Determining recommended acceptable intake limits for N-nitrosamine impurities in pharmaceuticals: Development and application of the Carcinogenic Potency Categorization Approach (CPCA).

N-Nitrosamine impurities, including nitrosamine drug substance-related impurities (NDSRIs), have challenged pharmaceutical industry and regulators alike and affected the global drug supply over the past 5 years. Nitrosamines are a class of known carcinogens, but NDSRIs have posed additional challenges as many lack empirical data to establish acceptable intake (AI) limits. Read-across analysis from surrogates has been used to identify AI limits in some cases; however, this approach is limited by the availability of robustly-tested surrogates matching the structural features of NDSRIs, which usually contain a diverse array of functional groups. Furthermore, the absence of a surrogate has resulted in conservative AI limits in some cases, posing practical challenges for impurity control. Therefore, a new framework for determining recommended AI limits was urgently needed. Here, the Carcinogenic Potency Categorization Approach (CPCA) and its supporting scientific rationale are presented. The CPCA is a rapidly-applied structure-activity relationship-based method that assigns a nitrosamine to 1 of 5 categories, each with a corresponding AI limit, reflecting predicted carcinogenic potency. The CPCA considers the number and distribution of α-hydrogens at the N-nitroso center and other activating and deactivating structural features of a nitrosamine that affect the α-hydroxylation metabolic activation pathway of carcinogenesis. The CPCA has been adopted internationally by several drug regulatory authorities as a simplified approach and a starting point to determine recommended AI limits for nitrosamines without the need for compound-specific empirical data.

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.

The lived experience of uncertainty in everyday life with MS.

PURPOSE: This article examines how issues of control, certainty, and uncertainty are experienced and managed in everyday life with multiple sclerosis (MS) and explores the ways in which people living with MS make sense of these experiences. MATERIALS AND METHODS: Qualitative interviews with 23 women and men diagnosed with MS and four relatives were carried out in Denmark. Drawing on the notion of "phenomenological uncertainty," a thematic approach was used to analyse the interview data. RESULTS: Three themes characterise participants' experience of uncertainty: the body and issues of control; symptom fluctuations and disease progression; understanding and interpreting embodied MS experiences. Shared, between the themes, is a focus on the body and multi-faceted bodily aspects of uncertainty across diverse temporalities. CONCLUSION: Phenomenological uncertainty shapes and pervades the everyday lived experience of MS in the present and future. Gaining a sense of control and certainty in the face of daily uncertainty demands ongoing self-surveillance, and the evaluation and reconciliation of fluctuating MS symptom expressions and disease progression with personal needs, abilities, and management strategies.IMPLICATIONS FOR REHABILITATIONRehabilitation professionals and physicians should consider the lived experience of uncertainty in everyday life with MS in all their contacts with people living with MS.The multi-faceted uncertainties experienced by people living with MS should be actively acknowledged and incorporated in discussions of MS rehabilitation options and when integrating MS guideline content into activities-of-daily-living advice.Discussions of MS medical treatment options should actively consider and integrate the multi-faceted uncertainties experienced by people living with MS.

Short-term natural history in women with symptoms indicative of pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: Information about the natural history of pelvic organ prolapse (POP) is scarce. METHODS: This was a prospective cohort study of 160 women (mean age 56 years), whose answers in a population-based survey investigation indicated presence of symptomatic prolapse (siPOP), and 120 women without siPOP (mean age 51 years). RESULTS: Follow-up questionnaire was completed by 87%, and 67% underwent re-examination according to pelvic organ prolapse quantification (POP-Q) system after 5 years. Among re-examining siPOP women, 47% had an unchanged POP-Q stage, 40% showed regression, and 13% showed progression. The key symptom "feeling of a vaginal bulge" remained unchanged in 30% of women with siPOP, 64% improved by at least one step on our four-step rating scale, and 6% deteriorated. Among control women, siPOP developed in 2%. No statistically significant relationship emerged between changes in anatomic status and changes in investigated symptoms. CONCLUSION: Only a small proportion of women with symptomatic POP get worse within 5 years.

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.

Nonobstetric risk factors for symptomatic pelvic organ prolapse.

