The University of Medicine and Pharmacy Craiova. Doctoral thesis

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1 The University of Medicine and Pharmacy Craiova Doctoral thesis Investigation of the Use of Growth Factor Receptors as Therapeutic Targets in Glioblastoma Scientific coordinators: Prof. Dr. Anica Dricu Prof. Dr. Maria Vrăbete PhD candidate: Eugenia Laura Drăguţescu The research that led to this dissertation has been supported by: The grant from FP5 EUROGENDIS (grant nr. QLGA-CT ) The grant from CNMP (grant nr ), Bucharest

2 Table of contents Introduction Abbreviations General part Chapter 1 : Brain tumors. Generalities. Classification of brain tumors 1.1 Brain tumors. Generalities. Incidence. Location 1.2 Classification of brain tumors Chapter 2 : The role of growth factor receptor in the pathogenesis of brain tumors 2.1 Brain tumors. Genetic and molecular aspects. 2.2 Growth factor receptors. Generalities. 2.3 Role of intracellular proteins in cancer pathogenesis Chapter 3 : Types of treatment for brain tumors 3.1 Types of classic and modern therapies in brain tumors 3.2 Targeted therapy of brain tumors Chapter 4 : Mechanism of Oxidative stress in glioblastomas 4.1 Oxidative stress. Generalities. 4.2 Modulation of intracellular signaling pathways by oxidative stress and ionizing radiation Special part Aim of the study Chapter 5 : Material and method Chapter 6 : Results Chapter 7 : Discussions Chapter 8 : Conclusions References Key words: glioblastoma, growth factor receptors, targeted therapy, ionizing radiation, oxidative stress 2

3 I. GENERAL PART Malignant gliomas represent the most common primary brain tumors of neuroepithelial tissue, arising from glia, astrocytes or oligodendrocytes. Astrocytic tumors are graded after WHO classification into four histological grades: pilocytic astrocytoma- grade I; diffuse astrocytoma- grade II and high grade gliomas- grade III and IV. High grade gliomas are the most aggressive brain tumors. Glioblastoma account 50-60% of astrocytomas and 20% of all brain tumours. This type of tumour is characterized by mitotic activity, microvascular proliferation and/or necrosis. Alterations in growth factor receptors have been reported in many tumours types, including brain tumours. The major alterations in growth factor receptors are: overexpression, constitutive activation, structural defects caused by gene and chromosomal alterations (i.e. deletions in the extracellular domain or alteration in the cytoplasmic domain of growth factor receptors). In addition to disturbances in growth and cell proliferation, pathological changes in the expression and activation of growth factor receptors affect response to chemo- and radiotherapy in glioblastomas. Therefore, inactivation of growth factor receptors might modulate tumor progression and response to chemotherapy and radiotherapy. So, the current study aims to investigate the potential of these transmembrane proteins as therapeutic targets in glioblastoma treatment. The idea of this work was encouraged by the use of new therapeutic methods in clinical practice. Several therapeutic methods such as gene therapy, immunotherapy (monoclonal antibodies, tumor vaccination), antisense oligonucleotides and growth factor receptors inhibitors have shown potential useful clinical responses. Among the therapies that target these molecular changes, growth factor receptors inhibitors are representing by a family of small molecules, called tyrphostins, which selectively inhibit receptors autophosphorylation and its activation. For this reason, in this study were investigated in vitro cytotoxic effects induced by inactivation of growth factor receptors alone or in combination with radiation therapy. Ionizing radiation induced oxidative stress in many types of cell through production of reactive oxygen species (ROS). In tumour cells, growth factor receptors activation by ROS was suggested to coincide with the proliferation of cancer cells. So, the molecular 3

