Marianne Schmidt

Up-regulation of matrix metalloprotease-9 in middle ear cholesteatoma – correlations with growth factor expression in vivo?

Abstract The role of matrix metalloproteases and their regulation in the pathology of middle ear cholesteatoma is still unclear. Recently we have demonstrated that incubation of keratinocytes with cholesteatoma debris and granulation tissue extracts causes induction of gelatinase B (matrix metalloproteinase-9, MMP-9) secretion in vitro. Antibodies against a variety of growth factors revealed some inhibitory effect on MMP-9 induction, caused by debris or granulation tissue extracts. In order to investigate the coherence of growth factor expression and matrix metalloproteinase activity in vivo in middle ear cholesteatoma, we performed quantitative gelatin zymographic analysis with tissue homogenates of 37 cholesteatoma and nine external ear canal skin (EACS) samples. Furthermore we quantified levels of the cytokines IL-1α, IL-1β, TNF-α, TGF-β and EGF present in tissue extracts, using enzyme-linked immunosorbent assays (ELISA), and correlated cytokine concentrations with gelatinolytic activities. Zymographic analysis revealed a highly heterogeneous expression of gelatinase A and B in cholesteatoma specimens. As shown previously, MMP-9, but not MMP-2, was increased in cholesteatoma when compared to EACS samples. ELISA studies revealed a significantly elevated ¶IL-1α level in cholesteatoma. Regression analysis involving gelatinolytic activity and cytokine concentrations in tissue homogenates showed no statistically significant correlation between expression of gelatinases and the cytokines IL1-α, IL1-β, TNF-α, TGF-β or EGF. The discrepancy between in vitro observations and the situation in vivo is discussed critically.

Increased levels of urokinase receptor in plasma of head and neck squamous cell carcinoma patients.

Urokinase-type plasminogen activator (uPA) is important for matrix degradation and motility of cancer cells. The binding of uPA to its cell surface receptor on cancer cells is essential for effective invasion. A soluble form of urokinase receptor (suPAR) has been described in serum and ascites of ovarian cancer patients and in plasma samples of non-small cell lung cancer patients. Plasma samples from 36 head and neck squamous cell carcinoma patients and 24 healthy control persons were analysed for the presence of suPAR using enzyme-linked immunosorbent assay (ELISA) and the expression levels were correlated with clinical and histopathological data. Significantly elevated levels of suPAR in blood plasma from head and neck cancer patients were observed (p = 0.000), and the suPAR plasma levels decreased after resection of the carcinoma in 8 of 11 patients. suPAR plasma levels of cancer patients showed no significant correlations with T staging, metastasis, recurrence or differentiation stage of the tumours. The significance of suPAR plasma levels in head and neck squamous cell carcinoma patients for prognosis of the disease is discussed.

Urokinase receptor up-regulation in head and neck squamous cell carcinoma

Urokinase‐type plasminogen activator is important for matrix degradation and motility of cancer cells. For effective invasion, urokinase has to be associated with its cell surface receptor.1

Induction of matrix metalloproteinases in keratinocytes by cholesteatoma debris and granulation tissue extracts

Abstract Although it is generally accepted that destruction and remodeling of temporal bone associated with middle ear cholesteatoma is mainly caused by the action of osteoclasts, it has been shown that neutral collagenases also play a role in predigesting the osteoid layer and exposing the mineralized bone to osteoclastic activity. Here we show that gelatinase B (matrix metalloproteinase-9) is over-expressed in cholesteatoma compared to external ear canal skin (EACS). Expression of MMP-9 in cholesteatoma mainly occurs in suprabasal layers, and more rarely in basal layers of cholesteatoma epithelium, as well as in inflammatory cells of the perimatrix. We further analyzed the influence of cholesteatoma debris, cholesteatoma granulation tissue, and cholesteatoma components such as keratin, cholesterol and bacterial endotoxin on the expression of MMPs in EACS keratinocytes. We show that cholesteatoma debris and granulation tissue extract both induced the secretion of MMP-9 by EACS keratinocytes, while keratin, bacterial lipopolysaccharide (LPS) or cholesterol did not show any effect. We further performed co-incubation and immunoprecipitation experiments using neutralizing interleukin-1α, EGF, TGF-β, TGF-α, interleukin-6 and TNF-α antibodies. Inhibition of MMP-9 up-regulation by debris or granulation tissue extract could be revealed with diverse cytokine antibodies. The results are discussed with regard to previously published studies.

