Lapatinib-induced mesenchymal-epithelial transition in squamous cell carcinoma cells correlates with unexpected alteration of ß-catenin expression.

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression, and may promote resistance of cancer cells to therapy. Inhibiting EMT appears to be crucial to inhibit drug resistance. The mesenchymal-epithelial transition (MET), which is the reverse program of EMT in metastases, is characterized by the upregulation of epithelial adhesive proteins such as E-cadherin, and downregulation of mesenchymal proteins such as vimentin. The sensitivity of cancer cells to epithelial growth factor receptor (EGFR) inhibitor may be increased by inducing MET in these cells. Therefore, it is of clinical importance to specify the phenotype of cancer cells in order to overcome the phenomenon of drug resistance. The aim of the present study was to investigate the expression pattern of specific markers in squamous cell carcinoma (SCC) cells following stimulation with lapatinib and gefitinib. For this purpose, the head and neck (HN) SCC cell lines HNSCC22B and HNSCC11A were incubated with 0.5 and 2 µg/ml lapatinib and gefitinib, and the levels of E-cadherin, vimentin, matrix metalloproteinase-14, c-kit and ß-catenin were detected by immunocytochemistry and enzyme-linked immunosorbent assay at 5, 24 and 96 h post-incubation. The results indicated that, compared with HNSCC22B cells, the protein expression levels of vimentin increased, whereas those of E-cadherin reduced, in non-stimulated HNSCC11A cells. In addition, the protein expression levels of ß-catenin were altered in the epithelial- and mesenchymal-associated SCC cell lines following treatment with lapatinib and gefitinib. Furthermore, lapatinib induced the downregulation of vimentin and upregulation of E-cadherin in HNSCC11A cells in a time-dependent manner. This suggests that the sensitivity of cancer cells to lapatinib may be improved by inducing MET in these cells. In summary, the results of the present study demonstrated that lapatinib-induced MET led to an unexpected alteration of the protein expression levels of ß-catenin in SCC cells. Further studies on the mechanistic role of MET are required in order to increase the sensitivity of cancer cells to EGFR inhibitor and block the EMT process in these cells.

MMP9, Cyclin D1 and ß-Catenin Are Useful Markers of p16-positive Squamous Cell Carcinoma in Therapeutic EGFR Inhibition In Vitro.

BACKGROUND/AIM: In the United States 53,640 new cases of head and neck cancer were estimated in 2013. Over 95% of these cases were evaluated as squamous cell carcinoma (SCC). At present, smoking, drinking alcohol, chewing betel and infection with high-risk types of human papilloma virus (HPV) are classified as risk factors of oropharyngeal squamous cancer cell carcinoma (OPSCC). It could be suggested that patients with HPV-positive OPSCC have a better response to chemoradiotherapy than patients without. In many studies, there was observed an inverse correlation between epithelial growth factor receptor (EGFR) expression and HPV status in p16-positive SCC. Therefore, it is of great clinical interest to specify the phenotype of cancer cells in order to further individualize treatment modalities. The aim of the study was to investigate the expression pattern of specific markers in p16-positive SCC cells after stimulation with lapatinib and gefitinib. MATERIALS AND METHODS: We incubated p16-positive CERV196 cells with lapatinib and gefitinib (2 µg/ml) and after 5, 24 and 96 h determined E-cadherin, vimentin, matrix metalloproteinase-9 (MMP9), cyclin D1 and ß-catenin by immunocytochemistry, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction (PCR). RESULTS: We found an increase of E-cadherin and a decrease of vimentin in unstimulated cells. We detected an alteration of expression of vimentin and E-cadherin level after treatment with lapatinib and gefitinib. We demonstrated a statistically significant lapatinib- and gefitinib-induced repression of cyclin D1, MMP9 and ß-catenin in CERV196 cells dependent on incubation time. CONCLUSION: Cyclin D1 and MMP9 expression profiles may represent an early measure of sensitivity and level of response to lapatinib and gefitinib. The presented cell culture model is, therefore, well-suited for further study of epigenetic regulation of molecular targeted-therapy by EGFR inhibition and prevention of mesenchymal transition in p16-positive SCC cells.

