and zeta potential, whereas Lf conjugation was confirmed by FT-IR and ¹H NMR and efficiency of prepared system was evaluated by in vitro, ex vivo and in vivo evaluations. The ex vivo cytotoxicity studies on human bronchial epithelial cell lines, BEAS-2B, revealed superior anticancer activity of Lf-coupled SLNs than plain SLNs and free PTX. In vivo biodistribution studies showed higher concentrations of PTX accumulated in lungs via Lf-coupled SLNs than plain SLNs and free PTX. These studies suggested that Lf-coupled PTX-loaded SLNs could be used as potential targeting carrier for delivering anticancer drug to the lungs with the minimal side effects.

Class I HDACs are mediators of smoke carcinogen-induced stabilization of DNMT1 and serve as promising targets for chemoprevention of lung cancer.

DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDAC) 1 to 3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell-cycle independent increased DNMT1 stability, and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter, and derepression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen-induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability. In summary, our study provides evidence for an important role of class I HDACs in controlling the stability of DNMT1 and suggests that HDAC inhibition could be an attractive approach for lung cancer chemoprevention.

Bronchogenic carcinoid tumours that are 18F-fluorodeoxyglucose avid on positron emission tomography.

OBJECTIVES: Bronchogenic carcinoid tumours are widely cited as non-fluorodeoxyglucose (FDG) avid. However, three case reports of FDG-avid bronchogenic carcinoid tumours have been published, leading to speculation as to which clinicopathological factors may be associated with increased activity on FDG-positron emission tomography. We reviewed a series of cases from our institution and compared them with the available reports in the literature, to attempt to identify the factors associated with FDG avidity in bronchogenic carcinoids. METHODS: We performed a single-institution retrospective review. RESULTS: One patient was identified at our institution who had a typical carcinoid tumour with a standardized uptake value (SUV) of 26, oncocytic features on histology and positive staining for glucose transporter 1 (GLUT1). Three additional patients were identified in the literature with typical bronchogenic carcinoids with SUVs of 39, 38 and 33. Two of these tumours stained positive for GLUT1, and the remaining patient was not tested. Two of these patients had oncocytic features on histology, and results on the remaining patient are not reported. Additionally, 4 patients at our institution were identified with bronchogenic carcinoids with average SUV of 2.6. All were GLUT1 negative, and none had oncocytic features. In the reported literature, excluding the four most FDG-avid tumours described above, atypical carcinoids had a higher mean SUV than typical carcinoids (5.7 vs 3.4, P = 0.02), but size was not correlated with SUV (r = 0.7, P = 0.3). CONCLUSIONS: FDG uptake is commonly associated with worse prognosis in malignancy; however, bronchogenic carcinoids, particularly oncocytic typical carcinoids, are a possible source of extremely high SUVs on FDG-PET.

Synergistic effects of erlotinib and everolimus on bronchial carcinoids and large-cell neuroendocrine carcinomas with activated EGFR/AKT/mTOR pathway.

BACKGROUND: Epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) are crucial targets in cancer therapy. Combined inhibition of both targets yielded synergistic effects in vitro and in vivo in several cancer entities. However, the impact of EGFR and mTOR expression and combined inhibition in neuroendocrine lung tumors other than small-cell lung cancer remains unclear. MATERIAL AND METHODS: Expression and activation of EGFR/AKT/mTOR pathway constituents were investigated in typical and atypical bronchial carcinoid (AC) tumors and large-cell neuroendocrine lung carcinomas (LCNEC) by immunohistochemistry in 110 tumor samples, and correlated with clinicopathological parameters and patient survival. Cytotoxicity of mTOR inhibitor everolimus and EGFR inhibitor erlotinib alone and in combination was assessed using growth inhibition assay in NCI-H720 AC and SHP-77 LCNEC cells. Cell cycle phase distribution was determined by FACS. Apoptosis-associated activation of caspase-3/7 was measured by Caspase-Glo® assay. Activity status of EGFR and mTOR pathway components was analyzed by immunoblotting. RESULTS: Activation of the EGFR/AKT/mTOR axis could be demonstrated in all entities and was significantly increased in higher grade tumors. Neoadjuvant chemotherapy correlated significantly with p-AKT expression and p-ERK loss. Erlotinib combined with everolimus exerted synergistic combination effects in AC and LCNEC cells by induction of apoptosis, while cell cycle phase distribution remained unaffected. These effects could be explained by synergistic downregulation of phospho-mTOR, phospho-p70S6 kinase and phospho-AKT expression by everolimus and erlotinib. CONCLUSIONS: Our study indicates that EGFR and mTOR are clinically important targets in bronchial neuroendocrine tumors, and further in vivo and clinical exploration of combined inhibition is warranted.

