Zerumbone acts as a radiosensitizer in head and neck squamous cell carcinoma.

INTRODUCTION: Zerumbone is a phytochemical compound of the ginger plant Zingiber zerumbet with cytotoxic effects in various cancer cell lines. To date, zerumbone has shown an antiproliferative effect in oral squamous cell carcinoma cells lines. However, the effect of combination with radiation or cisplatin in head and neck squamous cell carcinoma (HNSCC) is unclear. The aim of this study was to investigate the effect of zerumbone alone, and in combination with irradiation and cisplatin on HNSCC cell lines. METHODS: The three HNSCC cell lines SCC25, Cal27 and FaDu were treated with zerumbone, radiation and/or cisplatin. Cell viability and clonogenic assays were performed. The interaction between zerumbone and radiation or cisplatin was evaluated using the combination index. Apoptosis was measured by flow cytometry and cell migration was assessed using a wound healing assay. RESULTS: Treatment with zerumbone resulted in a dose dependent induction of cytotoxicity and apoptosis in all three cell lines. The combination with cisplatin revealed a synergistic to additive effect in Cal27. The clonogenic assay showed a significant radiosensitizing effect in all three cell lines. The wound healing assay showed a reduction of cell migration in Cal27. CONCLUSION: The natural compound zerumbone shows a cytotoxic and proapoptotic effect on HNSCC cell lines. Furthermore, zerumbone enhances the radiation effect in all three cell lines and thus may be a suitable candidate for combination therapy in HNSCC.

Quantitative proteome analysis of Merkel cell carcinoma cell lines using SILAC.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumour of the skin with growing incidence. To better understand the biology of this malignant disease, immortalized cell lines are used in research for in vitro experiments. However, a comprehensive quantitative proteome analysis of these cell lines has not been performed so far. METHODS: Stable isotope labelling by amino acids in cell culture (SILAC) was applied to six MCC cell lines (BroLi, MKL-1, MKL-2, PeTa, WaGa, and MCC13). Following tryptic digest of labelled proteins, peptides were analysed by mass spectrometry. Proteome patterns of MCC cell lines were compared to the proteome profile of an immortalized keratinocyte cell line (HaCaT). RESULTS: In total, 142 proteins were upregulated and 43 proteins were downregulated. Altered proteins included mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, several proteins of the histone family and their variants were highly abundant in MCC cell lines. CONCLUSIONS: The results of this study present a new protein map of MCC and provide deeper insights in the biology of MCC. Data are available via ProteomeXchange with identifier PXD008181.

6-shogaol induces apoptosis and enhances radiosensitivity in head and neck squamous cell carcinoma cell lines.

Ginger (Zingiber officinale Roscoe) is used for a wide array of conditions in traditional medicine in Asia, but little is known about the effect on head and neck cancer. In this study, the effect of two major pharmacologically active compounds of ginger, 6-gingerol and 6-shogaol, were studied on head and neck cancer cell lines. Furthermore, experiments in combination with established treatment methods for head and neck cancer were performed. Proliferation assays showed a dose-dependent reduction of cell viability. Flow cytometry analysis revealed the induction of apoptosis. Western blot analysis indicated that the antiapoptotic protein survivin was suppressed after treatment. Although a combination of 6-shogaol with cisplatin exhibited no synergistic effect, the combination with irradiation showed a synergistic reduction of clonogenic survival. In conclusion, ginger compounds have many noteworthy effects on head and neck cancer cell lines. In particular, the enhancement of radiosensitivity is remarkable.

Oral health status and dental care behaviours of head and neck cancer patients: a cross-sectional study in an Austrian tertiary hospital.

OBJECTIVES: This study aimed to assess the oral health status and dental care behaviours of patients treated for head and neck squamous cell carcinoma (HNSCC) in an Austrian tertiary hospital. MATERIALS AND METHODS: Dental care behaviours, oral hygiene level, caries, and periodontal parameters were assessed in 48 patients treated for HNSCC >6 months ago. RESULTS: Only 52 % requested a dental check-up after HNSCC diagnosis and prior to treatment, and of those, 80 % received some type of dental treatment. At time-point of clinical examination, 69 % of the patients had consulted a dentist within the last year, but 88 % still needed dental treatment; 75 % had at least one tooth with caries and 78 % had moderate to severe periodontitis. CONCLUSION: Although it was recommended, only half of the patients did consult a dentist prior to HNSCC treatment and oral health appeared, in general, low prioritized. CLINICAL RELEVANCE: About 90 % of the current group of head and neck squamous cell carcinoma cancer patients presented large treatment needs, both in regard with caries and periodontal disease, about 20 months after cancer treatment.