Targeting mTOR and AREG with everolimus, sunitinib and sorafenib in HPV-positive and -negative SCC.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is the most common neoplasm appearing in the upper aerodigestive tract and the sixth most common cancer worldwide. The five-year survival rate remains poor despite advances in surgery, radiation and chemotherapy. Furthermore, the incidence of human papillomavirus (HPV)-associated oropharyngeal cancer is rising. Thus, innovative therapy approaches are imperative in order to improve the situation. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) and sorafenib and sunitinib, multityrosine kinase inhibitors, have been notably effective in the therapy of different tumor entities. The modest side-effects and oral application of the drugs might improve patient compliance. Expression levels of mTOR and Amphiregulin (AREG) in p16-positive and -negative SCC (squamous cell carcinoma) and the effect of everolimus, sorafenib or sunitinib on the expression levels of these target proteins were assessed. As far as we are aware of, this is one of the first in vitro studies to evaluate the effect of these small-molecule drugs with regard to the p16 status of SCC cells. MATERIALS AND METHODS: p16-negative HNSCC 11A and 14C cells and p16-positive CERV196 cells were exposed to different concentrations of everolimus, sorafenib and sunitinib for 2-8 days. Expression levels of mTOR and AREG were determined by enzyme-linked immunosorbent assay (ELISA) and compared against a chemonaïve control. RESULTS: AREG and mTOR were expressed in all tested cell lines. CERV196 displayed a remarkable increase of mTOR expression compared to p16-negative HNSCC. On the contrary, AREG levels were reduced by 50% in CERV196. Everolimus, sorafenib and sunitinib significantly reduced mTOR expression. Everolimus significantly decreased AREG expression independently of the HPV status. Sunitinib and sorafenib increased AREG expression in HNSCC 11A and 14C but not in CERV196. CONCLUSION: The applied drugs showed remarkable suppression of mTOR expression, which might delay tumor progression. Interestingly, sorafenib and sunitinib increased AREG in HNSCC 11A and 14C, which could be a possible evasive mechanism following incubation with these drugs. On the contrary, p16-positive CERV196 showed increased susceptibility to sorafenib and sunitinib concerning suppression of AREG expression. Further studies are required to evaluate the HPV-dependent differences of therapy response and the possible consequences for treatment options.

Contribution of Ninjurin1 to Toll-like receptor 4 signaling and systemic inflammation.

Nerve injury-induced protein (Ninjurin [Ninj]) 1 is an adhesion molecule originally identified in Schwann cells after nerve injury, whereas it is also expressed in leukocytes, epithelium, endothelium, and various organs, and is induced under inflammatory conditions. Its contribution to inflammation was so far restricted to the nervous system and exclusively attributed to its role during leukocyte migration. We hypothesized a proinflammatory role for Ninj1 also outside the nervous system. To elucidate its impact during inflammation, we analyzed expression levels and its contribution to inflammation in septic mice and studied its effect on inflammatory signaling in vitro. The effect on inflammation was analyzed by genetic (only in vitro) and pharmacologic repression in septic mice (cecal ligation and puncture) and cell culture, respectively. Repression of Ninj1 by an inhibitory peptide or small interfering RNA attenuated LPS-triggered inflammation in macrophages and endothelial cells by modulating p38 phosphorylation and activator protein-1 activation. Inhibition of Ninj1 in septic mice reduced systemic and pulmonary inflammation as well as organ damage, and ameliorated survival after 24 hours. Ninj1 is elevated under inflammatory conditions and contributes to inflammation not only by mediating leukocyte migration, but also by modulating Toll-like receptor 4-dependent expression of inflammatory mediators. We assume that, owing to both mechanisms, inhibition reduces systemic inflammation and organ damage in septic mice. Our data contribute to a better understanding of the complex inflammatory mechanisms and add a novel therapeutic target for inflammatory conditions such as sepsis.

Small molecules alter VEGFR and PTEN expression in HPV-positive and -negative SCC: new hope for targeted-therapy.

