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Gliwice Scientific Meetings 2013 ~~~ Lecture abstracts ~~~
Session I: Regulation of gene expression
The role of microRNAs in B cell Hodgkin and non-Hodgkin lymphoma
Anke van den Berg, Izabella Slezak-Prochazka, Jan Lukas Robertus, Debora de Jong, Gertrud Kortman, Bea Rutgers, Jasper Koerts, Bart-Jan Kroesen and Joost Kluiver
Department of Pathology & Medical Biology, University of Groningen, University Medical Center Groningen, Netherlands.
MicroRNAs (miRNAs) are important regulator of B-cell development and maturation. B cells at the germinal center (GC) stage of differentiation display the most characteristic miRNA signature. These GC B cells are the normal counterparts of various B cell lymphoma subtypes including Burkitt lymphoma and Hodgkin lymphoma. Deregulation of miRNAs is one of the characteristics of B cell lymphoma and the oncogenic transcription factor MYC significantly contributes to the miRNA signature of B-cell lymphoma. To determine the functional relevance of deregulated miRNAs we studied the effect of miRNA overexpression and inhibition on proliferation and defined the miRNA-targetome for a subset of these miRNAs. Overexpression of ten MYC-repressed miRNAs in ST486 BL cells revealed for 6 miRNAs a more than 50% decrease in the percentage of miRNA overexpressing cells in a period of 18 days indicating tumor suppressive activity. One MYC repressed miRNA, i.e. miR-155, induced a more than 50% increase upon overexpression indicating a growth advantage. Inhibition of the miR-17~92 cluster, including the only MYC-induced miRNA, miR-18a, revealed no significant effect on cell growth in Burkitt lymphoma. Inhibition of six miRNAs highly expressed in Hodgkin lymphoma did show a significant effect on proliferation for two miRNAs. Based on the dual effects of miR-155 in Burkitt and Hodgkin lymphoma we performed Ago2-RIP-Chip in miR-155-transduced cells. In silico validation of the 54 experimentally identified miR-155 target genes indicated that 32% were predicted as miR-155 targets and 77% contained the 6-mer miR-155-binding motif in the 3’UTR. Using shRNA constructs we showed that inhibition of TBRG1 pheno-copied the effect of miR-155. Our data indicate specific miRNA signatures for different B cell lymphoma subtypes and support crucial roles for individual miRNAs in the pathogenesis of B cell lymphoma. Long Non-Coding RNAs: novel players in B Cell
Lymphoma J. Kluiver1, M. Winkle1, M. Tayari1, M. Terpstra2,
G. Kortman1, D. de Jong1,
1Department of Pathology and Medical Biology; 2Department
of Genetics, University of Groningen,
B-cell lymphoma is a common malignancy of the B cells, often characterized by translocations involving one of the immunoglobulin loci and an oncogene. A well-known example is MYC, an important oncogenic transcription factor overexpressed in B-cell lymphoma. MYC involved B cell lymphomas are associated with aggressive behavior and poor clinical outcome. A large number of genes are regulated by MYC, several of which are shown to contribute to the MYC induced phenotype. Long non-coding (lnc)RNAs have recently emerged as a novel class of regulatory RNAs acting at the epigenetic, transcriptional or posttranscriptional level. Aberrant expression of several lncRNAs has already been implicated in various aspects of tumorigenesis. It is currently unknown to what extend MYC can regulate lncRNA expression and whether these lncRNAs contribute to the pathogenesis of B-cell lymphoma. Using an inducible MYC B cell lymphoma model and a custom microarray we investigated the expression >10,000 lncRNA loci and identified 1,820 lncRNA probes that show a MYC regulated expression pattern. Of these, 355 responded already after 4h, indicating direct MYC regulation. To identify transcripts relevant to lymphoma pathogenesis, we determined if these 355 lncRNAs were differentially expressed between primary lymphoma cases with high and low MYC expression and in addition also between MYC-high lymphoma cell lines and normal germinal center B cells. This revealed an overlap of 176 lncRNAs that were MYC regulated, aberrantly expressed in B cell lymphomas and differentially expressed between MYC-high and MYC-low lymphomas. Differential expression patterns were validated by qRT-PCR. As a first indication for lncRNA function, we isolated RNA from nuclear and cytoplasmic fractions of B cell lymphoma cell lines and determined lncRNA enrichment in comparison to RNA isolated from the total cell lysates. Approximately 40% of all lncRNA transcripts showed specific subcellular localization, 80% nuclear and 20% cytoplasmic enriched. 31 of the 176 candidate lncRNAs were enriched in a specific cellular fraction. Furthermore, we analyzed which lncRNAs are enriched in Argonaute 2 containing complexes as an indication for lncRNA-miRNA interaction. For ~5% of all expressed lncRNAs we found evidence for miRNA-lncRNA interactions, including 8 of the 176 differentially expressed MYC-induced lncRNAs. This study identified 176 MYC responsive lncRNAs that are deregulated in B cell lymphoma. To establish a definitive role in B cell lymphoma pathogenesis a further characterization is warranted.
HETEROGENEITY
OF MELANOSPHERES: THERAPEUTIC IMPLICATIONS Małgorzata Czyż
Department of Molecular Biology of Cancer, Medical University of Lodz, Poland.
Multiple factors have been implicated in heterogeneity of human tumors. Tumor heterogeneity poses a major challenge for effective cancer treatment. In case of melanoma, a high tumor plasticity driven by changes in the microenvironment additionally contributes to complexity of this disease. Testing drug efficacies in vitro in two-dimensional, serum-driven monolayer cultures has a poor predictive value. Therefore, more reliable in vitro model is needed. Patient-derived melanospheres grown in anchorage independent manner in stem cell medium were investigated. Transcriptome profiles were generated to explore the molecules governing phenotypes of melanospheres and monolayers. We found that melanospheres better portrayed the original tumor than monolayers. Current results showing activities of selected anticancer drugs against cells from melanospheres will be outlined.
Acknowledgement: This research was supported by grants
N° SFI20121205624 from Association Dynamics of intracellular processes
Krzysztof Puszyński
Silesian University of Technology, Gliwice, Poland.
The rapid development of biological research techniques that we can observe in recent years leads to more detailed and complex data. With novel technology we are able not only to observe tissues or cells but also observe what happens inside a single cell. We can detect single molecules and their location changes, their phosphorylation status change and we can measure how the amount of particular molecules changes over time. With this knowledge we can explore the dependences network of the particular peptides and with this exploration it becomes obvious that the intracellular interactions between various molecules are not straightforward but more complex, with many mutual dependencies and feedback loops. To fully understand complex dependencies’ network and its function, one has to explore its dynamics. This can be done by construction of mathematical models for observed phenomena and then by their analysis. Mathematical models might be deterministic, based on ordinary differential equations or stochastic based on reaction propensities. The network of dependences among intracellular species is called signaling pathway. The examples of the signaling pathways models will be shown. Their dynamics will be discussed with focus on new hypothesis resulting from proper analysis of dynamics. This will reveal advantages offered by analysis of dynamics of intracellular processes.