4 interactions between ROS and growth factor receptors could represent a triggering factor in resistance to chemo- and radiotherapy. Another obiective of the present study was to determine the level of mitochondrial and intracellular ROS generation, following treatment with ionizing radiation, treatment with GFRs inhibitors or combined treatment with ionizing radiation and GFRs inhibitors. II. SPECIAL PART Aim of the study The aims of the present study were : 1. To investigate the membrane expression of growth factor receptors: epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), insulin growth factor(igf-1r) and vascular endothelial growth factor receptor (VEGFR) in two primary glioblastomas cell lines 11 and 15. The lever of the proteins were detected by using flow cytometry. 2. To study in vitro cytotoxic effect of four synthetic small molecules that inactivate growth factor receptors in glioblastoma(gb) cell lines 11 and To evaluate the response to radiation therapy of GB cell lines 11 and 15. Cells were exposed to a single dose irradiation of 2, 4, 6, 8 and 10 Gy and cells survival was measured by MTT assay, 3 and 7 days after irradiation. To determine intrinsic radiosensitivity for 11 and 15 GB cells line, the experiments were performed in exponentially growing cultures and cell survival was measured by a colony forming assay by the survival fraction determination to 2Gy(SF2). 4. To investigate the effect of combined treatment (ionizing radiation and growth factor receptors inhibitors) on GB cells. Cells survival were analyzed by using MTT assay after 3 and 7 days of treatment. 5. To investigate the capability of Helianthin treatment (an azo-dye compound) to induce cytotoxicity in these GB cell lines 11 and 15. Cells survival were analyzed by using MTT assay after 3 and 7 days of treatment. 4

5 6. To determinate the level of mitochondrial and intracellular ROS production, after treatment with ionizing radiation AG1024 or combined treatment (ionizing radiation and AG1024) in GB cell lines 11 and 15. Detection of intracellular and mitochondrial ROS was made by using probes DCFH (2,7 - dichlorodihydrofluorescein) and DHR 123(Dihydrorhodamine 123). V. Material and method Cells and cell cultures The primary glioblastoma cell cultures 11 and 15 used in this study were established from tumors at the Academic University Hospital of Uppsala according to standard procedures. The cell lines were cultured in MEM containing 10% fetal bovine serum (FBS), 2mM glutamine and antibiotic ( 100 UI/ ml penicillin and 100 UI/ml streptomycin). The cells were grown in tissue culture flasks maintained in a 95% air /5% CO2 atmosphere, at 37 C in a humidified incubator. EGFR, PDGFR, VEGFR and IGF- 1R were inhibited by 10, 20 and 30 μm of AG556, AG1433, TSU1498, respectively by AG1024, after 3 and 7 days of treatment. Appropriate control groups with diluents only were included. The ionizing radiation was given in a single dose of 2, 4, 6, 8 and10 Gy. Reagents The reagents were obtained from : Invitrogen/Life Technologies, Inc.(Rockville, MD, SUA), Santa Cruz, Sigma Aldrich, Amersham Bioscience, Dako, Jackson, Calbiochem (La Yolla, CA), Sigma (St. Louis, MO, USA), Roche Diagnostics Gmbh (Mannheim, Germany). Methods Irradiation Cells were incubated in a 95% air/5% CO2 atmosphere at 37 C in a humidified incubator, in culture medium until 70-80% confluence. Cells were then irradiated in a culture medium at room temperature with of 2, 4, 6, 8 and 10 Gy in a single dose, using a 137Cs radiation source. After 3 and 7 days after irradiation, in single 5

6 therapy and after combined treatment(ionizing radiation and growth factor receptors inhibitors), the cell survival was evaluated using by MTT assay. MTT assay ( Roche Diagnostic GmbH ) is based upon the cleavage of the yellow tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to purple formazan crystals by metabolically active cells. 1x 109 cells/w/200 μl medium were seeded in 96-well culture plates, incubated for 8 h and exposed to different concentrations of drugs. After 3 and 7 days of incubation 10 μl of MTT labeling reagent were added to each well and plates were incubated at 37 C for 4 h. Formazan products were solubilised and the optical density (OD) was measured at 595 nm. Results, relative cell viability were expressed as percentage of that in untreated control cultures. Clonogenic assay The celular sensitivity to radiation treatment was also determined from the loss of colony forming ability. GB cells were trypsinized, resuspended in complet medium and counted. The number known of cells was cultured on 60 mm agar coated tissue culture dishes. The experiment has been driven until surviving more than 50 cells and then after staining with methylene blue, the cells were counted. Surviving fraction determination(sf) Surviving fraction is a ratio of colonies produced to cell plated, with the correction for plating efficiency(pe). Determination of SF2 value was made by using a linear quadratic model(l-q model) with a computer program. SF2 value was calculated by the formula [SF = 1-(1-e-D/Do)N] (2]. Flow cytometry For detection of EGFR, PDGFR, IGF-1R and VEGFR, the human GB cells were detached, washed with FACS buffer (PBS containing 3% FBS and 0.02% NaN3), blocked in 10% FBS and incubated with anti-egfr Ab, anti- PDGFR Ab, anti- IGF-1R or anti- VEGFR Ab, working dilution 1:10 for min at 4 C. The cells were then stained with FITC-conjugated second antibody for 30 min on ice. Cells were analyzed using a FACS Calibur flow cytometer (BD Biosciences, Heidelberg, Germany) and the CellQuestTM software. For each measurement 100,000 events were acquired. Statistical analysis All data are represented as mean +/- SEM. Data were analyzed using ANOVA two-tailed t-test for analysis. P < 0.05 values were considered statistically significant. Interactions between drugs and radiation were calculated by the Multiplicative Method. 6