BACKGROUND/AIM: Prognosis for patients with head and neck squamous cell carcinoma (HNSCC) is poor in most cases and has not improved despite advances in therapy. Novel therapeutic approaches are mandatory in order to improve the situation. Everolimus, an inhibitor of mammalian target of rapamycin, as well as the multi-tyrosine kinase inhibitors sorafenib and sunitinib, has demonstrated a substantial therapeutic effect in various types of human cancer with moderate side-effects. Expression of vascular endothelial growth factor receptor (VEGFR) 1 and 2, and of the tumor-suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were evaluated in chemonaïve human papillomavirus (HPV)-positive and -negative squamous cell carcinoma (SCC) and after exposure to everolimus, sorafenib or sunitinib. MATERIALS AND METHODS: p16-positive CERV196 and p16-negative HNSCC 11A and 14C cells were incubated with different drug concentrations for 48-192 h. Expression of VEGFR1 and -2 as well as PTEN were determined by enzyme-linked immunosorbent assay and was compared to a chemonaïve control. RESULTS: VEGFR1 and -2, as well as PTEN, were expressed in all three cell lines. Sunitinib, sorafenib and everolimus significantly reduced the expression of VEGFR1 and -2, especially in p16-positive CERV196 cells. Sunitinib appeared to be more effective in reducing VEGFR1 and -2 expression than sorafenib and everolimus. PTEN levels were remarkably lower in HPV-positive CERV196 cells. PTEN expression increased significantly under sunitinib and sorafenib in HNSCC 11A and CERV196 cells. Everolimus, on the other hand, led to a significant decrease of PTEN expression in these cell lines. CONCLUSION: The tested drugs displayed a remarkable anti-angiogenic effect by inhibition of VEGFR1 and -2 expression. Sunitinib and sorafenib were able to increase PTEN expression, which might induce apoptosis of cancer cells. HPV-positive CERV196 cells were characterized by an increased susceptibility to these small-molecule drugs. Further studies are imperative to scrutinize HPV status-dependent differences in drug response and possible implications for future treatment options.

Incomplete epithelial-mesenchymal transition in p16-positive squamous cell carcinoma cells correlates with ß-catenin expression.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is suggested to be a crucial factor for the development of an invasive and metastatic cell phenotype, which is characterized by down-regulation of epithelial adhesive proteins (e.g. E-cadherin) and induction of mesenchymal proteins (e.g. vimentin). Therefore, there is a great clinical interest to specify this phenotype. Different growth factors induce EMT, such as epithelial growth factor (EGF) and transforming growth factor beta 1 (TGFß1). The role of EMT in human papilloma virus (HPV)-positive squamous cell carcinoma (SCC) is still not understood. The aim of this study was to investigate the expression pattern in p16-positive and -negative SCC cells of vimentin, ß-catenin and E-cadherin after stimulation with growth factors. MATERIALS AND METHODS: We incubated the p16-positive CERV196 and p16-negative HNSCC22B SCC cell lines with EGF and EGF/TGFß1 (10 ng/ml) and detected E-cadherin, vimentin and ß-catenin by immunocytochemistry and enzyme-linked immunosorbent assay after 5, 24 and 96 h. RESULTS: We found a low expression of vimentin in all studied tumor cell lines. The negative control of HNSCC22B cells showed a higher intrinsic level of membranous E-cadherin and ß-catenin. We found statistically significant EGF/TGFß1-induced expression of vimentin dependent on incubation time in p16-negative HNSCC22B cells. Particularly in the presence of EGF, we detected an increase of ß-catenin and vimentin expression in p16-positive SCC tumor cell lines in addition to induced cell scattering and unexpected expression of E-cadherin. CONCLUSION: In conclusion, E-cadherin, ß-catenin and vimentin expression are important features to characterize EMT-like events. We were able to show incomplete EGF-induced EMT with ß-catenin expression in p16-positive SCC. Extended studies are required to investigate the mechanistic role of EMT markers, especially in p16-positive SCC, in order to develop new anti-SCC therapies to block EMT progression.

Matrix metalloproteinase-2 and -14 in p16-positive and -negative HNSCC after exposure To 5-FU and docetaxel In Vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world. While the incidence of HNSCC associated with tobacco and alcohol abuse is falling, the incidence of HNSCC associated with human papilloma virus (HPV) is rising. Proliferation, cell migration and formation of metastases are dependent on interactions between the tumor cells, tumor stromal cells and the extracellular matrix (ECM). Degradation of the ECM is a crucial step in the process of local tumor infiltration and formation of locoregional and distant metastases. Matrix metalloproteinases (MMPs) are a family of enzymes that are able to degrade the ECM. Locally advanced HNSCC with cervical node metastases are treated with docetaxel in induction chemotherapy (ICT) combined with platinum-based chemotherapy and 5-fluorouracil (5-FU) as standard clinical anti-neoplastic regimens. This study evaluated the expression of MMP-14 and MMP-2 in HPV-positive (CERV196) and HPV-negative squamous cell carcinoma (HNSCC 11A and 14C) and the alteration of expression levels after exposure to either docetaxel or 5-FU. MATERIALS AND METHODS: Tumor cells were exposed to 5-FU or docetaxel in concentrations of 1.0 and 5.0 µmol/ml. MMP-protein expression was evaluated by enzyme-linked immunosorbent assay (ELISA) after 2, 3, 5, 8 and 10 days of incubation. RESULTS: Docetaxel exposure significantly decreased MMP-14 expression in HNSCC 11A and especially 14C but not in CERV196 apart from an apoptotic process. 5-FU had no significant effect on MMP-14 expression independent of the HPV-status. Significant alterations of MMP-2 could be detected in HNSCC 11A only. CONCLUSION: Although neither of the applied drugs were selective inhibitors of MMP-expression, surprisingly docetaxel significantly decreased MMP-14 in HNSCC 14C and 11A in this study. Interestingly, HPV-positive CERV196 was not sensitive to decreased MMP-14 or -2 expression following incubation with 5-FU or docetaxel.

