[Show abstract][Hide abstract] ABSTRACT: Purpose: Cancer stem cells are resistant to chemotherapy and radiotherapy and are implicated in tumor relapse and high patient mortality. Substances impairing cancer stem cell activity could be very useful as novel cancer therapeutics. Caffeic acid phenethyl ester (CAPE), a component of propolis, whose antitumor activity has been confirmed in several tumor types in vitro, has recently been shown to inhibit the growth of some cancer stem cell types. Methods: An ALDH1-positive fraction was isolated from UT-SCC-74A cells as cancer stem cells by magnetic-activated cell sorting. The isolated cells were characterized by sphere formation assay and biomarkers of cancer stem cells were analyzed by quantitative RT-PCR. To demonstrate the effect of CAPE on head and neck squamous cancer stem cells, XTT cell viability assays were performed. Results: Increasing concentrations of CAPE decrease the viability of head and neck squamous cancer stem cells. IC50 value was calculated from XTT results as 50.29 μg/ml. Conclusion: CAPE has a growth inhibitory effect on head and neck squamous cancer stem cells in vitro.
No preview · Article · Jan 2015 · Clinical and investigative medicine. Médecine clinique et experimentale
[Show abstract][Hide abstract] ABSTRACT: Purpose: A subpopulation of cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC) play a critical role in invasion, metastasis, and tumour recurrence posttherapy. 5-Fluorouracil (5-FU) is a pyrimidine analogue that is used in the treatment of HNSCC. Thymoquinone (TQ), found in Cumin (Nigella sativa) seed oil, has anti-diabetic, anti-oxidant, anti-inflammatory, and antitumour effects. The purpose of this study was to examine the effects of 5-FU and TQ on CSCs. Methods: ALDH1 (+) cells were isolated from the HNSCC cell line UT-SCC-74A using magnetic activated cell sorting (MACS) and sphere formation was confirmed. Real-time RTPCR revealed was used to measure expression levels of a number of stem cell markers, including OCT4, SOX2, and KLF-4. To investigate the effects of the therapeutic agents, 5-FU and TQ, concentration-dependent assay procedures were used and cell viability after treatment was measured using XTT assay. Results: ALDH1 (+) cells formed spheres while the ALDH1 (-) cells did not. Real-time RT-PCR revealed that a number of stem cell markers, including OCT4, SOX2, and KLF-4, were up-regulated in the CSCs. XTT results showed that, when given alone at a dose of 50 ng/mL, neither agent had a statistically significant cytotoxic effect on CSCs. However, when dosed in combination, TQ and 5-FU had a synergistic effect on CSC cytotoxicity. Conclusion: We report the effects of TQ and 5-FU on CSCs in HNSCC.
No preview · Article · Jan 2015 · Clinical and investigative medicine. Médecine clinique et experimentale
[Show abstract][Hide abstract] ABSTRACT: Radiotherapy remains the backbone of head and neck cancer therapy but response is sometimes impeded by tumor radioresistance. Identifying predictive biomarkers of radiotherapy response is a crucial step towards personalized therapy. The aim of this study was to explore gene expression data in search of biomarkers predictive of the response to radiotherapy in head and neck squamous cell carcinoma (HNSCC). Microarray analysis was performed on five cell lines with various intrinsic radiosensitivity, selected from a panel of 29 HNSCC cell lines. The bioinformatics approach included Gene Ontology (GO) enrichment profiling and Ingenuity Pathway Analysis (IPA). The GO-analysis detected 16 deregulated categories from which development, receptor activity and extracellular region represented the largest groups. Fourteen hub genes (CEBPA, CEBPB, CTNNB1, FN1, MYC, MYCN, PLAU, SDC4, SERPINE1, SP1, TAF4B, THBS1, TP53 and VLDLR) were identified from the IPA network analysis. The hub genes in the highest ranked network, (FN1, SERPINE1, THBS1 and VLDLR) were further subjected to qPCR analysis in the complete panel of 29 cell lines. Of these genes, high FN1 expression associated to high intrinsic radiosensitivity (p = 0.047). In conclusion, gene ontologies and hub genes of importance for intrinsic radiosensitivity were defined. The overall results suggest that FN1 should be explored as a potential novel biomarker for radioresistance.