7 VI. Results The expression of growth factor receptors in GB cells We examined the expression level of EGFR, VEGFR, IGF-1R and PDGFR membrane proteins in the GB cell lines 11 and 15, by using Flow cytometry. In GB cell lines 11, EGFR and PDGFR were moderate expressed and overexpressed in GB cell line 15. IGF- 1R was a low expressed in both GB cell lines. VEGFRs were overexpressed in GB cell lines 11 and The role of growth factor receptors inhibition in GB cells survival We investigated the cytotoxic effects of four small molecular drugs that specifically inhibit autophosphorilation of EGFR, PDGFR, VEGFR and IGF-1R activity, on GB cells in vitro. GB 11 and 15 cell lines were exposed to AG553, AG1433, TSU1498 and AG1024 drug inhibitors in concentration of 10, 20 and 30 μm and proliferation rates were evaluated after 3 and 7 days by MTT assay. Administration of AG1433, a PDGFR inhibitor, induced significant cytotoxic effect in concentration of 20 and 30 µm, after 7 days, in 11GB cell lines but did not showed any effect on 15GB cells viability. On the opposite, the inhibition of EGFR by AG556 induced cell death in 15GB cells, but did not demonstrate any effect in GB11 cells. The inhibition of VEGFR by TSU1498 was ineffective in both GB cell lines analysed. The inhibition of IGF-1R by AG1024 induced moderate cytotoxicity after 3 days of treatment, and a considerable citotoxicity after treatment 7 days with 30 μm AG1024, in both GB cell lines evaluated, compared with the untreated cells The effect of ionizing radiation on GB cells In this study we investigate the effect of radiation on the GB cell lines 11 and 15. The cells were exposed to single dose irradiation of 2, 4, 6, 8 and 10 Gy of γ- radiation and cell survival was measured by MTT assay, 3 and 7 days after irradiation. The results obtained showed that the GB cell linies 11 and 15 were resistant to ionizing radiation by dose irradiation of 2, 4, 6, 8 and 10 Gy, after 3 days. Seven days after irradiation, GB 7

8 cell lines were radiosensitive at irradiation doses of 6, 8 and 10 Gy,. The intrinsec radiorezistnce was also determined on these GB cells (11 and 15) by colony-forming assay. Cells were irradiated in a single dose from 2 to 10Gy and radiation sensitivities of cells were evaluated by the survival fraction determination to 2Gy(SF2) The effect of combined treatment on viability of GB cells To investigate whether the inhibition of these growth factor receptors or the disruption of their intracellular transduction pathway can sensitise the GB cells to radiation, pharmacologic inhibition of EGFR, PDGFR, VEGFR and IGF-1R were combined with ionizing radiation. The drug inhibitors AG556, AG1433, TSU1498 and AG1024 were added to the cell cultures and irradiation doses of 2, 4, 6, 8 and 10 Gy were given concomitantly. The cytotoxicity of the combined treatment was determined after 3 and 7 days by using MTT assay. Interactions between drugs and radiation were analyzed by the Multiplicative Method. We found that the combination of small molecules inhibitors with ionizing radiation was more effective in 11GB cells compared with 15GB cells. In 11GB cells, synergistic interaction was found in 20 combinations and additive interaction was found in 42 combinations of 120 analysed. The subadditive cytotoxicity was induced in 58 combinations of 120 analyzed in 11GB cells. In contrast, no synergistic interaction was found in 15GB cells, the interaction between small inhibitors and radiation was mostly subadditive (100 combinations of totally 120 analysed). Additive interaction was found in 20 of 120 analysed combinations The effect of Helianthin on GB cells viability In this study we investigate the effect of treatment with various concentrations of Helianthin (between 0,01 to 1µM) on GB cell lines 11 and 15. The cytotoxicity of the treatment was determined by MTT assay. Our results showed that Helianthin induced cytotoxicity in a dose-dependent manner in both GB cell lines. 8