Interleukin 4, interleukin 6 and osteopontin-serological markers of head and neck malignancy in primary diagnostics: A pilot study.

The progression of head and neck squamous cell carcinoma (HNSCC) is stimulated by various angiogenic peptides and growth factors. A correlation between tumor progression and the secretion of various serological mediators in patients with malignant tumors of the head and neck is of major interest for tumor diagnostics, evaluation of the therapy response and it may predict prognosis by specifying the individual tumor biology. Established chemotherapeutic regimes for head and neck tumors usually consist of platinum-based chemotherapeutic drugs and 5-fluorouracil (5-FU). The present pilot study sought to assess the eligibility of seven serological factors as biomarkers for malignant tumors of the head and neck: Platelet-derived growth factor, vascular endothelial growth factor, epidermal growth factor receptor, osteopontin, granulocyte-colony stimulating factor, interleukin-4 (IL-4) and IL-6. The serum levels of each factor in 20 patients receiving concomitant radiochemotherapy with cisplatin or carboplatin and 5-FU with curative intent were determined prior and subsequent to chemotherapy and were compared with 40 healthy controls. Another aim of the pilot study was to investigate whether the serum of patients showed significant differences in the concentrations of the analyzed factors at the start of concomitant radiochemotherapy compared with the controls, whether those markers indicated a neoplastic process and whether concomitant radiochemotherapy with cisplatin or carboplatin and 5-FU induced significant alterations of concentration compared with pre-therapeutic levels. The included patients were histopathologically diagnosed with HNSCC and the average age was 62.3 years. The serum samples of the patients were obtained during the course of regular pre- and post-chemotherapeutic blood draws one week prior to the start of radiochemotherapy and one week following the completion of chemotherapy. The healthy controls were collected from patients of the Sleep Laboratory of the Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital (Mannheim, Germany) without clinical evidence or laboratory signs of inflammation or history of a malignant disease. The average age was 50.3 years. The serological level of each factor was ascertained by enzyme-linked immunosorbent assay in duplicate. Serum levels of IL-4, IL-6 and osteopontin were significantly increased in patients with HNSCC compared with those in chemotherapy-naive healthy controls. IL-4 and osteopontin showed no significant therapy-associated alterations. Notably, IL-6 levels significantly increased post-therapeutically. Using logistic regression with osteopontin and IL-4, an individual risk-profile for random samples was calculated. IL-4, IL-6 and osteopontin appear to be suitable indicators of the neoplastic process as they are significantly increased in HNSCC patients compared with the control group. With the exception of IL-6, whose levels were in fact increased following therapy, a significant therapy-associated alteration of these factors was missing. Therefore, these serological markers failed to predict the therapy response, but they may be valuable as a screening instrument in primary diagnostics.