Cushing syndrome secondary to ectopic adrenocorticotropic hormone secretion: the University of Texas MD Anderson Cancer Center Experience.

BACKGROUND: Cushing syndrome (CS) secondary to ectopic adrenocorticotropic hormone (ACTH) secretion (EAS) has been described in association with a variety of tumors. The current experience with this syndrome was based on a few case series and individual case reports. Limited data were available about the tumors associated with CS-EAS in a cancer center setting. In this report, the authors have described their experience with CS-EAS at The University of Texas MD Anderson Cancer Center to further enhance the current understanding and management of this syndrome. METHODS: This was a retrospective review of 43 patients with CS-EAS who were diagnosed between 1979 and 2009 at The University of Texas MD Anderson Cancer Center. RESULTS: Different neuroendocrine tumors were associated with CS-EAS. Twenty-one patients (48.9%) had tumors located in the chest cavity, with bronchial carcinoid and small cell lung cancer representing the 2 most common causes. The ACTH source remained occult in 4 patients (9.3%) despite extensive workup. Clinical presentation varied, and the classic features of CS were not evident in some patients. Death occurred in 27 patients (62.8%), and the median overall survival was 32.2 months. Major morbidities included new-onset or worsening hyperglycemia (77%), symptomatic venous thromboembolism (14%), and infections (23%). CONCLUSIONS: In patients with CS-EAS who attended a comprehensive cancer center, tumors originating in the chest cavity were the leading tumors associated with this syndrome. The authors suspect that CS-EAS is under reported because of the atypical presentation in some patients. Thus, they suggest careful evaluation of patients with neuroendocrine tumors to avoid missing coexisting CS-EAS.

Immunohistochemical analysis of CYP2A13 in various types of human lung cancers.

Human CYP2A13, which is expressed in the respiratory tract, is the most efficient enzyme for the metabolic activation of tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The relevance of CYP2A13 in carcinogenicity and toxicity in the respiratory tract has been suggested, but the expression of CYP2A13 protein in lung cancer tissues remains to be determined. We first prepared a mouse monoclonal antibody against human CYP2A13. The antibody showed no cross reactivity with the other CYP isoforms including CYP2A6. Using the specific antibody, we performed immunohistochemical analysis for human lung carcinomas. In adenocarcinomas (n = 15), all specimens were positive for the staining and five samples showed strong staining. In squamous cell carcinomas (n = 15) and large cell carcinomas (n = 15), each 14 samples were positive for the staining and two and three samples showed strong staining, respectively. In small cell carcinoma samples (n = 15), eight samples were negative for the staining and five samples showed weak or moderate staining. In conclusion, we first found that the expression of CYP2A13 was markedly increased in non-small cell lung carcinomas. The high expression might be associated with the tumor development and progression in non-small cell lung carcinomas.

Expression of endothelin receptor subtypes in bronchial tumors.

The importance of endothelin-1 (ET-1) in cell growth, migration and stimulation of angiogenesis suggests that ET-1 may play a role in tumor progression. The expression of the ET-1 receptors ETA (ET(A)R) and ETB (ET(B)R) was analyzed by immunohistochemistry in fragments of human lung carcinomas. Samples were obtained from 11 patients with adenocarcinoma (ADK), 12 with squamous cell carcinoma (SCC) and 8 patients with small cell carcinoma (SCLC). Morphologically normal airway areas adjacent to the tumors served as controls. ADK and SCC samples had ET(A)R and ET(B)R levels similar to normal tissues; however, the ET(A)R/ET(B)R ratio was higher in ADK than in SCC. We also observed the presence of endothelin receptors in SCLC, although the ET(A)R levels and the ratio ET(A)R/ET(B)R were lower than in normal tissue and in other carcinomas. In conclusion, both ET(A)R and ET(B)R are present in lung carcinomas but at different levels, according to the histological type of tumor.