Session II: Cellular pathways driven Reactive oxygen
species in the nucleus and cell death Sebastian Student, Magdalena Skonieczna, Joanna Rzeszowska-Wolny
Biosystems Group, Institute of Automatic Control, Silesian
University of Technology,
Reactive oxygen species (ROS) are chemically reactive molecules that contain oxygen. ROS such as superoxide, hydrogen peroxide, hydroxyl radical or singlet oxygen are produced permanently in low quantities during cellular metabolism, but their levels can radically increase owing to the action of external factors such as ionizing or UV radiation. The main cellular producers of ROS are mitochondria, where oxidants are formed predominantly by complex I or III of the cytochrome chain [rev. in 1]. Another important source of intracellular oxidants are oxidases that catalyze reactions involving molecular oxygen as the electron acceptor, the best studied of which are the seven membrane-bound NADPH-dependent oxidases (Nox1–5 and Duox1–2) which are widely expressed and evolutionarily conserved [2,3]. Cellular ROS are important for redox-signaling pathways which, by oxidation of proteins, activate transcription factors, kinases, and phosphates [1,4,5] but also can interact with nucleic acids inducing DNA damage and mutations.
References: [1] Finkel T. (2011) Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7-15. [2] Brown D.I. Griendling K.K. (2009) Nox proteins in signal transduction. Free Radic. Biol. Med. 47, 1239-1253. [3] Aguirre J. Lambeth J.D. (2010) Nox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals. Free Radic. Biol. Med. 49, 1342-1353. [4] Kensler T.W. Wakabayash N. Biswal S. (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47, 89-116.
This work was supported by grants No. DEC-2012/05/B/ST6/03472 from NCN and BKM / 514 /RAU-1/2013.
RADIATION-INDUCED NON-TARGETED EFFECTS: HORIZONTAL AND VERTICAL TRANSMISSION OF GENETIC CHANGE MEDIATED BY OXIDATIVE STRESS?
Carmel Mothersill, Colin Seymour
Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada. mothers@mcmaster.ca, seymouc@mcmaster.ca
The "non-targeted effects" of ionizing radiation including bystander effects and genomic instability are unique in that no classic mutagenic event occurs in the cell showing the effect. In the case of bystander effects, cells which were not in the field affected by the radiation show high levels of mutations, chromosome aberrations, ROS and membrane signaling changes (horizontal transmission of mutations and information which may be damaging) while in the case of genomic instability, generations of cells derived from an irradiated progenitor appear normal but then lethal and non-lethal mutations appear in distant progeny (vertical transmission). The phenomena are characterized by high yields of mutations and distant occurrence of events both in space and time. This precludes a mutator phenotype or other conventional explanation and appears to indicate a generalized form of ROS mediated stress induced mutatgenesis which is well documented in bacteria. The nature of the signal travelling between irradiated and unirradiated cells and organisms is currently unknown but recent evidence suggests that there may be a physical component such as a vibration wave involved. UV mediated transmission has also been documented. This presentation will discuss the phenomenology of non-targeted effects both in vitro and in vivo, including recent data suggesting that ROS and neurochemicals are important in signal production while the cytokine mediated pathways determine response to the signal. By highlighting some key challenges and controversies, concerning the mechanisms and more importantly, the reason these effects exist, we will discuss current ideas about the wider implications of non-targeted effects in evolution and biology.
LIPID PEROXIDATION MODULATES DNA REPAIR AND SENSITIZES CELLS TO GENOTOXIC FACTORS
Barbara Tudek1,2, Alicja Winczura1, Konrad Kosicki2, Alicja Czubaty3
1Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland; 2Institute of Genetics and
Biotechnology, University
of Warsaw, Pawińskiego 5a, Poland;
Oxidative stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic exocyclic-DNA adducts, removed from DNA by Base Excision Repair (BER), Nucleotide Excision Repair (NER) and Homologous Recombination (HR) pathways. One of the major reactive aldehydes generated by LPO is trans-4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in vitro and in human wild type cells, as well as in cells lacking several Fanconi Anemia (FANC) proteins, and thus deficient in HR. The activity of purified ANPG and TDG glycosylases excising from DNA 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC), but not of APE1 endonuclease and 8-oxoGua-DNA glycosylase 1 (OGG1) were inhibited by high concentrations of HNE. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H2O2 and MMS, when compared to untreated cells. Examination of HNE influence on particular stages of BER in K21 cells revealed that HNE may decrease the rate of εA and εC excision, but not that of 8-oxoguanine. Simultaneously, HNE increased the rate of AP sites incision and blocked the re-ligation step after the gap-filling by DNA polymerases. Such imbalance resulted in the increased number of DNA single-strand breaks, as revealed by augmentation of the level of poly(ADP-ribose) foci. The DNA damage response was also affected by HNE. Treatment of HeLa cells with HNE resulted in inhibition of phosphorylation of SMC1, KAP1 and Chk1 proteins engaged in multiple cellular processes like regulation of cell cycle checkpoints, DNA repair, cell growth and proliferation, apoptosis and maintenance of cellular integrity. These results suggest that LPO products act not only by forming DNA adducts, but also have the ability to deregulate activities of BER enzymes, and DNA damage response. This may have an impact on cell survival and genome stability, and may be an additional mechanism of pro-carcinogenic effect of inflammations. Gene deletion of stefin B enhances mitochondrial ROS formation and Nlrp3 inflammasome activation
Katarina Maher1, Miha Butinar1, Georgy Mikhaylov1, Mateja Manček-Keber2, Barbara Jerič1 Olga Vasiljeva1, Boris Turk1, Nataša Kopitar-Jerala1
1Department of Biochemistry, Molecular and Structural
Biology, Jožef Stefan Institute, Jamova 39,
Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor localized in the cytosol and nucleus. Its expression is upregulated upon macrophage activation and cellular stress. Mutations in the gene of stefin B are associated with the neurodegenerative disease known as Unverricht-Lundborg disease. The NLRP3 inflammasome is a caspase-1-activating complex involved in the maturation of pro-inflammatory cytokines IL-1β and IL-18. We studied the role of stefin B in the regulation of IL-1β production and activation of pro-inflammatory caspases-1 and -11. Stefin B-deficient (StB KO) mice were significantly more sensitive to the LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1β and IL-18. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase activation determined in stefin B-deficient bone marrow-derived macrophages (BMDMs) resulted in enchanced IL-1β processing due to extensive destabilization of mitochondria and mitochondrial superoxide generation upon nlrp 3 inflammasome activation. Pretreatment of macrophages with a broad spectrum cathepsin inhibitor E-64d did not affect secretion of IL-1β, suggesting that the increased cathepsin activity determined in stefin B-deficient BMDMs is not essential for the inflammasome activation. Our study demonstrates that the LPS-induced sepsis in stefin B-deficient mice is dependent on caspase-11 and mitochondrial reactive oxygen species (mtROS), but is not associated with the lysosomal destabilization. We propose that the inflammatory signaling pathways determined in stefin B-deficient macrophages could be important in the pathology of the Unverricht-Lundborg disease.