9 6. 6 The influence of ionizing radiation, AG1024 treatment and combined treatment on ROS production in GB cells In this study we evalueted intracellular and mitochondrial ROS production after treatment with ionizing radiation, treatment with AG1024 and combined treatment (ionizing radiation and AG1024). The ROS generation were detected by flow cytometry, using probes DCFH-DA to detect cytosolic ROS and DHR 123 to detect mitochondrial ROS, measured fluorecence intensity. In GB cell line 11, both treatment with ionizing radiation in dose of 2Gy and treatment with 20µM AG1024, induced a increase of intracellular ROS with 0,3 fold. Combined treatment induced an increasing in the levels of intracellular ROS with 0,25 fold. In this cell line the treatment did not generate mitochondrial ROS. All treatment led to a low level of mitochondrial ROS with 0,15 fold. In GB cell line 15 the treatment with 20µM AG1024, increased the level of intracellular ROS with 0,75 fold, ionizing radiation in dose 2Gy, with 1,15 fold and the combined treatment determined increase of intracellular ROS with 1,05 fold compared with the untreated cells. VII. Discussions Glioblastomas are the most common type of malignant gliomas, with frequency of 60-70%, with poor prognosis and high rates of morbidity and mortality. Therefore standard therapy in glioblastoma, including surgical resection, radiotherapy and chemotherapy, have a reserved response. A good understanding of the genetic changes in glioblastomas may be important to finding potential new therapeutic targets. Because growth factor receptors modulate tumour progression, there is an increasing interest in investigating their inhibitor potential as novel therapeutic approaches in tumour cells. Both PDGF and PDGFR are overexpressed in secondary GB and in primary GB the specific molecular changes are amplification of EGFR and mutation of EGFR (EGFRvIII). Several small molecules targeted inhibitors of PDGF, Bcr-Abl and c-kit receptor tyrosine kinases have been used in treatment of cancer. Imatinib mesylate(gleevec) has shown clinical benefits in chronic myelogenous leukaemia and 9

10 gastrointestinal stromal tumors, but with minimal effect of recurrent malignant gliomas in a phase II clinical trials. Another inhibitor Geftinib(Iressa), is a selective inhibitor of EGFR approved by FDA for treatment of non small cell lung cancer and squamos cell carcinoma. The EGFR inhibitors as Geftinib(Iressa) and Erlotinib(Tarceva) have been used in clinical trials and were well tolerated but they have shown a limited responses in malignant gliomas. A recent monoclonal antibody against VEGF is Bevacizumab(Avastin) approved by FDA for metastatic colorectal cancer. Several studies phase I and II have demonstrated a good response in GBM and anaplastic gliomas. In this study we found the heterogeneous expression of EGFR, PDGFR, IGF-1R and VEGFR in GB cell lines 11 and 15. In GB cell lines 11, EGFR and PDGFR were moderate expressed and they were overexpressed in GB cell line 15. IGF-1R was low expressed in both GB cell lines. VEGFR were overexpressed in GB cell lines 11 and 15. For this reason, we investigated the cytotoxic effects of four small molecular drugs that specifically inhibit EGFR, PDGFR,VEGFR and IGF-1R activity, on glioblastoma cells in vitro. Administration of AG1433, a PDGFR inhibitor, induced significant cytotoxic effect in 11GB cell lines in concentration of 20 and 30 µm, 7 days after treatment, but did not showed any effect on 15GB cells viability. The inhibition of EGFR by AG 556 induced more pronounced cell death in 15GB cells than in GB11 cells. The inhibition of VEGFR by TSU1498 was ineffective in both GB cell lines analysed. Administration of AG1024 (an IGF-1R inhibitor) led to a moderate cytotoxic effect in both GB cell lines analysed. We also investigated the effect of ionizing radiation in single dose of 2, 4, 6, 8 and 10Gy as the single therapy or combined with receptors inactivation on GB cells. The radiosensitivity of cells was determined by colony-forming assay. The survival fraction after exposure to 2Gy (SF2) was 0,8 for GB cell line 11 and 0,55 for GB cell line 15. The platting efficency was 0,14 for GB cell line 11 and 0,12 for GB cell line 15. We found that the combination of growth factor receptors inhibitors with ionizing radiation was more effective in 11GB cells compared with 15GB cell line. In 11GB cells, synergistic interaction was found in 17% of combinations, additive interaction was found in 35% of combinations and subadditive cytotoxicity was induced in 48% of analysed combinations. In contrast, no synergistic interaction was found in 15GB cells, subadditive 10

