Australia, 6 The Sol Goldman Pancreatic Cancer Research Center, Sidney. Cancer Center and Johns Hopkins University, Baltimore, USA, 7 University. Hospital ...
S26
european journal of cancer 48, suppl. 5 (2012) S25–S288
105 In Vitro Evaluation of Antioxidant and Antitumoral Activities of Marine Algae Gelidium Sesquipedale and Fucus Spiralis 1
1
2
3
3
N. Grozdanic , T.P. Stanojkovic , Z. Kljajic , S. Etahiri , O. Assobhei , A. Konic-Ristic4 , T. Srdic-Rajic1 , N. Kardum4 , S. Backovic2 . 1 Institute for Oncology and Radiology of Serbia, Experimental Oncology, Belgrade, Serbia, 2 Institute of Marine Biology, Department of Chemistry Biochemistry and Molecular Biology, Kotor, Montenegro, 3 Universite´ Chouaib Doukkali, Faculte´ des Sciences, El Jadida, Morocco, 4 Institute for Medical Research, Department of Nutrition and Metabolism, Belgrade, Serbia Background: Over the past several decades, seaweeds and their extracts have generated an enormous amount of interest in the pharmaceutical industry as a fresh source of bioactive compounds with immense medicinal potential. In addition, natural antioxidants from algal extracts have attracted increasing interest due to their safety. Recent studies have been focused on finding novel antioxidants to combat and/or prevent ROS (reactive oxidative species) mediated diseases. Based on these facts, the aim of this work was to evaluate antioxidant properties and antitumoral activity of dichloromethane-methanol extracts of red alga Gelidium sesquipedale (Clemente) Thuret (Rhodophyta) and brown alga Fucus spiralis Linnaeus (Phaeophyceae). Material and Methods: Dichloromethane-methanol (50:50) extracts of red alga Gelidium sesquipedale and brown alga Fucus spiralis found on the Atlantic coast of Morocco were screened for the studies on their antitumor activities. In vitro cytotoxicity of the extracts was tested by MTT assay against the target cells: human cervix carcinoma (HeLa), and chronic myelogenous leukemia (K562). H2DCFDA (dichlorodihydrofluorescein diacetate)-cell-permeable indicator was used to measure intracellular ROS level in human platelets using flow cytometry. Results: The obtained results showed that both studied extracts expressed significant cytotoxic activity in vitro toward malignant HeLa, and K562 cell lines. The IC50 values in the MTT assay for Gelidium sesquipedale were 38.03±2.62 (mg/ml) for HeLa and 18.41±1.66 (mg/ml) for K562 cell lines. IC50 values for Fucus spiralis were ranged from 36.70±1.65 (mg/ml) for HeLa, and 17.34±1.12 (mg/ml) against K562 line. Also, the treatment of human platelets by the extract of Gelidium sesquipedale resulted in reduction of intracellular ROS level induced by the treatment of human platelets with arachidonic acid. Conclusions: Results obtained indicate the potential of these extracts for the antitumor action, making them particularly interesting for further in vitro and in vivo investigations. 106 Attenuated Total Reflectance − Fourier Transform Infrared Spectroscopy (ATR-FTIR) Reveals Increased Cholesterol Esters in Drug Resistant Laryngeal Carcinoma Cells M. Osmak1 , S. Rak1 , T. De Zan1 , O. Gamulin2 , M. Kovosic2 . 1 Rudjer Boskovic Insitute, Department of Molecular Biology, Zagreb, Croatia, 2 School of Medicine University of Zagreb, Department of Physics and Biophysics, Zagreb, Croatia Background: Infrared spectroscopy is developing as a new and more holistic approach to studying drug resistance, yielding a unique fingerprint of all molecules present in the cell. In our laboratory we have developed a human laryngeal carcinoma (HEp-2) subline resistant to carboplatin (7T cells) and cross resistant to the natural compound curcumin. In our previous work we have reported that following treatment with curcumin, 7T cells exhibited reduced accumulation of curcumin, induction of reactive oxidative species (ROS) and Hsp70 expression, as well as diminished lipid peroxidation, oxidative and basic DNA damage, G2/M phase arrest, and the induction of apoptosis, as compared to parental HEp2 cells. In order to shed more light on the molecular mechanism of curcumin resistance in 7T cells, we used FTIRATR spectroscopy. Materials and Methods: HEp-2 and 7T cells were independently grown and samples were collected 20 times. The infrared spectra of both, HEp-2 and 7T exponentially growing cells were recorded with the FTIR Perkin-Elmer GX spectrometer. 5×105 cells in 20 microL NS solution per sample were placed on the surface of HATR ZnSe crystal, and dried with N2 flux. 20 independent ATRFTIR spectra for each cell line were recorded and used for ATR-FTIR spectral analysis. Cell spectra were obtained in the 4000–480 cm−1 region, 200 scans and at a resolution with 4 cm−1 at room temperature. The data analysis was carried out using the MATLAB program with two add-ons, PLS_Toolbox and Kinetics. Results and Discussion: By comparing the spectra from parental HEp-2 and resistant 7T subline, we found that the most interesting difference was the increase in cholesterol ester content recorded in resistant 7T cells. According to the literature, cholesterol esters are localized in lipid droplets inside cells. Using the Oil Red O dye we confirmed the existence of lipid droplets in resistant 7T cells and also that their quantity and presence in the cell culture is serum dependent. However, almost none of these properties were found in HEp-2 cells. Conclusion: We can conclude that in addition to previous determined molecular mechanisms involved in resistance of 7T cells to curcumin, these