Effect of the coffee ingredient cafestol on head and neck squamous cell carcinoma cell lines.

BACKGROUND AND PURPOSE: Cafestol is a diterpene molecule found in coffee beans and has anticarcinogenic properties. The aim of the study was to examine the effects of cafestol in head and neck squamous cell carcinoma (HNSCC) cells. MATERIALS AND METHODS: Three HNSCC cell lines (SCC25, CAL27 and FaDu) were treated with increasing doses of cafestol. Then combination experiments with cisplatin and irradiation were carried out. Drug interactions and possible synergy were calculated using the combination index analysis. Clonogenic assays were performed after irradiation with 2, 4, 6 and 8 Gy, respectively, and the rate of apoptosis was measured with flow cytometry. RESULTS: Treatment of HNSCC cells with cafestol leads to a dose-dependent reduction of cell viability and to induction of apoptosis. Combination with irradiation shows a reduction of clonogenic survival compared to each treatment method alone. In two of the cell lines a significant additive effect was observed. CONCLUSION: Cafestol is a naturally occurring effective compound with growth-inhibiting properties in head and neck cancer cells. Moreover, it leads to a significant inhibition of colony formation.

Assessment of caroverine as a potential chemotherapeutical agent in HNSCC cell lines.

Since the prognosis of head and neck squamous cell carcinoma (HNSCC) still remains poor, identifying novel chemotherapeutic agents is of outmost importance. The anticancer potential of quinoxalines has been described in various tumor entities. Caroverine, also a quinoxaline derivative, has been shown to suppress tumor promotion factors. The aim of this study was to evaluate the effect of caroverine on HNSCC cell lines. The HNSCC cell lines SCC9, SCC25, CAL27, and FaDu were incubated with caroverine alone or in combination with cisplatin, 5-fluorouracil (5-FU) or cetuximab. Cell viability was measured using the CCK-8 assay. The murine 3T3 fibroblast cell line was used to address tissue specificity. Apoptosis was visualized by immunohistochemistry. Caroverine showed a dose-dependent growth inhibition in all cell lines, IC50 values ranged from 75.69 to 179.80 µM. This effect was increased when caroverine was combined with cetuximab or 5-FU. Immunohistochemistry displayed more apoptosis after caroverine treatment compared to controls. Furthermore, caroverine alone had no growth inhibitory effect on 3T3 cells. For the first time, this study provides evidence that caroverine may serve as a supportive drug in the treatment of HNSCC patients.

Radiosensitization of head and neck cancer cells by the phytochemical agent sulforaphane.

BACKGROUND AND PURPOSE: Sulforaphane is a naturally occurring compound found in broccoli and other cruciferous vegetables. Recently it gained attention because of its antiproliferative properties in many cancer cell lines. The aim of this study was to investigate whether sulforaphane could act as a radiosensitizer in head and neck squamous cell carcinoma cell lines. MATERIALS AND METHODS: Four head and neck squamous cell carcinoma cell lines (i.e., (HNSCC) SCC9, SCC25, CAL27, and FADU) were treated with sulforaphane and subsequently irradiated. Then proliferation and clonogenic assays were performed. Apoptosis was detected by flow cytometry. Possible regulation of Akt and Mcl-1 was investigated by western blotting. RESULTS: Sulforaphane and radiation in combination leads to stronger inhibition of cell proliferation and of clonogenic survival than each treatment method alone. Western blot analysis of Akt and Mcl-1 showed no changed expression. CONCLUSION: Sulforaphane is a promising agent in the treatment of head and neck cancer due to its antiproliferative and radio-sensitizing properties. A combination of sulforaphane and radiation decreases clonogenic survival. Apoptosis is not regulated through Akt or the Mcl-1 protein.

HS-173, a selective PI3K inhibitor, induces cell death in head and neck squamous cell carcinoma cell lines.

BACKGROUND: The selective PI3K (Phosphatidylinositol 3-kinase) inhibitor HS-173 has anticancer activity in non-small cell lung cancer and pancreatic cancer cells. Of all head and neck squamous cell carcinomas (HNSCC) 20% harbor specific mutations in the genome. The aim of this study was to investigate the effect of HS-173 on HNSCC cell lines. METHODS: The cell lines SCC25, CAL27 and FaDu were incubated with HS-173. Its antiproliferative effect was determined using the CCK­8 cell proliferation assay. Combined incubation with cisplatin was performed and combination index analysis was conducted. To investigate its effect on radiotherapy, cells were irradiated with 2, 4, 6 and 8 Gy, respectively. Synergistic effects of radiation and HS-173 were measured by proliferation assays and clonogenic survival. RESULTS: The use of HS-173 induced significant reduction of cell proliferation across all cell lines. Most interestingly, it showed a synergistic effect with cisplatin treatment. Clonogenic survival revealed a radiosensitizing effect in CAL27 and FaDu cells. The HS-173 caused significant induction of apoptosis in SCC25 and FaDu cells. CONCLUSION: The selective PI3K inhibitor HS-173 is a potent chemosensitizing and also radiosensitizing drug in treatment of HNSCC cell lines and could be an effective treatment in PI3K-mutated HNSCC.

