Anticancer effects of NSC­631570 (Ukrain) in head and neck cancer cells: In vitro analysis of growth, invasion, angiogenesis and gene expression.

NSC­631570 (Ukrain) is an aqueous extract of Chelidonium majus, a herbaceous perennial plant, one of two species in the genus Chelidonium, which has been demonstrated to selectively kill tumor cells without affecting non­malignant cells. In the present study, the components of NSC­631570 were examined by combined liquid chromatography/mass spectroscopy (LC­MS) and the effects of NSC­631570 on HNSCC cell lines, as well as primary cells, were analyzed with respect to growth, apoptosis, invasion, angiogenesis and gene expression. LC­MS identified chelerythrine and allocryptopine as the major alkaloids of the extract. Moreover, NSC­631570 suppressed the growth of all tested HNSCC cell lines, including a paclitaxel­resistant and P­glycoprotein (MDR1)­overexpressing cell line. Mucosal keratinocytes were also affected by the extract, while fibroblasts proved to be much more resistant. In contrast to allocryptopine, chelerythrine had toxic effects on HNSCC cell lines at low doses. NSC­631570 significantly induced apoptosis in the FaDu and HLaC78 cell lines. As analyzed by a spheroid­based invasion assay, cell migration was significantly suppressed by NSC­631570 in FaDu cells on gelatine, fibronectin, collagen, laminin and Matrigel®. Migration of the highly invasive cell line HLaC78 was also inhibited, albeit to a lesser extent (not significant on laminin). Microarray analysis revealed the downregulation of genes encoding key regulators, including EGFR, AKT2, JAK1, STAT3 and ß­catenin (CTNNB1), all of which are involved in cell proliferation, migration, angiogenesis, apoptosis as well as the radiation­ and chemo­resistance of HNSCC. The strongest upregulation occurred for cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), involved in the metabolism of xenobiotics. Upregulation of CYP1A1 was at least partially caused by chelerythrine and allocryptopine, as shown by RT­qPCR in two HNSCC cell lines. In addition, NSC­631570 showed a high anti­angiogenic action on the tube formation ability of human umbilical vein endothelial cells (HUVECs). In conclusion, this study highlights NSC­631570 as a promising therapeutic approach for HNSCC.

DL-Methadone as an Enhancer of Chemotherapeutic Drugs in Head and Neck Cancer Cell Lines.

BACKGROUND/AIM: The aim of this study was to analyze the effect of DL-methadone on enhancing the action of the chemotherapeutic drugs cisplatin, doxorubicin, 5-fluoruracil (5-FU) and paclitaxel on head and neck squamous carcinoma (HNSCC) cell lines. MATERIALS AND METHODS: The chemotherapeutic drugs were applied alone or in combination with DL-methadone and cytotoxicity was analyzed by XTT assays. Expression of the µ-opioid receptor and the drug transporter p-glycoprotein were analyzed by qRT-PCR. RESULTS: The effect of DL-methadone strongly depended on the respective chemotherapeutic agent. The basic expression of the µ-opioid receptor was not associated with the effect of DL-methadone, rather its induction by chemotherapeutic drugs. Expression or expression induction of p-glycoprotein was higher in weak-responder cell lines. CONCLUSION: Enhancement of the toxicity of chemotherapeutic drugs by DL-methadone depends on the drug and on the cell line used.

Effect of chelidonine on growth, invasion, angiogenesis and gene expression in head and neck cancer cell lines.

The greater celandine 'Chelidonium majus' and its main alkaloid chelidonine have previously been shown to exert high cytotoxicity against cancer cells. Furthermore, chelidonine is proposed to possess pro-apoptotic and anti-metastatic properties. Within the present study, the effects chelidonine on several HNSCC cell lines, as well as primary cells, were analyzed with respect to growth, migration, angiogenesis and apoptosis. Chelidonine suppressed the growth of all tested HNSCC cell lines, including a paclitaxel-resistant and P-glycoprotein (MDR1) overexpressing cell line, but not in a clear dose-dependent manner. Mucosal keratinocytes were also strongly affected by chelidonine, while fibroblasts proved to be much more resistant. Chelidonine failed to trigger apoptosis at physiological concentrations in HNSCC cell lines. Based on a spheroid invasion model chelidonine suppressed invasion of FaDu cells effectively on gelatin, fibronectin, collagen I, laminin and Matrigel®. However, invasion inhibition of the more aggressively invading cell line HLaC78 largely failed. Using the tube formation assay, chelidonine effectively inhibited angiogenesis. Expression analysis revealed an upregulation of the xenobiotic metabolism genes CYP1A1 and MDR1 by chelidonine. In summary, chelidonine appeared to exert only minor impact on head and neck cancer cells. Chelidonine did not produce clear dose-dependent and cell-type specific cytotoxicity nor did it trigger apoptosis strongly.

The influence of Osmunda regalis root extract on head and neck cancer cell proliferation, invasion and gene expression.