Differential gene expression in a paclitaxel-resistant clone of a head and neck cancer cell line

The anti-neoplastic drug paclitaxel (taxol), which is known to block cells in the G2/M phase of the cell cycle through stabilization of microtubules, is meanwhile commonly used for chemotherapy of advanced head and neck cancer. Chemotherapy is primarily used in order to preserve laryngeal and/or pharyngeal structures. Although paclitaxel generally seems to be a powerful agent, it failed to reach a loco-regional tumor control in a sufficient percentage of patients. In order to investigate molecular resistance mechanisms, we have established a paclitaxel-resistant subline originating from the larynx carcinoma cell line HLaC79, which seemed to be partially dependent on taxol. The original and the descendant cell line were characterized by growth inhibition assays. We used western blotting and the cDNA subtraction (SSH) technique to identify genes differentially expressed in the taxol-resistant cell clone. cDNA subtraction revealed increased expression of six genes, including clathrin heavy chain, α3-tubulin, a neuroblastoma-specific Thymosin β, the ribosomal protein L7a, HLA-B associated transcript 3 and collagen IIIα1 in the taxol-resistant cell line. Furthermore, western blots showed an overexpression of MDR-1 in the taxol-resistant clone, while α- and β-tubulins and p48/IRF9 were expressed in equal amounts in both cell lines.

Inducible promoters for gene therapy of head and neck cancer: an in vitro study

The aim of gene therapy includes the tight spatial and temporal control of transgenic expression. There are several approaches concerning externally inducible gene promoters used for the control of suicide genes. Two of the promoters that might play a role in head and neck cancer gene therapy are the hyperthermia-inducible human heat shock protein-70 (hsp70) promotor, as well as the radiation-inducible promoter of the early growth response-1 gene (egr-1). We tested the hsp-70 promoter as well as a promoter construct, containing synthetic radio-responsive elements of the egr-1 enhancer for the effect on reporter gene expression in two stably transfected head and neck carcinoma cell lines in vitro and measured the success of gene activation by FACS analysis, western blot analysis and fluorescence microscopy. With the hsp70 promoter we reached a 5.83-fold increase of reporter gene expression after hyperthermic treatment in one of the two cell lines tested. The radiation-inducible construct revealed only weak gene induction and was marked by high background expression. Both systems worked in a highly cell-type dependent manner. The possible clinical use of externally inducible transgene expression in head and neck carcinoma gene therapy is critically discussed.

Proteolytic patterns of head and neck squamous cell carcinoma.

Abstract The significance of plasminogen activators and matrix metalloproteases for clinical outcome, growth and metastatic behavior of head and neck squamous cell carcinoma (SCC) is still controversial. The majority of studies has been based on either immunohistological stainings, which provide only limited quantitative information, or in vitro experiments. We analyzed 44 head and neck SCC and 11 mucosa tissue samples for the expression of gelatinolytic or fibrinolytic proteases by quantitative zymographic analysis and compared lytic activities to clinical and histopathological data. We calculated activation ratios for matrix metalloproteinases-2 and –9 (MMP-2 and MMP-9) by separate evaluations of inactive and activated MMP forms. Increased gelatinolytic and fibrinolytic activity was found in head and neck SCC when compared to mucosa. Increased values were caused by MMP-9 and urokinase type plasminogen activator, respectively. No statistically significant correlations of either protease lytic activity or activation ratio could be related to T-stage, metastasis, tissue necrosis or the differentiation stage of tumors. The data recorded are compared with previously published reports.

Expression of bone morphogenetic protein-2 messenger ribonucleic acid in cholesteatoma fibroblasts.

Hyphothesis The aim of the study was to evaluate the role of bone morphogenetic protein-2 (BMP-2) in the pathology of middle ear cholesteatoma. Background Middle ear cholesteatoma is a chronic inflammatory disease associated with destruction of the temporal bone and marked by increased expression levels of diverse cytokines. Bone remodeling associated with this disease is mainly caused by the action of osteoclasts. It has been shown that BMP-2 expression is inducible by interleukin 1 in synovial fibroblasts and that BMP-2 in combination with interleukin 1&agr; is able to stimulate the formation of osteoclast-like multinucleated cells in co-cultures of osteoblast-like cells and hematopoietic cells. Methods By using Northern hybridizations, we examined the messenger ribonucleic acid expression of BMP-2 in keratinocytes and fibroblasts derived from normal external ear canal skin (EACS) and from cholesteatoma, respectively. Results We show that normal EACS fibroblasts do not express BMP-2, whereas keratinocytes of both EACS and cholesteatoma origin are positive for the BMP-2 transcript. In contrast to EACS fibroblasts, BMP-2 is clearly expressed in cholesteatoma perimatrix fibroblasts. Incubation of normal fibroblasts with cholesteatoma extracts caused the transcription of BMP-2. Interleukin 1&agr;, bacterial endotoxin, or bovine keratin, however, were not able to initiate BMP-2 expression in normal fibroblasts. Conclusion In view of the above data, it is tempting to speculate that BMP-2 expression might play a role in cholesteatoma pathology.

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.

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.