Imatinib-associated matrix metalloproteinase suppression in p16-positive squamous cell carcinoma compared to HPV-negative HNSCC cells in vitro.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. The growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM), which is modified by matrix metalloproteinases (MMPs). The MMP family is still relevant to cancer research, as it promotes malignant transformation, cell proliferation and modulation of angiogenesis even in the early stages of cancer. The proteolytic processing of bioactive molecules by MMP-14 (MT1-MMP) causes severe abnormalities in connective tissue, defective angiogenesis and premature death. MMP-2 (gelatinase A) and MMP-14 also play a role in degradation of basement membrane and cell carcinoma invasion. Imatinib blocks the PTK receptor c-kit and forestalls its PTK activity. The aim of the present study was to investigate the expression pattern of MMP-14 and MMP-2 in human papilloma virus (HPV)-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutic agents, imatinib and 5-fluorouracil (5-FU). We incubated the SCC cell lines with imatinib (18 and 30 µmol/ml) and 5-FU (1 and 5 µmol/ml) and detected MMP-14 and MMP-2 by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h. We detected expression of MMP-2 and MMP-14 in all incubated tumour cell lines. With imatinib in particular, we found a reliable trend towards decreased MMP-2 and MMP-14 expression levels in p16-positive and p16-negative SCC tumour cell lines in addition to an induced apoptotic effect. We found statistically significant imatinib-induced suppression of MMP-2- and MMP-14, dependent on the incubation time and the cell line. We detected a significant suppression of MMP-2 and MMP-14 especially in p16-negative HNSCC14C cells after prolonged treatment time with imatinib. Dose escalation of imatinib and 5-FU had no statistically significant effect on the expression of MMP-2 or MMP-14. The p16-positive SCC cells exhibited higher expression of total protein. We detected a significant suppression of MMP-2 and MMP-14 in all the incubated SCC cell lines, partially after treatment with imatinib. We found higher suppression of MMP-2 in the CERV196 cells after incubation with imatinib. We detected a reliable trend towards increased chemosensitivity of p16-positive tumour cells in vitro after treatment with imatinib. Extended studies and clinical trials are needed to further investigate these findings in HPV-associated HNSCC.

Interaction of a CD44+ head and neck squamous cell carcinoma cell line with a stromal cell-derived factor-1-expressing supportive niche: An in vitro model.

The cancer stem cell (CSC) theory implies that CSCs are surrounded by supportive stromal cells, which are known as the CSC niche. Stromal cell-derived factor-1 (SDF-1) shows a multitude of functional effects in head and neck squamous cell carcinoma (HNSCC) cells, including migration and polarization. Therefore, the SDF-1-CXCR4 axis may be involved in the pathophysiology of the progression, recurrence and metastasis of malignant diseases of the head and neck. In the present study, the CD44+ HNSCC UM-SCC-11A cell line was used as a model for CSCs. The interaction between the UM-SCC-11A cells and the supportive microenvironmental cells, including fibrocytes, human umbilical vein endothelial cells (HUVECs) and human microvascular vein endothelial cells (HMVECs) was evaluated. All the cell types that were tested were shown to secrete different concentrations of SDF-1 into the surrounding culture medium [mean (m)fibro, 1243.3±156.2 pg/ml; mHMVEC, 1061.4±23.2 pg/ml; mHUVEC, 849.6±110.9 pg/ml]. The migration of the UM-SCC-11A cells towards the supportive cells was increased by a higher supply of SDF-1 (contrfibro, 315.23±61.55 µm; mfibro, 477.73±143.7 µm; Pfibro=0.003; contrHMVEC, 123.41±66.68 µm; mHMVEC, 249.04±111.95 µm; PHMVEC=0.004; contrHUVEC, 189.7±93.26 µm; mHUVEC, 260.82±161.58 µm). The amount of the UM-SCC-11A cells that migrated towards the differentiated fibrocytes was significantly higher than that which migrated towards the HMVECs or HUVECs (Pfibro/HMVEC=2.12E-11; Pfibro/HUVEC=2.28E-5). Cell-cell interaction by podia formation of the UM-SCC-11A cells was observed in all the supportive cell types that were tested. Broadly based cell-cell contacts were observed. By contrast, digitiform podia formations presented by the UM-SCC-11A cells were determined using fluorescence microscopy. The SDF-1-CXCR4 axis is postulated to be a crucial pathway in the interaction between CSCs and their surrounding supportive cells. Understanding the cell-cell interactions in the CSC niche using in vitro models may aid in gaining further insight into these mechanisms and finding new strategies of therapy in this field.

Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.

Small molecule-based chemotherapeutic approach in p16-positive and -negative HNSCC in vitro.