OBJECTIVE: To identify possible nonobstetric risk factors for symptomatic pelvic organ prolapse in the general female population. METHODS: This was a population-based, cross-sectional study derived from a sample of 5,489 Stockholm women, 30 to 79 years old, who answered a validated questionnaire for the identification of symptomatic prolapse. The 454 women whose answers indicated the presence of such prolapse and the 405 randomly selected control participants with answers that gave no indication of prolapse received a 72-item questionnaire, which probed into a priori suspected risk factors. Only those women with intact uteri and no prior surgery for incontinence or prolapse were included. Multivariable logistic regression models estimated prevalence odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: In addition to age and parity, overweight (prevalence OR for body mass index [kg/m] 26-30 compared with 19-25 was 1.9, 95% CI 1.2-3.1), history of conditions suggestive of deficient connective tissue (varicose veins/hernia/hemorrhoids, prevalence OR for positive history compared with no history 1.8, 95% CI 1.2-2.8), family history of prolapse (prevalence OR for positive history compared with no history 3.3, 95% CI 1.7-6.4), heavy lifting at work (prevalence OR for 10 kg or more compared with no heavy lifting 2.0, 95% CI 1.1-3.6), and presence of constipation, hard stools, or difficult evacuation (prevalence OR relative to normal bowel habits 2.1, 95% CI 1.4-3.3) all were linked independently, significantly, and positively to the presence of symptomatic prolapse. CONCLUSION: In this nonconsulting population, age and parity were the dominating risk factors, but significant independent associations with markers suggestive of congenital susceptibility (family history and conditions signaling weak connective tissue) and nonobstetric strain on the pelvic floor (overweight/obesity, heavy lifting, and constipation) imply that individual predisposition and lifestyle/environment also may play an important role. The causal direction of the association with bowel habits remains uncertain, and the link to family history could be partly because of information bias.

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.

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.

Symptoms and pelvic support defects in specific compartments.

OBJECTIVE: To investigate whether the nature of the anatomic defects in pelvic organ prolapse (POP) correlates with the character of the symptoms. METHODS: This study was a cross-sectional investigation within a population-based sample. Two hundred eighty women who had completed a symptom questionnaire were examined according to POP quantification by two gynecologists blinded to symptom reports. RESULTS: An age- and parity-adjusted logistic regression model, controlling for POP in other compartments, revealed that the feeling of vaginal bulge was specific to prolapse but not to any particular compartment, although the association was strongest with anterior-wall prolapse (odds ratio [OR] for the symptom among women with stage II-IV relative to stage 0 was 5.8, 95% confidence interval [CI] 2.5-13.3). Urge urinary incontinence tended to be linked to POP in either the anterior or posterior wall, but the association was stronger with anterior-wall prolapse. Stress urinary incontinence was strongly linked to posterior-wall prolapse (stage II-IV OR 5.4, 95% CI 1.9-15.2). Self-reports of hard/lumpy stool and difficult or painful defecation tended to be associated with anterior-wall prolapse but without consistent relationships with stage. Painful defecation was the only bowel symptom significantly linked to posterior-wall prolapse (P=.05). CONCLUSION: Pelvic floor-related symptoms do not predict the anatomic location of the prolapse in women with mild to moderate prolapse.

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.

Obstetric risk factors for symptomatic prolapse: a population-based approach.

OBJECTIVE: The purpose of this study was to identify obstetric risk factors for symptomatic prolapse. STUDY DESIGN: This was a population-based case-control study of prolapse prevalence. RESULTS: Four hundred fifty-four women with self-reported symptomatic pelvic organ prolapse who were identified among 5489 women who participated in a population survey (cases) and 405 control subjects without symptoms were selected randomly from the same survey. All cases and control subjects received a mailed questionnaire with 72 questions about factors that were suspected to be linked to risk and that included obstetric history. The response rate was 76%. Among parous women, the odds for symptomatic pelvic organ prolapse increased with number of childbirths and were 3.3-fold higher among mothers of 4 than among mothers of 1. Indices of excessive stretching and tearing during labor (vaginal lacerations or episiotomies) were associated with increased risk for symptomatic pelvic organ prolapse. Instrumental delivery with forceps or vacuum did not seem to increase the risk of symptomatic pelvic organ prolapse, nor did length of delivery or maternal age at time for delivery. Abdominal deliveries appeared to be protective; the age- and parity-adjusted odds ratio of symptomatic pelvic organ prolapse after > or =1 abdominal deliveries was 0.5 (95% CI, 0.3-0.9), relative to women who had had only vaginal deliveries. A positive association with child birth weight in unadjusted analyses disappeared after adjustments for attained age and parity of the mother. CONCLUSION: Excessive stretching and tearing and multiple deliveries seem to be the main predisposing obstetric factors for symptomatic pelvic organ prolapse. Abdominal delivery emerged as a comparably strong protective factor.

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.

A short-form questionnaire identified genital organ prolapse.

