Sabine Bauer

NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells.

Background:The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.Methods:To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.Results:We show that tumour spheroids generate ‘circular chemorepellent-induced defects’ (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.Interpretation:These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell–cell contacts, migration, and the generation of CCID.

Outcome and functional capacity after prolonged intensive care unit stay.

ZusammenfassungSTUDIENHINTERGRUND: Viele kritisch kranke Patienten, die ihre akute Erkrankung überleben, verbleiben in einem abhängigen Zustand und benötigen für Wochen bis Monate eine Behandlung an der Intensivstation. Die Datenlage betreffend die Spitalsletalität und insbesondere betreffend die Langzeit-Letalität bzw. die -Morbidität bei überlebenden Patienten ist jedoch noch spärlich. Ziel dieser Studie war es, den klinischen Verlauf und Prognosefaktoren bei Langzeit-kritisch kranken Patienten zu untersuchen. METHODEN: Diese retrospektive Beobachtungs-Kohorten-Studie wurde an unserer gemischten kardiologischen 8-Betten-Intensivstation in einem 2200-Betten-Universitätsspital durchgeführt. Patientendaten wurden zwischen dem 1. März 1998 und dem 31. Dezember 2003 analysiert. Patienten mit einer Stations-Aufenthaltsdauer von ≥30 Tagen bildeten die Studiengruppe. Die Evaluation der Morbidität und funktionellen Kapazität wurde mittels Telefoninterview unter Verwendung des Barthel Mobilitäts-Scores durchgeführt. ERGEBNISSE: Die Anzahl der Patienten mit einer Stations-Aufenthaltsdauer ≥30 Tagen betrug 135 (10% der Gesamtpatienten). Diese Gruppe belegte 5962 Bettentage, welches 40,9% der gesamten Bettenkapazität entsprach. Im Vergleich zu Patienten mit einer Stations-Aufenthaltsdauer < 30 Tagen hatten die Patienten in der Langzeitgruppe einen signifikant höheren SAPS II Score innerhalb von 24 Stunden nach Aufnahme (54 [IQR 41–65] vs. 38 [IQR 27–56], p < 0,001). Trendmäßig überwogen die Männer in der Langzeitgruppe (98/135 [82,6%] vs. 782/1215 [64,4%], p = 0,05). Unterschiede in der Intensivstations- und Hospitalsletalität waren nicht signifikant (28/135 [20,7%] vs. 295/1215 [24,3%], p = 0,620, und 46/135 [34,1%] vs. 360/1215 [29,6%], p = 0,285). Die Sterblichkeit bei Patienten, die den Spitalsaufenthalt überlebten, betrug nach einem bzw. nach vier Jahren 14% und 26% in der Kurzzeitgruppe verglichen mit 31% und 61% in der Langzeitgruppe. Ein log-rank-Test erbrachte eine signifikant höhere Überlebenswahrscheinlichkeit in der Kurzzeitgruppe nach Spitalsentlassung (log rank = 34,3, p < 0,001). Eine multivariate Datenanalyse zeigte, dass die Notwendigkeit zu einer Nierenersatztherapie während des Intensivstationsaufenthaltes der einzig unabhängige Prädiktor für das Versterben im Krankenhaus und innerhalb eines Jahres nach Intensivstations-Entlassung war (OR = 2,88; 95%CI 1,12–7,41, p = 0,028 und OR = 3,66, 95%CI 1,36–9,83, p = 0,01). In 28/31 der Langzeit-Überlebenden Patienten (90%) mit einem Intensivstationsaufenthalt ≥30 Tagen zeigte der Barthel Index keine oder nur mäßige Einschränkungen bei Alltagstätigkeiten. SCHLUSSFOLGERUNG: Die Hospitals-Letalität bei kritisch kranken Patienten mit einer Intensivstationsaufenthaltsdauer <30 oder ≥30 Tagen ist vergleichbar. Die Notwendigkeit zu einer Nierenersatztherapie war der einzige unabhängige Prädiktor für das Versterben im Krankenhaus und für die 1-Jahres-Letalität bei Langzeit-Patienten. Kritisch kranke Patienten mit einer Intensivstationsaufenthaltsdauer ≥30 Tagen haben ein hohes und anhaltendes Sterberisiko nach Spitalsentlassung. Dennoch, eine bedeutsame Anzahl von diesen Patienten sind LangzeitÜberlebende mit keinen oder nur mäßigen Einschränkungen bei Alltagstätigkeiten.SummaryBACKGROUND: An important proportion of critically ill patients who survives their acute illness remains in a critical state requiring intensive care management for weeks to months. Nevertheless, data on risk factors for in-hospital mortality and especially for long-term mortality and functional capacity are scarce. This study investigated outcome and prognostic factors in long-term critically ill patients. METHODS: This retrospective observational cohort study was performed at our mixed adult 8-bed cardiologic ICU at a 2200-bed University Hospital. Patient data from our local database connected to an Austrian multicenter program for quality assurance in intensive care were analyzed. Data were collected between March 1st, 1998 and December 31st, 2003. Patients with an ICU stay ≥30 days formed the long-term study group. Morbidity and functional capacity were assessed using the Barthel mobility index in telephone interviews. RESULTS: Patients spending ≥30 days in the ICU numbered 135 (10%) and occupied 5962 bed-days, representing 40.9% of the total bed-days. Compared with patients with an ICU stay <30 days, patients in the long-term group had a significantly higher SAPS II score during the first 24 hours after ICU admission (54 [IQR 41–65] vs. 38 [IQR 27–56], p < 0.001). There was a trend towards male preponderance in the long-term group (98/135 [82.6%] vs. 782/1215 [64.4%], p = 0.05). Differences in ICU and in-hospital mortality were not significant (28/135 (20.7%) vs. 295/1215 (24.3%), p = 0.620 and 46/135 [34.1%] vs. 360/1215 [29.6%], p = 0.285, respectively). After 12 and 48 months, the overall cumulative rates of death in hospital survivors were 14% and 26%, respectively in the short-term ICU group and 31% and 61% in the long-term group. A log-rank test revealed a significantly higher probability of survival in the short-term group after hospital discharge (log rank = 34.3, p < 0.001). Multivariate analysis of hospital survivors and non-survivors in the long-term group showed that the need for renal replacement therapy during the ICU stay was the sole independent predictor for in-hospital death and death within 1 year after ICU discharge (OR = 2.88; 95%CI 1.12–7.41, p = 0.028 and OR = 3.66, 95%CI 1.36–9.83, p = 0.01, respectively). In 28/31 long-term survivors (90%) in the long-term ICU group, the Barthel index indicated no or only moderate disability during daily activities. CONCLUSION: Hospital mortality rates in critically ill patients with a stay <30 or ≥30 days were comparable. The necessity for renal replacement therapy was the sole independent predictor for in-hospital and 1-year mortality in long-term ICU patients. Critically ill patients with a stay ≥30 days have a high and ongoing risk of death after hospital discharge; however, a substantial number of these patients are long-term survivors with no or only moderate disability during daily activities.

Bay11-7082 inhibits the disintegration of the lymphendothelial barrier triggered by MCF-7 breast cancer spheroids; the role of ICAM-1 and adhesion.

Background:Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics.Methods:Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of ‘circular chemorepellent-induced defects’ (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs.Results:Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively.Conclusion:Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

Berberine and a Berberis lycium extract inactivate Cdc25A and induce α-tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis

Berberis lycium Royle (Berberidacea) from Pakistan and its alkaloids berberine and palmatine have been reported to possess beneficial pharmacological properties. In the present study, the anti-neoplastic activities of different B. lycium root extracts and the major constituting alkaloids, berberine and palmatine were investigated in p53-deficient HL-60 cells. The strongest growth inhibitory and pro-apoptotic effects were found in the n-butanol (BuOH) extract followed by the ethyl acetate (EtOAc)-, and the water (H(2)O) extract. The chemical composition of the BuOH extract was analyzed by TLC and quantified by HPLC. 11.1 microg BuOH extract (that was gained from 1mg dried root) contained 2.0 microg berberine and 0.3 microg/ml palmatine. 1.2 microg/ml berberine inhibited cell proliferation significantly, while 0.5 microg/ml palmatine had no effect. Berberine and the BuOH extract caused accumulation of HL-60 cells in S-phase. This was preceded by a strong activation of Chk2, phosphorylation and degradation of Cdc25A, and the subsequent inactivation of Cdc2 (CDK1). Furthermore, berberine and the extract inhibited the expression of the proto-oncogene cyclin D1. Berberine and the BuOH extract induced the acetylation of alpha-tubulin and this correlated with the induction of apoptosis. The data demonstrate that berberine is a potent anti-neoplastic compound that acts via anti-proliferative and pro-apoptotic mechanisms independent of genotoxicity.

In vitro anti-leukemic activity of the ethno-pharmacological plant Scutellaria orientalis ssp. carica endemic to western Turkey.

AIM OF THIS STUDY Within the genus Scutellaria various species are used in different folk medicines throughout Asia. Traditional Chinese Medicine (TCM) uses S. baicalensis (Labiatae) to treat various inflammatory conditions. The root shows strong anticancer properties in vitro and was suggested for clinical trials against multiple myeloma. Further, S. barbata was successfully tested against metastatic breast cancer in a phase I/II trial. Therefore, we investigated the anti-cancer properties of S. orientalis L. ssp. carica Edmondson, an endemic subspecies from the traditional medicinal plant S. orientalis L. in Turkey, which is used to promote wound healing and to stop haemorrhage. MATERIALS AND METHODS Freeze-dried plant material was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, and by investigating protein expression profiles specific for cell cycle arrest and apoptosis. RESULTS The strongest anti-leukemic activity was shown by the methanol extract, which contained apigenin, baicalein, chrysin, luteolin and wogonin, with an IpC50 of 43 microg/ml (corresponding to 1.3mg/ml of dried plant material) which correlated with cyclin D1- and Cdc25A suppression and p21 induction. At 132 microg/ml (=4 mg/ml of the drug) this extract caused genotoxic stress indicated by substantial phosphorylation of the core histone H2AX (gamma-H2AX) followed by activation of caspase 3 and signature-type cleavage of PARP resulting in a 55% apoptosis rate after 48 hours of treatment. CONCLUSIONS Here, we report for the first time that S. orientalis L. ssp. carica Edmondson exhibited potent anti-leukaemic properties likely through the anti-proliferative effect of baicalein and the genotoxic property of wogonin.

Pro- and anticarcinogenic mechanisms of piceatannol are activated dose dependently in MCF-7 breast cancer cells

Estrogenic procarcinogenic effects of piceatannol (PIC) contrast reports about anticarcinogenic activities of PIC. To explain this contradiction, we investigated PIC in estrogen-dependent MCF-7 breast cancer cells and elucidated those cellular mechanisms that correlated with the observed cell effects induced by PIC. Low PIC concentrations (50 nM) induced c-Myc that depended on progesterone receptor (PR) and estrogen receptor (ER). PR-mediated c-Myc induction by PIC was independent of nuclear PR activity but depended on mitogen-activated protein kinase (MAPK) signaling and was associated with an acceleration of cancer cell proliferation. In contrast, 25 μM PIC inhibited deoxynucleotide triphosphate synthesis, activated Chk2 and p38-MAPK and this was accompanied by an attenuation of cancer cell growth. Apoptosis was most probably inhibited due to activation of Akt; however, high PIC concentrations (>100 μM) permitted apoptosis-like cell death in consequence to disruption of orchestrated mitotic signaling. The presented results show for the first time that nanomolar PIC concentrations signal through PR and Erk1/2 and provide a mechanistic explanation why moderate wine consumption-but not other alcoholic beverages-increases the breast cancer risk in women. In contrast, higher PIC concentrations in the micromolar range are considered for adjuvant anticancer therapeutic concepts.

Fractionation of an Extract of Pluchea odorata Separates a Property Indicative for the Induction of Cell Plasticity from One That Inhibits a Neoplastic Phenotype

Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory.