Efstathia Giannopoulou

X-rays modulate extracellular matrix in vivo

X‐rays have an antiangiogenic effect in the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis. Our study demonstrates that X‐rays induce an early apoptosis of CAM cells, modulate the synthesis and deposition of extracellular matrix (ECM) proteins involved in regulating angiogenesis and affect angiogenesis induced by tumour cells implanted onto the CAM. Apoptosis was evident within 1–2 hr, but not later than 6 hr after irradiation. Fibronectin, laminin, collagen type I, integrin αvβ3 and MMP‐2 protein amounts were all decreased 6 hr after irradiation. In contrast, collagen type IV, which is restricted to basement membrane, was not affected by irradiation of the CAM. There was a similar decrease of gene expression for fibronectin, laminin, collagen type I and MMP‐2, 6 hr after irradiation. The levels of mRNA for integrin αvβ3 and collagen type IV were unaffected up to 24 hr after irradiation. The decrease in both protein and mRNA levels was reversed at later time points and 48 hr after irradiation, there was a significant increase in the expression of all the genes studied. When C6 glioma tumour cells were implanted on irradiated CAMs, there was a significant increase in the angiogenesis induced by tumour cells, compared to that in non‐irradiated CAMs. Therefore, although X‐rays have an initial inhibitory effect on angiogenesis, their action on the ECM enhances new vessel formation induced by glioma cells implanted on the tissue.

Angiogenesis and Anti-Angiogenic Molecularly Targeted Therapies in Malignant Gliomas

Angiogenesis is considered to be a regulating factor of vascular development and growth for malignant gliomas, including glioblastoma multiforme (GBM) and anaplastic astrocytomas. The mechanism of angiogenesis is primarily mediated by hypoxia through chronic activation of the HIF pathway leading to the production of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. Alternatively, it can be triggered by genetic factors. The VEGF/VEGFR-2 is the predominant angiogenic signalling pathway in malignant gliomas. Currently, anti-angiogenic molecularly targeted therapies, including administration of monoclonal antibodies or tyrosine kinase inhibitors (TKIs), are being increasingly adopted for treating GBMs. This approach is based on the ability of anti-VEGFRs monoclonal antibodies to decrease vascular permeability and perfusion, whereas the use of TKIs is mainly based on their capacity to interfere with cell communication, receptor signaling and growth of tumours. Our aim is to review current knowledge on angiogenesis as a molecular pathogenetic mechanism of malignant gliomas and to critically look at and discuss antiangiogenic molecularly targeted therapies for these brain malignancies. We also highlight areas of future research to pursue.

Cell-matrix interactions: focus on proteoglycan-proteinase interplay and pharmacological targeting in cancer.

Proteoglycans are major constituents of extracellular matrices, as well as cell surfaces and basement membranes. They play key roles in supporting the dynamic extracellular matrix by generating complex structural networks with other macromolecules and by regulating cellular phenotypes and signaling. It is becoming evident, however, that proteolytic enzymes are required partners for matrix remodeling and for modulating cell signaling via matrix constituents. Proteinases contribute to all stages of diseases, particularly cancer development and progression, and contextually participate in either the removal of damaged products or in the processing of matrix molecules and signaling receptors. The dynamic interplay between proteoglycans and proteolytic enzymes is a crucial biological step that contributes to the pathophysiology of cancer and inflammation. Moreover, proteoglycans are implicated in the expression and secretion of proteolytic enzymes and often modulate their activities. In this review, we describe the emerging biological roles of proteoglycans and proteinases, with a special emphasis on their complex interplay. We critically evaluate this important proteoglycan–proteinase interactome and discuss future challenges with respect to targeting this axis in the treatment of cancer.

Expression of the ribonucleases Drosha, Dicer, and Ago2 in colorectal carcinomas

The pathogenesis of colorectal carcinoma (CRC) is a complex process that involves the recruitment of both genetic and epigenetic mechanisms. Recent studies underline the cardinal role of small, noncoding RNA molecules, called microRNAs (miRs), in the pathobiology of numerous physiological and pathological processes, including oncogenesis. MiR biogenesis and maturation is mainly regulated by the nuclear ribonuclease Drosha and the cytoplasmic ribonucleases Dicer and Ago2. In the present study, we investigated the expression and distribution of these molecules in three colon cancer cell lines and in human CRC samples. Drosha, Dicer, and Ago2 mRNA and protein expression was assessed with real-time PCR, western blotting, and immunofluorescence. Our experiments showed that Drosha, Dicer, and Ago2 were expressed in all the cell lines and in the majority of the CRC samples examined. The mRNA levels of Dicer were significantly augmented in stage III compared to stage II tumors. Our results suggest that Drosha, Dicer, and Ago2 are possibly implicated in CRC pathobiology and that Dicer might have a role in the progression of these tumors to advanced stages.

