Clinical Oncology Laboratory, Department of Medicine, University Hospital of Patras, Rio, Greece
giannop@upatras.gr
Dr E. Giannopoulou (Chemist, MSc, PhD) received her MSc and PhD in Molecular Pharmacology from Department of Pharmacy at University of Patras, Greece, in 2003. Dr Giannopoulou is a senior researcher at Clinical Oncology Laboratory, Department of Medicine, University of Patras since 2003. Her scientific interests focus on the study of actions' mechanisms of drugs in the field of oncology. She is interested in the activation of alternative pathways of drugs in tumors as well as in the activation of pathways participated in chemoresistance. New agents are also studied for a possible antitumor effect. Further study is performed for neurotoxicity caused by chemotherapy or targeted therapy. Her scientific areas include HER network, estrogen receptors pathways and Notch signaling. The effect of antirumor agents in mechanisms that regulate cell invasion and metastasis are also studied.
Abstract: In the present study we assessed the expression and distribution of endoribonuclease Dicer in soft tissue tumors and correlated its cellular levels with clinicopathological parameters, including clinical outcome. Dicer was expressed in the tested cell line as well as in the majority of the sarcomas examined. Staining intensity was significantly higher in sarcomas compared with benign neoplasms and in high-grade compared with low-grade tumors. Elevated Dicer immunoreactivity was strongly associated with poor outcome and Dicer cellular levels were an independent negative prognostic factor.
Abstract: 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.
Abstract: The effect of different doses of X(-)rays on apoptosis, proliferation, epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP-2) expression was investigated in a human glioblastoma cell line.
Abstract: Radiological and nuclear medicine imaging modalities used for assessing bone metastases treatment response include plain and digitalised radiography (XR), skeletal scintigraphy (SS), dual-energy X-ray absorptiometry (DEXA), computed tomography (CT), magnetic resonance imaging (MRI), [(18)F] fluorodeoxyglucose positron emission tomography (FDG-PET) and PET/CT. Here we discuss the advantages and disadvantages of these assessment modalities as evident through different clinical trials. Additionally, we present the more established response criteria of the International Union Against Cancer and the World Health Organization and compare them with newer MD Anderson criteria. Even though serial XR and SS have been used to assess the therapeutic response for decades, several months are required before changes are evident. Newer techniques, such as MRI or PET, may allow an earlier evaluation of response that may be quantified through monitoring changes in signal intensity and standard uptake value, respectively. Moreover, the application of PET/CT, which can follow both morphological and metabolic changes, has yielded interesting and promising results that give a new insight into the natural history of metastatic bone disease. However, only a few studies have investigated the application of these newer techniques and further clinical trials are needed to corroborate their promising results and establish the most suitable imaging parameters and evaluation time points. Last, but not least, there is an absolute need to adopt uniform response criteria for bone metastases through an international consensus in order to better assess treatment response in terms of accuracy and objectivity.
Abstract: The aim of this study was to assess the antitumour effect of an anti-VEGFR (sunitinib) and the anti-EGFR multi-targeted agent (lapatinib), applied either alone or in combination on the migration capacity of two glioma cell lines. Furthermore, this study sought to evaluate the effect of lapatinib in the formation of EGFR-integrin β(1) complex, as well as the effect of sunitinib in the VEGFR-integrin β(3) and PDGFR-integrin β(3) complexes formation.
