Amir Abdollahi

MicroRNA expression after ionizing radiation in human endothelial cells

BackgroundEndothelial cells (EC) in tumor and normal tissue constitute critical radiotherapy targets. MicroRNAs have emerged as master switchers of the cellular transcriptome. Here, we seek to investigate the role of miRNAs in primary human dermal microvascular endothelial cells (HDMEC) after ionizing radiation.MethodsThe microRNA status in HDMEC after 2 Gy radiation treatment was measured using oligo-microarrays covering 361 miRNAs. To functionally analyze the role of radiation-induced differentially regulated miRNAs, cells were transfected with miRNA precursor or inhibitor constructs. Clonogenic survival and proliferation assays were performed.ResultsRadiation up-regulated miRNA expression levels included let-7g, miR-16, miR-20a, miR-21 and miR-29c, while miR-18a, miR-125a, miR-127, miR-148b, miR-189 and miR-503 were down-regulated. We found that overexpression or inhibition of let-7g, miR-189, and miR-20a markedly influenced clonogenic survival and cell proliferation per se. Notably, the radiosensitivity of HDMEC was significantly influenced by differential expression of miR-125a, -127, -189, and let-7g. While miR-125a and miR-189 had a radioprotective effect, miR-127 and let-7g enhanced radiosensitivity in human endothelial cells.ConclusionOur data show that ionizing radiation changes microRNA levels in human endothelial cells and, moreover, exerts biological effects on cell growth and clonogenicity as validated in functional assays. The data also suggest that the miRNAs which are differentially expressed after radiation modulate the intrinsic radiosensitivity of endothelial cells in subsequent irradiations. This indicates that miRNAs are part of the innate response mechanism of the endothelium to radiation.

Intercellular Communication by Exchange of Cytoplasmic Material via Tunneling Nano-Tube Like Structures in Primary Human Renal Epithelial Cells

Transfer of cellular material via tunneling nanotubes (TNT) was recently discovered as a novel mechanism for intercellular communication. The role of intercellular exchange in communication of renal epithelium is not known. Here we report extensive spontaneous intercellular exchange of cargo vesicles and organelles between primary human proximal tubular epithelial cells (RPTEC). Cells were labeled with two different quantum dot nanocrystals (Qtracker 605 or 525) and intercellular exchange was quantified by high-throughput fluorescence imaging and FACS analysis. In co-culture, a substantial fraction of cells (67.5%) contained both dyes indicating high levels of spontaneous intercellular exchange in RPTEC. The double positive cells could be divided into three categories based on the preponderance of 605 Qtracker (46.30%), 525 Qtracker (48.3%) and approximately equal content of both Qtrackers (4.57%). The transfer of mitochondria between RPTECs was also detected using an organelle specific dye. Inhibition of TNT genesis by actin polymerization inhibitor (Latrunculin B) markedly reduced intercellular exchange (>60%) suggesting that intercellular exchange in RPTEC was in part mediated via TNT-like structures. In contrast, induction of cellular stress by Zeocin treatment increased tube-genesis in RPTEC. Our data indicates an unexpected dynamic of intercellular communication between RPTEC by exchange of cytosolic material, which may play an important role in renal physiology.

Consensus Micro RNAs Governing the Switch of Dormant Tumors to the Fast-Growing Angiogenic Phenotype

Tumor dormancy refers to a critical stage in cancer development in which tumor cells remain occult for a prolonged period of time until they eventually progress and become clinically apparent. We previously showed that the switch of dormant tumors to fast-growth is angiogenesis dependent and requires a stable transcriptional reprogramming in tumor cells. Considering microRNAs (miRs) as master regulators of transcriptome, we sought to investigate their role in the control of tumor dormancy. We report here the identification of a consensus set of 19 miRs that govern the phenotypic switch of human dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors to fast-growth. Loss of expression of dormancy-associated miRs (DmiRs, 16/19) was the prevailing regulation pattern correlating with the switch of dormant tumors to fast-growth. The expression pattern of two DmiRs (miR-580 and 190) was confirmed to correlate with disease stage in human glioma specimens. Reconstitution of a single DmiR (miR-580, 588 or 190) led to phenotypic reversal of fast-growing angiogenic tumors towards prolonged tumor dormancy. Of note, 60% of angiogenic glioblastoma and 100% of angiogenic osteosarcoma over-expressing miR190 remained dormant during the entire observation period of ∼ 120 days. Next, the ability of DmiRs to regulate angiogenesis and dormancy-associated genes was evaluated. Transcriptional reprogramming of tumors via DmiR-580, 588 or 190 over-expression resulted in downregulation of pro-angiogenic factors such as TIMP-3, bFGF and TGFalpha. In addition, a G-CSF independent downregulation of Bv8 was found as a common target of all three DmiRs and correlated with decreased tumor recruitment of bone marrow-derived CD11b+ Gr-1+ myeloid cells. In contrast, antiangiogenic and dormancy promoting pathways such as EphA5 and Angiomotin were upregulated in DmiR over-expressing tumors. This work suggests novel means to reverse the malignant tumor phenotype into an asymptomatic dormant state and may provide promising targets for early detection or prevention of cancer.

Peroxisome Proliferator-Activated Receptor (PPAR)γ can inhibit chronic renal allograft damage

Chronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor known to have antidiabetogenic and immune effects, and PPARgamma forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARgamma-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 +/- 5.8 (controls) vs. 8.1 +/- 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 +/- 1.8 (controls) vs. 2.6 +/- 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-beta1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARgamma activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARgamma-dependent manner. The combination of PPARgamma- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARgamma activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist.

Prospective identification of glioblastoma cells generating dormant tumors.

Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant tumors have an impaired angiogenesis potential both in vitro and in vivo and have a slower invasion capacity. This work demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence, and changes in rate of tumor progression.

Celecoxib Enhances Radiation Response of Secondary Bone Tumors of a Human Non-Small Cell Lung Cancer via Antiangiogenesis In Vivo

AbstractPurpose:Cyclooxygenase-2 (COX-2) inhibitors mediate a systemic antitumor activity via antiangiogenesis and seem to enhance the response of primary tumors to radiation. Radiosensitizing effects of COX-2 inhibition have not been reported for bone metastases. Therefore, the aim of this study was the investigation of the radiosensitizing effects of the selective COX-2 inhibitor celecoxib in secondary bone tumors of a non-small cell lung carcinoma in vivo.Materials and Methods:Human A549 lung carcinomas were implanted into a cranial window preparation in male SCID mice (n = 24). Animals were treated with either celecoxib or radiation (7 Gy single photon dose) alone or a combination of celecoxib and radiation, respectively. Untreated animals served as controls. The impact of radiation and COX-2 inhibition on angiogenesis, microcirculation, and tumor growth was analyzed over 28 days by means of intravital microscopy and histological methods.Results:Monotherapies with radiation as well as celecoxib had significant antitumor effects compared to untreated controls. Both therapies reduced tumor growth and vascularization to a similar extent. The simultaneous administration of celecoxib and radiation further enhanced the antitumor and antiangiogenic effects of single-beam radiation. With the combined treatment approach, tumor vascularization and tumor size were decreased by 57% and 51%, respectively, as compared to monotherapy with radiation.Conclusion:The combined application of radiation therapy and COX-2 inhibition showed synergistic effects concerning the inhibition of tumor growth and tumor angiogenesis. Therefore, the combination of radiation with COX-2 inhibitor therapy represents a promising approach to improve the therapeutic efficacy of radiotherapy of bone metastases.ZusammenfassungZiel:Cyclooxygenase-(COX-)2-Inhibitoren vermitteln systemisch eine Wirkung gegen Tumoren indem sie die Tumorangiogenese hemmen und das Ansprechen von Primärtumoren auf Bestrahlung zu verbessern scheinen. Über strahlensensibilisierende Effekte einer COX-2-Hemmung bei der Behandlung von Knochenmetastasen wurde bisher nicht berichtet. Das Ziel dieser Studie war daher die Untersuchung der strahlensensibilisierenden Effekte des selektiven COX-2-Inhibitors Celecoxib bei sekundären Knochentumoren eines nicht-kleinzelligen Lungenkarzinoms in vivo.Material und Methoden:Humane A549 Lungenkarzinome wurden in eine Schädelfensterpräparation bei männlichen SCID- Mäusen (n = 24) implantiert. Die Tiere wurden entweder mit Celecoxib oder Strahlentherapie (7 Gy als Einzeldosis) alleine oder mit einer Kombination aus Celecoxib und Bestrahlung behandelt. Unbehandelte Tiere dienten als Kontrolle. Es wurden der Einfluss der Bestrahlung und der COX-2-Hemmung auf Angiogenese, Mikrozirkulation und Tumorwachstum über einen Zeitraum von 28 Tagen mit Hilfe der Intravitalmikroskopie und histologischer Methoden analysiert.Ergebnisse:Die Monotherapien in Form einer Einmalbestrahlung mit 7 Gy und einer Behandlung mit Celecoxib zeigten signifikante anti-tumorale Effekte im Vergleich zu unbehandelten Kontrollen. Beide Therapien reduzierten Tumorwachstum und Vaskularisation in ähnlichem Ausmaß. Die simultane Anwendung von Celecoxib und Bestrahlung steigerte die anti-tumoralen und die anti-angiogenen Effekte der Einmalbestrahlung. Mit dem kombinierten Behandlungsansatz wurde die Tumorvaskularisation und die Tumorgröße um 57% beziehungsweise 51% im Vergleich zur Strahlentherapie alleine verringert.Schlussfolgerung:Die kombinierte Anwendung von Strahlentherapie und COX-2-Hemmung zeigte synergistische Effekte hinsichtlich der Hemmung des Tumorwachstums und der Tumorangiogenese. Somit repräsentiert die Kombination von Strahlentherapie mit einer COX-2-Inhibiton einen vielversprechenden Ansatz, um die therapeutische Effektivität der Bestrahlung von Knochenmetastasen zu verbessern.

Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells.

BackgroundQuantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies.MethodsWe tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm.ResultsExpression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p ≤ 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly normalize expression data in the more variable A549 cells. Considering both variables tested, i.e. cell type and radiation qualities, 5 genes-- RPLP0, UBC, PPIA, TBP and PSMC4-- were identified as the consensus set of stable ECGs.ConclusionsCaution is warranted when selecting the internal control gene for the qRT-PCR gene expression studies. Here, we provide a template of stable ECGs for investigation of radiation induced gene expression.

A novel model for the investigation of orthotopically growing primary and secondary bone tumours using intravital microscopy

Here is reported the development of an experimental model using intravital microscopy as a tool to orthotopically investigate malignant bone tumours. Although up to 85% of the most frequently occurring malignant solid tumours, such as lung and prostate carcinomas, metastasize into the bone, and despite the knowledge that a tumours course may be altered by its surrounding tissue, there is no adequate experimental model available enabling the investigation of orthotopically grown bone tumours in vivo. Intravital microscopy is an internationally accepted experimental method, used in various acute and chronic animal models, that enables qualitative and quantitative analysis of the angiogenesis, microcirculation, growth behaviour, etc. of various benign and malignant tissues. Non-invasive investigations of up to several weeks are possible. Additionally, tissue samples can be taken after termination of the in vivo experiments for further ex vivo investigation (histology, immunohistochemistry, molecular biology, etc.), elucidating the mechanisms that underlie the in vivo observations. Severe combined immunodeficient mice were fitted with a cranial window preparation where the calvaria served as the site for orthotopic implantation of the solid human tumours Saos-2 osteosarcoma (primary) and A 549 lung carcinoma and PC-3 prostate carcinoma (secondary). In all preparations, the take rate was 100%. Histological assessment confirmed the data obtained in vivo, showing typical tumour growth with infiltration of the surrounding osseous and soft tissues. This novel model serves as a valuable tool in understanding the biology of primary and secondary bone tumours in physiological and pathophysiological situations, with implications for the most areas of tumour therapy such as chemotherapy, radiation and antiangiogenesis.

Radiosensitization by histone deacetylase inhibition in an osteosarcoma mouse model

BackgroundOsteosarcomas (OS) are highly malignant and radioresistant tumors. Histone deacetylase inhibitors (HDACi) constitute a novel class of anticancer agents. We sought to investigate the effect of combined treatment with suberoylanilide hydroxamic acid (SAHA) and radiotherapy in OS in vivo.MethodsClonogenic survival of human OS cell lines as well as tumor growth delay of OS xenografts were tested after treatment with either vehicle, radiotherapy (XRT), SAHA, or XRT and SAHA. Tumor proliferation, necrosis, microvascular density, apoptosis, and p53/p21 were monitored by immunohistochemistry. The CD95 pathway was performed by flow cytometry, caspase (3/7/8) activity measurements, and functional inhibition of CD95 death signaling.ResultsCombined treatment with SAHA and XRT markedly reduced the surviving fraction of OS cells as compared to XRT alone. Likewise, dual therapy significantly inhibited OS tumor growth in vivo as compared to XRT alone, reflected by reduced tumor proliferation, impaired angiogenesis, and increased apoptosis. Addition of HDACi to XRT led to elevated p53, p21, CD95, and CD95L expression. Inhibition of CD95 signaling reduced HDACi- and XRT-induced apoptosis.ConclusionOur data show that HDACi increases the radiosensitivity of osteosarcoma cells at least in part via ligand-induced apoptosis. HDACi thus emerge as potentially useful treatment components of OS.ZusammenfassungZielsetzungOsteosarkome (OS) sind hochmaligne und relative radioresistente Tumore. Histon-Deacetylase-Inhibitoren (HDACi) stellen eine neue Substanzklasse in der Tumortherapie dar, insbesondere in Kombination mit anderen antineoplastischen Therapiestrategien. Wir haben deshalb die Effekte der Kombinationstherapie von Suberoylanilide Hydroxamic Acid (SAHA) und konventioneller Radiotherapie im OS-Mausmodell untersucht.MethodenEs wurde die Tumorwachstumsverzögerung in OS-Xenografts nach Behandlung mit Radiotherapie (XRT), SAHA alleine oder XRT und SAHA untersucht. Die Tumorproliferation, Tumornekrose, Tumorvaskularisation, Apoptose und p53/p21-Expression wurden immunohistochemisch analysiert. Die Bestimmung der CD95-vermittelten Apoptose erfolgte durchflusszytometrisch sowie durch Messung der Caspase-(3-/7-/8-)Aktivität und funktionaler Inhibition des CD95-Liganden.ErgebnisseDie Kombinationsbehandlung führt zu einer signifikanten Reduktion des Tumorwachstums in vivo im Vergleich zur alleinigen Radiotherapie, reflektiert durch eine verminderte Tumorproliferation sowie reduzierte Angiogenese und gesteigerte Apoptoseinduktion. Die Kombination von HDACi und XRT resultiert in einer gesteigerten p53-, p21-, CD95- und CD95L-Expression. Eine Inhibition des CD95-Liganden führt zu einer reduzierten HDACi- und XRT-induzierten Apoptoserate nach Kombinationstherapie.SchlußfolgerungDie Ergebnisse zeigen, dass eine HDACi die Radiosensitivität von Osteosarkomzellen signifikant erhöht. Dies erfolgt – zumindest teilweise – durch die CD95-abhängige Apoptoseinduktion. Die Autoren sind daher der Meinung, dass HDACi eine potenziell wirksame Therapiestrategie beim OS darstellt.