BACKGROUND: Incidence of oropharyngeal head and neck squamous cell carcinoma (HNSCC) induced by the human papilloma virus (HPV) is rising. HNSCC is the sixth most common neoplasia worldwide. The survival rate remains poor, thus innovative therapy approaches are necessary. Everolimus, an inhibitor of the mammalian target of rapamycin, as well as the multi-tyrosine kinase inhibitors sorafenib (targeting vascular endothelial growth factor receptor, platelet-derived growth factor receptor and RAF) and sunitinib (targeting vascular endothelial growth factor receptor, platelet-derived growth factor receptor, stem cell factor receptor, RET proto-oncogene and colony-stimulating factor), have shown a remarkable antitumor effect against various tumor entities, with moderate side-effects. These drugs are administered orally, which should lead to higher patient compliance and less hospitalisation. AIM: This study sought to evaluate the expression of PDGFR α/ß and hypoxia-inducible factor-1α (HIF-1α) and their alterations induced by everolimus, sorafenib and sunitinib in chemonaïve HPV-positive and HPV-negative HNSCC. To our knowledge, this is the first in vitro study to investigate such cases. MATERIALS AND METHODS: We incubated HPV-positive CERV196 and HPV-negative HNSCC 11A and 14C cells for 2 to 8 days with increasing concentration of drugs. Expression of PDGFR α/ß and HIF-1α was measured by enzyme-linked immunosorbent assay and compared to a chemonaïve controls. RESULTS: Our study showed that PDGFR α/ß and HIF-1α were expressed in all three cell lines. Incubation with everolimus, sorafenib or sunitinib led to a decrease in PDGFR α/ß and HIF-1α expression, depending on the HPV status. A statistically significant alteration of PDGFR α/ß was detected in CERV196 only. Thus, HPV-positive HNSCC exhibited a higher sensitivity to the drugs used compared to HPV-negative HNSCC 11A and 14C tumor cells. A significant reduction of HIF-1α was measured for HNSCC 11A and 14C only. An escalation of drug concentration had no significant effect. CONCLUSION: We showed that these novel agents led to a significant reduction of PDGFR and HIF-1α, depending on the HPV status. HPV positivity is associated with increased chemosensitivity and may be associated with better locoregional control and overall patient survival compared to HPV negativity. Further studies are necessary to investigate the efficacy and safety of these agents in the treatment of HPV-positive and -negative HNSCC in vivo.

Unexpected alteration of ß-catenin and c-KIT expression by 5-FU and docetaxel in p16-positive squamous cell carcinoma compared to HPV-negative HNSCC cells in vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. In several tumour entities, the tyrosine kinase receptor c-KIT is associated with tumour transformation in the epithelial tissue in cases of aberrant expression. Furthermore, tumour development and dissemination are a result of dysregulated cellular pathways such as the WNT/ß-catenin pathway. ß-Catenin is a multifunctional protein within the canonical WNT signalling pathway and a pivotal factor for the stabilization of cell-cell interactions. In malignant tissues, ß-catenin triggers tumour proliferation and progression. The aim of this study is to investigate the expression patterns of c-KIT and ß-catenin in human papillomavirus-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutical agents docetaxel and 5-fluorouracil (5-FU). MATERIALS AND METHODS: We incubated the tumour cell lines with docetaxel (5 µmol/ml) and 5-FU (1 µmol/ml) and detected ß-catenin and c-KIT by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h. RESULTS: We found a reliable trend towards decreased ß-catenin expression levels in p16-positive and p16-negative tumour cell lines when incubated with docetaxel, in addition to induced apoptotic effect. At best, 5-FU had a slight influence on the alteration of the expression of ß-catenin. Dose escalation of docetaxel and 5-FU had no statistically significant effect on the expression of ß-catenin or c-KIT. In HPV-negative HNSCC, a reduced expression level of ß-catenin and c-KIT was detected in an incubation period-dependent manner. p16-transformed SCC (CERV196) cells were characterized by a reduced susceptibility to docetaxel induced alteration of ß-catenin expression. CONCLUSION: We were unable to confirm the clinically-substantiated increased chemosensitivity of p16-positive tumour cells in vitro. Extended studies and clinical trials are needed to investigate these findings further in HPV-associated HNSCC.

