Alexandra Giatromanolaki

Democritus University of Thrace, Komotina, East Macedonia and Thrace, Greece

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Publications (355)1308.21 Total impact

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    Daniel J Klionsky · Kotb Abdelmohsen · Akihisa Abe · Md Joynal Abedin · Hagai Abeliovich · Abraham Acevedo Arozena · Hiroaki Adachi · Christopher M Adams · Peter D Adams · Khosrow Adeli · [...] · Xiao-Feng Zhu · Yuhua Zhu · Shi-Mei Zhuang · Xiaohong Zhuang · Elio Ziparo · Christos E Zois · Teresa Zoladek · Wei-Xing Zong · Antonio Zorzano · Susu M Zughaier ·
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    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
    Full-text · Article · Jan 2016 · Autophagy
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    ABSTRACT: LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.
    Full-text · Article · Sep 2015 · PLoS ONE

  • No preview · Article · Aug 2015 · Cancer Research
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    ABSTRACT: We investigated the role of lysosomal biogenesis and hydrolase activity in the clinical behavior and postoperative outcome of lung cancer. Using immunohistochemistry we investigated the expression of the transcription factor EB (TFEB) which orchestrates lysosomal biogenesis, the lysosome membrane protein LAMP2a and of the lysosomal hydrolase cathepsin D in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery alone. In vitro experiments with the A549 and H1299 lung cancer cell lines were also performed. Overexpression of TFEB, LAMP2a and Cathepsin D was noted in 47/98 (47.9%), 43/98 (43.9%) and 39/98 (39.8%) cases, respectively, and were significantly correlated with each other and with adenocarcinomas. High LAMP2a was related to high histology grade. Linear regression analysis confirmed significant association of TFEB with BNIP3 (p=0.0003, r=0.35) and LC3A with LAMP2a expression (p=0.0002, r=0.37). An inverse association of Cathepsin D expression with stone-like structures (SLS) was recorded (p=0.02, r=0.22). On univariate analysis all three lyososomal variables were associated with poor prognosis (p=0.05, 0.04 and 0.01, for TFEB, Cathepsin D and LAMP2a, respectively). Multivariate analysis showed that the SLS number (p=0.0001, HR5.37), Cathepsin D expression (p=0.01, HR=2.2) and stage (p=0.01, HR=1.5) were independent prognostic variables. Silencing of TFEB with siRNAs in the A549 and H1299 lung cancer cell lines did not affect proliferation but resulted in reduced migration ability. Lysosomal biogenesis is linked to autophagosomal protein expression in NSCLC and characterizes subgroups of high risk patients after complete surgical lung tumor resection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
    Full-text · Article · Jul 2015 · Lung cancer (Amsterdam, Netherlands)
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    ABSTRACT: In this case report, we describe the successful treatment of a small-bowel intussusception, which was caused by a 3cm solitary hamartomatous polyp, with single-incision laparoscopic surgery. Single-incision laparoscopic surgery is a minimally invasive surgical procedure with important advantages that allows the reduction of the intussusception and the resection of the polyp. This case report contributes to the medical literature by describing the advantages of this surgical technique that warrant its consideration as a treatment of choice in similar cases. We report a case of a 19-year-old Greek woman who complained about intermittent, non-specific abdominal pain in her left lateral abdomen. She had been admitted to the hospital because of incomplete obstructive ileus. Ultrasound and computed tomography were carried out, which revealed an intussusception of the small bowel. This pathogenic situation was treated by single-incision laparoscopic surgery. Her pathology report revealed a benign, hamartomatous excised polyp of the Peutz-Jeghers type. The patient had a quick recovery without any post-operative complications. We recommend single-incision laparoscopic surgery for the safe excision of solitary hamartomatous polyps and the management of their complications, as it represents a potential advance in minimally invasive approaches.
    Full-text · Article · Jun 2015 · Journal of Medical Case Reports
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    ABSTRACT: Predictive assays for acute radiation toxicities would be clinically relevant in radiation oncology. We prospectively examined the predictive role of the survival fraction at 2 Gy (SF2) and of γH2AX (double-strand break [DSB] DNA marker) expression kinetics in peripheral blood mononuclear cells (PBMCs) from cancer patients before radiation therapy. SF2 was measured with Trypan Blue assay in the PBMCs from 89 cancer patients undergoing radiation therapy at 4 hours (SF2[4h]) and 24 hours (SF2[24h]) after ex vivo irradiation. Using Western blot analysis and band densitometry, we further assessed the expression of γH2AX in PBMC DNA at 0 hours, 30 minutes, and 4 hours (33 patients) and 0 hour, 4 hours, and 24 hours (56 patients), following ex vivo irradiation with 2 Gy. Appropriate ratios were used to characterize each patient, and these were retrospectively correlated with early radiation therapy toxicity grade. The SF2(4h) was inversely correlated with the toxicity grade (P=.006). The γH2AX-ratio(30min) (band density of irradiated/non-irradiated cells at 30 minutes) revealed, similarly, a significant inverse association (P=.0001). The DSB DNA repair rate from 30 minutes to 4 hours, calculated as the relative RγH2AX-ratio (γH2AX-ratio(4h)/γH2AX-ratio(30min)) showed a significant direct association with high toxicity grade (P=.01). Our results suggest that SF2 is a significant radiation sensitivity index for patients undergoing radiation therapy. γH2AX Western blot densitometry analysis provided 2 important markers of normal tissue radiation sensitivity. Low γH2AX expression at 30 minutes was linked with high toxicity grade, suggesting that poor γH2AX repair activity within a time frame of 30 minutes after irradiation predicts for poor radiation tolerance. On the other hand, rapid γH2AX content restoration at 4 hours after irradiation, compatible with efficient DSB repair ability, predicts for increased radiation tolerance. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Apr 2015 · International journal of radiation oncology, biology, physics
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    ABSTRACT: Radiotherapy is an equivalent alternative or complement to radical prostatectomy, with high therapeutic efficacy. High risk patients, however, experience high relapse rates, so that research on radio-sensitization is the most evident route to improve curability of this common disease. In the current study we investigated the autophagic activity in a series of patients with localized prostate tumors treated with radical radiotherapy, using the LC3A and the LAMP2a proteins as markers of autophagosome and lysosome cellular content, respectively. The role of autophagy on prostate cancer cell line resistance to radiation was also examined. Using confocal microscopy on tissue biopsies, we showed that prostate cancer cells have, overall, high levels of LC3A and low levels of LAMP2a compared to normal prostate glands. Tumors with a 'highLC3A/lowLAMP2a' phenotype, suggestive of intensified lysosomal consumption, had a significantly poorer biochemical relapse free survival. The PC3 radioresistant cell line sustained remarkably its autophagic flux ability after radiation, while the DU145 radiosensitive one experiences a prolonged blockage of the autophagic process. This was assessed with aggresome accumulation detection and LC3A/LAMP2a double immunofluorescence, as well as with sequestrosome/p62 protein detection. By silencing the LC3A or LAMP2a expression, both cell lines became more sensitive to escalated doses of radiation. High base line autophagy activity and cell ability to sustain functional autophagy define resistance of prostate cancer cells to radiotherapy. This can be reversed by blocking up-regulated components of the autophagy pathway, which may prove of importance in the field of clinical radiotherapy. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Apr 2015 · Biochemical and Biophysical Research Communications
  • A. Karpouzis · A. Giatromanolaki · A. Bounovas · E. Sivridis