A novel study investigating the therapeutic outcome of patients with lytic, mixed and sclerotic bone metastases treated with combined radiotherapy and ibandronate

PurposeTo investigate the therapeutic response of patients with different types of bone metastases treated with combined radiotherapy and bisphosphonates.Patients and methodsBy using computed tomography 52 patients were grouped into groups of lytic, mixed and sclerotic bone lesions. All patients were treated with concomitant radiotherapy and ibandronate (10 monthly cycles) and underwent clinical and radiological evaluations prior to therapy and at 3, 6 and 10 months of follow up.ResultsAt baseline there were statistically significant differences between the three groups for all the evaluated parameters. From 3 months onwards differences were leveled out. Statistically significant improvements were noted at all time points of evaluation for all groups in parameters such as pain (0–10), quality of life (QOL-physical functioning, 0–100) and Karnofsky performance status (KPS). The average pain score for the lytic group was reduced from 8.1 to 1.5 points at 3 months. The corresponding reductions for the mixed and sclerotic groups were from 6.2 to 0.5 and from 4.4 to 0.3 points respectively. Complete pain responses were >76.4% at all time points for all groups. Opioid consumption was also markedly reduced. Overall, the highest clinical response was noted for the lytic group, even though the mean values of pain, QOL and KPS were worse than those of the two other groups at all time points (apart from pain score at 10 months). The percentage of patients of the lytic group experiencing a complete pain response was the least of the three groups during follow up. At 10 months bone density was almost tripled for the lytic and almost doubled for the mixed group.ConclusionsEven though the therapeutic outcome for the three groups was similar, the degree of clinical response and reossification differed.

Serglycin is implicated in the promotion of aggressive phenotype of breast cancer cells.

Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients’ material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of ∼250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (∼87%), 6-sulfated (∼10%) and non-sulfated (∼3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.

Antiproliferative effect of exemestane in lung cancer cells

BackgroundRecent evidence suggests that estrogen signaling may be involved in the pathogenesis of non-small cell lung cancer (NSCLC). Aromatase is an enzyme complex that catalyses the final step in estrogen synthesis and is present in several tissues, including the lung. In the current study we investigated the activity of the aromatase inhibitor exemestane in human NSCLC cell lines H23 and A549.ResultsAromatase expression was detected in both cell lines. H23 cells showed lower protein and mRNA levels of aromatase, compared to A549 cells. Exemestane decreased cell proliferation and increased apoptosis in both cell lines, 48 h after its application, with A549 exhibiting higher sensitivity than H23 cells. Aromatase protein and mRNA levels were not affected by exemestane in A549 cells, whereas an increase in both protein and mRNA levels was observed in H23 cells, 48 h after exemestane application. Moreover, an increase in cAMP levels was found in both cell lines, 15 min after the administration of exemestane. In addition, we studied the effect of exemestane on epidermal growth factor receptor (EGFR) localization and activation. Exemestane increased EGFR activation 15 min after its application in H23 cells. Furthermore, we demonstrated a translocation of EGFR from cell membrane, 24 h after the addition of exemestane in H23 cells. No changes in EGFR activation or localization were observed in A549 cells.ConclusionOur findings suggest an antiproliferative effect of exemestane on NSCLC cell lines. Exemestane may be more effective in cells with higher aromatase levels. Further studies are needed to assess the activity of exemestane in NSCLC.

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane.

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and alpha-tubulin. Quantification of actin and alpha-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.

Pleiotrophin expression and role in physiological angiogenesis in vivo: potential involvement of nucleolin

BackgroundPleiotrophin (PTN) is a heparin-binding growth factor with significant role(s) in tumour growth and angiogenesis. Although implication of endogenous PTN has been studied in several in vivo models of tumour angiogenesis, its role in physiological angiogenesis has not been addressed. In the present work, we studied expression and functional significance of endogenous PTN during angiogenesis in the chicken embryo chorioallantoic membrane (CAM).MethodsUsing molecular, cellular and biochemical assays, we studied the expression pattern of PTN in CAM and human endothelial cells and its possible interaction with nucleolin (NCL). CAM cells were transfected with a pCDNA3.1 vector, empty (PC) or containing full length cDNA for PTN in antisense orientation (AS-PTN). Angiogenesis was estimated by measuring total vessel length. In vitro, human endothelial cells migration was studied by using a transwell assay, and down-regulation of NCL was performed by using a proper siRNA.ResultsEndogenous PTN mRNA and protein levels, as well as protein levels of its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) were maximal at early stages, when CAM angiogenesis is active. Application of AS-PTN onto CAM at days of active angiogenesis was not toxic to the tissue and led to dose-dependent decreased expression of endogenous PTN, ERK1/2 activity and angiogenesis. Interestingly, endogenous PTN was also immunolocalized at the endothelial cell nucleus, possibly through interaction with NCL, a protein that has a significant role in the nuclear translocation of many proteins. Down-regulation of NCL by siRNA in human endothelial cells significantly decreased nuclear PTN, verifying this hypothesis. Moreover, it led to abolishment of PTN-induced endothelial cell migration, suggesting, for the first time, that PTN-NCL interaction has a functional significance.ConclusionsExpression of endogenous PTN correlates with and seems to be involved in angiogenesis of the chicken embryo CAM. Our data suggest that NCL may have a role, increasing the number of growth factors whose angiogenic/tumorigenic activities are mediated by NCL.

Oxidative stress due to radiation in CD34+ Hematopoietic progenitor cells: protection by IGF-1

Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34+ irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34+ from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.