Astrid Katzer

Plasma osteopontin levels in patients with head and neck cancer and cervix cancer are critically dependent on the choice of ELISA system

BackgroundThe tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma surrogate marker of tumor hypoxia and as an indicator of the presence of pleural mesothelioma in asbestos-exposed individuals. The clinical introduction of plasma OPN measurements requires the availability of a reliable enzyme-linked immunosorbence assay (ELISA).MethodsWe compared previously described and currently available ELISA systems on 88 archival plasma samples obtained from patients with head and neck or cervix cancer between 20 days before and 171 after the start of radiotherapy.ResultsMedian (range) plasma OPN levels were 667 (148.8–2095) ng/ml and 9.8 (3.5–189.5) ng/ml for a previously described and a newly marketed assay, respectively. Although results for different assays were significantly correlated (r = 0.38, p < 0.05, Spearman rank test), between-assay factors ranged from 2.0 to 217.9 (median 74.6) in individual patients. OPN levels in cervix cancer patients were comparable to those of head and neck cancer patients.ConclusionCommercially available OPN ELISA systems produce different absolute plasma OPN levels, compromising a comparison of individual patient data with published results. However, different assays appear to have a similar capacity to rank patients according to plasma OPN level. A review of literature data suggests that plasma OPN levels measured even with identical ELISA systems can only be compared with caution.

Modulation of Carbonic Anhydrase 9 (CA9) in Human Brain Cancer

Hypoxia is a crucial factor in tumour aggressiveness and its treatment resistance, particularly in human brain cancer. Tumour resistance against radiation- and chemo- therapy is facilitated by oxygenation reduction at tumour areas. HIF-1α regulated genes are mostly responsible for this type of resistance. Among these genes, carbonic anhydrase isoform 9 (CA9) is highly overexpressed in many types of cancer especially in high grade brain cancer like GBM. CA IX contributes to tumour environment acidification by catalyzing the carbon dioxide hydration to bicarbonate and protons, leading to the acquisition of metastasic phenotypes and chemoresistance to weakly basic anticancer drugs and therefore to inadequate application of radio-therapeutic or chemotherapeutic anti-cancer treatment strategies. Inhibition of this enzymatic activity by application of specific chemical CA9 inhibitors (sulphonamide derivative compounds) or indirect inhibitors like HIF-1α inhibitors (chetomin) or molecular inhibitors like CA9-siRNA leads to reversion of these processes, leading to the CA9 functional role inhibition during tumourigenesis. Hypoxia significantly influences the tumour microenvironment behaviour via activation of genes involved in the adaptation to the hypoxic stress. It also represents an important cancer prognosis indicator and is associated with aggressive growth, malignant progression, metastasis and poor treatment response. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Sulfonamide derivative compounds with CA9 inhibitory characteristics represent one of the optimal treatment options beside other CA9 inhibitory agents or chemical inhibitory compounds against its main regulating transcription factor which is the hypoxia induced HIF-1α when applied against human cancers with hypoxic regions like GBM, bearing potential for an effective role in human brain tumour therapeutic strategies. Glycolytic inhibitors, when added in controlled doses under hypoxia, lead to a reduced accumulation of HIF-1α and can function as indirect hypoxia regulated genes inhibitors like CA9. These may be used as alternative or in conjunction with other direct inhibitors like the sulphonamide derivate compounds, chetomin or specific siRNAs, or other different chemical compounds possessing similar functionality making them as optimal tools for optimized therapy development in cancer treatment, especially against human brain cancer. Further experimental analysis towards the tumour stage specific inhibitory CA9 characteristics determination are necessary to find the optimal therapeutic solutions among the different available modalities; whether they are direct or indirect chemical, molecular or natural inhibitors to be able to set up successful treatment approaches against the different human tumour diseases.

Radiosensitivity in breast cancer assessed by the histone γ-H2AX and 53BP1 foci

BackgroundHigh expression of constitutive histone γ-H2AX, a sensitive marker of DNA damage, might be indicative of defective DNA repair pathway or genomic instability. 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor. This study explores the relationship between the clinical radiosensitivity of tumor patients and the expression/induction of γ-H2AX and 53BP1 in vitro.MethodsUsing immunostaining, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53 BP1 in peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=57) undergoing radiotherapy (RT). Cells from apparently healthy donors (n=12) served as references.ResultsNon-irradiated cells from controls and unselected BC patients exhibited similar baseline levels of DNA damage assessed by γ-H2AX and 53BP1 foci. At the same time, the γ-H2AX assay of in vitro irradiated cells revealed significant differences between the control group and the group of unselected BC patients with respect to the initial (0.5 Gy, 30 min) and residual (2 Gy, 24 h post-radiation) DNA damage. The numbers of 53BP1 foci analyzed in 35 BC patients were significantly higher than in controls only in case of residual DNA damage. A weak correlation was found between residual foci of both proteins tested. In addition, cells from cancer patients with an adverse acute skin reaction (grade 3) to RT showed significantly increased radiation-induced γ-H2AX foci and their protracted disappearance compared to the group of BC patients with normal skin reaction (grade 0–1). The mean number of γ-H2AX foci after 5 clinical fractions was significantly higher than that before RT, especially in clinically radiosensitive patients.ConclusionsThe γ-H2AX assay may have potential for screening individual radiosensitivity of breast cancer patients.Trial registrationhttp://www.krebshilfe.de/wir-foerdern.html