    No preview · Article · Feb 2015 · Annales de Dermatologie et de Vénéréologie
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    ABSTRACT: Aims Neutrophil extracellular traps (NETs) are chromatin filaments released by activated polymorphonuclear neutrophils (PMNs) and decorated with granule proteins with various properties. Several lines of evidence implicate NETs in thrombosis. The functional significance and the in vivo relevance of NETs during atherothrombosis in humans have not been addressed until now. Methods and results Selective sampling of thrombotic material and surrounding blood from the infarct-related coronary artery (IRA) and the non-IRA was performed during primary percutaneous revascularization in 18 patients with ST segment elevation acute myocardial infarction (STEMI). Thrombi isolated from IRA contained PMNs and NETs decorated with tissue factor (TF). Although TF was expressed intracellularly in circulating PMNs of STEMI patients, active TF was specifically exposed by NETs obtained from the site of plaque rupture. Treatment of NET structures with DNase I abolished TF functionality measurement. In vitro treatment of control PMNs with plasma obtained from IRA and non-IRA was further shown to induce intracellular up-regulation of TF but not NET formation. A second step consisting of the interaction between PMNs and thrombin-activated platelets was required for NET generation and subsequent TF exposure. Conclusion The interactionof thrombin-activatedplateletswith PMNs atthe site of plaque rupture during acuteSTEMI resultsin local NET formation and deliveryof active TF. The notion that NETs represent a mechanism by which PMNs release thrombogenic signals during atherothrombosis may offer novel therapeutic targets.
    Full-text · Article · Feb 2015 · European Heart Journal
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    ABSTRACT: The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression. A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy. Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines. The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands.
    Full-text · Article · Jan 2015 · PLoS ONE
  • M.I. Koukourakis · D. Kalamida · I. Karagounis · A. Giatromanolaki

    No preview · Article · Sep 2014 · International journal of radiation oncology, biology, physics
  • A. Karpouzis · E. Sivridis · A. Bounovas · A. Giatromanolaki
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    ABSTRACT: Aim: Syringoma is a term that represents the histological features of the benign micropa-pular entity, denominated hidradenoma. Eyelid syringoma and eruptive syringoma are the more frequent forms while the rare scalp localization appears in form of cicatricial alopecia. Methods: A fair-sized infiltrated plaquelike growth, skin coloured with grey-mauve areas, first appeared 10 years ago in the sincipital region of the scalp, was described in an old-aged woman. After total excision, synin-goma diagnosis was established. Conclusion: Our case growth anatomoclini-cal pattern has to be differentiated from both clear cell myoepithelioma of the skin and chondroid syringoma. Our case constitutes the first case of scalp plaquelike non chondroid syringoma as well as the first case of non-chondroid syringoma appeared in old age, in the literature. Therefore, the spectrum of syringoma lesion morphology-topography tends to be broadened.
    No preview · Article · Sep 2014
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    ABSTRACT: ABSTRACT Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.
    No preview · Article · Aug 2014 · Cancer biology & therapy
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    Dimitra Kalamida · Ilias V Karagounis · Alexandra Giatromanolaki · Michael I Koukourakis
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    ABSTRACT: Objectives Vasculature damage is an important contributor to the side-effects of radiotherapy. The aim of this study is to provide insights into the radiobiology of the autophagic response of endothelial cells. Methods and Materials Human umbilical vascular endothelial cells (HUVEC) were exposed to 2 Gy of ionizing radiation (IR) and studied using confocal microscopy and western blot analysis, at 4 and 8 days post-irradiation. The role of autophagy flux in HUVEC radio-sensitivity was also examined. Results IR-induced accumulation of LC3A+, LC3B+ and p62 cytoplasmic vacuoles, while in double immunostaining with lysosomal markers (LAMP2a and CathepsinD) repression of the autophagolysosomal flux was evident. Autophagy-related proteins (ATF4, HIF1α., HIF2α, Beclin1) were, however, induced excluding an eventual repressive effect of radiation on autophagy initiating protein expression. Exposure of HUVEC to SMER28, an mTOR-independent inducer of autophagy, enhanced proLC3 and LC3A, B-I protein expression and accelerated the autophagic flux. Pre-treatment of HUVEC with SMER28 protected against the blockage of autophagic flux induced by IR and conferred radio-resistance. Suppression of LC3A/LC3B proteins with siRNAs resulted in radio-sensitization. Conclusions The current data provide a rationale for the development of novel radioprotection policies targeting the autophagic pathway.
    Full-text · Article · Jul 2014 · PLoS ONE
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    ABSTRACT: Neutrophil activation by inflammatory stimuli and the release of extracellular chromatin structures, (neutrophil extracellular traps - NETs), have been implicated in inflammatory disorders. Herein, we demonstrate that NETs released by neutrophils treated either with fibrosis-related agents, such as cigarette smoke, magnesium silicate, bleomycin or generic NET inducers, such as phorbol 12-myristate 13-acetate, induced activation of lung fibroblasts (LFs) and differentiation into myofibroblast (MF) phenotype. Interestingly, the aforementioned agents or IL-17 (a primary initiator of inflammation/fibrosis) had no direct effect on LF activation and differentiation. MFs treated with NETs demonstrated increased connective tissue growth factor expression, collagen production and proliferation/migration. These fibrotic effects were significantly decreased after degradation of NETs with DNase1, heparin or myeloperoxidase inhibitor, indicating the key role of NET-derived components in LF differentiation and function. Furthermore, IL-17 was expressed in NETs and promoted the fibrotic activity of differentiated LFs but not their differentiation, suggesting that priming by DNA and histones is essential for IL-17-driven fibrosis. Additionally, autophagy was identified as orchestrator of NET formation, as shown by inhibition studies using bafilomycin A1 or wortmannin. The above findings were further supported by the detection of NETs in close proximity to alpha-smooth muscle actin (α-SMA) expressing fibroblasts in biopsies from patients with fibrotic interstitial lung disease or from skin scar tissue. Together these data suggest that both autophagy and NETs are not only involved in inflammation but also in the ensuing fibrosis and thus may represent potential therapeutic targets in human fibrotic diseases.