No preview · Article · Oct 2014 · Cancer biology & therapy
[Show abstract][Hide abstract] ABSTRACT: OFP-12-001
Early stage oral tongue cancer: A prognostic model for survival
A. Almangush*, R. Coletta, I. Bello, C. Bitu, J. Hagström, Y. Soini, P. Koivunen, R. Grénman, T. Salo, I. Leivo
*Haartman Institute, Dept. of Pathology, Helsinki, Finland
Objective: Oral tongue squamous cell carcinoma (OTSCC) is the most common and most aggressive cancer diagnosed within the oral cavity and characterized by poor prognosis even at early stage (cT1-2N0). We introduced a simple prognostic model for early stage OTSCC based on two well-defined histopathologic parameters, tumor budding and depth of invasion.
Method: Three hundred and eleven cases treated for early stage (cT1-T2N0) OTSCC were included in this multicenter retrospective study. Tumor budding (B) and depth of invasion (D) were scored on hematoxylin-eosin stained cancer slides. The scored parameters were combined in a predictive model (BD model).
Results: In the multivariate analysis, cases with high risk score (BD score 2) were showed to have more loco-regional recurrence (hazard ratio [HR], 2.49; 95 % confidence interval [CI], 1.41–4.39) and more deaths from OTSCC (HR: 6.52; 95 % CI: 2.68–15.83).
Conclusion: BD model is a promising indicator for patients’ survival in early OTSCC. Early stage OTSCC cases with high BD score (score 2) might benefit from multimodality treatment. Further validation of BD
model is recommended.
[Show abstract][Hide abstract] ABSTRACT: Background
Here, the hypoxic regulation of the PERK/ATF4/LAMP3-arm of the UPR in head and neck squamous cell carcinoma (HNSCC) was examined.MethodsLAMP3 expression was determined in patient biopsies by immunohistochemistry and correlated to clinicopathological parameters. mRNA and protein expression for PERK, ATF4 and LAMP3 was evaluated after hypoxic exposure of HNSCC cell lines.ResultsIn HNSCC patients, high LAMP3 expression correlated with N-stage (P=0.019) and the occurrence of distant metastases during follow-up (P=0.039). Patients with high LAMP3 levels had a worse metastasis-free survival (P=0.008). Intriguingly, LAMP3 expression was localized exclusively in normoxic areas of tumors and xenografts. Expression of PERK, p-PERK, p-eIF2α, ATF4 and LAMP3 was not universally induced in hypoxic HNSCC cell lines. Exposure to ER-stress stimulated PERK, ATF4 and LAMP3 expression.ConclusionLAMP3 is relevant for prognosis in HNSCC. However, the PERK/ATF4/LAMP3-arm of the UPR responds differently to hypoxia in HNSCC compared to other tumor types. Head Neck, 2014
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Wood has been used as a model material for the development of novel fiber-reinforced composite bone substitute biomaterials. In previous studies heat treatment of wood was perceived to significantly increase the osteoconductivity of implanted wood material. AIM: The objective of this study was to examine some of the changing attributes of wood materials that may contribute to improved biological responses gained with heat treatment. METHODS: Untreated and 140 degrees C and 200 degrees C heat-treated downy birch (Betula pubescens Ehrh.) were used as the wood materials. Surface roughness and the effect of pre-measurement grinding were measured with contact and non-contact profilometry. Liquid interaction was assessed with a dipping test using two manufactured liquids (simulated blood) as well as human blood. SEM was used to visualize possible heat treatment-induced changes in the hierarchical structure of wood. RESULTS: The surface roughness was observed to significantly decrease with heat treatment. Grinding methods had more influence on the surface contour and roughness than heat treatment. The penetration of the human blood in the 200 degrees C heat-treated exceeded that in the untreated and 140 degrees C heat-treated materials. SEM showed no significant change due to heat treatment in the dry-state morphology of the wood. DISCUSSION: The results of the liquid penetration test support previous findings in literature concerning the effects of heat treatment on the biological response to implanted wood. Heat-treatment has only a marginal effect on the surface contour of wood. The highly specialized liquid conveyance system of wood may serve as a biomimetic model for the further development of tailored fiber-composite materials.
No preview · Article · May 2014 · Bio-medical materials and engineering
[Show abstract][Hide abstract] ABSTRACT: The presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. alphaB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether alphaB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces alphaB-crystallin expression in vitro and in this way may confer hypoxic cell survival.