BACKGROUND: According to only a handful of historical sources, Osmunda regalis, the royal fern, has been used already in the middle age as an anti-cancer remedy. To examine this ancient cancer cure, an ethanolic extract of the roots was prepared and analysed in vitro on its effectiveness against head and neck cancer cell lines. METHODS: Proliferation inhibition was measured with the MTT assay. Invasion inhibition was tested in a spheroid-based 3-D migration assay on different extracellular matrix surfaces. Corresponding changes in gene expression were analysed by qRT-PCR array. Induction of apoptosis was measured by fluorescence activated cell sorting (FACS) with the Annexin V binding method. The plant extract was analysed by preliminary phytochemical tests, liquid chromatography/mass spectroscopy (LC-MS) and thin layer chromatography (TLC). Anti-angiogenetic activity was determined by the tube formation assay. RESULTS: O. regalis extract revealed a growth inhibiting effect on the head and neck carcinoma cell lines HLaC78 and FaDu. The toxic effect seems to be partially modulated by p-glycoprotein, as the MDR-1 expressing HLaC79-Tax cells were less sensitive. O. regalis extract inhibited the invasion of cell lines on diverse extracellular matrix substrates significantly. Especially the dispersion of the highly motile cell line HlaC78 on laminin was almost completely abrogated. Motility inhibition on laminin was accompanied by differential gene regulation of a variety of genes involved in cell adhesion and metastasis. Furthermore, O. regalis extract triggered apoptosis in HNSCC cell lines and inhibited tube formation of endothelial cells. Preliminary phytochemical analysis proved the presence of tannins, glycosides, steroids and saponins. Liquid chromatography/mass spectroscopy (LC-MS) revealed a major peak of an unknown substance with a molecular mass of 864.15 Da, comprising about 50% of the total extract. Thin layer chromatography identified ferulic acid to be present in the extract. CONCLUSION: The presented results justify the use of royal fern extracts as an anti-cancer remedy in history and imply a further analysis of ingredients.

Spheroid-based 3-dimensional culture models: Gene expression and functionality in head and neck cancer.

In the present study a panel of 12 head and neck cancer (HNSCC) cell lines were tested for spheroid formation. Since the size and morphology of spheroids is dependent on both cell adhesion and proliferation in the 3-dimensional (3D) context, morphology of HNSCC spheroids was related to expression of E-cadherin and the proliferation marker Ki67. In HNSCC cell lines the formation of tight regular spheroids was dependent on distinct E-cadherin expression levels in monolayer cultures, usually resulting in upregulation following aggregation into 3D structures. Cell lines expressing only low levels of E-cadherin in monolayers produced only loose cell clusters, frequently decreasing E-cadherin expression further upon aggregation. In these cell lines no epidermal growth factor receptor (EGFR) upregulation occurred and proliferation generally decreased in spheroids/aggregates independent of E-cadherin expression. In a second approach a global gene expression analysis of the larynx carcinoma cell line HLaC78 monolayer and the corresponding spheroids was performed. A global upregulation of gene expression in HLaC78 spheroids was related to genes involved in cell adhesion, cell junctions and cytochrome P450-mediated metabolism of xenobiotics. Downregulation was associated with genes controlling cell cycle, DNA-replication and DNA mismatch repair. Analyzing the expression of selected genes of each functional group in monolayer and spheroid cultures of all 12 cell lines revealed evidence for common gene expression shifts in genes controlling cell junctions, cell adhesion, cell cycle and DNA replication as well as genes involved in the cytochrome P450-mediated metabolism of xenobiotics.

Galium verum aqueous extract strongly inhibits the motility of head and neck cancer cell lines and protects mucosal keratinocytes against toxic DNA damage.

Galium verum, also known as Lady's Bedstraw, is an herbaceous plant native to Europe and Asia, and has been used in traditional medicine as an anticancer medicine applied in most cases as a decoction. The influence of a Galium verum decoction on the head and neck cancer cell lines HLaC78 and FADU was analyzed and proved to be toxic in high doses on both cell lines. Cytotoxicity appeared to be influenced by expression of p-glycoprotein (MDR-1) in the carcinoma cell lines. Mucosal keratinocytes, although void of MDR-1 expression, showed only low sensitivity against high Galium concentrations. Sublethal doses of Galium extract acted as strong inhibitors of motility, as shown by a spheroid-based invasion analysis on Matrigel-coated surfaces. Inhibition of invasion was significantly more pronounced in the invasive HLaC78 cell line. mRNA expression analysis of matrix metalloproteinases MMP-2 and MMP-9 and their inhibitors TIMP-1/-2 revealed significant TIMP-1 upregulation after an 8-h Galium exposition in FADU cells. Gelatinolytic activity, however, was not influenced by Galium extract in HLaC78, in the FADU cells MMP-2/-9 activity was slightly increased after incubation with Galium extract. In primary mucosal keratinocytes, Galium decoction protected DNA against benz[a]pyrene, one of the most DNA toxic agents in cigarette smoke. In conclusion Galium extract may be useful as a preventive and/or a concomitant therapeutic approach in head and neck cancer.

Effect of Galium verum aqueous extract on growth, motility and gene expression in drug-sensitive and -resistant laryngeal carcinoma cell lines.

Galium verum, also known as Lady's Bedstraw, is a herbaceous perennial plant of the family Rubiaceae, native to Europe and Asia and used in traditional medicine as an anticancer medicine. It is used as a decoction in most traditional recipes, applied externally as well as internally. We produced a Galium verum decoction and applied it in vitro to chemosensitive (Hep-2 and HLaC79) and chemoresistant, P-glycoprotein-overexpressing (Hep2-Tax, HLaC79-Tax) laryngeal carcinoma cell lines. It could be demonstrated that Galium aqueous extract is cytotoxic for all cell lines. A detailed spheroid-based 3D invasion analysis of Hep2 and Hep2-Tax in semisolid collagen gels and on different extracellular matrix coatings was performed, which showed an inhibition of invasion by sublethal concentrations of Galium decoction and proved to be even more pronounced in the more aggressively invading chemoresistant Hep2-Tax cell line. Gelatinolytic activity of MMP-2 was downregulated in three of the four cell lines. Angiogenesis (endothelial tube formation) in contrast, was not affected by Galium aqueous extract. Gene expression array on HLaC79 and Hep2 cell lines treated with Galium decoction vs. untreated controls revealed no unique pathway activation patterns in these cells. Results are discussed with respect to the use of herbal drugs as a preventive and/or a concomitant therapeutic approach in head and neck cancer.

Cytotoxicity of herbal extracts used for treatment of prostatic disease on head and neck carcinoma cell lines and non-malignant primary mucosal cells.

Previously, a growth inhibiting effect of PC-Spes on head and neck carcinoma cell lines had been demonstrated. In order to determine the toxic impact of particular herbs in the mixture, we exposed the head and neck cancer cell lines FADU, HLaC79 and its Paclitaxel-resistant subline HLaC79-Clone1 as well as primary mucosal keratinocytes to increasing concentrations of the herbal mixture Prostaprotect, which has a similar formulation as PC-Spes, as well as its single herbal components Dendranthema morifolium, Ganoderma lucidium, Glycyrrhiza glabra, Isatis indigotica, Panax pseudo-ginseng, Rabdosia rubescens, Scutellaria baicalensis and Pygeum africanum. Growth inhibition was measured using the MTT assay. Expression of P-glycoprotein (P-GP), multidrug resistance protein-1 (MRP-1), multidrug resistance protein-2 (MRP-2), breast cancer resistance protein (BCRP) and androgen receptor (AR) were examined by western blot analysis. Pygeum africanum extract clearly turned out as the main cytotoxic component of the Prostaprotect prescription mixture, and initated apoptosis in sensitive cell lines. All other extracts had only minor toxic effects. Western blot analysis revealed increased expression of P-GP in HLaC79-Clone1 cells, while HLaC79 and FADU cells were negative. All three cell lines were negative for MRP-1 and BCRP but positive for MRP-2. HLaC79 and its descendant HLaC79-Clone1 both expressed AR, as verified by western blotting and immunofluorescence staining. Primary mucosal keratinocytes were negative for all multidrug resistance markers as well as for AR. Growth inhibition rates of the single herbal extracts were compared with previously published results in prostate carcinoma cell lines. The relationship between expression levels of AR and multidrug resistance markers in relation to the measured toxicity of herbal extracts in our head and neck cancer cell system is critically discussed.

The effects of PC-Spes on chemosensitive and chemoresistant head and neck cancer cells and primary mucosal keratinocytes.

PC-Spes is a preparation of eight Chinese herbs, which exhibits antiproliferative and antitumour activity in diverse cancer types in vivo and in vitro. We exposed the head and neck squamous carcinoma cell lines (HNSCC) FADU, HLaC79 and HLaC79-clone1, which is a paclitaxel-resistant descendant of HLaC79, as well as primary cultured mucosal keratinocytes to increasing concentrations of paclitaxel and/or PC-Spes. Growth inhibition was measured using the MTT assay. While FADU and HLaC79 were growth inhibited by paclitaxel, HLaC79-clone1 cells proved to be resistant against paclitaxel up to doses of 100 nM, whereas all three cell lines were growth inhibited by PC-Spes. Interestingly primary keratinocytes were less sensitive to PC-Spes, they even showed better survivel at low PC-Spes doses. Furthermore, we analyzed cell cycle distribution, apoptosis and tubulin expression level and polymerization status in the HNSCC cell lines. PC-Spes caused a slight decrease of cells in S/G2 phase in HLaC79-clone1. In FADU and HLaC79 cells the cell cycle was shifted towards S/G2 phase as expected. Apoptosis was initiated in all three cell lines by PC-Spes, in mucosal keratinocytes, however, it was triggered less distinctively. In summary, PC-Spes revealed distinct growth inhibition in a paclitaxel-resistant cell line, whereas primary mucosal keratinocytes were less sensitive. PC-Spes might therefore provide a therapeutical approach in chemoresistant head and neck cancer.