Hypoxia induced CA9 inhibitory targeting by two different sulfonamide derivatives including Acetazolamide in human Glioblastoma

HIF-1α regulated genes are mainly responsible for tumour resistance to radiation- and chemo-therapy. Among these genes, carbonic anhydrase isoform IX (CA9) is highly over expressed in many types of cancer especially in high grade brain cancer like Glioblastoma (GBM). Inhibition of the enzymatic activity by application of specific chemical CA9 inhibitor sulphonamides (CAI) like Acetazolamide (Aza.), the new sulfonamide derivative carbonic anhydrase inhibitor (SU.D2) or indirect inhibitors like the HIF-1α inhibitor Chetomin or molecular inhibitors like CA9-siRNA are leading to an inhibition of the functional role of CA9 during tumorigenesis. Human GBM cells were treated with in vitro hypoxia (1, 6, or 24 h at 0.1%, O2). Aza. application was at a range between 250 and 8000 nM and the HIF-1α inhibitor Chetomin at a concentration range of 150-500 nM. Cell culture plates were incubated for 24 h under hypoxia (0.1% O2). Further, CA9-siRNA constructs were transiently transfected into GBM cells exposed to extreme hypoxic aeration conditions. CA9 protein expression level was detectable in a cell-type specific manner under normoxic conditions. Whereas U87-MG exhibited a strong aerobic expression, U251 and U373 displayed moderate and GaMG very weak normoxic CA9 protein bands. Aza. as well as SU.D2 displayed inhibitory characteristics to hypoxia induced CA9 expression in the four GBM cell lines for 24 h of hypoxia (0.1% O2) at concentrations between 3500 and 8000 nM, on both the protein and mRNA level. Parallel experiments using CA9-siRNA confirmed these results. Application of 150-500 nM of the glycolysis inhibitor Chetomin under similar oxygenation conditions led to a sharply reduced expression of both CA IX protein and CA9 mRNA levels, indicating a clear glucose availability involvement for the hypoxic HIF-1α and CA9 expression in GBM cells. Hypoxia significantly influences the behaviour of human tumour cells by activation of genes involved in the adaptation to hypoxic stress. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Aza., SU.D2, Chetomin or CA9-siRNA possesses functional CA9 inhibitory characteristics when applied against human cancers with hypoxic regions like GBM. They may be used as alternative or in conjunction with other direct inhibitors possessing similar functionality, thereby rendering them as potential optimal tools for the development of an optimized therapy in human brain cancer treatment.

Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

Background:Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells.Material and Methods:HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER´) was calculated as the ratio of the doses to achieve the same survival at 0.1% O2 as at ambient oxygen tensions. OER´ was obtained at cell survival levels of 50%, 37%, and 10%.Results:HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro.Conclusion:Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro.Hintergrund und Ziel:Hypoxia-inducible Factor-1 (HIF-1) wurde als potentielles therapeutisches Target identifiziert. Ziel der Arbeit war es, zu untersuchen, ob die selektive HIF-1-Inhibition mittels Small Interfering RNA (siRNA) gegen HIF-1α die Strahlensensibilität von hypoxischen HT-1080-Zellen beeinflussen kann.Material und Methodik: Die HIF-1α-Expression in humanen HT-1080-Fibrosarkomzellen wurde mittels RNA-Interferenz nach Transfektion der Zellen mit siRNA unter hypoxischen Bedingungen (0,1%, O2, 12 h), bzw. Normoxie (20% O2) in vitro inhibiert. Die HIF-1α-Genexpression wurde mit quantitativer Realtime-Polymerasekettenreaktion (qRT-PCR), das HIF-1α-Protein mittels Western Blot quantifiziert. Das klonogene Überleben wurde nach Bestrahlung unter Hypoxie und Normoxie bestimmt und daraus eine Oxygen Enhancement Ratio (OER´) bei den Überlebensniveaus 50%, 37% and 10% berechnet.Resultate:HIF-1α-siRNA erhöht die Strahlensensibilität unter hypoxischen Bedingen, verglichen mit HT-1080-Zellen, die mit Kontroll-siRNA behandelt wurden. Die OER` war bei allen Überlebensniveaus reduziert. Schlussfolgerung:Eine selektive Inhibition der HIF-1-Aktivierung durch HIF-1α-siRNA wirkt synergistisch mit einer Bestrahlung und erhöht die Strahlensensitivität hypoxischer Tumorzellen in vitro.