In 38 HNSCC biopsies, the overlap between immunohistochemically stained alphaB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of alphaB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of alphaB-crystallin on cell survival under hypoxic conditions.
In all biopsies alphaB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased alphaB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, alphaB-crystallin expression was increased. alphaB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced alphaB-crystallin upregulation. Moreover, it was found that decreased alphaB-crystallin levels reduced cell survival under hypoxic conditions CONCLUSIONS: We provide the first evidence that hypoxia stimulates upregulation of alphaB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of alphaB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.
[Show abstract][Hide abstract] ABSTRACT: Kinases downstream of growth factor receptors have been implicated in radioresistance and are, therefore, attractive targets to improve radiotherapy outcome in head and neck squamous cell carcinoma (HNSCC) patients.
An antibody-based array was used to quantify the expression levels of multiple phospho-kinases involved in growth factor signaling in nine untreated or irradiated HNSCC lines. Radiosensitivity was assessed with clonogenic cell survival assays and correlated with the expression levels of the phospho-kinases. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to increase radiosensitivity in the 3 most radioresistant HNSCC lines.
The basal expression of phosphorylated Yes, Src and STAT5A, and the expression after radiotherapy of phosphorylated AKT, MSK1/2, Src, Lyn, Fyn, Hck, and STAT6, were correlated with radiosensitivity in the panel of HNSCC lines. In combination with radiotherapy, inhibitors of AKT, p38 and Src Family Kinases (SFK) were variably able to reduce survival, whereas MEK1/2, STAT5 and STAT6 inhibition reduced survival in all cell lines. The combined effect of radiotherapy and the kinase inhibitors on cell survival was mostly additive, although also supra-additive effects were observed for AKT, MEK1/2, p38 and STAT5 inhibition.
Kinases of the AKT, MAPK, STAT and SFK pathways correlated with radiosensitivity in a panel of HNSCC lines. Particularly inhibitors against MEK1/2, STAT5 and STAT6 were able to decrease survival in combination with radiotherapy. Hence, inhibitors against these kinases have the potential to improve radiotherapy outcome in HNSCC patients and further research is warranted to confirm this in vivo.
[Show abstract][Hide abstract] ABSTRACT: Background:
This study investigated the use of 3 different established cell-sorting strategies to isolate and characterize stem cells from head and neck cancer cell lines.
Five low-passage cell lines were subjected to cell sorting based on Hoechst side population, Aldefluor, and CD44 expression. Isolated cell populations were studied for gene expression, radiosensitivity, and chemosensitivity to cisplatin and paclitaxel.
Each sorting method identified a different set of genes associated with different gene ontology categories, with mitosis being the only common category. CD44-associated gene changes were almost exclusively associated with cell cycle and in particular mitosis. There were no significant differences in radiosensitivity or cisplatin sensitivity of stem or non-stem cells, but CD44-isolated stem cells were more resistant to paclitaxel.
This study suggested that CD44 may be the most promising cell-sorting strategy to isolate and investigate the impact of stem cells in head and neck squamous cell cancer (HNSCC).
[Show abstract][Hide abstract] ABSTRACT: Cancer cells can obtain their ability to invade and metastasise by undergoing epithelial-to-mesenchymal transition (EMT). Exploiting this mechanism of cellular plasticity, malignant cells can remodel their actin cytoskeleton and down-regulate proteins needed for cell-cell contacts. The mechanisms of cytoskeletal reorganisation resulting in mesenchymal morphology and increased invasive potential are poorly understood. Actin nucleating formins have been implicated as key players in EMT. Here, we analysed which formins are altered in squamous cell carcinoma related EMT. FHOD1, a poorly studied formin, appeared to be markedly upregulated upon EMT. In human tissues FHOD1 was primarily expressed in mesenchymal cells, with little expression in epithelia. However, specimens from oral squamous cell cancers demonstrated consistent FHOD1 upregulation in mesenchymally transformed cells at the invasive edge. This upregulation was confirmed in an oral squamous carcinoma model, where FHOD1 expression was markedly increased upon EMT in a PI3K signalling dependent manner. In the EMT cells FHOD1 contributed to the spindle-shaped morphology and mesenchymal F-actin organization. Furthermore, functional assays demonstrated that FHOD1 contributes to cell migration and invasion. Finally, FHOD1 depletion reduced the ability of EMT cancer cells to form invadopodia and to degrade extracellular matrix. Our results indicate that FHOD1 participates in cytoskeletal changes in EMT. In addition, we show that FHOD1 upregulation occurs during cancer cell EMT in vivo, which indicates that FHOD1 may contribute to tumour progression.
[Show abstract][Hide abstract] ABSTRACT: To investigate possible differences in the effects of soluble factors from oral squamous cell carcinoma (SCC) cells (UT-SCC-87) and normal oral keratinocytes (NOK) on fibroblast expression of genes involved in tumor stroma turnover.
Transwell co-cultures with fibroblasts in collagen gels, and SCC cells or NOK in inserts were carried out. Fibroblast gene expression was measured with real-time polymerase chain reaction (PCR).
The expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) was up-regulated in co-cultures with SCC cells but not with NOK. In contrast, both SCC cells and NOK regulated matrix metalloproteinase-1 (MMP1) and -3, and tissue inhibitor of metalloproteinases-2 (TIMP2) and -3 to a similar extent, while MMP2 and TIMP1 were largely unaffected. Interleukin 1 alpha (IL1α) up-regulated both MMP1 and MMP3 and down-regulated PAI-1, TIMP2 and -3.
SCC and NOK regulate fibroblast expression of genes involved in tumor stroma turnover differentially in vitro. These observations may contribute to a better understanding of the mechanisms behind extracellular matrix turnover in tumors.
No preview · Article · Aug 2013 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: To validate the association of phosphorylated (p)AKT with lymph node metastasis in an independent, homogeneous cohort of patients with larynx cancer.
Seventy-eight patients with laryngeal cancer were included. Epidermal growth factor receptor, pAKT, vimentin, E-cadherin, hypoxia, and blood vessels were visualized in biopsy material using immunohistochemistry. Positive tumor areas and spatial relationships between markers were assessed by automated image analysis. In 6 laryngeal cancer cell lines, E-cadherin and vimentin messenger RNA was quantified by real-time polymerase chain reaction and by immunohistochemistry before and after treatment with the pAKT inhibitor MK-2206.
A significant correlation was found between low pAKT in the primary tumor and positive lymph node status (P=.0005). Tumors with lymph node metastases had an approximately 10-fold lower median pAKT value compared with tumors without lymph node metastases, albeit with large intertumor variations, validating our previous results. After inhibition of pAKT in laryngeal cancer cells with MK-2206, up-regulation of vimentin and a downregulation of E-cadherin occurred, consistent with epithelial-mesenchymal transition.
Low pAKT expression in larynx tumors is associated with lymph node metastases. Further, inhibition of pAKT in laryngeal cancer induces epithelial-mesenchymal transition, predisposing for an increased metastatic risk.
Full-text · Article · Jul 2013 · International journal of radiation oncology, biology, physics
[Show abstract][Hide abstract] ABSTRACT: Background
αB-crystallin is able to modulate vascular endothelial growth factor (VEGF) secretion. In many solid tumors VEGF is associated with angiogenesis, metastasis formation and poor prognosis. We set out to assess whether αB-crystallin expression is correlated with worse prognosis and whether this is related to VEGF secretion and cell motility in head and neck squamous cell carcinoma (HNSCC).
αB-crystallin expression was determined immunohistochemically in tumor biopsies of 38 HNSCC patients. Locoregional control (LRC) and metastasis-free survival (MFS) of the patients were analyzed in relation to αB-crystallin expression. Additionally, the effects of αB-crystallin knockdown on VEGF secretion and cell motility were studied in vitro.
Patients with higher staining fractions of αB-crystallin exhibited a significantly shorter MFS (Log-Rank test, p < 0.005). Under normoxic conditions αB-crystallin knockdown with two different siRNAs in a HNSCC cell line reduced VEGF secretion 1.9-fold and 2.1-fold, respectively. Under hypoxic conditions, a similar reduction of VEGF secretion was observed, 1.9-fold and 2.2-fold, respectively. The effect on cell motility was assessed by a gap closure assay, which showed that αB-crystallin knockdown decreased the rate by which HNSCC cells were able to close a gap by 1.5- to 2.0-fold.