Session III: DNA repair in aging Spot on the DNA damage
response: from lesion recognition
Leon H.F. Mullenders
Department of Toxicogenetics, Leiden University Medical Center, Leiden, the Netherlands.
A wide variety of genome-caretaking mechanisms (collectively referred to as the DNA damage response or DDR) can counteract the deleterious effects of DNA damage and are essential to prevent toxicity, mutagenesis, genomic instability and disease. The DDR comprises a complex signal transduction cascade that can sense DNA damage, coordinate distinct repair activities and enforce cell cycle arrest ensuring timely and efficient removal of DNA damages from the genome. A corrupted DDR drives mutagenesis and genomic instability with ultimately pathological consequences. In my presentation I will focus on nucleotide excision repair (carried out by a multiprotein complex) a multistep process involved in removal of structurally diverse DNA lesions including ultraviolet (UV) light-induced photolesions. I will discuss a variety of aspects: the molecular mechanisms of DNA damage recognition, DNA damage signaling via NER-dependent and NER-independent process, NER related human syndromes, the role of NER in controlling mutagenesis. Finally, I will address NER related opportunities for cancer treatment.
BLM COLLABORATES WITH TOPBP1 IN PROMOTING CHECKPOINT ACTIVATION AND REPLICATION FORK STABILITY
Andrew N. Blackford1,2, Jadwiga
Nieminuszczy1, Rebekka A. Schwab1,
1WIMM, Oxford University, United Kingdom; 2Wellcome Trust/Cancer Research, Cambridge, United Kingdom.
Maintenance of DNA replication fork stability is essential for dividing cells to preserve genomic integrity in the face of endogenous and exogenous DNA damage. Critical to these responses is ATR kinase, which promotes checkpoint activation to stabilize stalled forks and to prevent late origin firing. Current models for ATR activation suggest a multistep process that involves initial recruitment of ATR to DNA lesions via its cognate binding partner ATRIP and the subsequent recruitment of topoisomerase 2-binding protein 1 (TopBP1). The mechanisms that regulate TopBP1 recruitment to stalled forks are not yet clear. Recently, we have identified FANCM as a master regulator of replication stress-response. In line with this, we showed that FANCM-/- cells are impaired in stable association of TopBP1 on chromatin and subsequent activation ATR signaling following replication stress. Here I will provide evidence that a ternary complex of TopBP1, FANCM and BLM is required to maintain fork stability upon replication stress, in part by promoting ATR signaling. Phosphorylation of BLM at a CDK consensus site creates a binding site for tandem BRCT domains of TopBP1, thereby promoting assembly of the ternary complex. Disruption of the TopBP1/BLM interaction in cells leads to ATR signaling defects in response to replication stress, including reduced Chk1 phosphorylation and increased origin firing. Moreover, these cells also display increased sister chromatid exchanges, that we hypothesize arise as a result of unscheduled homologous recombination associated with stalled replication forks. Taken together, these results provide a mechanistic basis for the fork stability defects previously observed in both FANCM and BLM-deficient cells, and may also help to explain why many Fanconi anemia and Bloom’s syndrome patients display symptoms such as short stature and microcephaly that are also found in ATR-Seckel patients. DNA REPAIR HELICASE – THE STORY WITH AN UNEXPECTED END Zuzana Šestáková1, Ján Gurský1,4, Dana Cholujová2, Ivan Chalupa3, Miroslav Piršel1
1Laboratory of Molecular Genetics; 2Laboratory
of Tumor Immunology; 3Laboratory of Mutagenesis
The ERCC3/XPB DNA helicase is the subunit of transcription factor II H (TFIIH) which is involved directly in transcription and nucleotide excision repair and via its Cdk- activating kinase (CAK) complex in cell cycle control. The TFIIH structure is known to be affected by truncation mutations of its subunits. Here, we showed that there is a two-fold and three-fold lower amount of TFIIH in Chinese hamster UV68 and UV24 ERCC3 nucleotide excision repair deficient mutants, respectively, as compared with the parental wild type CHO AA8 cells. The degree of this instability is proportionally reflected in the extent of reduction of the cell cycle progression after UV-irradiation measured by pulse- and pulse-chase labeling of the cells by ethynyldeoxyuridine. In fact, the cell cycle profiles of UV68 and UV24 mutants are comparable after 12 Jm-2 and 6 Jm-2 of UV-irradiation, respectively. The slower S-phase transition in UV24 cells resulted in the higher expression of phosphorylated form of the histone H2AX – the marker of DNA double-strand breaks’ presence. This creates an additional apoptotic signal (resulting in about 20% more apoptotic cells), contributing to the overall UV-sensitivity of the UV24 mutant caused primarily by the nucleotide excision repair deficiency.
The authors acknowledge financial support from Slovak Research and Development Agency (APVV-0208-07) and VEGA Grant Agency of the Slovak Republic (2/0051/10, 2/0150/11). DNA DAMAGE
RESPONSE IN CANCER CELL SENESCENCE Grażyna Mosieniak, Małgorzata Śliwińska, Anna Strzeszewska, Halina Waś,
Laboratory of Molecular Bases of Aging, Nencki Institute of Experimental Biology, PAS, 02-093 Warsaw, Pasteura 3 St., Poland.
It is believed that anticancer therapy relies on cytotoxic treatment strategies that would lead to complete destruction of tumor. These approaches, which induce cells’ death within solid tumors can also cause severe side effects in patient due to high doses of drugs or irradiation during therapy. An alternative strategy to restrict tumor growth, and eventually eliminate cancer cells, which presents limited toxicity to patients is therapy-induced senescence. Cellular senescence was primarily described as the effect of gradual exhaustion of replicative potential in normal cells cultured in vitro that leads to permanent growth arrest. Currently, it is recognized as a stress response of both normal and tumor cells that is induced by a variety of exogenous and endogenous factors. It was also shown, both in vitro and in vivo, that senescence sustains an anticancer barrier during carcinogenesis, as well as restricts tumor growth upon radio- or chemotherapy. The main cause of senescence is induction of unrepairable double-strand DNA damage (DBS) within telomeric regions that persist in senescent cells. We have shown that human colon cancer HCT116 cell undergo cellular senescence upon treatment with low, non-toxic doses of DNA damaging agent – doxorubicin. They ceased to proliferate (Ki-67 negative), became enlarged and showed increased activity of senescence-associated β-galactosidase, which are hallmarks of senescence. Activation of DNA damage response (DDR) pathway, namely activation of ATM kinase and upregulation of p53 and p21 proteins were observed. Cancer cell senescence was also induced when cells were treated with a natural polyphenol (curcumin). Contrary to doxorubicin, curcumin lead to accumulation of cells in mitosis due to improper mitotic spindle formation. Prolonged mitotic arrest resulted in DSB, which persisted in cells that exit mitosis, leading to DDR activation and induction of senescence. Interestingly, both doxorubicin and curcumin were able to induce permanent growth arrest in HCT116 cells that do not express p53 (p53-/- cells), which is one of the main components of DDR signaling pathway. Moreover, p53-independent induction of p21 protein was observed. Altogether our results prove that DNA damage-induced senescence could be obtained in both p53-proficient and deficient tumor cells, however different signaling pathways must be activated in order to sustain growth arrest of those cells.