Chemotherapeutic alteration of VEGF, PDGF and PDGFRα/ß expression under 5-FU vs. docetaxel in HPV-transformed squamous cell carcinoma compared to HPV-negative HNSCC in vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the most common malignant epithelial tumor in the upper aerodigestive tract. The incidence of HNSCC induced by the oncogenic human papilloma virus (HPV) is rising, indicating a growing importance of the viral etiology. Cell proliferation, migration and tumor vascularization are regulated by a set of angiogenic peptides such as PDGF (platelet-derived growth factor), PDGFRα/ß (platelet-derived growth factor receptor α/ß) and VEGF (vascular endothelial growth factor). In locally advanced HNSCC docetaxel is used for induction chemotherapy (ICT) combined with platinum-based chemotherapy and 5-fluorouracil (5-FU). This study sought to evaluate the expression of angiogenic factors (VEGF, PDGF and PDGFRα/ß) in HPV-positive (CERV196) and HPV-negative squamous cell carcinoma (HNSCC 11A and 14C) and the efficacy of chemotherapy with docetaxel as a potential treatment modality, compared to 5-FU as a single-drug application. MATERIALS AND METHODS: Tumor cell lines were incubated with 5-FU or docetaxel at a concentration of 1.0 and 5.0 µmol/ml. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical analyses were carried out after 48, 72, 120, 192 and 240 hours, in order to identify changes in protein expression of VEGF, PDGF and PDGFRα/ß. RESULTS: We demonstrated a significant reduction of VEGF and PDGFRß expression after incubation with docetaxel by ELISA and of PDGF by immunohistochemistry, irrespective of the HPV status, whereas the application of 5-FU had a significantly weaker impact on the expression of angiogenic peptides. HPV-positive CERV196 cells were characterized by a reduced susceptibility to a docetaxel-altered expression. CONCLUSION: Although neither of the applied drugs are selective anti-angiogenic agents, docetaxel surprisingly was demonstrated to cause a significant decrease of angiogenic factors in this study.

SDF-1-CXCR4 axis: cell trafficking in the cancer stem cell niche of head and neck squamous cell carcinoma.

Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. CXCR4 has been identified as its corresponding receptor. The SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells in the cancer stem cell niche. We evaluated the expression of CD44 as a cancer stem cell marker and of CXCR4 in human HNSCC tissue samples. Afterwards, we monitored the concentration of SDF-1 in peripheral blood samples of HNSCC patients and healthy donors. We showed that CD44 and CXCR4 are expressed in human HNSCC tissues. Markedly, CD44 showed a high expression in HNSCC cells bordering cancer stromal cells. CXCR4 was mainly expressed in HNSCC tumor nests, but not in the surrounding stromal cells. No significant difference was noted between the SDF-1 concentration in the peripheral blood of HNSCC patients compared to healthy donors. We showed that CD44, as a stem cell marker in HNSCC, is located mainly at the borderline of HNSCC tumor nests with the surrounding cells. In addition, we demonstrated that CXCR4 as the corresponding receptor to SDF-1 is highly expressed in HNSCC tumor nests, but not in the tumor stroma. We collected evidence that SDF-1-CXCR4 interaction may be a crucial pathway in cell trafficking in the cancer stem cell niche of HNSCC, while SDF-1 was not detected in the peripheral blood of HNSCC patients. The SDF-1-CXCR4 axis may play an important role in the cancer stem cell theory of HNSCC. As SDF-1α also exhibits a multitude of functional effects on HNSCC cells, such as migration and polarization, it may be possible that the SDF-1-CXCR4 axis is also involved in the pathophysiology of the progression, recurrence and metastasis of malignant disease. Understanding these interactions may help to gain further insight into these mechanisms and as such help to discover new strategies of therapy.

Functional effects of SDF-1α on a CD44(+) CXCR4(+) squamous cell carcinoma cell line as a model for interactions in the cancer stem cell niche.

Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. It is known to have selective effects on cell migration, morphology, survival and cell homing. As such the SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells, the so-called (cancer) stem cell niche. We evaluated the expression of CD44 as a cancer stem cell (CSC) marker and the expression of CXCR4 in the head and neck squamous cell carcinoma (HNSCC) cell line UM-SCC 11A. In addition, we monitored proliferation, formation of podia and migration of UM-SCC 11A cells under the influence of SDF-1α. Whereas SDF-1α induced the formation of podia of CD44(+) CXCR4(+) UM-SCC 11A cells in a dose-dependent manner and the maximum number of cells exhibiting the formation of podia was observed under the influence of 10 ng/ml SDF-1α (P=5.3x10(-6)), the highest number of migrating cells was noted using a concentration of 100 ng/ml (P=0.027). Proliferation and survival were not affected by SDF-1α. We showed that UM-SCC 11A cells could be a target for SDF-1α by CXCR4 expression and these cells also showed characteristics of HNSCC CSCs via CD44 expression. We demonstrated that SDF-1α is a chemoattractant for UM-SCC 11A cells, and a maximum directed migration was achieved under the influence of 100 ng/ml SDF-1α. Changes in cell morphology by presenting filopodia or a prominent uropod were noted following treatment of 10 ng/ml SDF-1α. The SDF-CXCR4 axis may play a crucial role in the interaction between CSCs and their supportive cells in the CSC niche. Understanding these interactions may help to gain further insight into the pathophysiology of the progression and recurrence of malignant diseases and thus help to develop novel strategies for therapy.