OBJECTIVE: We constructed a simple questionnaire that, with a minimum of questions, could accurately and reliably identify women with genital organ prolapse. STUDY DESIGN AND SETTING: Two hundred women with confirmed genital organ prolapse and 199 outpatients with various gynecologic symptoms but no objective prolapse answered 13 questions perceived to be valuable for the diagnosis. With stepwise backward logistic regression, the discriminatory ability of a successively abbreviated set of questions was assessed. The resulting short questionnaire was tested in a new population-based sample of 282 women participating in a screening survey. RESULTS: A final five-item questionnaire retained 94% of the predictive value of all 13 questions and had 92.5% sensitivity and 94.5% specificity in the first group of women. When the questionnaire was used in the subsequent population-based survey, the sensitivity and specificity values were 66.5% and 94.2%, respectively. Most missed cases had stage I prolapse. CONCLUSION: Although the sensitivity of the test was moderate, the specificity, and hence the ability to rule in cases, was satisfactory. The test is suitable for case finding in epidemiologic studies.

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

HYPOTHESIS: 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 1alpha 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 1alpha, 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.

[Meropenem therapy in children with febrile neutropenia]. / Lázas, neutropeniás gyermekek kezelése meropenemmel.

OBJECTIVES: Infectious complications are the major causes of morbidity and mortality in children receiving chemotherapy for malignant diseases. Granulocytopenia carries the risk of bacterial infection, and also, if prolonged, of fungal infection. The aim of this study was to evaluate the clinical effectiveness of meropenem in immunocompromised children in association with isolated bacteria from blood cultures and clinical background. METHODS: Retrospective study of all febrile episodes when meropenem was used in neutropenic children between January 1998 and December 2002 in the haemato-oncological units of the authors hospital. During the study period meropenem was used in 87 febrile events diagnosed in 55 patients (mean age 10 years 5 months), and 328 bacterial cultures were performed. Blood samples were taken from each patient with granulocytopenia (< 0.5 G/l) and fever (> or = 38 degrees C), prior to the start of any antibiotic therapy. For the microbiological process Bactec 9050 (Becton Dickinson) blood culture systems were used. RESULTS: Microorganisms were detected and identified in 64 (19.5%) from the 328 hemocultures. There was a predominance of Gram-positive strains, 67% (43/64)--the most common bacteria being coagulase negative Staphylococcus (cnS). From the 87 periods in 43 cases (49.4%) the infection was documented microbiologically. In 16 additional cases the infection was proven clinically (based on the clinical course, laboratory and radiologic results) and 32.2% (28/87) of the febrile neutropenic episodes were considered to be fever of unknown origin. Meropenem was used in a mean dose of 60.8 (30-120) mg/kg/die, for 9.3 (2-24) days. The success rate of the meropenem therapy -excluding the proven fungal (n = 13) or cnS (n = 15) infections-was 72.9%. No severe side effects occurred in any regimens. CONCLUSION: The results demonstrate that meropenem is effective and well-tolerated when used for the treatment of feverish neutropenic cancer children.

[Frequent infections of neutropenic pediatric patients and therapeutic modalities] / Neutropéniás gyermekek leggyakoribb fertôzései és a kezelés lehetôségei.

Neutropenia, resulting from intensive chemotherapy is a common problem. The appearance of fever in neutropenic patients should always raise the suspicion of infection and should be followed by an intensive diagnostic evaluation and start of antibacterial treatment. The authors analyzed the association between isolated bacteria from blood cultures and the clinical background of all febrile episodes that occurred in neutropenic children in a two-year long period. Comparable to the international trends, our results suggest an increased prevalence of the Gram-positive organisms causing bacteriaemia. The clear majority of the isolated bacteria was coagulase-negativ Staphylococcus (cnS), which is a multiresistant strain, and sensitive only to the glycopeptide antibiotics. This latter fact can be a consequence of the frequent use of central venous catheters. The empirical therapy, the therapy used in microbiologically and clinically proved infections, and the supplementary and prophylactic methods of treatment are presented.

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.

Suicide gene therapy in head and neck carcinoma cells: an in vitro study.

The aim of gene therapy includes the tight spatial and temporal control of transgenic expression. There are several approaches concerning the externally inducible gene promoters used for the control of suicide genes. We have tested the mifepristone-dependent system GeneSwitch to regulate the expression of a deletion mutant of Pseudomonas exotoxin A in the hypopharyngeal carcinoma cell line, FADU. The GeneSwitch system consists of two plasmids, the regulatory plasmid, pSwitch, and the pGene/V5-His plasmid, in which we cloned the toxin mutant (pGene/V5-His-ETA). We stably transfected FADU cells with pSwitch and subsequently transiently separated pSwitch clones with pGene/V5-His-ETA. We tested the inductive capacities of single pSwitch clones, the influence of experimental variations in transfection, the inductive capacities without antibiotic selection pressure, the inductive capacity after re-induction, as well as the background expression levels. In FADU cells the GeneSwitch-ETA combination worked precisely and effectively. Our in vitro study revealed that the use of toxin genes in combination with the GeneSwitch system is a promising approach for gene therapy in head and neck cancer.