    Full-text · Article · Jul 2014 · The Journal of Pathology
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    ABSTRACT: Background: Radiotherapy for lung cancer may induce pneumonitis. However, histological effects of radiotherapy on normal lung tissue are unknown. Transbronchial biopsy (TBB) is safe and accurate in monitoring parenchymal lesions in lung-transplanted patients. The aim of this prospective study was to evaluate whether histological changes of the healthy lung parenchyma after radiotherapy are present on TBB biopsies. Patients and methods: Twelve patients with lung cancer necessitating radiation therapy participated in the study. Serial TBBs were obtained from lung parenchyma contra-lateral to the tumor before, just after radiotherapy, and at six months post-irradiation. Evaluation of each specimen was based on the presence of congestion, inflammation, hemorrhage and fibrosis. Results: A significant increase of interstitial fibrosis (thickness) and congestion was observed between the point prior to radiotherapy and after completion of radiotherapy (p=0.047), as well as between the pre-radiotherapy point and at six months after radiotherapy (p=0.014). Six patients (50%) showed intra-alveolar fibroblastic growth after radiotherapy. No patient showed clinical or radiographic findings of radiation pneumonitis. Conclusion: Even in the absence of clinical or radiographic findings, the lung parenchyma contra-lateral to the tumor suffers early histological lesions after radiation therapy, as monitored by serial TBBs.
    No preview · Article · Jun 2014 · Anticancer research
  • A. Karpouzis · E. Sivridis · A. Bounovas · A. Giatromanolaki
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    ABSTRACT: Cylindroma is a benign tumour, characterized by basaloid cell islands, surrounded by a thick zone of hyaline. Entity is multiple or solitary, localized in head and neck as a rule. Only one solitary small nose lesion has been reported in the literature. An impressively giant, corrugated, red-blue and traversed by telangiectases, growth of the nose, histologicaly consistent with cylindroma, is reported. Our case constitutes the first literature report of giant solitary nose cylindroma and therefore, this pattern is involved in the differential diagnosis among nose skin tumours.
    No preview · Article · Jun 2014
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    ABSTRACT: Background Idiopathic eosinophilic pleural effusions (IEPEs) comprise the eosinophilic pleural effusions for which a specific aetiology cannot be established. There are no reports investigating IEPE on the basis of a systematically applied pleuroscopy approach and entailing an appropriate patient follow-up till the final outcome is established; existing series rather combine clinical and thoracocentesis criteria to establish the idiopathic character of the diagnosis. Objectives The aim of our study was to assess the clinical outcome of patients with IEPE, who underwent a systematic diagnostic approach by pleuroscopy. Methods We studied 10 patients with IEPE among 175 consecutive patients who underwent pleuroscopy for undiagnosed pleural effusion. Pleural biopsies were obtained from observed lesions. All patients were followed up by means of clinical examination and imaging. ResultsThe diagnosis of IEPE was established in 10 patients (median age was 50.5years, range 35-91). Macroscopic examination of the pleura showed diffuse thickening with pleural plaques in eight patients, consistent with diffuse pleural eosinophilic inflammation histologically proven. In two patients, macroscopic examination showed scattered nodules associated with non-caseating granulomas histologically. In all 10 patients, a specific aetiology could not be established. Follow-up was available for all patients ranging from 24-102months (median 60months). No patient received a specific treatment during the follow-up period. No relapse of a pleural effusion was documented during this period. Conclusion Pleuroscopy is mandatory in diagnosing IEPE. Negative histology and a long follow-up showed a benign course. These findings suggest that we should call these effusions indeterminate'.
    No preview · Article · May 2014 · The Clinical Respiratory Journal