Alteration of MMP-2 and -14 expression by imatinib in HPV-positive and -negative squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is known to be the most common neoplasm appearing in the upper aerodigestive tract. The poor 5­year survival rate has remained unchanged in the last decades even though improved techniques in surgery, radiation and chemotherapy have been established. In contrast to the overall decreasing incidence of head and neck cancer in the US, the incidence of HPV-associated oropharyngeal cancer is increasing, indicating the importance of viral etiology. Furthermore, growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM). Matrix metalloproteinases (MMP) have been shown to play key roles in the remodeling of the ECM. Imatinib (STI 571) was originally designed to inhibit the BCR-ABL tyrosine kinase in chronic myeloid leukaemia. But it also has an inhibitory impact, e.g., on the protein-tyrosine-kinase (PTK) receptor c-kit and on its PTK activity in HNSCC. In this study, we incubated the HNSCC cell lines HNSCC 11A and 14C and the p16-positive SCC line CERV196 with increasing concentrations of imatinib or carboplatin. After an incubation time of up to 10 days, we evaluated MMP-2 and -14 expression by ELISA techniques and immunohistochemistry. MMP-2 and -14 expression was demonstrated in all incubated tumor cell lines. Especially incubation with imatinib resulted in a significant decrease in MMP expression in incubated cell lines. Our results indicate that the expression of MMP-2 and -14 is suppressed in the presence of imatinib. Thus, imatinib may exert in part its inhibitory effect on malignant cell growth via the blockage of the signal transduction of PTK receptors. Further studies are warranted, especially keeping in mind the moderate toxicity of imatinib.

Impact of static magnetic fields on human myoblast cell cultures.

Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches.

CD44 as a stem cell marker in head and neck squamous cell carcinoma.

In the recent past, evidence is increasing indicating the existence of a subpopulation of resistant tumor cells in head and neck squamous cell carcinoma (HNSCC) that cannot be eradicated by established antineoplastic treatments. These cancer stem cells (CSCs) have features of somatic stem cells such as selfrenewal, proliferation and differentiation. CD44+ cells in tumors of the head and neck are referred to as CSCs of HNSCC. Expression profiling of CD44 in 29 HNSCC tumors was performed by fluorescence microscopy. ELISA analysis was performed to detect concentration of soluble CD44 in the peripheral blood of 29 HNSCC patients and 11 healthy controls. Expression of CD44 was determined in all HNSCC tissue samples (n=29). In all samples a surface staining pattern was found. The concentration of CD44 in the peripheral blood of HNSCC patients was significantly higher compared to a healthy control group (mHNSCC =13.5 ± 0.5 ng/ ml; mCont = 9.3 ± 0.6 ng/ml; P=0.6 x 10(-12)). The role of CD44 as a marker for CSCs in HNSCC remains to be ascertained. Further experiments might reveal its role as a diagnostic and prognostic factor, and possibly as a therapeutic target.

Do lifestyle factors and general health predict dropout among recently qualified eldercare workers? A two-year follow-up study.

BACKGROUND: The eldercare sector in Denmark as in many industrialised countries is characterised by difficulties in retaining labour. Research suggests a possible imbalance between lifestyle and health among eldercare trainees and the demanding work encountered as eldercare employees. The aim of the present study was to determine the predictive effect of lifestyle and self-rated health on dropout from the Danish eldercare sector two years after qualification. METHODS: We included 4,526 female eldercare trainees in the analyses of lifestyle parameters and 5,023 in the analyses of self-rated health. The participants in this prospective study were recruited from 27 of the 28 Danish colleges for eldercare. We linked survey data with national register data to obtain information about labour market attachment two years after qualification. RESULTS: The results of the present study showed that the poorer self-rated health, the higher the risk for dropout from the labour market (p < 0.0001). However, the results were less consistent regarding the predictive effect of a detrimental lifestyle. We found that overweight/obesity (p = 0.0021 and p = 0.0012) as well as smoking (p = 0.0017) decreased the risk of dropout from eldercare into education. We found no support for increased likelihood of dropout among physically inactive. CONCLUSIONS: The results of the present study show that a poorer self-rated health is a predictor for dropout, not only from the eldercare two years after qualification, but from the labour market as a whole. However, the results were less consistent regarding the predictive effect of a detrimental lifestyle on dropout.