Modulation of Glucose Metabolism Inhibits Hypoxic Accumulation of Hypoxia-Inducible Factor-1α (HIF-1α)

Background and Purpose:The hypoxic accumulation of the transcription factor subunit hypoxia-inducible factor-1α (HIF-1α), a potential endogenous hypoxia marker and therapeutic target, has recently been shown to strongly depend on glucose availability. The aim of this study was to investigate the underlying mechanism of this effect.Material and Methods:HIF-1α protein levels were studied by Western blotting in HT 1080 human fibrosarcoma cells and in a hypoxia-responsive element green fluorescent protein (HRE-GFP) reporter assay in stably transfected HT 1080 cells treated with hypoxia (0.1% O2, 12 h) and glycolysis inhibitors 2-deoxyglucose (2-DG) or iodoacetate (IAA). HIF-1α mRNA expression was quantified via real-time polymerase chain reaction (RT-PCR).Results:Both inhibitors drastically reduced hypoxic HIF-1α accumulation (2-DG + hypoxia 2% mean HIF-1α protein level vs. 59% hypoxia alone; IAA + hypoxia 13% mean HIF-1α protein level vs. 96% hypoxia alone), an effect not rescued by the addition of pyruvate and confirmed in an HRE-GFP reporter assay in stably transfected HT 1080 cells. RT-PCR under identical conditions showed no effect of glycolysis inhibition on HIF-1α mRNA levels, suggesting a translational or posttranslational mechanism.Conclusion:The effect of glycolysis modulation on the HIF-1α levels in tumor cells may provide a novel approach to therapeutically target HIF-1α.Hintergrund und Ziel:Die hypoxische Akkumulation des Hypoxia-inducible factor-1α (HIF-1α), eines potentiellen Hypoxiemarkers und therapeutischen Targets, ist in hohem Maße von der Glucoseverfügbarkeit abhängig. Ziel dieser Arbeit war es, den zugrundeliegenden Mechanismus zu untersuchen.Material und Methodik:Das Protein HIF-1α wurde in humanen HT-1080-Fibrosarkomzellen nach Behandlung der Zellen mit den Glykolyseinhibitoren 2-Desoxy-D-Glucose (2-DG) und Iodacetat (IAA) unter hypoxischen Bedingungen (0,1% O2, 12 h) mittels Western-Blot und mit einem HRE-GFP-(„hypoxia-responsive element green fluorescent protein“-)Reporter-Assay in stabil transfizierten HT-1080-Zellen detektiert. Die Expressionsrate der HIF-1α-mRNA wurde mit Real-Time-Polymerase-Kettenreaktion (RTPCR) gemessen.Ergebnisse:Nach Zugabe von Glykolyseinhibitoren zeigte sich eine deutlich verringerte hypoxische HIF-1α-Akkumulation (Abbildungen 1, 2 und 5: 2-DG + Hypoxie: 2% mittleres HIF-1α-Protein-Level vs. 59% Hypoxie ohne 2-DG; IAA + Hypoxie: 13% mittleres HIF-1α-Protein-Level vs. 96% Hypoxie ohne IAA), die durch die Zugabe von Pyruvat nicht rückgängig gemacht werden konnte (Abbildung 3). Diese Ergebnisse wurden mit einem HRE-GFP-Reporter-Assay in stabil transfizierten HT-1080-Zellen bestätigt (Abbildung 4). Die HIF-1α-RT-PCR zeigte keine veränderten Expressionsraten von HIF-1α-mRNA nach Zugabe von IAA bzw. 2-DG (Abbildung 6). Diese Daten deuten auf einen translationalen bzw. posttranslationalen Mechanismus der Inhibition der hypoxischen HIF-1a-Akkumulation durch Glykolyseinhibitoren hin.Schlussfolgerung:Der Einfluss einer Modulation der Glykolyse auf HIF-1α-Level in Tumorzellen könnte einen neuen Ansatz einer HIF-1α-gerichteten Therapie darstellen.

Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines

This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.

Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia

NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone γH2AX. The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone γH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.

Cell surface area and membrane folding in glioblastoma cell lines differing in PTEN and p53 status.

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C m = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C m values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.

Differential response of human glioblastoma cell lines to combined camptothecin and ionizing radiation treatment.

In order to enhance the cytotoxicity of radiation, camptothecin (CPT), an inhibitor of DNA topoisomerase I, was added to the cultured glioma cell lines before irradiation (IR). Radiation responses of five glioblastoma cell lines (U87-MG, U373-MG, GHE, GaMG and SNB-19) treated with CPT were analyzed in terms of cell and colony counts, cell cycle progression, expression of histone γH2AX, DNA repair protein Rad50, survivin, cleaved caspase 3, p53 and of topoisomerase I. CPT enhanced the radiotoxicity in U87-MG and SNB-19 cell lines if cell and colony counts were used as the end-points. In contrast, pre-treatment with CPT of U373-MG, GHE and GaMG cell lines did not enhance cytotoxicity of IR in terms of cell and colony counts but accelerated DNA damage repair assessed by Rad50 foci. CPT treated glioma cells revealed at least two subpopulations with respect to the expression of histone γH2AX, a marker of DNA double-strand breaks. The cell lines tested also differed in the expression of survivin, cleaved caspase 3, p53 and of topoisomerase I. The failure of CPT to enhance the radiotoxicity of glioma U373-MG, GHE and GaMG cell lines in terms of cell and colony counts was found to correlate with accelerated DNA damage repair, and with low expression of topoisomerase I, a target of CPT.