11 effect was found in 83% of combinations and additive interaction was found in 17% of analysed combinations. The treatment with various concentrations of Helianthin (between 0,01 to 1µM) on GB cell lines 11 and 15 induced cytotoxicity in a dose-dependent manner. In this study we evaluated intracellular and mitochondrial ROS production after treatment with ionizing radiation, treatment with AG1024 and combined treatment (ionizing radiation and AG1024). We found that in GB cell line 11, ionizing radiation in dose of 2Gy, the treatment with 20µM AG1024, and combined treatment increased the level of intracellular ROS. In this cell line, the treatment generated low levels of mitochondrial ROS. In GB cell line 15, the treatment with 20µM AG1024, with ionizing radiation in dose 2Gy and the combined treatment determined increasing levels of intracellular ROS and increasing of mitochondrial ROS levels, compared with the untreated cells. Our study indicate that human glioblastoma-derived cells show differences in their response to the different combinations of treatments as inactivation of growth factor receptors or to combined treatment with growth factor receptors inhibitors and ionizing radiation and thus may offer differential therapeutic opportunities. Therefore a good understanding of molecular heterogencity of glioblastomas can be a continuous challenge in the develop of individualized therapies in malignant gliomas. VIII. Conclusions 1. Because GB cell lines 11 and 15 expressed growth factor receptors EGFR, VEGFR, PDGFR and IGF-1R, our studies allowed to investigate the importance of these transmembrane proteins, as targets for glioblastomas treatment. 2. Inactivate of growth factor receptors by using small molecule inhibitors belonging the tyrphostins family has a modest cytotoxic effect on GB cells. 3. Helianthin, a compound belonging to the class of azo-die substances, had a cytotoxic effect on GB cell lines. 4. Both GB cell linies 11 and 15 were resistant to ionizing radiation. GB cell linies 11 mostly radioresistant than 15 lines. 11

12 5. The combined treatment with growth factor receptors inhibitors and radiotherapy was more efficient than the single therapy in GB cell lines. 6. In GB cell lines 11 and 15 the ionizing radiation led to decrease in intracellular ROS production. The mitochondrial ROS were decreased only in GB cell line 15. The inhibiton of IGF-1R with or without ionizing radiation in dose of 2Gy induced decrease in intracellular ROS production in both GB cell lines. The combined treatment AG1024 and ionizing radiatiaon and single therapy with IGF-1R inhibitor led to a decrease of mitochondrial ROS levels in GB cell line 15 and an increase of mitochondrial ROS levels in GB cell line Although the two primary GB cell lines have the same histological type, they responded differently to all types of treatments considered in this study, suggesting the need for individualized therapy for patients with glioblastoma. 12

13 CURRICULUM VITAE PERSONAL DATES: Name: DRĂGUŢESCU Surname: EUGENIA LAURA Marital status- Married Date and place of birth: December 29th, 1975, Craiova, Romania. Address: Eroilor Street, No.19, Craiova, Romania. PROFESSIONAL ACTIVITY: University of Medicine and Pharmacy Craiova Licence of Free Practice - Physician in General Medicine Physician on probation in The Regional Emergency Hospital no.1 Craiova Fellowship - Marie Curie Training - "The Genetic Basis of Diseases"- Department for Oncology-Pathology, Karolinska Institut, Karolinska University Hospital, Stockholm, Sweden present PhD student - University of Medicine and Pharmacy of Craiova SCIENTIFIC PAPERS : 1. Oana Alexandru, Laura Drăguţescu, Ligia Tătăranu, Vasile Ciubotaru, Ani Sevastre, Ada Maria Georgescu, Oana Purcaru, Suzana Dănoiu, L. Magnus Backlund, Anica Dricu; Helianthin induces antiproliferative effect on human glioblastoma cells in vitro; Journal of Neurooncology, Jul 16;2010. [Epub ahead of print]. 2. Mia Carapancea, Oana Alexandru, Ani S Fetea, Laura Drăguţescu, Juan Castro, Ada Georgescu, A. Popa-Wagner, L. Magnus Backlund, Anica Dricu; Growth factor receptors signaling in glioblastoma cells: therapeutic implications; Journal of Neurooncology, 92(2); ; PUBLICATIONS : First author - 4 Joint author