Session IV: Cancer proteomics and metabolomics (session co-organized by Polish Society of Proteomics)
MALDI IMAGING IN CLINICAL RESEARCH: SEARCHING FOR THE HOLY GRAIL
Corinna Henkel, Hanna Diehl, Julian Elm, Birte Beine, Helmut E. Meyer
Medizinisches Proteom-Center, Ruhr-University Bochum, Germany.
Mass spectrometry imaging (MSI) has become a powerful and successful tool in the context of biomarker detection especially in recent years. This emerging technique is based on the combination of histological information of a tissue and its corresponding mass spectrometric information. The range of samples to be analyzed is wide and includes besides proteins, peptides, lipids, drugs and their metabolites also glycans and other posttranslational modifications. For data acquisition a tissue section covered with matrix is moved in two dimensions within the mass spectrometer whereas a mass spectrum is recorded for each position. The obtained spatially resolved information of for example protein or peptide abundances can be used to detect differences between healthy and diseased tissue. Hence the method is able to generate peaks specific for histologically defined tissue areas. As an example for a protein MALDI imaging experiment, a bladder cancer study is presented, in which the differentiation of various papillary tumor grades were feasible. In general the identification of such differential peaks is still the methods bottleneck. An alternative approach to get closer to the final goal of biomarker identification, offers peptide imaging. A brief method development of peptide MALDI imaging is also shown. As a conclusion, the “holy grail” of biomarker research is not tangible by MALDI imaging yet, but promising attempts in the field seem to gain strength to the technique as will be presented in some examples.
Concluding, MALDI imaging is not yet the “holy grail” of biomarker research, but promising results in this field will be shown to illustrate the great potential of this technique and where it might lead to in the future.
References: [1] Oezdemir R.F., Gaisa N.T., Lindemann-Docter K., Gostek S., Weiskirchen R., Ahrens M., […] Henkel C. (2012) Proteomic tissue profiling for the improvement of grading of noninvasive papillary urothelial neoplasia. Clinical Biochem. 45, 7–11.
PROTEINS AND PROTEIN-PROTEIN INTERACTING MOTIFS - THERAPEUTIC TARGETS OF METASTASIS
Angels Sierra
Molecular and Translational Oncology Group, IDIBAPS, Barcelona, Spain
Cancer metastasis is produced by perturbations affecting several genes and pathways. Environmental stimuli trigger uncontrolled cell growth and invasion of other tissues. Understanding cancer progression requires a profound knowledge of the pathways involved in the communication between proteins and genes at a systems level. Consequently, protein-protein interaction networks play an important role in delineating cancer related pathways. Our understanding of cancer has evolved towards the co-operation of groups of genes that constitute pathways. We have described the characteristics of genes involved in breast cancer metastasis and the relationships between them in the context of the protein-protein interaction network. We carried out a systems-level study of the mechanisms underlying organ-specific metastases of breast cancer (Guney et al., 2012). We followed a network-based approach using
microarray expression data from human breast cancer metastases to select
organ-specific proteins that exert a range of functions allowing cell survival
and growth in the microenvironment of distant organs. MinerProt, In conclusion, the network-based approach
is useful to filter information by selecting key protein functions as
metastatic markers or therapeutic targets (Stresing et al, 2012; Santana et al.,
2013). Towards developing effective network-based therapeutics, we give details
of identifying dysregulation patterns using protein-protein interaction
networks
References [1] Sanz-Pamplona, R. et al. (2012) A taxonomy of organ-specific breast cancer metastases based on a protein–protein interaction network. Mol. BioSyst. 8, 2085–2096. [2] Guney, E. et al. (2012) Springer Book on Systems Biology and Cancer. Part II Understanding Cancer Progression Using Protein Interaction Networks. [3] Stresing, V. et al. (2012) Peroxiredoxin 2 specifically regulates the oxidative and metabolic-stress response of human breast cancer cells. Oncogene 0950-9232/12. DOI: 10.1038/onc. [4] Santana-Codina N. et al. (2013) Transcriptome-proteome integrated network Identifies endoplasmic reticulum thiol oxidoreductase (ERp57) as a Hub that mediates bone metastasis. Mol. Cell. Proteom. 10.1074/mcp.M112.022772, 1–15. Metabolic PROFILING OF BREAST CANCER
Tone F. Bathen
Dept. of Circulation and Medical Imaging, NTNU, Norway.
Metabolism comprises the integrated network of biochemical reactions that supports life in a living organism, and metabolomics is the systematic study of small-molecular compounds from metabolism. The metabolism of cancer cells is changed compared to normal cells due to high proliferation rates and malignant transformation. Metabolomics, using high resolution magic angle spinning MR spectroscopy (HR MAS MRS), may establish detailed tumor portraits reflecting diagnostic status or therapeutic response, thus potentially leading to the discovery of useful biomarkers in a clinical context. HR MAS MRS enables investigation of tissue samples with minimal sample preparation and keeps the sample intact after analyses. This talk will cover some of the practical issues of HR MAS MRS, and results from published and ongoing studies will be presented.
WEB: http://www.ntnu.edu/isb/mr-cancer TARGETING SELECTIVELY THE UNIQUE ENERGY METABOLISM OF CANCER WITH A SMALL MOLECULE INHIBITOR
André Goffeau1, Young H. Ko2, Peter L. Pedersen3,
Margarida Casal4,
1Institut des Sciences de la Vie, Université Catholique de
Louvain-la-Neuve, Place de, l`Université, 1348 Louvain-la-Neuve, Belgium; 2KoDiscovery,
UMBioPark, Innovation Center, 801 West Baltimore Street, Baltimore, MD, USA; 3Departments
of Biological Chemistry
The “Warburg Effect” is the most common biochemical phenotype of many cancer types. It is due to elevated tumor glycolysis even in the presence of oxygen. (Under such conditions normal cells convert glucose mainly to carbon dioxide and water and not to lactic acid.) Most cancers show a positive Positron Emission Tomography (PET) scan that monitors the “Warburg Effect”. Such cancers export the produced lactic acid via specific lactic acid transporters called “monocarboxylate transporters” as the lactic acid inside the cell is accumulating. In contrast, normal cells produce little or no lactic acid under physiological conditions and have very few functioning lactic acid transporters within their cell membrane. Utilizing this knowledge, several agents were screened for their capacity to selectively kill cancer cells. A lactic acid analog, 3-bromopyruvate (3BP) was found to be the best among the agents tested [1-3]. Because 3BP is a structural analog of lactic acid, cancer cells readily take it up, i.e., the 3BP enters these cells via lactic acid transporters, the same transporters that allow lactic acid to exit. Once the 3BP enters the cancer cells it targets and destroys their two energy (ATP) production factories, mitochondria and glycolysis. In contrast, little or no 3BP enters normal cells as they have very few lactic acid transporters. This explains why 3BP is highly specific in killing cancer cells. Although there are several other proteins that are targeted by 3BP inside the cancer cells, they are not immediately involved in cell death. “Proof of principle” of 3BP as a potent anticancer agent has been established earlier in a Case Report [4, 5]. In summary, 3BP is a new class of anticancer agent. It is quite different from the currently available chemo-drugs that target one or more of the following: DNA replication, cell cycle/growth, cellular signal transduction pathways, angiogenesis, and receptors. Significantly, 3BP gains specific entry to cancer cells via a monocarboxylate transporter and targets their energy metabolism, thus inhibiting their ATP production and depleting their energy sources (reserves). Therefore, 3BP is a potent “Energy Blocker” of cancer cells and is very effective in killing such cells exhibiting the “Warburg Effect” with little or no effect on normal cells. This unique nature of 3BP as a potent anticancer agent warrants continued development towards clinical treatment of cancer patients. Currently, 3BP is being actively researched via global collaborative efforts [5-8].