Gene expression signatures in the peripheral blood after radiosurgery of human cerebral arteriovenous malformations

Purpose:To unravel biological mechanisms potentially resulting in the obliteration process after radiosurgery (RS) of human cerebral arteriovenous malformations (AVMs) by investigating molecular signatures on the transcriptomic level in peripheral blood of patients.Patients and Methods:Venous blood samples were obtained at definite points of time before and after RS. The samples were tested for radiation-induced changes regarding biological markers (mRNA) using cDNA and oligo-microarray technology. The corresponding expression profiles were correlated with clinical data and obliteration signs in radiologic imaging.Results:The proof of principle that RS outcome can be successfully correlated with transcriptomics of cellular blood components as disease parameter was demonstrated. The authors identified 76 differentially regulated genes (p < 0.001) after RS. Interestingly, in particular genes with known roles in antiangiogenic and procoagulative pathways were identified as potentially relevant. In particularly, the authors found a significant downregulation of neuropilin-2, protein C inhibitor and cyclin-dependent kinase 6. They also found that low pretreatment blood mRNA levels of TLR4 (toll-like receptor 4) and STAT3 (signal transducer and activator of transcription 3) correlated with fast obliteration of AVMs.Conclusion:The authors report on a novel technique for molecular biological analysis of blood from patients with cerebral AVM treated with RS. Differential regulation of genes in peripheral blood was successfully correlated with RS and time to obliteration of AVMs. The identified genes indicate a potential new methodology to monitor RS, which may result in an individualized therapy and optimized follow-up.Ziel:Untersucht wurden die molekularen Mechanismen der Wirkung ionisierender Strahlung auf das pathologische Gefäßkonvolut bei zerebralen arteriovenösen Malformationen (AVM) im peripheren Blut von Patienten nach Radiochirurgie (RS).Patienten und Methodik:Bei Patienten mit zerebraler AVM wurde zu definierten Zeitpunkten vor und nach RS eine venöse Blutprobe gewonnen und mittels cDNA- und Oligo-Microarray-Technologie auf strahleninduzierte Veränderungen hinsichtlich biologischer Marker (mRNA) getestet. Die entsprechenden Expressionsprofile wurden mit dem Obliterationsverlauf in der radiologischen Bildgebung verglichen.Ergebnisse:Erstmals konnte gezeigt werden, dass eine Korrelation zwischen klinischen Parametern nach RS zerebraler AVM und Genaktivität von peripheren Blutbestandteilen möglich ist. Es konnten 76 Gene als signifikant (p < 0,001) reguliert identifiziert werden. Insbesondere wurden antiangiogene Mechanismen der Strahlenwirkung sowie prokoagulatorische Effekte als funktionell relevant identifiziert. Beispielsweise zeigte sich eine signifikant verminderte Expression von Neuropilin-2 (NRP-2), Protein-C-Inhibitor (PCI) und „cyclin-dependent kinase 6“ (CDK6). Darüber hinaus konnten eine positive Korrelation zwischen niedrigen prätherapeutischen mRNA-Werten von TLR4 („toll-like receptor 4“) und STAT3 (signal transducer and activator of transcription 3) im Blut der Patienten und schneller Obliteration der AVM nachgewiesen werden.Schlussfolgerung:Durch genomweite Untersuchungen an Blutproben von Patienten mit zerebraler AVM vor und nach RS konnten erstmals molekulare Signaturen im peripheren Blut identifiziert werden. Diese erlauben möglicherweise ein biologisches Therapiemonitoring. Die unterschiedliche Genregulation im peripheren Blut nach RS konnte erfolgreich mit der Zeit bis zur Obliteration der AVM korreliert werden. Weiterführende Untersuchungen der identifizierten Gene und Proteine sollen in Zukunft eine optimierte Verlaufskontrolle nach Radiotherapie und eine individuell optimierte Behandlung ermöglichen.