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    ABSTRACT: Glioblastoma is the most aggressive tumor of central nervous system. Aytophagy is an intracellular pathway that aims at recycling of damaged proteins and organelles via a process of engulfing them in autophagosomes and fusing with lysosomes. The basic proteins that take part in this pathway is LC3A and LC3B (aytophagosomes’ proteins), Lamp2a (lysosomal membrane protein), Cathepsin D (lysosomal protease) and p62 (carries damaged organelles to the autophagosomes). In this study, we investigated the expression pattern of these proteins in normal tissue and two glioblastoma cell lines with Western Blot Analysis. Moreover, we studied the localization of LC3A and LC3B in glioblastoma cells after incubation with Bafilomycin A (late autophagy inhibitor) using confocal microscopy. Finally, we used immunohistochemistry to study the expression pattern of LC3 in cancer cells exposed to various stress factors that simulate tumor environment. Overall, normal brain expressed proLC3A and LC3A-I, but not LC3A-II. This later form was intensively present in U87MG cells and to a lower extent in T98G. In contrast, none of the LC3B forms was expressed in normal brain and T98G cells, whilst an intense expression was evident in U87MG. Moreover, LC3A and B autophagosomes were distinct between them, as confirmed by the lack of co-localization in immunofluorescence, even in the presence of Bafylomycin A. Finally, immunohistochemical findings showed induction of autophagic vesicles and stone-like structures (SLS) in both cancer cell lines under 2DG exposure, but their expression were increased only in U87MG under low pH exposure. All the other autophagic proteins showed the following pattern: a) p62, weak expression in normal brain, intense expression in cancer cell lines, b) Lamp2a, Cathepsin D, intense expression in U87MG cell line, weak expression in T98G cell line and lack of expression in normal tissue.
    No preview · Conference Paper · May 2014
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    ABSTRACT: Glioblastoma is the most aggressive tumor of central nervous system. Aytophagy is an intracellular pathway that aims at recycling of damaged proteins and organelles via a process of engulfing them in autophagosomes and fusing with lysosomes. The basic proteins that take part in this pathway is LC3A and LC3B (aytophagosomes’ proteins), Lamp2a (lysosomal membrane protein), Cathepsin D (lysosomal protease) and p62 (carries damaged organelles to the autophagosomes). In this study, we investigated the expression pattern of these proteins in normal tissue and two glioblastoma cell lines with Western Blot Analysis. Moreover, we studied the localization of LC3A and LC3B in glioblastoma cells after incubation with Bafilomycin A (late autophagy inhibitor) using confocal microscopy. Finally, we used immunohistochemistry to study the expression pattern of LC3 in cancer cells exposed to various stress factors that simulate tumor environment. Overall, normal brain expressed proLC3A and LC3A-I, but not LC3A-II. This later form was intensively present in U87MG cells and to a lower extent in T98G. In contrast, none of the LC3B forms was expressed in normal brain and T98G cells, whilst an intense expression was evident in U87MG. Moreover, LC3A and B autophagosomes were distinct between them, as confirmed by the lack of co-localization in immunofluorescence, even in the presence of Bafylomycin A. Finally, immunohistochemical findings showed induction of autophagic vesicles and stone-like structures (SLS) in both cancer cell lines under 2DG exposure, but their expression were increased only in U87MG under low pH exposure. All the other autophagic proteins showed the following pattern: a) p62, weak expression in normal brain, intense expression in cancer cell lines, b) Lamp2a, Cathepsin D, intense expression in U87MG cell line, weak expression in T98G cell line and lack of expression in normal tissue.
    Full-text · Conference Paper · May 2014

Publication Stats

11k Citations
1,308.21 Total Impact Points

Institutions

  • 1998-2015
    • Democritus University of Thrace
      • • School of Medicine
      • • Α΄ Πανεπιστημιακή Παθολογική Κλινική
      Komotina, East Macedonia and Thrace, Greece
    • University of Crete
      • Department of Radiotherapy
      Retimo, Crete, Greece
  • 2014
    • University General Hospital Alexandroupoli
      Alexandroúpoli, East Macedonia and Thrace, Greece
  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 1997-2007
    • Oxford University Hospitals NHS Trust
      • • Nuffield Department of Clinical Laboratory Sciences
      • • Molecular Oncology Laboratory
      Oxford, England, United Kingdom
  • 2003
    • University of Leicester
      Leiscester, England, United Kingdom
  • 1998-2001
    • University Hospital of Heraklion
      • Department of Gastroenterology
      Irákleio, Attica, Greece
  • 1996-1999
    • Saint Savvas Hospital
      Athínai, Attica, Greece
  • 1995-1999
    • University of Oxford
      Oxford, England, United Kingdom
  • 1994
    • Red Cross Hospital, Athens
      Athínai, Attica, Greece