References [1] Ko Y.H., Pedersen P.L., Geschwind J.F. (2001) Cancer Lett. 173, 83-91. [2] Ko Y.H., Smith B.L., Wang Y., Pomper M.G., Rini D.A., Torbenson M.S., Hullihen J., Pedersen P.L. (2004). Biochem. Biophys. Res. Commun. 324, 269-275. [3] Pedersen P.L. (2007) J. Bioenerg. Biomemb. 39, 211-222. [4] Pedersen P.L. (2012) J. Bioenerg. Biomembr. 44, 1-6. [5] Ko Y.H., Verhoeven H.A., Lee M.J., Corbin D.J., Vogl T.J., Pedersen P.L. (2012) J. Bioenerg. Biomemb. 44, 163-170. [6] Lis P., Zarzycki M., Ko Y.H., Casal M., Pedersen P.L., Goffeau A., Ułaszewski S. (2012) J. Bioenerg. Biomemb. 44, 155-161. [7] Queirós O., Preto A., Pacheco A., Pinheiro C., Azevedo-Silva J., Moreira R., Pedro M., Ko Y. H., Pedersen P., Baltazar F., Casal M. (2012) J. Bioenerg. Biomemb. 44 141-153. [8] Dyląg M., Lis P., Niedźwiecka K., Ko Y.H., Pedersen P.L., Goffeau A., Ułaszewski S. (2012) Biochem. Biophys. Res. Commun. 434, 322-327.
Session V: Dietary factors in cancer prevention
DIET AND CANCER: FROM EPIDEMIOLOGICAL DATA TO MECHANISM-BASED CANCER PREVENTION
Wanda Baer-Dubowska
Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznań, Poland.
The ideas that
cancer might be preventable, and that food and nutrition might affect The traditional approach to cancer prevention has consisted of attempts to eliminate carcinogenic agents and to detect and remove precancerous lesions. Currently, efforts are increasingly focused on interrupting, reversing or delaying the neoplastic process. This approach not only complements therapeutic modalities currently in use but may provide alternatives for combating tumors that are unresponsive to treatment. Hence, cancer chemoprevention or reversal of carcinogenesis in the premalignant phase can be defined as the use of natural, or synthetic chemicals to suppress, delay or prevent the process of carcinogenesis. Food is a rich source of potential anticarcinogenic agents. Thus it is reasonable to postulate that dietary components may prevent cancer from reaching its invasive and metastatic stages. Alternatively, components of diet may reduce the risk of second primaries or modify the behavior of established cancer. Interest in this area of research has markedly increased with the improved understanding of the biology of carcinogenesis and led to mechanism–based cancer prevention approaches. THE TUMOUR MICROENVIRONMENT AS TARGET FOR CANCER THERAPY AND PREVENTION BY DIETARY COMPONENTS
Adriana Albini
IRCCS “Tecnologie Avanzate e Modelli Assistenziali in Oncologia” - Arcispedale S. Maria Nuova - Reggio Emilia, Italy; Douglas Noonan, Oncology Research, Polo Scientifico e Tecnologico, Fondazione Onlus MultiMedica, Milano, Italy.
The tumour microenvironment (TME) is a "complex society" that involves many “actors” including the malignant cells, but also stromal cells, inflammatory components, the endothelium and the recent discovered cancer stem/initiating cells. All these non-malignant cells of the TME have a dynamic and often tumour-promoting function at all stages of carcinogenesis, including angiogenesis. In this view, targeting the TME represent a valid and necessary strategy in cancer therapy both in intervention and prevention. The ability of some dietary components, like terpenoids (CDDO-Me, CDOO-Im), Resveratrol, Green tea catechins, quercetin, curcumin, to inhibit tumor progression and angiogenesis, both in vitro and in vivo, has been highly documented. Many of these compounds exert anti-oxidant, anti-proliferative, anti-angiogenic and pro-apoptotic effects on a variety of malignancies, including leukemia, prostate, breast, colon, brain, melanoma, and pancreatic cancer. In addition, these compounds are well tolerated and often found in food products that can be added to diet. Furthermore, most phytochemicals could be taken on a long-term basis to either prevent primary tumor formation or tumor recurrence. We demonstrated the ability of phytochemicals to efficiently target several component of the TME, including the inflammatory component (neutrophils, Natural Killer Cells), the endothelium1 and interestingly also the “dormant” component represented by the cancer stem cells. Taken together, the TME complex entity provides many more targets available for cancer therapy than do tumor cells alone and phytochemicals and dietary components represent a valid tool both for intervention and prevention approaches.
References: [1] Albini A., Tosetti F., Li V.W., Noonan D.M., Li W.W. (2012) Cancer prevention by targeting angiogenesis. Nat. Rev. Clin. Oncol. PMID: 22850752.
Development of resveratrol
for cancer chemoprevention Karen Brown
Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicster, LE2 7LX, UK.