Enkephalin, its precursor, processing enzymes, and receptor as part of a local opioid network throughout the respiratory system of lung cancer patients.

Evidence is accumulating regarding the local opioid regulation of physiologic respiratory functions. However, anatomical evidence for a local opioid network of the respiratory system is scarce. In this study, tissue samples from 12 lung cancer patients undergoing lobectomy or pneumonectomy were examined immunohistochemically for the expression of the opioid network components met-enkephalin, the respective precursor proenkephalin, the key processing enzymes prohormone convertases 1 and 2, carboxypeptidase E, and the delta opioid receptor in different areas of human lung. Colocalization of proenkephalin with met-enkephalin, prohormone convertase 1, prohormone convertase 2, and carboxypeptidase E was demonstrated by double-immunofluorescence confocal microscopy in alveolar macrophages, submucosal glands, cancerous cells, and pulmonary neuroendocrine cells of bronchial epithelium. Corresponding delta opioid receptor was identified on cells of all these functionally relevant anatomical structures and on substance P-immunoreactive sensory nerve fibers arborizing within bronchial epithelium. Our findings provide evidence of a local opioid network, that is, the exact anatomical localization of proenkephalin, its functionally active peptide met-enkephalin, and the key processing enzymes as well as corresponding delta opioid receptor, linked to functionally important structures of the respiratory system. These findings encourage future studies to examine the functional role of local opioid peptides within the respiratory system.

Altered Ca2+-homeostasis of cisplatin-treated and low level resistant non-small-cell and small-cell lung cancer cells.

BACKGROUND: Chemotherapy often leads to encouraging responses in lung cancer. But, in the course of the treatment, resistance to chemotherapy ultimately limits the life expectancy of the patient. We aimed at investigating if treatment with cisplatin alters the intracellular Ca2+-homeostasis of lung cancer cells and how this may be related to cisplatin resistance. METHODS: The squamous cell lung carcinoma cell line EPLC M1 and the small cell lung cancer cell line H1339 were exposed to cisplatin analogue to the in vivo pharmacokinetics. Changes in the cytoplasmic Ca2+-concentration ([Ca2+]c) were recorded using fluorescence microscopy. Protein expression was quantified using immuno-fluorescence and Western Blot analysis. Changes in gene expression were accomplished by small-interfering (si) RNA techniques. RESULTS: Four "cycles" of cisplatin led to low level resistance in EPLC and H1339 cells. In the low level resistant cell clones, the Ca2+-content of the endoplasmic reticulum (ER) was decreased. In low level resistant EPLC cells, this was correlated with an increased expression of the inositol-1,4,5-trisphosphate receptor (IP3R). Inhibiting the increased expression of IP3R using siRNA, the low level resistance could be reversed. In low level resistant H1339 cells, the decreased Ca2+-content of the ER was correlated with a decreased expression of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). Decreasing the expression of SERCA in naïve H1339 cells resulted in low level cisplatin resistance. CONCLUSION: We conclude that cisplatin therapy leads to a decreased Ca2+-content of the ER thereby inducing low level resistance. This is caused by upregulation of the IP3R in EPLC and decreased expression of SERCA in H1339 cells.

High expression of dopamine receptor subtype 2 in a large series of neuroendocrine tumors.

