Harun M. Said

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.

GAPDH is not regulated in human glioblastoma under hypoxic conditions.

BackgroundGene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.ResultsWe observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.ConclusionGAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.

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.

Absence of GAPDH regulation in tumor-cells of different origin under hypoxic conditions in - vitro.

BackgroundGene expression studies related to cancer diagnosis and treatment are important. In order to conduct such experiment accurately, absolutely reliable housekeeping genes are essential to normalize cancer related gene expression. The most important characteristics of such genes are their presence in all cells and their expression levels remain relatively constant under different experimental conditions. However, no single gene of this group of genes manifests always stable expression levels under all experimental conditions. Incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of GAPDH expression regulation in Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines as well as in human lung adenocarcinoma epithelial cell line (A-549) in addition to both HT-29, and HCT-116 colon cancer cell lines, under hypoxic conditions in vitro in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches was comparatively examined in vitro on both protein and mRNA level, by western blot and semi quantitative RT-PCR, respectively.FindingsNo hypoxia-induced regulatory effect on GAPDH expression was observed in the cell lines studied in vitro that were; Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines, Human lung adenocarcinoma epithelial cell line (A-549), both colon cancer cell lines HT-29, and HCT-116.ConclusionAs it is the case for human hepatocellular carcinoma, mouse hepatoma, human colon cancer, and human lung adenocarcinoma, GAPDH represents an optimal choice of a housekeeping gene and/(or) loading control to determine the expression of hypoxia induced genes in tumors of different origin. The results confirm our previous findings in human glioblastoma that this gene is not an attractive target for tumor therapeutic approaches because of the lack of GAPDH regulation under hypoxia.

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.

Elevated tumor and serum levels of the hypoxia-associated protein osteopontin are associated with prognosis for soft tissue sarcoma patients

BackgroundOsteopontin (OPN) overexpression is correlated with a poor prognosis for tumor patients. However, only a few studies investigated the prognostic impact of expression of OPN in soft tissue sarcomas (STS) yet.MethodsThis study is based on tumor and serum samples from 93 adult STS patients. We investigated OPN protein levels in serum (n = 86) and tumor tissue (n = 80) by ELISA and OPN mRNA levels in tumor tissue (n = 68) by quantitative real-time PCR.ResultsNo correlation was found between OPN levels in serum and tumor tissue. Moreover, an elevated OPN protein level in the serum was significantly associated with clinical parameters such as higher stage (p = 0.004), higher grade (p = 0.003), subtype (p = 0.002) and larger tumor size (p = 0.03). OPN protein levels in the tumor tissue were associated with higher stage (p = 0.06), higher grade (p = 0.003), subtype (p = 0.07) and an increased rate of relapse (p = 0.02). In addition, using a Coxs proportional hazards regression model, we found that an elevated OPN protein level in the serum and tumor tissue extracts is a significant negative prognostic factor for patients with STS. The relative risks of tumor-related death were 2.2 (p < 0.05) and 3.7 (p = 0.01), respectively.ConclusionOur data suggest OPN protein in serum as well as in tumor tissue extracts is an important prognostic factor for soft tissue sarcoma patients.

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.

Hypoxia and cytokines regulate carbonic anhydrase 9 expression in hepatocellular carcinoma cells in vitro

AIM To study the expression of carbonic anhydrase (CA) 9 in human hepatocellular carcinoma (HCC) cells. METHODS We studied CA9 protein, CA9 mRNA and hypoxia-inducible factor-1 alpha (HIF-1α) protein levels in Hep3B cells exposed in different parallel approaches. In one of these approaches, HCC cells were exposed to extreme in vitro hypoxia (24 h 0.1% O(2)) without or with interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) stimulation for the same hypoxic exposure time or exposed to normoxic oxygenation conditions without or with cytokine stimulation. RESULTS The tumour cell line analysed showed a strong hypoxic CA9 mRNA expression pattern in response to prolonged severe hypoxia with cell-line specific patterns and a marked induction of CA9 protein in response to severe hypoxia. These results were paralleled by the results for HIF-1α protein under identical oxygenation conditions with a similar expression tendency to that displayed during the CA9 protein expression experimental series. Continuous stimulation with the cytokines, IL-1, IL-6, TNF-α and TGF-β, under normoxic conditions significantly increased the carbonic anhydrase 9 expression level at both the protein and mRNA level, almost doubling the CA9 mRNA and CA9 and HIF-1α protein expression levels found under hypoxia. The findings from these experiments indicated that hypoxia is a positive regulator of CA9 expression in HCC, and the four signal transduction pathways, IL-1, IL-6, TNF-α and TGF-β, positively influence CA9 expression under both normoxic and hypoxic conditions. CONCLUSION These findings may potentially be considered in the design of anti- cancer therapeutic approaches involving hypoxia-induced or cytokine stimulatory effects on expression. In addition, they provide evidence of the stimulatory role of the examined cytokine families resulting in an increase in CA9 expression under different oxygenation conditions in human cancer, especially HCC, and on the role of the CA9 gene as a positive disease regulator in human cancer.

Egr-1 is not upregulated in response to hypoxic and oxygenation conditions in human glioblastoma in vitro

The early growth response factor 1 (Egr-1) gene (also known as krox24, NGFI-A, TIS8 or zif268) belongs to a family of immediate early response genes. This family of proteins contains a conserved zinc finger DNA-binding domain and can bind to a GC-rich sequence in the promoter region of target genes. Egr-1 expression is rapidly and transiently activated in many different cell types during development. In adult tissues, a variety of signals, including serum, growth factors, cytokines and hormones, stimulate Egr-1 expression. In several studies, it was demonstrated that the transcription factor Egr-1 is regulated by hypoxia, and it is hypothesized that Egr-1 is responsible for the hypoxia-induced regulation of the N-Myc downregulated gene 1 (NDRG1) in human tumor cells. In the present study, Egr-1 regulation was examined in the human glioblastoma cell lines U373, U251, GaMG and U87-MG under extreme hypoxic aeration conditions (0.1% O2) for 1, 6 and 24 h, 24-h extreme hypoxia with reoxygenation for 24 and 48 h, respectively, as well as oxygenated conditions (21% O2 and 5% CO2) in vitro. Protein and mRNA levels were detected in the lysates by Western blotting and RT-PCR, respectively. Egr-1 expression under hypoxic conditions was compared with the well-known and characterized hypoxia-induced gene regulator hypoxia-inducible factor-1α (HIF-1α) in parallel experimental sets. Cells incubated for 24 h with 100 µM desferroxamine served as a positive control for hypoxia, and β-tubulin and β-actin were used as loading controls. The experimental data indicate that Egr-1 was not upregulated under extreme hypoxic conditions (0.1% O2) or by reoxygenation after hypoxia in different glioblastoma cells in vitro. In conclusion, the regulation of Egr-1 in reaction to hypoxic development, at both the protein and mRNA levels, is not a general phenomenon. In contrast to previously published data, no Egr-1 regulatory events were observed in glioblastoma under hypoxic conditions in vitro. We suggest that Egr-1 regulation in human tumors in reaction to hypoxia could be a cell-specific post-translational event. Therefore, at least in glioblastoma, HIF-1α can be considered a major regulator of NDRG1 under hypoxic conditions. Further extensive analysis of tumor cells from different origins under similar physiological conditions is necessary to increase our knowledge of the conditions and functional role of Egr-1 in the regulation of hypoxia-induced gene expression.