Prognostic Relevance of Thyroid-Hormone-Associated Proteins in Adenoid Cystic Carcinoma of the Head and Neck.

The proteins sodium iodide symporter (NIS), µ-crystallin (CRYM), and thyroid hormone receptor beta (THRB) have been associated with prognosis in various cancer entities. While NIS and THRB may serve as possible therapeutic targets, the role of CRYM in cancer is still unclear. Protein levels of 44 patients with adenoid cystic carcinoma of the head and neck were analyzed using immunohistochemistry and correlated with clinicopathological data and outcome. NIS was positive in 72%, CRYM was positive in 55%, and THRB was positive in 39% of the patients. CRYM-positive adenoid cystic carcinomas were associated with a better cause-specific survival. Thus, our data indicate that CRYM might be a suitable positive prognostic marker in adenoid cystic carcinoma of the head and neck. Furthermore, expression of NIS was present in most patients and therefore evaluation of the use of radioiodine treatment is recommended.

Intraparotid and cervical lymph nodes metastasis in primary parotid gland carcinoma-impact on clinical outcome.

OBJECTIVE: The aim of this study was to investigate the prognostic value of evaluation of intraparotid and cervical lymph node metastases in primary parotid cancer. STUDY DESIGN: A retrospective medical chart review and histopathologic evaluation of all patients surgically treated for primary parotid cancer during the period 1993 to 2010 was performed. The presence and ratio of intraparotid and cervical lymph node metastases were assessed and determined as primary predictor variables. Overall survival (OS) and disease-free survival (DFS) were defined as primary outcome variables. RESULTS: In total, 50 patients were included. The presence of pathologic cervical lymph nodes (P = .005) and a high cervical lymph node ratio (LNR) (P = .0001) had a significant association with worse OS. Worse DFS was found in patients with a high cervical LNR (P = .001) and intraparotid lymph node metastases (P = .029). In high-grade carcinoma, a high LNR showed worse DFS (P = .05). A high cervical LNR (P = .012) and resection margin status (P = .002) were identified as independent prognostic markers for OS and the presence of intraparotid lymph nodes for DSS (P = .05). CONCLUSIONS: Evaluation of patterns of lymph node metastases provides additional prognostic value in patients with primary parotid gland cancer.

A Nomogram to Predict the Outcome of Fine Needle Aspiration Cytology in Head and Neck Masses.

Fine needle aspiration cytology (FNAC) is an important diagnostic tool for tumors of the head and neck. However, non-diagnostic or inconclusive results may occur and lead to delay in treatment. The aim of this study was to evaluate the factors that predict a successful FNAC. A retrospective search was performed to identify all patients who received an FNAC at the Department of Otorhinolaryngology, Head and Neck Surgery, Medical University of Vienna. The variables were patients' age and sex, localization and size of the punctured structure, previous radiotherapy, experience of the head and neck surgeon, experience of the pathologist and the FNAC result. Based on these parameters, a nomogram was subsequently created to predict the probability of accurate diagnosis. After performing 1221 FNACs, the size of the punctured lesion (p = 0.0010), the experience of the surgeon and the pathologist (p = 0.00003) were important factors for a successfully procedure and reliable result. FNACs performed in nodes smaller than 20 mm had a significantly worse diagnostic outcome compared to larger nodes (p = 0.0004). In conclusion, the key factors for a successful FNAC are nodal size and the experience of the head and neck surgeon and the pathologist.

ELMO3 - a Negative Prognostic Marker in Minor Salivary Gland Carcinoma.

Engulfment and cell motility 3 protein (ELMO3) is a protein that is involved in cell migration and promotes the remodeling of the cytoskeleton. Moreover, it is described as a prognostic marker in several cancers. The aim of this study was to evaluate ELMO3 expression in patients with minor salivary gland carcinoma. The expression of ELMO3 was examined by immunohistochemistry. The intensity of staining was evaluated and data was correlated to clinical outcome. Forty-six patients with complete clinical data were included into statistical analysis. ELMO3 expression was observed in 85% of the cases. High staining intensity of ELMO3 correlated with a significantly worse disease free survival (p = .0495) and a higher recurrence rate (p = .0071). In conclusion, it is still difficult to predict the clinical outcome of patients with minor salivary gland carcinoma. Evaluation of ELMO3 might serve as a clinical prognostic marker in future.