Our data suggest that αB-crystallin expression is associated with distant metastases formation in HNSCC patients. This association might relate to the chaperone function of αB-crystallin in mediating folding and secretion of VEGF and stimulating cell migration.
[Show abstract][Hide abstract] ABSTRACT: Invasion is an important hallmark of cancer involving interactions between the tumor microenvironment and the cancer cells. Hypoxia, low oxygen level, is related to increased invasion and metastasis in many cancers. The aim was to elucidate the effect of hypoxia on invasion of oral squamous cell carcinoma cells (OSCC), and the applicability of a novel 3-dimentional myoma organotypic invasion model in hypoxia experiments. OSCC cell lines (primary oral carcinoma derived cellsUT-SCC-43A, recurrent oral carcinoma cells UT-SCC-43B and aggressive tongue carcinoma cells HSC-3) were studied for their migration and invasion capabilities under normoxia, hypoxia, and in the presence a hypoxia-mimicker cobalt chloride. As expected, the recurrent UT-SCC-43B cells were significantly more aggressive than the primary tumor derived cells. In contrast to tongue carcinoma HSC-3 cells, they only mildly responded to hypoxia in the migration or invasion assays, indicating a cell line specific response of hypoxia on the invasive potential. The modification of the organotypic human tissue-derived matrix via the removal of various yet unidentified soluble factors by rinsing the tissue resulting in stripped matrixsubstantially changed the invasion pattern of HSC-3 cells and the outcomes of hypoxic treatments. Only in the stripped tissue hypoxia significantly increased invasion, whereas in native intact tissue the induced invasion was not observed. This demonstrates the importance of the soluble factors to the invasion pattern andto the hypoxia response. A metastasis and poor prognosis marker, hypoxia-regulated lysyl oxidase (LOX), was present in the myoma tissue, but could be removed by rinsing. The inhibition of LOX resulted in a decrease in invasion area, but only very mildly in invasion depth. Thus, it may have a role in the modulation of the invasion pattern. Another hypoxia-related poor prognosis marker carbonic anhydrase 9 (CAIX) was induced in HSC-3 cells both by the hypoxic exposure andinterestingly in invading HSC-3 cells inside the tissue even in normoxic conditions. In conclusion,this suggests that the intact myoma organotypic model offersoptimally hypoxicsurroundings, thus being an excellent human tumor microenvironment mimicker.
Full-text · Article · Dec 2012 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: Background
Only a minority of cancer patients benefits from the combination of EGFR-inhibition and radiotherapy in head and neck squamous cell carcinoma (HNSCC). A potential resistance mechanism is activation of EGFR and/or downstream pathways by stimuli in the microenvironment. The aim of this study was to find molecular targets induced by the microenvironment by determining the in vitro and in vivo expression of proteins of the EGFR-signaling network in 6 HNSCC lines. As hypoxia is an important microenvironmental parameter associated with poor outcome in solid tumors after radiotherapy, we investigated the relationship with hypoxia in vitro and in vivo.
Six human HNSCC cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo). Expression levels were determined via western blot analysis and localization of markers was assessed via immunofluorescent staining. To determine the effect of hypoxia and pAKT-inhibition on cell survival, cells were incubated at 0.5% O2 and treated with MK-2206.
We observed strong in vitro-in vivo correlations for EGFR, pEGFR and HER2 (rs=0.77, p=0.10, rs=0.89, p=0.03) and rs=0.93, p=0.02, respectively), but not for pAKT, pERK1/2 or pSTAT3 (all rs<0.55 and p>0.30). In vivo, pAKT expression was present in hypoxic cells and pAKT and hypoxia were significantly correlated (rs=0.51, p=0.04). We confirmed in vitro that hypoxia induces activation of AKT. Further, pAKT-inhibition via MK-2206 caused a significant decrease in survival in hypoxic cells (p<0.01), but not in normoxic cells.
These data suggest that (p)EGFR and HER2 expression is mostly determined by intrinsic features of the tumor cell, while the activation of downstream kinases is highly influenced by the tumor microenvironment. We show that hypoxia induces activation of AKT both in vitro and in vivo, and that hypoxic cells can be specifically targeted by pAKT-inhibition. Targeting pAKT is thus a potential way to overcome therapy resistance induced by hypoxia and improve patient outcome.