Abstract: AIM: We sought to assess the effect of sunitinib and lapatinib applied either alone or in combination, on U87 and M059K glioma cells. METHODS: Both cell lines were cultured as recommended by the manufacturer. Sunitinib and lapatinib were applied, either separately or in combination, in the cultured cells after cell attachment at doses of 10 nM, 100 nM, 1 microM and 10 microM. To determine whether the agents affect the proliferation of glioma cells, the 3-[4,5-dimethylthiazol-2-yl]-2,5 dimethyltetrazolium bromide assay was used. Apoptosis was detected using annexin V/propidium iodide detection assay, migration assay was performed in 24-well microchemotaxis chambers. The release of MMPs into the culture medium of U87 and M059K cells was measured by zymography. RESULTS: Both agents, administered either alone or in combination, decreased cell proliferation in a dose-dependent manner 48 h after their application in both cell lines. The inhibition of their combination was statistically different than the inhibition of each agent alone. Apoptosis was increased and migration of U87 and M059K cells was inhibited either by each agent alone or their combination. MMPs levels remained unaffected by the application of both agents in U87 cells. However, MMP-9 and MMP-2 levels were decreased 48 h after treatment of M059K cells with sunitinib either alone or in combination with lapatinib. CONCLUSION: Sunitinib and/or lapatinib appear to exhibit significant effects on proliferation, apoptosis and migration of glioma cells. When applied alone, sunitinib appears to be a more potent inhibitor than lapatinib.
Abstract: BACKGROUND: Recent 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. RESULTS: Aromatase 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. CONCLUSION: Our 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.
Abstract: BACKGROUND: Panitumumab, a fully-human monoclonal antibody raised against epidermal growth factor receptor (EGFR), has been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for the treatment of patients with EGFR-expressing metastatic colorectal carcinoma (mCRC) after failure of standard chemotherapy. Additionally, the guideline of the EMEA includes the use of panitumumab in patients with wild-type KRAS. The goal of the current study was to evaluate the effect of panitumumab on colon cancer cells, proliferation, apoptosis, necrosis, cell cycle arrest and autophagy. The effect of panitumumab on the redox status of the cells was also studied. MATERIALS AND METHODS: The cell lines Caco-2, DLD-1 and HT-29 which differ in their expression of EGFR and HER-2 were used. Cell proliferation and apoptosis/necrosis were measured by methyl tetrazolium (MTT) assay and annexin V/propidium iodide assay, respectively. Cell cycle arrest was estimated by propidium iodide assay and autophagy was detected using Western blot analysis. Spectrophotometrical quantification of glutathione (GSH) levels and an analysis of KRAS sequence were applied. RESULTS: Panitumumab reduced proliferation only in the DLD-1 cells despite the mutated KRAS in this cell line. However, panitumumab did not affect DLD-1 cell apoptosis, necrosis or cell cycle progression. Interestingly, immunoblotting analysis revealed that panitumumab increased protein levels of beclin-1, a marker of autophagy. In addition, an increase in the GSH level was noted following panitumumab treatment reflecting an imbalance in the redox status of the cells. CONCLUSION: Panitumumab affects colon cancer cell proliferation independently of KRAS mutations and EGFR protein levels, possibly through the induction of autophagy.
Abstract: PURPOSE: A number of studies have revealed that coexpression of EGFR and HER-2 has been found in a subset of colon cancers and may cooperatively promote tumor cell growth and survival. In the present work, two tyrosine kinase inhibitors, gefitinib and lapatinib, together with trastuzumab, raised a monoclonal antibody against HER-2 were evaluated in two colon cancer cell lines, DLD-1 and Caco-2. The aim of the study was to investigate their effect on tumor cell proliferation and apoptosis. METHODS: Cell proliferation was assessed using the MTT assay and apoptosis was evaluated by DNA fragmentation and the Annexin V binding assay. EGFR and HER-2 protein and mRNA levels were evaluated by immunoblotting and quantitative RT-PCR, respectively. RESULTS: Treatment of cells with each agent alone resulted in inhibition of cell proliferation after 48 h in a dose-dependent manner except for trastuzumab, which did not alter cell proliferation of DLD-1. Apoptosis increased in DLD-1 cells, after 24 h treatment with gefitinib. None of the tested agents altered apoptosis in Caco-2 cells. HER-2 and EGFR protein levels did not follow the changes of mRNA levels after treatment with the tested agents. CONCLUSIONS: Tauhe inhibitory effect of these agents on cell proliferation and the induction of apoptosis differ for the two colon cancer cell lines under consideration. Further studies are necessary to investigate the way they exert their antitumor effect.
Abstract: In the present work, a series of conjugates of amino acids with all-trans-retinoic acid (ATRA) and shorter polyene chain analogues were rationally designed, synthesized by coupling the succinimidyl active esters of the acidic retinoids with appropriately protected amino acids or peptides followed by deprotection, and examined for their possible effect on viability of human prostate cancer LNCaP cells. In contrast to ATRA, all conjugates bearing amino acids with polar side chains showed no inhibitory effect on LNCaP cell proliferation, while conjugates with alpha-amino acids with lipophilic side chain, such as 7, or linear amino acids, such as 9, significantly decreased prostate cancer LNCaP cell number. Interestingly, while the effect of ATRA was RARalpha-dependent, the effect of its active analogues was not inhibited by a selective RARalpha antagonist. Cell cycle analysis showed no effect on cell cycle, while quantitative analysis by annexin V-propidium iodide staining revealed that neither ATRA nor its analogues affected LNCaP cell apoptosis or necrosis. These results demonstrate that compounds 7 and 9 are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.
Abstract: 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.
Abstract: Aim: Oval cells are liver stem cells involved in liver regeneration following liver damage. Previous studies have shown that pretreatment with a hepatocyte inhibitor is required to allow full oval cell activation. This study investigates whether oval cells develop and proliferate in a model of experimental liver fibrosis without pretreatment with a known hepatocyte inhibitor. Methods: The study comprised 66 male Wistar rats divided into two groups: A (n = 6): controls; and B (n = 60): CCl(4) injection (intraperitoneally 2 mL/kg bodyweight 1:1 volume in corn oil twice weekly). Rats were sacrificed at four, eight and 12 weeks. Liver tissues were evaluated for the degree of fibrosis (Masson's trichrome), cell proliferation (Ki67 antigen), expression of alpha-fetoprotein (AFP) mRNA (RT-PCR and in situ hybridization), AFP protein (Western blot) and cytokeratin-19. Cells with morphologic features of oval cells that were cytokeratin 19 (CK19)+ and AFP mRNA+ were scored in morphometric analysis. Results: Oval cells were present in all 66 specimens; their percentage was higher in group B compared to group A (P < 0.001). AFP mRNA and protein expression increased as fibrosis advanced. Similarly, the numbers of CK19+, AFP mRNA+ and Ki67+ oval cells were higher in advanced fibrosis stages. Conclusion: This study demonstrates that oval cells develop and proliferate in a model of experimental liver fibrosis without pretreatment with a known hepatocytic inhibitor. However, further research is warranted in order to identify the exact molecular mechanisms involved in this process.
Abstract: PURPOSE: To investigate the therapeutic response of patients with different types of bone metastases treated with combined radiotherapy and bisphosphonates. PATIENTS AND METHODS: By 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. RESULTS: At 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. CONCLUSIONS: Even though the therapeutic outcome for the three groups was similar, the degree of clinical response and reossification differed.
Abstract: BACKGROUND: Hypoxia-inducible-factor-1 (HIF-1) is present at high levels in human tumors and plays a crucial role in tumor promotion by up-regulating several target genes. HIF-1 stimulates the production of NO through the induction of inducible NO synthase (iNOS). PATIENTS AND METHODS: Sixty-three human astrocytic gliomas were analyzed by immunohistochemistry for HIF-1alpha and iNOS using formalin-fixed paraffin-embedded material. In 39 cases, the results of immunohistochemistry were correlated with the clinical outcomes. RESULTS: HIF-1alpha was detected only in astrocytic gliomas grades III and IV, both in the nucleus and in the cytoplasm. The iNOS expression was increased in astrocytic gliomas grades I, II and III and was statistically significantly decreased in astrocytic gliomas grade IV. iNOS was localized round the capillary vessels as well. Statistical analysis showed that the HIF-1alpha and iNOS expressions did not correlate with patient survival. CONCLUSION: We believe that HIF-1alpha and iNOS expressions merit further investigations in order to understand the biology of astrocytic gliomas. More data are needed from prospective studies.