Sunday 8 − Tuesday 10 July 2012
cells exhibit increased content of cholesterol esters suggesting an alteration in their cellular metabolism. In addition, our results clearly show the advantages of FTIR-ATR spectroscopy as a new technique in cellular or molecular biology research. 107 Clinical Value of Proliferation and Apoptosis Markers in Osteogenic Sarcoma Tumor Cells D. Polatova1 , K. Abdikarimov1 , S. Urunbaev1 . 1 National Research Center of Oncoclogy, General Oncology, Tashkent, Uzbekistan Background: To find out prognostic role of expression of apoptosis and proliferation in osteogenic sarcoma. Materials and Methods: 46 patients to 30 ages have been examined, 30 − males and 16 − females (T2 N0−1 M0 ). Expression of genes mtp53, Bcl-2 and Ki67 in tumor cells have been studied by the method of immunohistochemistry with monoclonal antibodies (Novocastra). Visualization have been executed with using of Streptavidin − HRP and DAB (Dakocytomation). Results: Organ-conserving operations have been performed in 16 patients (10 males, 6 females). Full effect and partial effect were marked in these patients, but expression of genes mtp53 and Ki-67 was low (1+) or absent. Expression of genes bcl-2 was also low (1+). Crippling operations have been performed in 20 males and 10 females with osteogenic sarcoma, in 9 patients of them was detected metastasises further. Partial effect and stabilization of tumoral process was marked in all patients of this group, but the expression of genes mtp53 and Ki-67 was high and moderate (3+, 2+), and expression of genes bcl-2 was moderate (2+) and high (3+). Conclusion: By study of the expression level of genes mtp53, Ki-67 and bcl-2 in tumor cells, we can use conducted therapy for effective prognosis in the choice of osteogenic sarcoma treatment. 108 The Deubiquitinase USP9X Suppresses Pancreatic Ductal Adenocarcinoma P.A. Perez-Mancera1 , A.G. Rust2 , A.V. Biankin3 , L.F.A. Wessels4 , S.A. Wood5 , C.A. Iacobuzio-Donahue6 , C. Pilarsky7 , D.A. Largaespada8 , D.J. Adams2 , D.A. Tuveson1 . 1 Cambridge Research Institute Cancer Research UK, Department of Oncology, Cambridge, United Kingdom, 2 Wellcome Trust Sanger Institute, Experimental Cancer Genetics, Hinxton, United Kingdom, 3 The Kinghorn Cancer Centre Cancer Research Program, Garvan Institute of Medical Research, Sydney, Australia, 4 Bioinformatics and Statistics, The Netherlands Cancer Institute, Amsterdam, The Netherlands, 5 Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Australia, 6 The Sol Goldman Pancreatic Cancer Research Center, Sidney Cancer Center and Johns Hopkins University, Baltimore, USA, 7 University Hospital Dresden, Department of Surgery, Dresden, Germany, 8 Masonic Cancer Center, University of Minnesota, Minneapolis, USA Introduction: Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite tremendous progress in its molecular characterization. Indeed, the biological sequelae of PDA has been partially attributed to frequent and well characterized mutations in KRAS (>90%), CDKN2A (>90%), TP53 (70%) and SMAD4 (55%), and a plethora of lower frequency genetic events of uncertain significance. Recent genome-wide analyses have uncovered numerous additional somatic genetic alterations, although the functional relevance of most remains uncertain. Therefore, the identification of novel ‘drivers’ will allow a better understanding of the pathogenesis of this disease. Material and Method: We have used Sleeping Beauty (SB) transposonmediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic KrasG12D to accelerate tumorigenesis and promote progression. A cohort of 117 KCTSB13 (KrasLSL−G12D ; Pdx1-cre; T2/Onc; Rosa26-LSL-SB13) mice was monitored for tumor development. KCTSB13 mice rapidly progressed and succumbed to invasive pancreatic neoplasms. A total of 198 distinct primary tumors and metastases were subjected to histological and molecular analysis. Splinkerette PCRs were performed to identify common insertion sites (CISs) associated with candidate cancer genes. Results and Discussion: Our screen has revealed new candidates and confirmed the importance of many genes and pathways previously implicated in human PDA. Interestingly, the most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumors. We found that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and mRNA expression in PDA correlates with poor survival following surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2 deoxycytidine elevates USP9X expression in human PDA cell lines to suggest a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with KrasG12D to rapidly accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. Conclusion: We propose USP9X as a major new tumor suppressor gene with prognostic and therapeutic relevance in PDA. More generally, the identification