Clinical trials of tamoxifen, finasteride and aspirin have provided proof of principle that cancer chemoprevention in healthy high-risk populations is feasible. However, safety is paramount in this context and the serious side effects of pharmaceutical interventions when taken chronically have hampered their further use. Chemicals derived from the diet, such as resveratrol, are an attractive alternative, with those that are consumed frequently likely to have a favourable safety profile. Such agents are typically multi-targeted and capable of interfering with all stages of carcinogenesis in preclinical models. Unfortunately, despite extremely promising preclinical data, the outcome of clinical chemoprevention trials of isolated micronutrient supplements such as selenium and vitamin E has been disappointing. These failures may be attributed, at least partly, to a lack of sufficient human pharmacokinetic and pharmacodynamic information required to optimise the trial design. This presentation will review our current clinical data relating to the use of resveratrol for cancer chemoprevention, with a focus on potential low dose effects and the role of resveratrol metabolites in mediating efficacy. In addition the major translational challenges in the development of resveratrol for widespread use in human populations will also be addressed. CHEMOPREVENTION AGAINST CANCER BY ISOTHIOCYANATES Albena T. Dinkova-Kostova1,2
1Division of Cancer Research, Medical Research
Institute, University of Dundee, Dundee, Scotland, United Kingdom; 2Department
of Pharmacology and Molecular Sciences, Johns Hopkins University School
All aerobic cells are equipped with elaborate networks of highly inducible proteins which protect against the cumulative damaging effects of reactive oxygen species, toxic electrophiles, and misfolded proteins, the major causes of malignancy and chronic degenerative diseases. These cytoprotective proteins are transcriptionally regulated via the Keap1/Nrf2 pathway. Keap1, a substrate adaptor protein for Cullin3/Rbx1 ubiquitin ligase, continuously targets transcription factor Nrf2 for ubiquitination and degradation, but loses this ability in response to various chemical and phytochemical agents (inducers), all of which have sulfhydryl reactivity. We have developed a quantitative Förster Resonance Energy Transfer (FRET)-based system using multiphoton fluorescence lifetime imaging microscopy (FLIM) and found that, under homeostatic conditions, the interaction between Keap1 and Nrf2 follows a cycle whereby the complex sequentially adopts two distinct conformations: “open”, in which Nrf2 interacts with one molecule of Keap1, followed by “closed”, in which Nrf2 binds to both members of the Keap1 dimer. Inducers disrupt this cycle by causing accumulation of the complex in the “closed” conformation, without release of Nrf2. Consequently, free Keap1 is not regenerated, newly-synthesized Nrf2 is stabilized, and the expression of cytoprotective proteins is upregulated. Plant isothiocyanates are prominent inducers of the Keap1/Nrf2 pathway. Due to the electrophilic central carbon of the isothiocyanate group, they react with critical cysteine residues of Keap1, disrupting the cycle of Nrf2 ubiquitination, and leading to activation of the transcription factor. The isothiocyanate sulforaphane was first isolated as the principal inducer of the Keap1/Nrf2 pathway from broccoli extracts, and has been shown to be an effective protective agent in numerous preclinical models of gastric, intestinal, prostatic, pulmonary, cutaneous and bladder cancers in animals, and in xenograft models of human tumors. Sulforaphane-rich broccoli preparations have been and currently are in several human studies, ranging from healthy human subjects to populations at high-risk for developing diseases conditions. CHEMOPREVENTION OF PROSTATE CANCER BY ORGANOSULFUR COMPOUNDS FROM GARLIC
Jędrzej Antosiewicz, Andżelika Borkowska
Department of Bioenergetics and Physiology
of Exercise, Medical University of Gdansk, Dębinki 1,
Epidemiologic data strongly suggests that dietary intake of Allium family vegetables, including garlic, may be protective against the risk of prostate cancer. The risk of prostate cancer was shown to be significantly lower in men consuming >10g/d of total Allium vegetables than in men with total Allium vegetable intake of <2.2 g/d in a population-based case-control study. Anticarcinogenic effect of Allium vegetables is attributed to organosulfur compounds (OSC) such as dially trisulfide (DATS), dially disulfide and many others. Evidence is accumulating to indicate that DATS and other OSC can inhibit prostate cell cycle arrest and induced apoptosis and that this is mediate by reactive oxygen species (ROS). In this study we provide experimental evidence to indicate that in DATS treated prostate cancer cells iron-dependent ROS formation is mediated by JNK1-ITCH-p66shc signaling pathway. Ferritin is an iron storage protein and its concentration is inversely related to the level of labile iron pool (LIP). Increased ferritin degradation or down regulation of its biosynthesis leads to an increase of LIP which is able to participate in redox reaction and generation of free radicals. Thus, signaling pathways which regulate ferritin degradation may exert an impact on intracellular ROS formation. However, the mechanism of the ferritin degradation has not been fully elucidated so far. In cells stably expressing a dominant negative mutant of p66Shc (p66ShcS36A), DATS did not induce ROS formation. Previously, we were able to demonstrate that JNK signaling pathway regulates ferritin ubiquitination and proteasomal degradation. As p66Shc and ubiquitin ligase Itch are activated by JNK, their role in ferritin degradation has been studied. We were able to demonstrate that DATS-induced ferritin degradation in prostate cancer cells expressing inactive form of p66Shc (p66ShcS36A) neither increased ferritin H degradation nor increased LIP. In addition, cells transfected with inactive form of Itch were more resistant to cytotoxicity exerted by DATS and showed lower DATS-induced ferritin degradation. In conclusion, our results suggest that ferritin degradation is a regulated process where JNK1, p66Shc and Itch play important roles. In addition, the data suggest a role of p66Shc in posttranslational modification of ferritin H, which needs additional work to be elucidated.
PROMISES & CHALLENGES IN EPIGENETIC REMODELLING
Katarzyna Szarc vel Szic1, David Scherf2, Ilse M. Beck3, Marc Bracke3, Tim De Meyer4, Clarissa Gerhauser2, Wim Vanden Berghe1
1Laboratory of Protein Chemistry, Proteomics and
Epigenetic Signalling, Department of Biomedical Sciences, University of Antwerp
(UA), Universiteitsplein 1, Campus Drie Eiken, 2610, Wilrijk, Belgium; 2Workgroup
Cancer Chemoprevention and Epigenomics, Division of Epigenomics and Cancer Risk Factors, German
Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg,
Germany; 3Laboratory
A vast majority of cancer patients succumb to metastatic disease. Intriguingly, interfering with metastatic spread remains one of the main challenges in cancer therapy. Metastasis development includes several discrete steps: local invasion, intravasation (or dissemination in lymph nodes or body cavities), circulation and cell survival, extravasation, growth at distinct sites and angiogenesis, all of which occur in a context of tumour-promoting microenvironment. It is now becoming apparent that these cell-microenvironment interactions are highly susceptible to epigenetic regulation, both by internal and external cues. Here we show that essential components of metastasis development, including urokinase plasminogen activator, ADAM8 metallopeptidase, and tumour promoting cytokine TNFSF12 are regulated epigenetically by DNA methylation in breast cancer as revealed by 450k Illumina BeadChipArray, MCIp and EpiTyper MassArray. Moreover, Withaferin A, a natural compound derived from Withania somnifera decreases breast cancer invasion by increasing methylation of these genes leading to lowered gene expression as revealed by RT-qPCR. Discoveries of
experimental chemoprevention -
Agnieszka Bartoszek, Barbara Kusznierewicz, Anna Lewandowska, Anna
Piekarska, Tadeusz Pilipczuk, Dominik Kołodziejski, Izabela Koss, Piotr
Konieczka,
Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland.
Translational research is usually associated with the development of new therapeutic treatments as summarized by the popular phrase - from lab bench to bedside. However, in the case of health promoting foods, the transfer of knowledge from research laboratories to industry - from lab bench to shop shelf - may actually occur much more efficiently as evidenced by the plethora of so-called functional foods available on the market. These are usually food items or dietary supplements fortified with a single or couple of natural compounds whose health benefits had been experimentally demonstrated. Recently, a more scientifically advanced food synergy concept has been proposed by Jacobs et al. (Pub. Hlth. Rev., 2012) in which a reductionist research focused on single food components shifts to natural combinations of nutrients and non-nutrients as they may play a concerted role in influencing health. Such a philosophy of designing new food products with enhanced health quality has been practised by our group over the past decade. Examples of commercialized, and being currently elaborated, anticarcinogenic foods will be presented along with some scientific background behind their development.
Session VI Cancer killing BIOMARKERS IN COLORECTAL CANCER
Sun Xiao-Feng
Linkoping University, Linkoping, Sweden.
The presentation includes three parts: firstly, an introduction of the RESEARCH TEAM. Secondly, the AIMS of this translational study (bedside-bench-bedside), i.e. 1) detection of biomarkers for making early diagnosis and finding high risk individuals; 2) identification of biomarkers associated with therapy response for designing individual therapy; and 3) identification of the mechanisms behind the relationships between biomarkers and clinicopathological variables. Thirdly, the RESULTS AND IMPORTANCE of the study will be presented: 1) Early diagnosis; The detection of early-stage tumour is a prerequisite for improving therapy response and prognosis. By studying the process of tumour development from normal mucosa to adenoma, primary cancer and metastasis, we may find biomarkers for early diagnosis. The study of polymorphisms will provide information for identifying high risk individuals; 2) Therapy response; Radio-, chemo- and/or antibody therapy have improved patient survival but patients elicit a large variation in therapy response. Certain molecular mechanisms are involved in therapy response. It is crucial to search for biomarkers used for individual therapy in order to improve prognosis and limit side-effects; and 3) Prognosis; TNM staging is a rather broad classification since cancers at the same stage result in different survival rates. The determination of biomarkers may clarify prognosis for the patients within the same TNM stage.
SALINOMYCIN – EXPERIMENTAL DRUG WITH PREFERRED
TOXICITY TOWARDS CANCER STEM CELLS, THE ROLE OF AUTOPHAGY
Marek J. Łos1, Jaganmohan Reddy Jangamreddy1, Saeid Ghavami2, Artur Cieslar-Pobuda1,3
1Depart. Clinical and Experimental Medicine (IKE), Division of Cell Biology, Integrative Regenerative Medicine Center (IGEN), Linköping University, Sweden; 2Department of Human Anatomy and Cell Science, Univ. Manitoba, Canada; 3Biosystems Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland
Salinomycin is an antibiotic and potassium ionophore. It has recently been identified as preferentially killing breast cancer stem cells. The molecular mechanism of Salinomycin's toxicity is not fully understood, as it cannot be explained solely by its ionophore activity. Various studies reported that Ca2+, cytochrome c, and caspase activation play a role in Salinomycin-induced action. Furthermore, Salinomycin may target Wnt/beta-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer Rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell-protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increases mitochondrial membrane potential in a subpopulation of cells. Salinomycin strongly decreases, in time-dependent manner, cellular ATP level. In contrast, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggest possible clinical application of Salinomycin in combination with autophagy inhibitors (i.e. clinically-used Chloroquine). NUMERICAL STUDY OF CANCER
INVASION OF EXTRACELLULAR MATRIX Nikolaos Sfakianakis
University of Mainz, Germany
In this work we studied high order numerical methods resolving a class of reaction-diffusion-taxis systems modeling the dynamics of cancer tumor invasion on the extracellular matrix. The complication of this type of systems and the dynamics that arise make the analytical study of the system almost impossible and the numerical study extremely difficult. We propose in this work a numerical method to analyse this type of systems to extend their complexity. SENESCENCE, POLYPLOIDY, AND STEMNESS – THREE
COMPONENTS
Jekaterina Erenpreisa1, Anda Huna1, Thomas Jackson2, Kristine Salmina1, Paul Townsend2, Mark Cragg2
1Latvian Biomedical Research & Study Centre, Riga, Latvia; 2Cancer Sciences Unit, University of Southampton, Southampton, UK.
All aggressive tumours display signature of embryonal stem cells which is responsible for resistance to genotoxic treatments and cancer progression. Furthermore, recent data from several tumour types shows that stemness is actually induced by genotoxic treatments in differentiated tumour cells [1,2] and that this response is associated with induced polyploidy and its reverse [3,4]. Intriguingly, genotoxic treatments concurrently induce senescence in the same cells, akin to what happens in induced pluripotent stem cells (iPSC) during somatic cell reprogramming. Therefore, we studied the genotoxic response of the TP53- and telomerase-competent ovarian cancer cell line PA1 [5] and showed a TP53-dependent activation of both stemness-promoting (OCT4A) and senescence-promoting (p21cip1) regulators. Mechanistic and dynamic studies of individual cells show that high level of two regulators keep each other in balance and the cell in a bi-potential state within the G2 arrest favouring DNA damage repair and vitality, whilst preventing premature exit, which triggers terminal senescence and aberrant cell division. This bi-potential state is leaky for tetraploidy coupled to persistent DNA damage, thus carrying the risk of cancer progression. Our conclusion is that the induction of stemness is inevitably coupled to senescence and DNA damage signalling and that involvement of tetraploidy leads to overcoming the genome-guarding functions of TP53. The same mechanism may be induced during artificial reprogramming for iPSC, raising the possibility of cancer development from these cells.
References: [1] Lee G.Y. et al.(2011) Stochastic acquisition of a stem cell-like state and drug tolerance in leukemia cells stressed by radiation. Int. J. Hematol. 93, 27-35. [2] Ghisolfi L. et al. (2012) Ionizing radiation induces stemness in cancer cells. PloS one 7, e43628. [3] Salmina K. et al. (2010) Up-regulation of the embryonic self-renewal network through reversible polyploidy in irradiated p53-mutant tumour cells. Exp. Cell Res. 316, 2099-2112. [4] Lagadec C. et al. (2012) Radiation induced reprogramming of breast cancer cells. Stem Cells 30, 833-844. [5] Jackson T. et al. (2013) DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells. Cell Cycle 12, 430-441.
P-GLYCOPROTEIN TRANSPORT OF THE ACTIVE IMATINIB METABOLITE, CGP74588, IN CHRONIC MYELOID LEUKEMIA CELLS
Kourosh Lotfi1,2 Karin Skoglund1,
Samuel Boiso Moreno1, Jan-Ingvar Jönsson3,
1Division of Drug Research/Clinical Pharmacology, Department of Medical and Health Sciences, Linköping University, Sweden; 2Department of Hematology, Linköping University Hospital, Sweden; 3Division of Cell Biology, Department of Clinical and Experimental Medicine, Linköping University, Sweden; 4Science for Life Laboratory, Division of Gene Technology, Royal Institute of Technology, Solna, Sweden.
The tyrosine kinase inhibitor (TKI) imatinib is, despite the introduction of second generation TKI’s, the standard first-line therapy in chronic myeloid leukemia (CML). Early response to therapy is correlated to long-term therapeutic effect in CML. To be able to switch to second generation TKI’s in an early stage of CML, it would be advantageous to identify predictive markers for failure on imatinib. Imatinib is metabolized by hepatic CYP3A4 and CYP3A5, forming approximately 30 metabolites. The main metabolite, CGP74588, is pharmacologically active with similar potency to that of imatinib. CGP74588 is present in ~20% of imatinib plasma concentrations but with a large inter-individual variation. We have previously reported that high CYP3A activity is associated with a better therapeutic outcome of imatinib, indicating a clinical significance of imatinib metabolites (Gréen et al. 2010). Furthermore, imatinib is a substrate for the efflux transporter P-glycoprotein which is the product of the ABCB1 gene. Several ABCB1 polymorphisms have been described and some have been shown to influence the therapeutic response to imatinib in CML patients. The aim of this study was to investigate the effects of ABCB1 over-expression on the in vitro resistance to imatinib and its CYP3A metabolite CGP74588. ABCB1 wild type human cDNA was infected to the CML cell line K562 using a retroviral system. Co-expression of ABCB1 and the reporter gene for enhanced yellow fluorescent protein (EYFP) was used for the assessment of ABCB1 expression by the analysis of EYFP in FACS. The influence of ABCB1 expression on the cytotoxic effects of imatinib and CGP74588 was assessed using MTT assays. FACS analysis of EYFP confirmed an over-expression of ABCB1 in infected K562 cells with a mean fluorescence intensity of 23.5 compared to 0.55 in parental K562 cells. ABCB1-expressing cells (K562/ABCB1) were slightly, but not significantly, more resistant to imatinib than K562 cells (IC50 K562 = 0.41µM, K562/ABCB1 = 0.50µM). However, ABCB1 expression induced a 12-fold increase in resistance when cells were treated with the imatinib metabolite CGP74588 (IC50 K562 = 0.72µM, K562/ABCB1 = 8.53µM, p = <0.000). Recent reports have shown that CGP74588 accumulates in cell lines with acquired multi-drug resistance and high P-gp expression. Our studies on cells with ABCB1 expression as a single resistance mechanism prove that P-gp is indeed conferring resistance to CGP74588 in CML cells. Furthermore, CGP74588 cytotoxicity is affected by ABCB1 expression to a greater extent than imatinib itself, indicating that ABCB1 activity could be important for CGP74588 plasma and target cell concentrations in vivo and might contribute to the large intra-individual variation seen in patients’ plasma concentrations. Future studies will be needed to determine the clinical significance of P-gp activity and CGP74588 pharmacokinetics in relation to TKI therapy of CML. EVALUATION OF NOVEL, ANTICANCER, TARGETED FUSION
BIOMOLECULE WITH HIGH ANTIANGIOGENIC AND CYTOTOXIC ACTIVITY – SUMMARY
Jerzy Pieczykolan1, Piotr Rózga1,
Anna Pieczykolan1, Sebastian Pawlak1,
1Drug Discovery Department, Adamed, Pieńków 149, 05-270 Czosnów, Poland; 2Department of Medical Biotechnology, Jagiellonian University, Poland.
One of the most important mechanisms of tumorgenesis is formation of new blood vessels and tissue remodeling. Vascular endothelial growth factor (VEGF) is one of the most important and widely studied proangiogenic factor that has an important role in many physiological and pathological processes. Blockade of VEGF pathway has been shown to inhibit both pathological angiogenesis and tumor growth. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been considered a promising anticancer agent due its remarkable ability to induce apoptosis in cancer cells without harming normal cells. However, in clinical practice TRAIL efficacy is too low to become the effective single therapy agent. Here, we present a novel fusion protein based on TRAIL/Apo2L, equipped with an antiangiogenic, effector peptide, fused to its N- terminus. As effector, a 7-amino acid long peptide is used that binds to natural VEGF receptors competing with their natural ligand. The proposed fusion protein AD-O51.4 consists of extracellular soluble portion of TRAIL linked to two tandemly arranged copies of effector peptide separated by the sequence containing the motif recognized by tumor-specific proteases (MMP's, uPa). TRAIL targeted peptide is able to bind and sequester the VEGF receptors on malignant (cancer) and endothelial cells. As a consequence, peptide blocks the binding of the VEGF ligand, preventing new vessels’ formation. There is also a lot of evidences confirming expression of the VEGF receptors on the surfaces of different cancer cell lines. Due to the presence of VEGF receptors those cell lines can be also targeted by VEGF receptors blocking peptide what makes them susceptible for TRAIL-induced apoptosis. AD-O51.4 showed in vitro specific cytotoxic effect on various cancer cell lines at the level of IC50 below 0.1 ng/mL without toxic effects on normal cells. Cytotoxic activity of TRAIL was significantly lower in comparison to AD-O51.4. We demonstrated that TRAIL/Apo2L-VEGF-antagonist is a strong activator of caspase 3 and Bid processing. Additionally, its direct antiangiogenic activity was confirmed with ring aortic assay and HUVEC spheroid assay. Strong antitumor activity of novel fusion molecule was also confirmed on xenograft model of multidrug resistant human uterine sarcoma MES-SA/Dx5 where our protein caused complete tumor remission and showed much higher efficacy than TRAIL alone. The similar effect was observed with human colorectal adenocarcinoma Colo205, human pancreatic carcinoma MIA PaCa-2 and orthotropic model of human lung carcinoma NCI-H460-luc2. Therefore, we postulate that the use of this fusion proteins can open a new and exciting filed in anticancer compounds’ development.
Additional Lecture Abstract:
The instrumental analysis of
novel polymeric materials
Andrzej Swinarew1,2, Sylwia Golba1, Beata Swinarew2,3, Jadwiga Gabor1, Beata Kopek1, Sylwia Burczyk1
1Institute of Materials Science, University of Silesia,
40-007 Katowice, Poland; 2SHIM-POL A.M. Borzymowski
E.Borzymowska-Reszka A. Reszka Spółka Jawna; 3Institute of
Engineering, Polymer Materials and Dyes,
Some star-shaped polymers as well as molecularly imprinted polymers (MIPs) are polymeric materials with specific recognition sites complementary in shape, size and functional groups to the template molecule, involving an interaction mechanism based on molecular recognition. These recognition sites mimic the binding sites of biological entities such as antibodies and enzymes. Their stability, ease of preparation and low cost for most of the target analytes make them attractive for numerous applications. The use of star-shaped polymers as stationary phases for HPLC is one of the best studied application of novel polymeric materials, largely because it provides a convenient method for quantitative assessment of the quality of imprints produced by a particular strategy. A wide range of chemical compounds have been separated successfully, ranging from small molecules, such as drugs, to large proteins and cells. The best results have been obtained for molecules with molecular weights in the range of 200–1200Da. The resulting polymers are robust, inexpensive and, in many cases, possess affinity and specificity that is suitable for industrial applications. In this announcement we present an investigation of new polymeric stationary phase for pathogens detection using the Nexera UHPLC equipped with a Pinnacle DB PAH 1.9 µm, 50 x 2.1 mm column and the multiple DAD and FLD detectors supported with GCMS HS techniques enabling detection of trace-level components. The described method allows for selective absorption of pathogen molecules.
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