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.

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.