Abstract: Amifostine is a broad-spectrum cytoprotective agent, selective for normal tissues. It is a pro-drug metabolised to the free thiol WR-1065 that may act as a scavenger of free radicals, generated in tissues exposed to chemotherapeutic agents or radiation. WR-1065 can be further oxidized to its symmetric disulfide WR-33278 or degraded to hydrogen peroxide (H2O2). Both WR-1065 and WR-33278 resemble endogenous polyamines. Although amifostine is used in some cases in the clinic, there are only few studies concerning its actions at the cellular level. We have previously shown that amifostine inhibits angiogenesis in vivo, affecting the expression of several angiogenic genes. In the present work, we studied the effect of amifostine on human umbilical vein endothelial cell (HUVEC) functions in vitro, in order to further clarify its mechanism(s) of action. Amifostine increased HUVEC proliferation, an effect that was reversed by the intracellular H2O2 scavenger sodium pyruvate, agents that increase intracellular cAMP levels and L-valine. On the other hand, amifostine decreased HUVEC migration, an effect that was reversed by L-valine or L-arginine but not sodium pyrouvate. The decrease in migration was in line with decreased tube formation on matrigel and decreased amounts of metalloproteinase-2 released into the culture medium of HUVEC. Finally, amifostine reduced tyrosine nitration of the cytoskeletal proteins actin and alpha-tubulin in a time dependent manner. This last action could be due to the reduced production of nitric oxide (NO) or to other not yet identified mechanisms. Collectively, our results suggest that amifostine acts on endothelial cells through pathways that affect the redox status of the cells, either by producing H2O2 or by modulating NO production.
Abstract: Amifostine (WR-2721) is an inorganic thiophosphate-cytoprotective agent developed to selectively protect normal tissues against the toxicity of chemotherapy and radiation. We have previously shown that amifostine protects both chicken embryo chorioallantoic membrane (CAM) vessels and cells from the effects of X-rays. In the present work, we studied the effect of amifostine on angiogenesis in vivo, using the CAM model. Amifostine decreased the number of CAM vessels in a dose-dependent manner, without being toxic for the tissue. It also decreased the mRNA levels of both vascular endothelial growth factor (VEGF) isoforms VEGF(165) and VEGF(190), 6 and up to 48 h after its application onto the CAM. Similarly, it decreased the mRNA levels of inducible nitric-oxide synthase, 24 and 48 h after drug application. Furthermore, amifostine decreased the deposited amounts of laminin and collagen I 24 h after its application, without affecting the expression of the corresponding genes. The protein amounts and activity of matrix metalloproteinase-2 were not affected, whereas the expression of the corresponding gene was decreased up to 48 h after drug application. Finally, the activity of plasmin was increased 6 h after amifostine application and remained increased at later time points. These findings suggest that amifostine alters the expression of several molecules implicated in the angiogenesis process and affects the composition of the extracellular matrix in a way that leads to inhibition of angiogenesis. Such an antiangiogenic action of amifostine, together with its radioprotective effects, further supports its use in combination with radiotherapy for increased therapeutic efficacy.
Abstract: Amifostine (WR-2721) is a well-known radioprotective drug, selective for normal cells. The purpose of the present study was to define whether amifostine protects the vascular network from the effects of X-rays. We used the in vivo system of chicken embryo chorioallantoic membrane (CAM) as a model of angiogenesis. Amifostine reversed the early X-rays- induced decrease in the number of CAM blood vessels and reversed the early radiation-induced apoptosis of CAM cells. It also inhibited the increase in tyrosine nitration of actin and a-tubulin, which was observed 6 hours after CAM irradiation, when there was a significant decrease in non-protein SH groups. Furthermore, C6 rat glioma cells were inoculated on CAM and tumor growth, as well as tumor-induced angiogenesis, was estimated on haematoxylin-eosin-stained paraffin sections. Amifostine inhibited the post irradiation increase of C6 tumor-induced angiogenesis. These data suggest that amifostine protects CAM cells and blood vessels from the effects of X-rays, through mechanisms that do not depend solely on its free radical scavenging properties.
Abstract: 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.
Abstract: 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 alpha(v)beta3 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 alpha(v)beta3 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.