Overexpression of LAPTM4B-35 is a negative prognostic factor in head and neck squamous cell carcinoma.

Overexpression of LAPTM4B-35 (lysosomal-associated transmembrane protein 4ß-35) is associated with a poor prognosis in numerous malignant tumours. Expression patterns and effects of LAPTM4B-35 on head and neck squamous cell carcinomas (HNSCC) are unknown. The aim of this study was to investigate the prognostic relevance of LAPTM4B-35 in HNSCC. Tissue microarrays were constructed with primary tumours and associated lymph node metastases isolated from 127 patients. The expression of LAPTM4B-35 was investigated by immunohistochemistry and the results were correlated with survival data. LAPTM4B-35 in the primary tumour was highly expressed in 47.2% of the patients (60/127). LAPTM4B-35 expression was significantly associated with tumour stage. Moreover, overexpression of LAPTM4B-35 correlated with a significantly worse disease-free survival (10.23 years vs. not reached) and a higher recurrence rate (40.7% vs. 25%). High expression of LAPTM4B-35 in lymph node metastasis was found in 29.2% of cases. In 19.4% of cases, high LAPTM4B-35 expression was observed in both the primary tumour and corresponding lymph node metastases. In conclusion, our data indicates that overexpression of LAPTM4B-35 is associated with poor prognosis and may therefore serve as a new prognostic marker in HNSCC.

AZD5582, an IAP antagonist that leads to apoptosis in head and neck squamous cell carcinoma cell lines and is eligible for combination with irradiation.

CONCLUSION: On the one hand, AZD5582, an inhibitor of inhibitor of apoptosis family proteins (IAP), leads to cellular growth arrest and induction of apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines. On the other hand, it is a viable candidate for combination therapy with irradiation. OBJECTIVES: The aim and purpose of this study was to evaluate the effects of AZD5582 on HNSCC cell lines. METHODS: HNSCC cell lines SCC25, Cal27, and FaDu were used for all cell culture experiments. Proliferation assays were used to assess a potential inhibitory effect of AZD5582 and a combination therapy of the IAP inhibitor and irradiation. Colony forming assays were used to determine long-term effects of a combined treatment. Apoptosis was measured via flow cytometry and wound-healing assays were performed. RESULTS: All three cell lines showed a dose-dependent cytotoxic effect after treatment with AZD5582. It was possible to observe a synergistic and additive effect after short-term treatment of AZD5582 and irradiation in Ca27 and FaDu cells, respectively. All test cell lines showed a significant inhibition of colony formation after combined treatment. Treatment of AZD5582 resulted in apoptosis in SCC25, Cal27, and FaDu cells.

Effect of thymoquinone on head and neck squamous cell carcinoma cells in vitro: Synergism with radiation.

Thymoquinone (TQ) is the main bioactive constituent present in black seed oil (Nigella sativa); it has shown anti-inflammatory and anti-neoplastic effects in various cancer cell types. The aim of the present study was to investigate the effects of TQ on head and neck squamous cell carcinoma (HNSCC) cell lines, on its own and in combination with radiation and cisplatin, respectively. The SCC25 and CAL27 HNSCC cell lines were treated with TQ alone and in combination with cisplatin or radiation, respectively. Proliferation assays and clonogenic assays were performed. Apoptosis was detected by flow cytometry. TQ exhibited dose-dependent cytotoxicity via apoptosis in the investigated cell lines. In combination with cisplatin, TQ resulted in no significant increase in cytotoxicity. Combined with radiation, TQ significantly reduced clonogenic survival compared with each treatment method alone. TQ is a promising agent in the treatment of head and neck cancer due to its anti-proliferative and radiosensitizing properties. However, the combination of TQ with cisplatin showed no therapeutic benefit in vitro.

Effect of the histone deacetylase inhibitor resminostat on head and neck squamous cell carcinoma cell lines.

BACKGROUND: Carcinogenesis is determined by various epigenetic events, such as histone deacetylation. The purpose of this study was to investigate the effect of the new histone deacetylase inhibitor resminostat on head and neck squamous cell carcinoma (HNSCC) cell lines. METHODS: The cytotoxicity of resminostat and cisplatin on HNSCC cell lines SCC25, CAL27, and FaDu was determined using CCK-8 cell proliferation assay and combination index analysis. Cells were irradiated with 2 to 8 Gray. Apoptosis was measured using flow cytometry and expression of Mcl-1, p-AKT, and survivin was investigated. RESULTS: Treatment with resminostat showed a decrease of cell proliferation of HNSCC cell lines. In addition, a synergistic effect with cisplatin as well as with radiation treatment could be observed. Induction of cell death and dose-dependent downregulation of survivin was evident in all cell lines. CONCLUSION: Resminostat is a promising treatment of HNSCC because of its antiproliferative, chemosensitizing, and radiosensitizing effects. © 2017 Wiley Periodicals, Inc. Head Neck 39: 900-907, 2017.

ELMO3 expression indicates a poor prognosis in head and neck squamous cell carcinoma - a short report.

PURPOSE: Previously, the engulfment and cell motility 3 (ELMO3) protein has been reported to be involved in cell migration and cytoskeletal remodeling. As of yet, nothing is known about the role of ELMO3 in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to asses ELMO3 expression in postoperatively irradiated HNSCC patients and to evaluate a possible correlation between this expression and patient survival. METHODS: 125 postoperatively irradiated HNSCC patients were included in this study. ELMO3 expression was assessed using immunohistochemistry (IHC). The expression of ELMO3 in the respective HNSCC tumor tissues and its lymph node metastases was correlated with patient survival using Kaplan-Meier curve analyses. RESULTS: Through IHC, ELMO3 expression was detected in 71.2% of the HNSCC cases tested. We found significantly increased overall and disease-free survival rates and decreased recurrence rates in patients with no detectable ELMO3 expression. In reverse, we found that ELMO3 expression served as an independent marker for a decreased overall and disease-free survival. CONCLUSION: Our data indicate that in the surgically treated and postoperatively irradiated patients tested, ELMO3 expression serves as a predictive marker for reduced survival.

Role of cancer stem-cell marker doublecortin-like kinase 1 in head and neck squamous cell carcinoma.

BACKGROUND: So far, no data is available on the role of the tumor stem cell marker doublecortin-like kinase 1 (DCLK1) in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to evaluate DCLK1 expression in HNSCC patients that underwent surgery and postoperative radiotherapy, and to assess its potential as a therapeutic target in vitro. METHODS: We immunohistochemically stained for DCLK1 in 127 sections of HNSCC samples obtained during surgery of HNSCC patients and correlated the expression to patients' overall- and disease-free survival, as well as human papilloma virus (HPV) status. Additionally, we compared our survival data with data obtained from The Cancer Genome Atlas (TCGA). The effects of the DCLK1 inhibitor LRRK-2-in-1 on HNSCC cell lines alone and in combination with irradiation. RESULTS: Expression of DCLK1 in 127 patients was associated with poor survival. In particular, DCLK1 expression had a significant impact on survival of oropharyngeal carcinoma patients. Specifically, DCLK1+/HPV- patients had the worst prognosis after simultaneous assessment of DCLK1 and HPV status in comparison to the other three possible DCLK1/HPV constellations. Higher levels of DCLK1 mRNA were also associated with poor clinical outcome. Inhibition of DCLK1 in our HNSCC cell lines led to growth arrest and induction of apoptosis. The combination of DCLK1 inhibition with irradiation had a synergistic effect. CONCLUSION: Firstly, DCLK1 is a prognostic biomarker for shortened survival. Secondly, through inhibition of DCLK1, it may serve as a therapeutic target as well.

Arsenic trioxide enhances the cytotoxic effect of cisplatin in head and neck squamous cell carcinoma cell lines.

Arsenic trioxide (ATO) has been approved for the treatment of relapsed acute promyelocytic leukaemia. The aim of this study was to determine whether ATO would lead to cell death in head and neck squamous cell carcinoma (HNSCC) cell lines and whether it was able to enhance the cytotoxicity of cisplatin, a standard chemotherapeutic agent. The four HNSCC cell lines SCC9, SCC25, CAL27 and FADU were treated with ATO or cisplatin alone or with ATO and cisplatin in combination. Cytotoxicity assays, immunohistochemistry, western blot analysis and flow cytometry were carried out. Possible interactions between the two drugs were calculated using the Chou-Talalay equation. Ther results demonstrated a synergistic cytotoxic effect of the combination of ATO and cisplatin at high doses. The two agents induced apoptosis in all four HNSCC cell lines. In conclusion, this study showed that ATO is a promising therapeutic drug with cytotoxic effects in HNSCC. We demonstrated a synergistic effect in the combined treatment with cisplatin at high doses.