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.

Transformation of pleomorphic adenoma to carcinoma ex pleomorphic adenoma of the parotid gland is independent of p53 mutations.

BACKGROUND AND OBJECTIVES: This retrospective study was performed to evaluate the status of p53 in pleomorphic adenomas and carcinomas ex pleomorphic adenoma in the parotid gland. As loss or mutation of p53 can cause malignant transformation, the possible degeneration of pleomorphic adenomas to carcinomas ex pleomorhic adenoma was investigated by mutational analysis. METHODS: Twenty-five Patients including 14 patients with pleomorphic adenomas and 11 patients with carcinoma ex pleomorphic adenoma of the parotid gland were examined for p53 status. DNA was extracted out of paraffin-embedded tissue and PCR was performed for the coding exons 2-11. Denaturing gradient gel electrophoresis (DGGE) was carried out for mutational analysis and DNA sequencing was performed in case of a suspected mutation. RESULTS: Fourteen pleomorphic adenomas and 11 carcinomas ex pleomorphic adenoma were screened for p53 status and potent mutations. Subsequent sequencing of the distinct exons showed no mutation. CONCLUSION: We could not detect mutations of p53 neither in benign nor malignant parotid tumors and we therefore assume that p53 plays no role in the transformation from pleomorphic adenoma to carcinoma ex pleomorphic adenoma.

7beta-hydroxycholesterol induces apoptosis and regulates cyclooxygenase 2 in head and neck squamous cell carcinoma.

OBJECTIVE: To determine whether treatment with 7beta-hydroxycholesterol (7beta-HC) would trigger cell death in head and neck squamous cell carcinoma (HNSCC) cell lines in a dose-dependent fashion. DESIGN: In vitro study. SUBJECTS: The study included HNSCC cell lines SCC9, SCC25, CAL27, and FaDu. INTERVENTION: We treated HNSCC cell lines with increasing doses of 7beta-HC. Proliferation assays were performed to assess cell viability after treatment. Western blots were carried out to evaluate cyclooxygenase (COX)-1 and -2 expression levels. RESULTS: Using proliferation assays and immunocytochemical analysis, we detected significant growth inhibition via apoptosis in 4 different HNSCC cell lines after treatment with 7beta-HC (P < .001). The 50% inhibitory concentration levels were between 13.19 and 20.79 micromol/L after 72 hours. Western analysis indicated that COX-2, but not COX-1, levels were suppressed after treatment. CONCLUSIONS: Treatment with 7beta-HC resulted in suppression of HNSCC growth in vitro. Our data warrant further investigations for the potential use of 7beta-HC as a cytotoxic agent in head and neck cancer.

Down-regulation of Mcl-1 with antisense technology alters the effect of various cytotoxic agents used in treatment of squamous cell carcinoma of the head and neck.

Antisense oligonucleotides have recently been identified as new anticancer agents. Since human head and neck cancer cells highly express the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1), the aim of this study was to explore the efficacy of the Mcl-1 suppression in combination with various cytotoxic agents in the head and neck cancer cell line SCC9. After oligonucleotide transfection and/or treatment with cisplatin, 5-fluorouracil (5-FU), gemcitabine, paclitaxel or cetuximab, proliferation assays were performed to determine cell viability. The expression patterns of Mcl-1, Bax and Bak were assessed by Western blot analysis and the apoptotic cells were determined by immunohistochemistry using the M30 antibody. A combined Mcl-1 antisense oligonucleotide treatment with paclitaxel, cetuximab and gemcitabine led to a significant reduction in the viable cells. However, the combination with cisplatin and 5-FU showed only moderate synergistic cytotoxic effects. According to the cytotoxic data, distinct apoptosis rates were observed after the combined treatment with the different substances. Western blot analysis also showed a significant suppression of the Mcl-1 synthesis. Our data show that the Mcl-1 antisense oligonucleotide in combination with certain cytotoxic agents has the potential to significantly decrease cell viability in vitro.

Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix.

Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct.

Nimesulide and indomethacin induce apoptosis in head and neck cancer cells.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit the enzyme cyclooxygenase (COX). There are two isoforms of the enzyme. Recent investigations indicate that both isoforms, COX-1 and COX-2, are involved in carcinogenesis. METHODS: We investigated the effects of nimesulide, a COX-2 selective and indomethacin, a non-selective NSAID on the head and neck squamous cell carcinoma (HNSCC) cell lines SCC-9 and SCC-25. Effects on cell numbers and apoptosis were assayed by cell counting, immunofluorescence and fluorescence activated cell sorting (FACS). COX expression was examined by Western blotting. RESULTS: The investigated cell lines express COX-1 and COX-2. Nimesulide and indomethacin induce apoptosis and cause a reduction of cell number. Incubation with NSAIDs upregulated COX-2 expression. CONCLUSION: The results of our study on HNSCC cells together with data from different studies showing anti-cancer activity of NSAIDs suggest that COX inhibitors could play a role in HNSCC treatment and prevention.