AIM: To evaluate by immumohistochemistry the presence of DR subtype 2 (D2R) in well differentiated NETs of different sites and in normal islet cells. BACKGROUND: Recent data in vitro and in vivo support that dopaminergic drugs might exert an inhibitory effect on hormone secretion and, possibly, on tumor growth in neuroendocrine tumors (NET)s. Their potential therapeutic role needs the demonstration of dopamine receptors (DR) in tumor cells. Little is known on the expression of DR in NETs. RESULTS: 85% of samples (100% of bronchial carcinoids and 93% of islet cell tumors) showed positivity for D2R; intensity of immunoreaction in NETs was similar or higher than in pituitary (54% and respectively 31% of cases). D2R positivity in more than 70% of tumor cells was observed in 46% of samples. Same intensity of D2R-immunoreactivity was found in pituitary and normal islet cells. No differences in D2R expression were recorded on considering tumor grading, size, proliferative activity, presence of metastases, endocrine activity and gender. A significant difference (62.5% vs 96.4%, p = 0.039) was observed in the prevalence of D2R expression between patients with more aggressive tumors and patients without recurrence/progression of disease during follow-up. METHODS: 46 NET samples from 44 patients and normal endocrine pancreatic tissue were studied. D2R-staining was performed on NETs and compared with six non-secreting pituitary adenomas and related to clinical-pathological data. CONCLUSION: The present data demonstrate a high expression of D2R in NETs; this finding is of clinical relevance in view of the potential role of dopaminergic drugs in inhibiting secretion and/or cell proliferation in NETs.

Inflammatory markers in exhaled breath condensate following lung resection for bronchial carcinoma.

BACKGROUND AND OBJECTIVE: Pulmonary resection may cause inflammatory changes with subsequent injury to the remaining lung and deterioration in respiratory function. This study investigated the pattern of serum inflammatory markers and exhaled breath condensate (EBC) in patients undergoing major lung resection due to bronchial carcinoma compared with minimally invasive thoracic surgery. METHODS: The pro-inflammatory markers IL-1-beta, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured preoperatively (day -1) and on three postoperative days (day 1, 3, 7) in serum and EBC in patients after lobectomy or pneumonectomy due to bronchial carcinoma (test group) and in patients undergoing thoracoscopy with minimal wedge resection (control group). RESULTS: All mediators were detectable in serum and all but IL-8 were detectable in EBC. No patient suffered postoperative respiratory failure. In the test group, serum IL-6 was significantly higher postoperatively compared with day -1 (P < 0.001). For EBC (test group), the postoperative values of IL-1-beta were significantly higher compared with day -1 (P = 0.005). In EBC (test group), day -1 TNF-alpha and sICAM-1 were significantly higher compared with controls (P < 0.029 and P = 0.032, respectively). There was no correlation between the levels of mediators and the extent of resection. CONCLUSIONS: Pro-inflammatory markers are detected in EBC following pulmonary surgery. Mediators are detectable in both serum and EBC in patients with bronchial carcinoma undergoing pulmonary resection, but the levels are higher in EBC.

[Expression of Hsp70 Grp94 and IgG in human lung carcinoma].

AIM: To explore the expression and significance of heat shock protein 70 (Hsp70), glucose regulated protein 94 (Grp94) and immunoglobulin G (IgG) in human lung carcinoma. METHODS: The expression of Hsp70, Grp94 and IgG in 40 human lung carcinomas was studied using immunohistochemical technique and image analysis. The localization among Hsp70, Grp94 and IgG was analyzed by double labeling immunofluorescent staining and laser scanning confocal microscopy. RESULTS: Hsp70, Grp94 and IgG in Human lung carcinomas showed high expression. The positive rate of Hsp70, Grp94 and IgG was 65% (26/40), 45% (18/40), and 82.5% (33/40), respectively. The average value of optical density was 5.10 +/- 0.32, 3.52 +/- 0.35, and 8.12 +/- 0.31, respectively. Hsp70 was localized in nucleus and cellular cytoplasm while Grp94 and IgG were mainly localized in cellular cytoplasm. Ten cases showed Hsp70 was co-localized with IgG and Eighteen cases showed Grp94 was co-localized with IgG. CONCLUSION: High expression of Hsp70, Grp94 and IgG in Human lung carcinomas suggested that Hsp70, Grp94 and IgG might play an important role in the development of human lung carcinoma. IgG is co-localized with Hsp70 or Grp94. The study will lay a theorical basis for further research on anti-tumor immunotherapy.

The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation.

BACKGROUND AND PURPOSE: Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr). METHODS: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of gammaH(2)AX focus assay. RESULTS: As determined by dose-response curves, treatment of cells with P-Tyr for 16h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation. CONCLUSIONS: Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival.