Gliwice Scientific Meetings 2009
20-21 November

 

~~~ Poster abstracts ~~~

 

 

1. Image processing based method used in automated analysis of cell glycosylation patterns

D. Bednarczyk, K. Wieczorek, A. Lalik, A. Bal

System Engineering Group, Institute of Automation, Silesian University of Technology, 44-100 Gliwice, Poland.

Glycosylation is involved in many biological processes and its alterations is associated with some diseases. Quantification of oligosaccharide molecules and estimated theirs cell surface localization is important to understanding one of mechanisms of  diseases development.

The aim of this work was to develop algorithms for automatic recognition FITC-labeled lectin bound to K562 cells in fluorescence microscopy images. The mean fluorescence intensity of each cell, corresponding to amount of cell-bound lectins, was measured too.

For detecting of cells regions in the image the new adaptive global image thresholding method was developed. After segmentation stage numbers of image processing operations are used for improvement of image segmentation results.

The new method was using to estimate the level of glycosylation in irradiated and control (non-exposed) K562 cells at different time points after exposure to 4 Gy of X-rays. Obtained results showed changes in protein glycosylation elicited by ionizing radiation. The data were compared with the level of K562 cell glycosylation estimated with CometScore software.

This work was supported by the grant PBZ/MEiN/01/2006/49

 

2. CHEMOSENSITIVITY OF BLADDER CANCER CELLS AFTER CHEMICAL OR RNAi-mediated modulation of multidrug resistance (MDR) genes expression

Ilona Bednarek¹, Daniel Sypniewski¹, Sabina Gałka¹, Grzegorz Machnik¹, Tomasz Loch¹, Michał Tkocz², Maciej Kupajski², Magdalena Duda², Wiesław Duda²

¹Department of Biotechnology and Genetic Engineering, Medical University of Silesia, Katowice, Poland;

²Division of Urology, Małachowski Hospital, Strzelecka 9, Katowice, Poland.

Multidrug resistance (MDR) is the lack or the loss of sensitivity of cancer cells to chemotherapeutic agents and it is the main obstacle in successful treatment in most patients. Bladder cancer is one of the malignant cancers where MDR contributes significantly to treatment failure. The origin of most MDR mechanisms lies in the overexpression of cell membrane transporters, such as ABC (ATP-binding cassette) proteins, involved in xenobiotic efflux.

The aim of our study was to develop an efficient molecular method, based on RNA interference, capable of modulating expression of genes responsible for multidrug resistance in bladder cancer. Six MDR genes overexpressed in bladder cancer were subjected in the study: MDR1 (P-gp), MRP-1, -2, -3, -5, and LRP (lung resistance protein). Two strategies of post-transcriptional silencing were used: enzymatically prepared esiRNA (endoribonuclease-based siRNA) and vector-based strategy of short-hairpin RNA prepared by cloning shRNA sequences into pSUPER plasmids. Experiments were carried out in vitro using primary cancer cells isolated from bladder cancer resection tissue obtained from patients of Urology Division, (Małachowski Hospital, Katowice), and established T24 cell line (ATCC: HTB-4).

Lipid-based transient transfection of both primary, and T24 cultures revealed suppression of the target genes expression at the mRNA level as was confirmed by Real-Time RT-PCR. mRNA levels were reduced by 80% (MRP1) - 99% (MRP5). Stronger effects were observed in T24 cultures than in primary cultures; also shRNA modulation was more effective than esiRNA. In the next step, different chemotherapeuticals were used as substrates of MDR proteins: etoposide, doxorubicin, bleomycin, vinblastine, cytarabine. Results showed significantly reduced viability in T24 cultures compared with the non-transfected: from 8,45% (LRP + vinblastine) to 43,6% (MDR1 + etoposide).Finally, both esiRNA, and shRNA constructs were tested for their efficacies as molecular sensitizers in etoposide treatment. The results were compared with chemical modulators of MDR (cyclosporine A and probenecid). IC₅₀ values showed significant reduction in etoposide concentrations necessary to induce cancer cells death. In T24 cultures resistance-modifying factor (RMF) values were from 24.07 for MDR1 to 6.29 for LRP (shRNA modulation), and from 6.25 for MDR1 to 2.35 for LRP (esiRNA). In primary cells these values ranged between 31.2-6.53 (shRNA), and 7.06-2.68 (esiRNA), respectively. Chemical modulation by cyclosporine A and probenecid showed significantly lower RMF values (only 1.16 – 1.69).

Altogether, these results show that both types of our RNAi designs significantly suppress expression of all studied multidrug resistance genes in a sequence-specific manner. PTGS with esiRNA helps to obtain rapid effects, while that mediated by shRNA in situ expression enables to perform stable suppression (especially when neo selection is used). This molecular modulation allows us to decrease concentrations of chemotherapeutic drugs and it is significantly more efficient than using chemical modulators of MDR genes.

The study was supported by the funds of Medical University of Silesia (KNW-1-145/08, KNW-1-117/09).

Results were partially shown in I. Bednarek postdoctoral habilitation thesis (ISBN: 978-83-7509-094-9).

 

3. ZINC OCTACARBOXYPHTHALOCYANINE – A POSSIBLE PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY

B.Boroń¹; A. Szurko^1,3; M. Rams¹; A.Sochanik³; R. Wrzalik¹; Z. Ujma¹; J. Nackiewicz², K. Balin¹; A. Ratuszna¹

¹A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland;

²Institute of Chemistry, University of Opole, Opole, Poland;

³Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice,

 Poland.

            Photodynamic therapy (PDT) is a form of cancer treatment which requires three factors: molecular oxygen, light of specific wavelength and a photosensitizer - a substance which is activated by this light. The activated photosensitizer produces reactive oxygen species and free radicals as a result of energy transfer from its excited triplet state to ground state of molecular oxygen or organic substrate (tissue).

            Treatment of different kinds of cancer requires various photosensitizers. This is why chemists, physicists and biologists continue search for new substances which can act as photosensitizers and are activated by light of different wavelengths. Longer waves can penetrate deeper through the tissue and can activate photosensitizers which accumulate not only directly under the skin but deeper.

            Phthalocyanines are interesting candidates for PDT because their chemical structure is closely related to that of porphyrins. Phthalocyanines have four nitrogen atoms in meso positions and bind four pyrrole rings forming one molecule. Phthalocyanines have also four benzene rings bound to pyrrole rings (one benzene ring is bound to one pyrrole ring) which shift their absorption spectrum towards longer wavelengths. Zinc octacarboxyphthalocyanine has, in addition, two carboxyl groups bound to each benzene ring. Their presence makes this compound soluble in polar solvents. Zinc atom causes the quantum yield of singlet oxygen to be twice as high as that of unsubstituted phthalocyanines.

Zinc 2,3,9,10,16,17,23,24-octacarboxyphthalocyanine, ZnPcOC (Fig. 1), was obtained according to the general procedure described in Ref. [1]. We synthesised free-base octacyanophthalocyanine (1) by the cyclotetramerization of 1,2,3,4-benzenetetracarbonitrile and purified it, exactly as described in this procedure. Next, from 1 and purified zinc, we synthesised the 2,3,9,10,16,17,23,24-octacyanophthalocyanine, ZnPc(CN)₈ (2). Finally, we hydrolyzed 2 to ZnPcOC. A portion of 2 was added to the deoxidized ethylene glycol-water solution containing NaOH. The reaction was carried out in dark, at 110–120°C, with constant nitrogen bubbling. When ammonia stopped to evolve, the reaction mixture was cooled down, diluted with deoxidized water and filtered. The product was precipitated by addition of concentrated HCl, purified according to Ref. [1] and dried. Finally, Soxhlet extraction with acetone and methanol was performed.

Fig. 1 Zinc 2,3,9,10,16,17,23,24-octacarboxyphthalocyanine, ZnPcOC

            In the study, efficiency of zinc octacarboxyphthalocyanine in the absence of light was measured. The studies were carried on the cell line HCT 116- Human Colon Carcinoma. The solutions with concentrations of octacarboxyphthalocyanine: 0.4 [μM/l]; 0.75 [μM/l]; 1 [μM/l]; 1.5 [μM/l] were administered to the cells. The chemical compound is soluble in DMSO. Maximum concentration of DMSO in solutions was no higher than 0.3% volume. After 24 hours of incubation the MTS test was measured. The percentage of survival cells makes the zinc octacarboxyphthalocyanine a good candidate as photosensitizer for PDT.

            The image of surface of this compound was obtained by the use of an electron microscope (SEM). The chemical composition was also measured by the SEM and it shows that obtained percentage of the chemical composition confirms the probable composition of zinc octacarboxyphthalocyanine.

 [1] Wőhrle D, Meyer G. Reaktive oktafunktionelle phthalocyanine aus1,2,4,5-tetracyanbenzol. Makromol Chem 1980;181:2127–35.

This work was supported by the Polish Ministry of Science and Higher Education (grant No. 0538/R/T02/2007/03). 

 

4. FUZZY ANALYSIS IN RISK FACTORS OF CANCER

Gabriela Dudek, Anna Strzelewicz, Monika Krasowska, Aleksandra Rybak, Zbigniew J. Grzywna

Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, Strzody 9, 44-100 Gliwice, Poland.

Cancer is a major public health problem. Currently, one in four deaths in the United States is due to the cancer. In many papers we can find information about risk factors which show how significant problem it is. A risk factor is a variable associated with an increased risk of disease. Factors that increase cancer risk can be external such as personal lifestyle choices or substances such as chemicals and asbestos, present in the environment, those are known to cause cancer. Some people also have internal risk factors for cancer such as a genetic predisposition or those that develop as a result of aging.

Using fuzzy set theory, we have created a system which predicts the risk factors for different kind of cancer. We have taken into account lung, colon, breast, cervical and prostate cancer. We used the Mamdani model which is implemented in the Fuzzy Logic Toolbox in Matlab. As inputs in our system we have taken following risk factors: genetic, biological (race, age, sex) and behavioral (overweight, alcohol consumption, tabacco smoke). Better estimates of cancer risk probability will direct more intensive clinical services and research. By means of the fuzzy set theory this risk factors of cancer can be chosen very quickly.

 

5. STAT3 IS A PHOSPHO-SPECIFIC BINDING PARTNER FOR FIBROBLAST GROWTH FACTOR RECEPTOR THAT IS ACTIVATED BY AMPLIFIED RECEPTOR EXPRESSION

Anna A. Dudka¹, Steve M. M. Sweet², John K. Heath¹

¹CRUK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.;

²Present address: Department of Chemistry, University of Illinois, Urbana, IL  61801, USA.

            Fibroblast growth factor receptors (FGFRs) play many roles in development, cell proliferation, differentiation and physiological function. Potentially aberrant manifestations of FGFR signaling have been implicated in a variety of tumor cell types including the acquisition of FGFR amplicons and potentially elevated levels of FGFR expression. However the oncogenic mechanisms associated with amplified receptor expression are not known. FGFRs possess intrinsic tyrosine kinase activity which, via direct or indirect phosphorylation of target substrates, leads to activation of other signaling proteins, formation of multiprotein signaling complexes and concomitant downstream responses. Using a proteomics approach we here identify signal transducer and activator of transcription 3 (STAT3) as a phosphorylation-dependent partner for Tyr677 of FGFR1. Association of STAT3 with activated FGFR is essential for the subsequent tyrosine phosphorylation of STAT3, nuclear translocation and activation of downstream targets. Tyrosine phosphorylation of STAT3 is also dependent upon the concomitant FGFR-dependent activity of SRC and JAK kinases. We also show that tyrosine (but not serine) phosphorylation of STAT3 requires amplified FGFR protein expression generated either by forced expression or associated with gene amplification in tumor cells lines such as SUM-52PE.  These findings show that engagement of the STAT3 pathway resulting from amplified FGFR expression merits consideration as a potential therapeutic and diagnostic target.

 

6. IDENTIFICATION OF NOVEL (HYPOTHETICAL) PROTEIN PARTNERS OF THE MAJOR APOPTOTIC NUCLEASE DFF; YEAST TWO-HYBRID BASED STUDY

Hanus J., Kalinowska-Herok M., Ponge L., Widłak P.

Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch; Poland.

The major apoptotic nuclease, DNA fragmentation factor (DFF), also termed Caspase-activated DNase (CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45/ICAD) and a 40 kD latent nuclease subunit DFF40/CAD. Activation of the nuclease depends on caspase-3-mediated cleavage of DFF45/ICAD inhibitor and formation of DFF40/CAD homooligomers. Caspase-activated DFF40/CAD homo-oligomers can further interact with additional activators or inhibitors; however, only few of them had been identified so far.

Here we used a yeast-two and -three hybrid system in aim to identify novel proteins that potentially interact with DFF40/CAD. S. cerevisiae AH105 strain was transformed with human DFF40 (cloned into pGBT9 vector) and then mated with S. cerevisiae Y187 strain carrying human brain embryo or HeLa cell cDNA libraries. Alternatively, to identify proteins potentially interacting with the DFF heterodimer, S. cerevisiae AH105 strain was transformed with both human DFF40 and DFF45 (cloned into bi-cistronic pBridge vector); an approach called a yeast-three hybrid system. In addition, S. cerevisiae AH105 strain was transformed with human DFF45 to search for possible partners of this protein alone.

The screening revealed DFF45 as the only partner of DFF40 when expressed separately. However, screening of HeLa cDNA library revealed that DNA sequences present in chromosome 10 (ESTs CV366200.1 and DB313950) potentially encode for protein that interact with the DFF heterodimer. Interestingly, silencing of corresponding transcript in HeLa cell by shRNA affected proliferation rate and resistance to apoptosis. In addition, the screening revealed several proteins that potentially interact with DFF45, including GFAP, FHL1, FBXO28, FOSL1, PGK1 and PCNT.

This work was supported by the Ministry of Science, Grant N301 058 31/1763.

 

7. A COMBINATION OF ENDOGLIN-BASED DNA VACCINE AND INTERLEUKIN-12 GENE ENHANCES ANTIANGIOGENIC AND ANTITUMOR EFFECTS

Magdalena Jarosz, Joanna Jazowiecka-Rakus, Wioleta Marut, Tomasz Cichoń, Iwona Mitrus, Ryszard Smolarczyk, Andrzej Smagur, Aleksander Sochanik, Stanisław Szala Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, ul. Wybrzeże Armii Krajowej 15, 44-101 Gliwice, Poland.

Endoglin (CD105), a marker of endothelial cells, is a receptor for transforming growth factor β. CD105 is overexpressed on proliferating endothelial cells in tumor blood vessels and thus it offers an attractive target for antiangiogenic therapy.

In this study we made use of an oral DNA vaccine encoding murine endoglin, carried by attenuated Salmonella typhimurium SL7207 (aroA^-). It has been reported that CD8+ T cell-mediated immune response induced by this vaccine effectively suppressed tumor growth by eliminating proliferating endothelial cells in the tumor vasculature. Our own results showed that this endoglin-based DNA vaccine effectively inhibited growth of murine renal carcinoma as well as murine melanoma tumors, both in prophylactic and therapeutic settings.

            To improve the therapeutic effects achieved in murine tumor models we combined oral administration of this endoglin-based DNA vaccine with direct intratumoral injection of plasmid DNA vector encoding the murine IL-12 gene, an immunomodulatory cytokine with antiangiogenic properties. Interleukin 12 plays a prominent role in activating the immune system, inter alia by enhancing the cytotoxicity of CD8+ T cells. We observed that a combination of endoglin-based DNA vaccine and IL-12 gene significantly inhibited growth of established murine melanoma, leading to complete regression of tumors (without recurrence) in 33% of cases, compared to mice receiving single-agent therapy. As a consequence, lifespan of animals treated with the combination was significantly extended. In addition, the combined therapy appeared more effective at reducing the density of tumor microvessels than either treatment alone.

In summary, a combination of endoglin-based DNA vaccine carried by Salmonella typhimurium and interleukin-12 gene therapy showed a synergistic effect in suppressing tumor angiogenesis, leading to significant tumor regression. 

 

8. endothelial cells ARE ReSISTANT tO RADIATION-INDUCED BYSTANDER EFFECT

Jelonek K., Walaszczyk A., Pietrowska M., Gabryś D., Widłak P.

¹Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland.

Radiation-induced damage of cardiovascular system is one of reported side effects of radiotherapy. Heart failure related to radiotherapy most possibly involves long-term effects of damage of cardiac microcirculation. Here we aimed to analyze radiosensitivity of cardiac endothelial cells.

Three types of endothelial cells were used: primary cells isolated from hearts of C57BL/6J mice, H5V cells (isolated from mouse embryo heart) and b.END3 cells (isolated from mouse brain). In addition, primary cardiomyocytes were isolated from the same mice strain. Cells were irradiated in vitro with 2 Gy dose of ionizing radiation. Radiosensityvity of cells was measured using gH2A.X staining, induction of apoptosis and clonogenic survival. We have analyzed effect of either direct irradiation or so called bystander effect (in different combination of cell types). In addition, permeability of EC monolayer was analyze to determine possible effect of irradiation upon inter-cellular interaction. We observed that in vitro radiosensitivity of tested endothelial cells was similar to radiosensitivity of other cell types. However, endothelial cells were resistant to radiation- induced bystander effect.

This work was supported by the FP7/EURATOM Grant CARDIORISK.

 

9. ANALYSIS OF FREQUENCY CHEK2, P53, NOD2/CARD15 AND RET GENE POLYMORPHISMS IN POLISH PATIENTS WITH DIFFERENTIATED THYROID CANCER

Marta Kaczmarek-Ryś¹, Justyna Hoppe-Gołębiewska¹, Oliwia Zakerska¹, Ludwika Jakubowska-Burek², Katarzyna Ziemnicka³, Jerzy Sowiński³, Ryszard Słomski^1,4

¹Institute of Human Genetic Polish Academy of Science, Poznań, Poland;

²Department of Gastroenterology, Human Nutrition and Internal Diseases, University School of Medical Sciences, Poznań, Poland;

³Department of Endocrinology and Internal Diseases, University School of Medical Sciences, Poznań, Poland;

⁴Department of Biochemistry and Biotechnology, University of Life Sciences, Poznań, Poland.

Thyroid carcinomas amounts to 1% of general and are the most often carcinomas of endocrine system with still growing up frequency. The most often papillary and follicular thyroid cancer occurs (80-90%), which belongs to group of tumors well prognoses, slowly progress and benignity. Very serious problems in this cancer disease are recurrences and regional or remote metastasis. There were observed numerous cases of osteolytic, cerebral and pulmonary metastasis and moreover, well differentiated thyroid cancer can progress to malignant anaplastic.

In this focus, very important seems to be searching for molecular markers of disease course, good or poor prognosis and response on medical treatment as well. It is expected that SNP polymorphisms research in genes demonstrating association with neoplastic diseases will be helpful in understanding of molecular mechanisms of thyroid gland tumors development and allow improving diagnosing. 

The dependence of differential thyroid cancer occurrence on DNA variation: I157T in CHEK2 gene, R72P in P53 gene, 1007fs in NOD2/CARD15 gene and synonymic G2497T substitution in RET protooncogene was examined. 296 patients with differentiated thyroid cancer and 200 individuals from population group were examined. I157T, G2497T and R72P variants were analyzed by pyrosequencing and 1007fs by PCR-SSCP and DNA sequencing.

There were no significant differences in allele or genotype frequencies in analysis of RET G2497T substitution and R72P in P53 gene but polymorphic allele frequencies of 1007fs and I157T was 8,95% and 4,90% in patients with thyroid cancer, compared with 2,92% and 2,1% in control individuals respectively. Frequencies between patient and control groups were tested using Pearson’s χ² statistics. This analysis shows that 1007fs and I157T mutations are associated with susceptibility to differential thyroid cancer. Our findings indicates that particular characteristics of cancer risk genes on RNA level as well as DNA changes, which may influence on transcription is necessary. Additionally a summary effect of different SNP changes as a cancer predisposing factor is possible, so further analysis will be performed.

 

10. INTERFERENCE BETWEEN THE HEAT SHOCK RESPONSE AND NFkB-DEPENDENT SIGNALING PATHWAY

Kalinowska-Herok M.¹, Pakuła M.¹, Kashchak N.¹, Janus P.¹, Szołtysek K.¹, Pigłowski W.¹, Widłak W.¹, Kimmel M.², Widłak P.¹

¹Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice, Poland; ²Silesian University of Technology, Gliwice, Poland.

NFkB is a family of transcription factors that regulate numerous genes important for pathogen- or cytokine-induced inflammation, immune response and cell proliferation. NFκB also activates several genes that promote cell survival, which contributes to aggressive tumor growth and resistance to chemotherapy and radiation in cancer treatment. HSF1 is the primary transcription factor responsible for cellular response to different forms of stress (e.g., a heat shock), which upon stress-induced activation binds regulatory DNA elements, termed heat shock elements (HSE), present in promoters of heat shock proteins (HSPs) genes, and activates their expression. In general, HSPs function as molecular chaperones in regulation of cellular homeostasis and promoting survival. HSPs over-expression is frequently found in many types of cancer, and is usually associated with poor prognosis. On the other hand, however, hyperthermia is an adjuvant treatment used to sensitize cancer cells to radio- and chemotherapy, possibly affecting pathways that promote cell survival.

Here we aimed to address possible mechanisms by which hyperthermia and HSF1-dependent signaling interfered with NFkB-dependent pathways. The U₂OS osteosarcoma human cell line was used as an experimental model. The heat shock response was induced by mean of hyperthermia (incubation at 43^oC for one hour). Alternatively, cells were transfected with mutated constitutively active HSF1 with deletions in regulatory domain (HSF1DRD) to activate HSF1-dependent signaling in the absence of the heat shock. Cells were incubated with TNFa cytokine to activate the NFkB pathway, and then expression of NFkB-regulated genes was assessed by RT-PCR. The activation of the NFkB signaling pathway was monitored by mean of degradation of IkBa inhibitor and appearance of active DNA-binding NFkB forms in nuclear extracts.  

We have observed that TNFa-induced activation of NFkB was inhibited in cells subjected to hyperthermia, and four hours recovery in physiological temperature was necessary to allow full activation of NFkB. On the other hand, NFkB remained to be fully activatable by TNFa treatment in cells containing constitutively active mutated HSF1 at normal temperature. Interestingly, however, expression of several TNFa-activated and NFkB-dependent genes, including genes encoding TNFa and IL-6, was down-regulated in the presence of active HSF1. Our findings clearly indicates functional interference among hyperthermia, HSF1- and NFkB-dependent signaling pathways.

This work was supported by the Ministry of Science and Higher Education, Grant PBZ-MNiI-2/1/2005.

 

11. Antitumor potential of novel synthetic genistein glycoconjugates

K. Kędzior¹, A. Rusin¹, A. Gogler¹, M. Głowala-Kosińska¹, A. Gruca¹, J. Zawisza, W. Szeja², G. Grynkiewicz³, Z. Krawczyk¹

¹Department of Tumor Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland;

² Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of Chemistry, Silesian University of Technology, Gliwice, Poland;

³ Pharmaceutical Research Institute, Warsaw, Poland.

Genistein have been found to block the uncontrolled cell growth associated with cancer via different molecular mechanisms, which encourages chemical modification of this leader compound for further drug development. There is evidence that some sugar derivatives of genistein may have stronger inhibitory effect than genistein itself.

We decided to investigate biological effect of new synthesized genistein sugar derivatives on human prostate cancer cells DU145 and human colon cancer cells HCT116. Antiproliferative activity of a series of novel synthetic genistein glycoconjugates was studied in vitro with use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Genistein glycoconjugates showed diverse antiproliferative potency against tested human tumor cell lines. The structure activity relationship is not clear yet, and the role of a sugar moiety for molecular targeting still remains to be found. However, the series with genistein combined with acetylated rhamnal seems to be the most potent. Among analyzed derivatives the most active compound revealed Ram-3 which inhibit growth of cancer cell lines through different cellular mechanisms: inhibition of EGFR phosphorylation, cell cycle inhibition in the G2/M phase, mitotic arrestment and induction of apoptosis.

The results show that the compound Ram-3 demonstrates strong antiproliferative activity against human cancer cells. Further chemical modifications and structure – activity relationship studies should be performed in order to find optimized molecule for chemotherapeutical use.

 

12. SYNTHESIS OF THE DERIVATIVES OF VITAMIN C AND THEIR LIPOSOMES FOR A POSSIBLE ANTICANCER TREATMENT

M. Knaś¹, A. Mrozek-Wilczkiewicz^1,2, A. Szurko^2,3, A. Ratuszna², J. Polański¹

¹University of Silesia, Institute of Chemistry, ul. Szkolna 9, 40-006  Katowice, Poland;

²University of Silesia, Institute of Physics, ul. Uniwersytecka 4, 40-007 Katowice, Poland;

³Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, ul. Wybrzeże Armii Krajowej 15, 44-101 Gliwice, Poland.

Observational studies showed that ascorbate at pharmacologic concentrations generates hydrogen peroxide-dependent cytotoxicity toward a variety of cancer cells in vitro without adversely affecting normal cells [1]. 6-O-ascorbic acid derivatives were synthesized with the main purpose of preparing vitamin C analogues that combine the prooxidant properties of ascorbic acid and its anticancer activity with good solubility in lipophilic media [2]. Compared to ascorbic acid, solubility of 6-O-ascorbic acid analogues in oils and fats is greatly enhanced [3]. These compounds (Scheme 1) were synthesized and studied for their anticancer activity.

Scheme 1.

Products were obtained in good yields, and structures were confirmed by spectral data (NMR, MS). Antiproliferative activity was estimated on HCT 116 cell line (human colon carcinoma) and anticancer activity was evaluated using MTS - reduction colorimetric survival assay. However first results indicated that compounds under study if tested alone did not indicate cytotoxic effect. The next thing we wanted to achieve was to obtain liposomes containing 6-O-ascorbic acid analogues to evaluate their antiproliferative activity. The results showed that this compounds did not form liposomal structures.

References:

[1] Q. Chen, M.G. Espey, A.Y. Sun, Ch. Pooput, K.L. Kirk, M.C. Krishna, D. B. Khosh, J. Drisko, M. Levine,“Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice”, PNAS, 2008, 105, 32, 11105 – 11109.

[2] S. Palma, A. Jimenez-Kairuz, L. Fratoni, P. Lo Nostro, R. Manzo, D. Allemandi, „Coagels from alkanoyl-6-O-ascorbic acid derivatives as drug carriers: structure and rheology”, Il Farmaco, 2003, 58, 1271 – 1276.

[3] S. Palma, R.H. Manzo, D. Allemandi, L. Fratoni, P. Lo Nostro, „Solubilization of hydrophobic drugs in octanoyl-6-O-ascorbic acid micellar dispersions”, J. Pharm. Sci., 2002, 91, 8, 1810 – 1816.

The study has been financed by a grant from the Ministry of Science and Higher Education, No. N N 405 1787 35, 0538/R/T02/2007/03 and PB-W-03-036-00-08.

 

13. The Use of MoStBioDat for Rapid Screening of Molecular Diversity

Agata Kurczyk, Andrzej Bąk , Tomasz Magdziarz, Jarosław Polański

Institute of Chemistry, University of Silesia, Szkolna 9, 40007 Katowice, Poland;

MoStBioDat is a uniform data storage and extraction system with an extensive array of tools for structural similarity measures and pattern matching which is essential to facilitate the drug discovery process. Structure-based database screening has recently become a common and efficient technique in early stages of the drug development, shifting the emphasis from rational drug design into the probability domain of more or less random discovery. The virtual ligand screening (VLS), an approach based on high-throughput flexible docking, samples a virtually infinite molecular diversity of chemical libraries increasing the concentration of molecules with high binding affinity. The rapid process of subsequent examination of a large number of molecules in order to optimize the molecular diversity is an attractive alternative to the traditional methods of lead discovery. This poster presents the application of the MoStBioDat package not only as a data management platform but mainly in substructure searching. In particular, examples of the applications of MoStBioDat are discussed and analyzed.

 

14. Dose-dependent glycosylation changes in irradiated human K562 and Me45 cancer cells

A.Lalik¹ and J.Rzeszowska-Wolny^1,2

¹System Engineering Group, Institute of Automation, Silesian University of Technology, 44-100 Gliwice, Poland;

²Department of Experimental and Clinical Radiobiology,  Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, 44-100 Gliwice, Poland.

Aberrant glycosylation is associated with a number of diseases, including all cancer types. One of the most popular tools for characterizing cell surface glycoconjugates are lectins – ubiquitous proteins which specifically bind defined oligosaccharide structures.

The main aim of this work was to investigate the influence of different X-ray radiation doses  on radiation-induced changes in cell surface glycosylation. For these studies, we were used two human cancer cell lines (K562 and Me45) and several fluorescently-labeled lectins (LCA, PHA-E, UEA, PNA, ConA, DBA, WGA, RCA, PSA, PHA-L, SWGA, GSL-I). Control (non-irradiated) cells were compared with cells irradiated with different doses of ionizing radiation (in the range 4-16Gy).  Samples of cells were collected 7 days after exposure, incubated with a lectin, and the mean fluorescence intensity of each sample was measured using a fluorescence microplate reader (Infinite M200, Tecan). The results suggest that exposure to ionizing radiation had profound effect on the level of all of antigens recognized by lectins mentioned above and irradiation had different effect on glycosylation in both cell lines. What’s more, we observed that ionizing radiation can change sugar chain antigens in cancer cells in dose-dependent manner.

This work was supported by the grant PBZ/MEiN/01/2006/49

 

15. APPLICATION OF PROTON MICROBEAM TO STUDIES OF APOPTOSIS AND NECROSIS IN PC-3 CELL LINE

E. Lipiec¹, J. Dulińska-Litewka², J. Lekki¹, A. Wiecheć¹, J. Wiltowska-Zuber¹, W. M. Kwiatek¹

¹Polish Academy of Sciences, The Henryk Niewodniczanński Institute of Nuclear Physics, ul. Radzikowskiego 152, 31-342 Cracow, Poland;

²Jagiellonian University Medical College, Cracow, Poland..

            Scientific research carried out using the ion microprobes delivers valuable data for clinical oncology. Microprobe enables targeted irradiation of small and well-defined area of tissue (or even single cells) by the fully controlled number of ions (down to a single ion). Therefore, the results are not biased with statistical effects inherently present in traditional, random irradiation experiments and can be useful for optimization of radiotherapy or for medical bioimaging.

            The human prostate adenocarcinoma PC-3 line is derived from bone metastases. The studies of death (apoptosis or necrosis) induced in the PC-3 cell line by a controlled number of protons are presented. The high degree of invasive characteristics of this cell line makes it an interesting subject of study. The 2 MeV horizontal focused proton microbeam from the Van de Graaff accelerator at the Institute of Nuclear Physics Polish Academy of Sciences was used as the irradiation source. For comparison, the cellular response to damage induction by UVA, UVC, and staurosporine was also examined. Necrotic and apoptotic cells were generally visualized using fluorescence microscopy.

            The results show that „older” PC-3 cells after about 50th passage are much more sensitive to proton irradiation than „younger” ones (few passages only). Already 50 protons per cell (~1,3 Gy dose) causes apoptosis in the „older” PC-3 cells. Induction of apoptosis in younger cells was not successful. Necrosis occurs at 800 protons per cell (~20,9 Gy dose) in „older” PC-3 cells and 3200  protons per cell (~83,6 Gy dose) in cells after about 11th passage. In complementary experiments it was proved that incubation with 2,5 µM staurosporine causes apoptosis in this cell line. Necrosis occurs after 10 min of UVC (23,6 mW/cm²) irradiation in „older” PC-3 cells and after about 20 min in „younger” cells.

 

16. ANTIPROLIFERATIVE STUDIES OF THIOSEMICARBAZONE DERIVATIVES

A. Mrozek-Wilczkiewicz^1,2, A. Szurko^2,3, R. Musioł¹, D. Richardson⁴, A. Sochanik³, M. Rams², A. Ratuszna², J. Polański¹

¹University of Silesia, Institute of Chemistry, ul.  Szkolna 9, 40-006 Katowice, Poland;

²University of Silesia, Institute of Physics, ul. Uniwersytecka 4, 40-007 Katowice, Poland;

³Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, ul. Wybrzeże Armii Krajowej 15, 44-101 Gliwice, Poland;

⁴Children’s Cancer Institute Australia for Medical Research, New South Wales 2031, Sydney, Australia.

A series of di-2-pyridyl ketone and 2-benzoylpyridine thiosemicarbazone ligands showed a broad antitumour activity in vitro and in vivo against a wide spectrum of tumours [1]. This class of iron chelators could overcome resistance to established antitumor agents [2]. Recent research points to antifungal effects of this family of compounds [3]. Iron is critical for cell cycle progression and DNA synthesis. Thiosemicarbazones derivatives are tridentate active Fe chelators with high Fe mobilization efficacy and low toxicity [4]. These compounds were synthesized and evaluated for their anticancer activity. The products were obtained in good yields, and the identification of the structure was based on spectral data (NMR, MS). Antiproliferative activity was estimated on HCT 116 cell line (human colon carcinoma), LLC cell line (murine Lewis lung carcinoma) and anticancer activity was evaluated using MTS - reduction colorimetric survival assay. The latter purpose of our studies was to obtain liposomes containg thiosemicarbazone derivatives and comparison anticancer activity of free ligands or ligands locked in liposomes.

References:

[1] D.R. Richardson, D.S. Kalinowski, S. Lau, P.J. Jansson, D.B. Lovejoy, Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents, Biochim. Biophys. Act., 2008, Apr 27.

[2] M. Whitnall, J. Howard, P. Ponka, D. R. Richardson, A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics PNAS, October 3, 2006, 103(40), 14901 - 14906.

[3] V. Opletalová, D. S. Kalinowski, M. Vejsová, J. Kunes, M. Pour, J. Jampilek, V. Buchta, D. R. Richardson, Identification and Characterization of Thiosemicarbazones with Antifungal and Antitumor Effects: Cellular Iron Chelation Mediating Cytotoxic Activity, Chem. Res. Toxicol., 2008, 21, 1878 – 1889.

[4] D.S. Kalinowski, P.C. Sharpe, P.V. Bernhardt, D.R. Richardson, Structure-activity relationships of novel iron chelators for the treatment of iron overload disease: the methyl pyrazinylketone isonicotinoyl hydrazone series, J. Med. Chem., 2008, 51, 331 - 344 .

The study has been financed by a grant from the Ministry of Science and Higher Education, No. N 405 1787 35, 0538/R/T02/2007/03 and PB-W-03-036-00-08.

 

17. GENE EXPRESSION SIGNATURE OF HYPOXIA IN MELANOMA CELLS IN VIVO AND IN VITRO

Magdalena Olbryt¹, Anna Habryka¹, Tomasz Tyszkiewicz², Aleksandra Rusin¹, Tomasz Cichoń³, Katarzyna Lisowska¹, Zdzisław Krawczyk¹

¹Departament of Tumor Biology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch,44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland;

²Departament of Nuclear Medicine and Endocrine Oncology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch,44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland;

³Departament of Molecular Biology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch,44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland.

Melanoma is the most aggressive skin cancer largely refractory to current therapies. Lack of effective therapeutic strategies and shortcomings of traditional classification systems result in high mortality among melanoma patients. One of the most important features of tumor microenvironment is low oxygen tension. Transformation of melanocytes and melanoma progression in vivo indeed appears to be influenced by hypoxia. In our previous study we had determined global gene expression profile of B16-F10 murine melanoma cells cultured under hypoxic conditions and identified 430 hypoxia-responsive genes [Olbryt et al (2006) Gene Expression 13:191-203].

In the present study we have investigated whether expression of 23 selected genes from the above-mentioned signature is also hypoxia-affected in murine melanoma experimental tumors (B16-F10) as well as in human melanoma cell lines exposed to hypoxic conditions in vitro.

Localization pattern of hypoxic areas in B16-F10 tumors was detected immunohistochemically using pimonidazole. The hypoxic areas (perinecrotic regions), as well as normoxic ones (in the vicinity of blood vessels), were isolated from frozen tumor slices using laser microdissection technique. For in vitro study, six melanoma cell lines were cultured under hypoxic conditions (48h at 1% O₂). Expression of the selected genes in cell cultures as well as microdissected material was analyzed by quantitative real-time RT-PCR and/or semiquantitative RT-PCR.

We found excellent correlation between the hypoxic environment-induced expression patterns of majority of the studied genes (22), both in vitro and in vivo. The results obtained using human cell lines revealed significant variability in expression patterns of the analyzed genes. Nevertheless, 10 genes were proved to be hypoxia-regulated in most of the studied cell lines (³4). Among them are those with well-documented links to melanoma biology (NME1, STAT3, MXI1, FN1) as well as known hypoxia-responsive genes (BNIP3, ADM, NPPB, NDRG1)

To sum up: 1) molecular response to hypoxia under in vitro conditions appears to reflect the response observed in vivo, at least for the same cell line; 2) the selected genes (NME1, FN1, STAT3, ADM, BNIP3, MXI1, CCNG2, CDC6, NPPB, NDRG1) are new hypoxia-responsive genes in melanoma; 3) B16-F10 murine melanoma tumor model seems to be appropriate for further analyses of the selected genes.

 

18. CYTOGLOBIN OVEREXPRESSION EXERTS TUMOUR SUPPRESSOR ACTIVITY IN LUNG CANCER CELL LINEs

Urszula Oleksiewicz, Triantafillos Liloglou, John K. Field, George Xinarianos

Roy Castle Lung Cancer Research Programme, The University of Liverpool, School of Cancer Studies, L3 9TA Liverpool, 200 London Road, United Kingdom.

Cytoglobin (CYGB) is a novel globin, whose molecular function remains unclear. It has been shown that CYGB is engaged in cellular response to hypoxic and oxidative stress conferring cytoprotective features. In human cancers, CYGB level is frequently reduced due to promoter hypermethylation and loss of heterozygosity. Recent data suggested that CYGB might act as a tumour suppressor gene (TSG). However, more thorough investigations are needed in order to elucidate the involvement of CYGB in tumour biology. The aim of this study was to evaluate the impact of CYGB on cell phenotype (growth & death rate, migration, invasion and transformation abilities) in the lung cancer setting.

Two lung cancer cell lines: Calu1 and H358 were utilised to obtain stable tranfectants overexpressing CYGB. Cell proliferation was measured with MTT assay and haemocytometer counting method. Cellular death was assessed by quantification of ADP:ATP ratio. Transformation abilities were studied with the soft agar assay, migration with the wound healing assay and invasion properties were analysed with the Matrigel-coated chambers.

Our data showed that restoration of CYGB expression did not trigger cellular death (neither apoptosis nor necrosis). Haemocytometer cell counting showed no change in the growth rate of Calu1/cygb+ clone. However, we observed higher values in the parallel MTT assay. This potentially suggests a switch from glycolysis to oxidative ATP production. In case of H358 cygb+ clones, both MTT assay and haemocytometer counting revealed diminished growth rate. CYGB overexpression reduced migratory potential of all Calu1 and H358 clones in the wound healing assay. Also, their invasive properties were diminished up to 80% (p<0.02). We observed striking depletion (up to 98%, p<0.02) in transformation efficiency of CYGB+ clones as assessed in the soft agar assay.

These results strongly support the hypothesis that CYGB acts as a TSG and indicate that CYGB might be considered as a potential therapuetic target. CYGB knock-down clones, as well as in vivo studies, will  further improve our understanding of this novel TSG. It would be also interesting to test CYGB influence on tumour behaviour after exposure to hypoxia and radicals outburst.

 

19. SYNTHESIS AND DETEMINATION OF PHOTOCHEMICAL AND PHOTOPHYSICAL PROPERTIES OF SOME MESO - SUBSTITUTED PORPHYRINS AS POTENTIAL PDT AGENTS

A. Pasewicz^1,2.,  P. Kuś², A. Karocki³, G. Stopa³, J.M. Dąbrowski³, M. Rojkiewicz², G. Zięba², V. Kozik², A. Ratuszna¹

¹A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland;

²Institute of Chemistry; University of Silesia, Katowice, Poland;

³Faculty of Chemistry, Jagiellonian University, Krakow, Poland..

            Porphyrin derivatives have recently been the subject of numerous theoretical and experimental studies owing to their potential usefulness as sensitizers in photodynamic therapy (PDT). Examination of photophysical and photochemical properties of porphyrins is an essential part of research preceding biological applications. The purpose of our presentation is to show details of synthesis and investigation of properties of several meso-substituted porphyrin derivatives that appear suitable for PDT.

            The investigated compounds were obtained according to the classical Adler-Longo condensation procedure. Extraction and chromatographic methods were used to purify the products. All samples were characterized by mass spectrometry (ESI-MS) and nuclear magnetic resonance (¹H NMR) to confirm purity and structure.

            Electronic absorption spectra of the investigated compounds in diluted solutions were recorded on a Genesis Spectrophotometer using 1-cm path length cuvettes. Measurements in the 350-800 nm range enabled detecting long-wave characteristics of free-base porphyrins: Q bands and, a much more intensive short-wave Soret band. Molar extinction coefficients and localization of UV/VIS bands  are valuable parameters characterizing prospective PDT agents.

            Singlet oxygen quantum yields were estimated by flash photolysis using an Applied Photo-physics LKS.60 Laser Flash Photolysis Spectrometer and a Nd:YAG laser. The decay curves of singlet oxygen emission were recorded at room temperature following third harmonic (355nm) laser excitation. Phosphorescence of singlet molecular oxygen was detected at 1280 nm for oxygenated toluene solutions (absorbance at 355 nm was equal to 0.35 cm^-1). Actual values of singlet oxygen efficiencies were estimated by comparing decay curves interpolated to zero time flash and areas under corrected decay curves with values obtained for a reference sensitizer.

            The obtained results for singlet oxygen quantum yields lie in the 0.59-0.62 range. These values are similar to pophyrin singlet oxygen efficiencies that were reported in the literature and are comparable to those of agents used in clinical practice.

This work was supported by the Polish Ministry of Science and Higher Education (grant No. 0538/R/T02/2007/03). 

 

20. NON-HOMOLOGOUS DNA END JOINING AND DNA REPAIR GENES EXPRESSION IN MO59 HUMAN GLIOMA CELLS AFTER ANTICANCER AGENTS TREATMENT

Elzbieta Pastwa¹, Urszula Lewandowska², Richard Idem Somiari³

¹Department of Molecular Genetics, Medical University of Lodz, 92-215 Lodz, Mazowiecka 6/8, Poland;

²Department of Medical Enzymology, Medical University of Lodz, 92-215 Lodz, Mazowiecka 6/8, Poland;

³Functional Genomics & Proteomics, ITSI-Biosciences, 633 Napoleon Street, Johnstown, PA 15901, USA..

            Cisplatin and etoposide provide considerable synergy during treatment of glioma, although the mechanism of this synergy is not well defined. We previously showed that wortmannin, the catalytic subunit of DNA-dependent protein kinase (DNA-PK_cs) inhibitor, enhances cytotoxic effect of these drugs in combination in MO59K cells (reduction factor R=4,5). No such effect was observed in MO59J cells, lacking DNA-PK_cs expression, the main protein in non-homologous DNA end joining (NHEJ). The goal of this study was to test the hypothesis that chemosensitization of cancer cells by wortmannin is due to their effect on DNA repair system, especially NHEJ.

            Investigation of DNA repair efficiency was done by in vitro NHEJ assay. Wortmannin at the concentration of 5 µM did not significantly decrease NHEJ repair after combined cisplatin and etoposide treatment in comparison to untreated control. This effect corresponded to a decrease in DNA-PK_cs gene expression at the mRNA level as determined by the QuantiGene Plex branched DNA method. Under the same conditions Ku80 and Ku70 NHEJ genes expression was increased. In MO59J cells we did not observe NHEJ activity suggesting that our plasmid-based NHEJ assay is apparently dependent on DNA-PK_cs.            

            These results suggest that sensitization by wortmannin in human glioma cells following combined treatment with etoposide and cisplatin could be attributed to an inhibition of DNA-PK_cs.

This work was supported by grant 401-117-32 from the Polish Ministry of Science and Higher Education and by grants 502-19-677 and 503-00-78-3 from the Medical University of Lodz.

 

21. Random Walk model of potassium ion transport through biological membrane

Krzysztof Pawełek, Krzysztof Małysiak, Zbigniew J. Grzywna

Silesian University of Technology, Faculty of Chemistry, Section of Physics and Applied Mathematics, 44-100 Gliwice, Ks. M. Strzody 9, Poland.

We are presenting a random walk model of a potassium ion transport through  mammalian voltage gated potassium ion channels. Potassium channels are one of the most widely distributed type of ion channels. They can be found in virtually all kinds of living organisms from simple bacteria to eukaryotic cells of mammalian organisms and play an important role in many biophysical processes. X-ray crystallographic measurements have shown atomic structures of many different potassium ion channels. Three main processes seem to control a potassium ion transport: voltage gating, conduction of current through a selectivity filter and inactivation mechanism (ball on chain model). All these factors are taken into account in our model and allow us to compare obtained results with experiment data.

 

22. Mass spectrometry-based serum proteome pattern analysis allows identifying blood components specific for patients with non-small cell lung cancer

Pietrowska M.¹, Suwiński R.¹, Walaszczyk A.¹, Domińczyk I.¹, Marczak Ł.², Stobiecki M.², Polańska J.³, Polański A.³, Widłak P.¹

¹Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch Gliwice, Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland;

²Polish Academy of Science, Institute of Bioorganic Chemistry, Poznan, Poland;

³System Engineering Group, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland.

Mass spectrometry-based analysis of the blood proteome is an emerging method of clinical proteomics and cancer diagnostics. Although no single peptide is expected to be a reliable bio-marker in such analyses, multi-peptide sets of markers selected in numerical tests have been already shown in a few studies to have potential values in cancer diagnostics. Here we performed mass spectrometry-based serum proteome pattern analysis aimed at identifying features specific for patients with non-small cell lung cancer (NSCLC).

Blood samples were collected before the start of therapy from 49 patients with locally advanced NSCLC, 39 patients with head & neck cancer and  36 patients with colon cancer, as well as in a group of age and sex-matched healthy controls (48 donors). Serum was isolated after blood clotting and the low-molecular-weight proteome fraction (2-14 kDa) was analyzed using MALDI-ToF mass spectrometry. Registered mass spectra were analyzed using bioinformatic tools created and optimized in our group.

We have identified cancer classifiers built of multiple spectral components (peptide [M+H]⁺ ions) that differentiated sera from analyzed groups. Classifiers built of 16-18 components differentiated healthy persons and patients with NSCLC with about 90% specificity and sensitivity. Furthermore, we have compared similarity of proteome patterns specific for analyzed types of cancer. Peptide profiles characteristic of samples from blood of patients with NSCLC and head & neck cancer were the most similar, while samples of colon cancer were the most different. Spectral components that are the most characteristic of specific types of cancer have been also identified. We concluded that MALDI-ToF MS-based serum proteome pattern analysis has an obvious potential to differentiate samples between NSCLC patients and healthy controls or patients with other type of cancers.

 

23. Searching for dynamics’ patterns in time-course gene expression profiles

Joanna Polanska¹, Joanna Rzeszowska^1,2, Michal Marczyk¹, Michelle McRobbie³, Peter O'Neill³, Piotr Widlak², Andrzej Polanski^4,5

¹System Engineering Group, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland;

²Department of Experimental and Clinical Radiobiology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch Gliwice, Wybrzeze Armii Krajowej 15,44-101 Gliwice, Poland;

³Gray Institute for Radiation Oncology & Biology, University of Oxford, Mount Vernon Hospital, Middlesex HA6 2JR, UK;

⁴Institute of Informatics, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland;

⁵Polish-Japanese Institute of Information Technology, Koszykowa 86, 02-008 Warszawa, Poland.

Measurements of gene expression profiles in certain time points are commonly used for modeling the dynamics of cell reaction to a predefined factors. The application of a modified Gaussian mixture modeling (GMM) technique to the analysis of this type of data was proposed. The modification includes the assumption that parameters of Gaussian components, means and variances, can differ between time points, but the gene composition of components must be unchanged. The number of obtained components is related to the number of subgroups of genes with similar dynamics of gene expression. Solution to such problem requires the adaptations of a standard Expectation-Maximization (EM) algorithm and the reformulation of a likelihood function. The evaluation of the obtained subgroups of genes was done by a comparison of gene ontology terms related to the genes classified as members of particular subgroup. The hypothesis on non-random functional composition of gene subgroups was checked with the use of hypergeometric test.

We validate our method by applying it to two different datasets. The first one contains 45 Affymetrix DNA microarray data of human fibroblasts cells from three different subjects irradiated with different doses and measured in three time points. Second dataset include the data on cellular responses of human leukemic cells K562 to irradiation in five time points.

Both studies proved that the modified GMM outperforms standard decomposition techniques with respect to statistical meaning of the obtained gene groups, measured by p-values of the statistical tests. The application of our technique to time course data allows for precise division of genes into functional classes.

The work was partially supported by GENEPI low-RT FP6-036452 project.

 

24. DETERMINING SINGLET OXYGEN QUANTUM YIELD OF PHOTODYNAMIC AGENTS BY FLASH PHOTOLYSIS MEASUREMENTS

M. Rams¹, A. Szurko^1,2, A. Pasewicz^1,3, A. Sochanik² G. Stopa⁴, A. Karocki⁴, M. Rojkiewicz³, P. Kuś³, A. Ratuszna¹

¹A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland;

²Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland;

³Institute of Chemistry; University of Silesia, Katowice, Poland;

³Faculty of Chemistry, Jagiellonian University, Krakow, Poland.

Singlet oxygen is a reactive oxygen species that may be generated in biological systems. In photodynamic therapy singlet oxygen is generated by photoexcitation of sensitizers resulting in intracellular oxidative stress and induction of apoptosis. The efficiency of photodynamic therapy strongly depends on photochemical properties of photosensitizers and, in particular, on the quantum yield of molecular singlet oxygen formation.

The aim of our study was to determinate the molecular singlet oxygen quantum yield of three novel porphyrin-type compounds evaluated as sensitizers for photodynamic therapy (PDT). The quantum yield of molecular singlet oxygen formation was measured by detecting the phosphorescence decay curves in the presence of oxygen via flash photolysis measurements. The singlet oxygen quantum yield value was estimated using comparative method with tetraphenylporphyrin (TPP) as a reference.

Samples of the measured compounds were excited with laser pulses from Nd:YAG laser using third harmonic (355 nm) of laser irradiation and the phosphorescence intensity at 1275 nm was measured. The oxygenated solutions were prepared in toluene containing the sensitizer at a concentration suitable to produce absorbance around 0.35 at the excitation wavelength (355 nm). The phosphorescence decay curves for reference were detected under the same conditions. To confirm the sample’s stability, absorption spectra of the studied compounds were compared before and after each flash photolysis experiment. Three kinetic curves of phosphorescence decay were detected in the measurements. The presented data show their mean values. After correction (light filter 880 nm) the experimental datapoints were extrapolated and plotted as a function of phosphorescence intensity vs. time. The quantum yield of molecular singlet oxygen formation was determinated by comparing the areas under emission spectra and the initial phosphorescence intensity for the studied porphyrins and TPP.

The quantum yields of the molecular singlet oxygen formation calculated based on these flash photolysis measurements were similar for all the studied porphyrins. The obtained values, ca. 0.60, are comparable with those of photosensitizers used in clinical practice.

This work was supported by the Polish Ministry of Science and Higher Education (grant No. 0538/R/T02/2007/03). 

25. PRELIMINARY BIOLOGICAL INVESTIGATION OF NOVEL WATER-SOLUBLE CHLORINS

M. Rams¹, A. Szurko^1,2, A. Sochanik², D. Bauer³, F.-P. Montforts³, A. Mrozek-Wilczkiewicz¹, B. Boroń¹, A. Ratuszna¹

¹A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland;

²Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland;

³Institute of Organic Chemistry, University of Bremen, Bremen, Germany.

In recent years, photodynamic therapy (PDT) has been extensively investigated as a possible treatment modality for various types of cancer. This approach is based on the combined use of light and photosensitizing agent in the presence of molecular oxygen. The energy transfer from the activated photosensitizer to oxygen present results in the formation of cytotoxic agents, such as singlet oxygen and free radicals. The efficiency of tumor destruction is crucially influenced by type of applied photosensitizing compound. A large number of photosensitizers have been investigated, but only few have shown applicable properties. Therefore, present studies have focused on the development of new photosensitizers with desirable combinations of chemical, photophysical and biological properties.

Chlorins, a class of tetrapyrrolic photosensitizers, are some of the most promising candidates for application in PDT. They are the subject of intense research due to favorable photophysical properties approaching those of ideal photosensitizers (long wavelength absorption, high molar extinction and efficient singlet oxygen formation).

In this paper we present the results of our preliminary, in vitro investigations of two novel water-soluble chlorins. Cytotoxicity, phototoxicity as well as subcellular localization of the novel derivatives were studied using human colon carcinoma cultured cells (HCT 116 p53+/+). The obtained results, demonstrate low cytotoxicity and high phototoxicity of the studied compounds and  make them very promising  agents for further PDT studies.

This work was supported in part by the Polish Ministry of Science and Higher Education (grant No. 0538/R/T02/2007/03). 

26. Investigation of possible medical applications of magnetic membranes

Aleksandra Rybak, Monika Krasowska, Gabriela Dudek, Anna Strzelewicz, Zbigniew J. Grzywna

Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, Strzody 9, 44-100 Gliwice, Poland.

Air separation, or in less ambitious case, an air enrichment in oxygen, are both very important problems in medicine, industry, as well as in everyday life. Many biochemical reactions in the body depend on oxygen utilisation. Although oxygen is normally present in the air, higher concentrations are required to treat many disease processes. Oxygen therapy is a form of treatment that uses oxygen in elemental or compound forms to heal various disease conditions. It is one of the most powerful and versatile therapies known, mostly in Europe and other parts of the world. It can revitalize the practice of medicine with alternative therapies that work because of its antibacterial, anti-fungal, anti-inflammatory, anti-parasitic, anti-tumor and antiviral. We propose a new concept of air enrichment in oxygen by polymer membranes filled with neodymium powder and magnetized (“magnetic membranes”). The idea of implementing some external fields as a principal reason for gas mixtures separation (air in our case) is very promissing. The idea of “magnetic membranes” is based on the observation that oxygen and nitrogen have quite different magnetic properties i.e. oxygen is paramagnetic whereas nitrogen diamagnetic, what gives a real chance for their separation. We have got an oxygen enrichment up to the 56% in permeate for magnetic induction 2,25 mT.

 

27. ROLE OF DNA-REPAIR PATHWAY IN DOXORUBICIN-INDUCED CELL DAMAGE

Yury V.Saenko¹, Alexander M. Shutov², Eugenia V. Rastorgueva¹, Anna G. Maslakova¹

¹Department of Pharmacology, Ulyanovsk State University, Ulyanovsk, Russia;

²Department of Internal Medicine, Ulyanovsk State University, Ulyanovsk, Russia.

Background: The clinical efficacy of Doxorubicin (DOX) is greatly restricted due to the development of a severe form of cardiomyopathy and nephropathy due to induce of oxidative stress. DNA-repair and related redox-sensitive pathway may be involved in those negative effect of DOX. RNR3-lacZ reporter gene construction consist of ribonucleotide reductase promoter fusion with b- galactosidase (LacZ) and reflect the activity of DNA-repair pathway. TRX2-lacZ consist of thioredoxin gene promoter fusion with LacZ gene and may reflect the activity of related redox-sensitive pathway. The aim of our study was to investigate influence DNA-repair and related redox-sensitive pathway activation on DOX-induced oxidative stress by using eukaryotic cell model Saccharomyces cerevisiae.

Methods: S. cerevisiae strain YPH499 was transform by RNR3-lacZ and TRX2-lacZ reporter gene construction and incubate with different amount of DOX (10,30,50 mm) in SD media. The influence of DOX on strain’s survival, proliferation, intracellular glutathione reduced (GSH), malondialdehyd concentration was studied. The rate of expression  RNR3-lacZ and TRX2-lacZ reporter gene construction assessed through b- galactosidase activity.

Results: DOX induced dose-dependent inhibition of cell proliferation (95.5±1.5; 78.1±1.1; 70.5±0.9 per cent from control at concentration DOX in media 10,30,50 mm, respectively). Concentration of GSH was 2.82±0.5 mM//mg dry weight in control group. DOX at 10,30,50 mm increased GSH: 4.4±0.27 (p<0.01); 5.19±0.25 (p<0.001); 5.31±0.35 (p<0.001), resp. DOX is not significantly impact on malondialdehyde concentrations. DOX induced RNR3-lacZ and TRX2-lacZ expression in none dose-dependent manner. GSH concentration correlated with expression RNR3-lacZ (r=0.69;р<0.01), but not correlate with expression of TRX2-lacZ (r=0.23;p>0.05).

Conclusions: The results show that doxorubicin increase GSH concentration in cells of S. cerevisiae and this effect due to activation of DNA-repair pathway. We suggested that mechanisms of DOX-induced cell damage more complexes than simplicity oxidation of molecules.

 

28. THE NEW APPROACH TO DESIGN ANTITUMOUR IRON CHELATORS. USE OF THE KOHONEN NEURAL NETWORKS FOR MODELLING NOVEL BIOLOGICAL ACTIVE AGENTS

Maciej Serda, Tomasz Magdziarz, Jarosław Polański

Institute of Chemistry, University of Silesia, Szkolna 9, PL-40006-Katowice, Poland.

            Iron is one of  the essential parts of human biochemical system. It plays role in a variety of physiological cellular processes such as oxygen transport, energy metabolism, electron transport and DNA synthesis [1]. The rate limiting step of DNA synthesis involves the Fe-dependent enzyme, ribonucleotide reductase (RR), which converts ribonuclotides to deoxyribonucleotides. To facilitate rapid replication neoplastic cells have significantly higher levels of RR and transferrin receptor 1 (TfR1) [2]. The higher Fe utilization by cancer cells than their normal counterparts is a good reason for modelling and synthesis  the novel selective antitumour iron chelators.

              In the present investigation we chose quinoline derivatives with semicarbazide scaffold as a potential Fe chelators in tumour cells.  Functionalised quinoline derivatives are used as potential inhibitors of many enzymes (i.e. HIV integrase) [3]. 8-hydroxyquinoline  is a well-known iron chelator, therfore we decided to design new class of iron chelators based on its moiety [4].

            In our present work we used Kohonen maps of eletrostatic potencial to characterize 16 well-known iron chelators. This technique allowed us to visualize the electrostatic potencials on the molecular surfaces as a planar square map. Before building the neural map all the molecules had to be systemically aligned. The Kohonen system allowed us to compare those parts of the molecule surfaces that can be superimposed. If the surface couldn't be superimposed on reference molecule we  indicated it by white colour. In these electrostatic maps we can see also red areas which indicates the most electronegative regions and the purple- the most electropositive areas.

            In addition we calculated autocorrelation vectors for the set of 16 Fe chelators. It helped us to create Self Organized Maps capable of clustering active and inactive chemical compounds. The created SOM was afterwards used to screen chemical database containing 200 structures of quinoline derivatives.

References:

[1] D. Kalinowski, D.R. Richardson, Pharmacol. Rev. 2005, 57, 547.

[2] D. R. Richardson, Biochim. Biophys. Acta 2009, 1790, 702.

[3] J. Polański, J. Gasteiger, J.Med. Chem. 2002, 45, 4647.

[4] P. Ponka, C. Mouralian, Biochem. Pharmacol. 2005, 71, 214.

 

29. PHOTODYNAMIC REACTION IN COMBINATION WITH ELECTROPORATION IN HUMAN BREAST ADENOCARCINOMA CELLS IN VITRO

Nina Skołucka^1,4, Mariola Nowak², Małgorzata Kotulska², Iwona Kamińska^2,3, Anna Stecka^2,3 Jolanta Saczko¹, Julita Kulbacka¹, Agnieszka Chwiłkowska¹, Anna Choromańska^1,4

¹Department of Medical Biochemistry, Wroclaw Medical University, 50-368 Wroclaw, Chalubinskiego 10, Poland;

²Institute of Biomedical Engineering and Instrumentation, Wroclaw University of Technology, 50-370 Wroclaw,  Plac Grunwaldzki 13, Poland;

³The Group of Bio-Nanopor, Wroclaw University of Technology, 50-370 Wroclaw, Plac Grunwaldzki 13, Poland;

⁴The Group of Biomed, Wroclaw Medical University, 50-368 Wroclaw, Chalubinskiego 10, Poland.

The photodynamic therapy (PDT) is the method of selective tumor treatment, which uses the photosensitive substance and the suitable wavelength of light. Photosensitizer accumulation is dependent on the type of malignant cells, physical and chemical properties of the dye. It localizes in the plasma or subcellular membranes, making these structures especially sensitive to the photooxidative damage.

The application of the cell membrane electroporation in combination with cytotoxic drugs could increase their transport into cells. The combination of electroporation and application of drugs with inhibited transport is known as electrochemotherapy (ECT). ECT and PDT are low- invasive and targeted methods. Proper therapy conditions could limit necessity of surgical interventions, as well as give the better prognoses in treatment the tumors.

The current study examined an effect of combining both methods applied in vitro. Photodynamic reaction enhanced by electroporation was tested on the human breast cancer cells (MCF-7/DOX and MCF-7/WT). The photodynamic activity of an electro-photodynamic reaction with the hematoporphyrin derivative (HpD) was evaluated in relation to the standard photodynamic method by MTT assay as an examination of mitochondrial cell function. The experiments proved that electroporation effectively supports photodynamic method.

 

30. REGULATION REGULATION OF THE HUMAN GLUTATHIONE S-TRANSFERASE P1 GENE TRANSCRIPTION

Andriy Slonchak¹, Agata Chwieduk², Joanna Rzeszowska-Wolny², Maria Obolenska¹

¹Department of Mechanisms of Translation of Genetic Information, Institute of Molecular Biology and Genetics NAS of Ukraine, 03143 Kyiv, Zabolotnogo150, Ukraine;

²Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland.

Glutathione S-transferase P1-1 is a widely distributed enzyme which plays a key role in protection of the cells from genotoxic damages. The aim of the present finding was to clarify the mechanisms responsible for the different levels of GSTP1 expression observed in Me45, Hbl-100 and BeWo cells analyzing cis- and trans-acting factors implicated in regulation of its transcription. The aim of this work was to identify cis- and trans- acting factors responsible for the cell-specific levels of the human GSTP1 gene expression and to investigate molecular mechanisms responsible for the changes in GSTP1 expression during carcinogenesis utilizing human placenta and choriocarcinoma cells as a model system.

Level of GSTP1 mRNA was assessed be quantitative RT-PCR and level of GSTP1 protein was assessed by Western-blotting. Truncated promoter fragments for the transient transfection assay were obtained by PCR and cloned into pGL3basic. The Me45, Hbl-100 and BeWo cells were transfected with each of recombinant plasmids and luciferase activities were measured. Transcription factors interacting with GSTP1 promoter elements were assessed by competitive EMSA and supershift analysis. Promoter methylation was analyzed by MSP.

The highest level of GSTP1 mRNA and protein was detected in Hbl-100 and the lowest in BeWo. Transient transfection assay provided the evidence for two positive (ARE and NF-κB) and two negative (CRE and NF-κB-like) regulatory elements similarly acting in GSTP1 promoter in three cell types. Results of competitive EMSA and supershift analysis indicated that ERβ together with unidentified protein binds to ARE and together with Fos binds to CRE sites in all cell types, The structure of ERβ/Fos complex in Hbl-100 differs from that in Me45 and BeWo. In addition NF-κB interacting with NF-κB site was identified as a p50/p50 homodimer in BeWo and p50/p65 heterodimer in Hbl-100 and Me45cells. MSP revealed partial GSTP1 promoter methylation in Me45 and BeWo cells and no methylation in Hbl-100.

According to the results of qRT-PCR and sqWB GSTP1 expression is down-regulaed in BeWo cells comparing to placenta.

To identify transcription factor responsible for carcinogenesis-associated GSTP1 down-regulation in trophoblast cells competitive EMSA and supershift assay were applied. In consequence with the results ARE and NF-kB sites in normal trophoblast and choriocarcinoma cells interact with the same TFs however, CRE-protein complexe in BeWo cells is different from that in normal trophoblast. Besides ERb and Fos it also contains transcription factor Jun.

Thus, we analyzed for the first time the molecular mechanisms responsible for the steady-state level of GSTP1 transcription in Hbl-100, Me45 and BeWo cells. We assume that the absence of NF-κB p65 accounts for the lower GSTP1 expression level in BeWo comparing to Hbl-100 cells, and the structural changes in CRE-site complex is responsible for down-regulation observed in Me45. We also demonstrated that GSTP1 promoter becomes methylated in cell with low transcription of the gene.

 

31. GEFITINIB INCREASES CYTOTOXICITY OF BLEOMYCIN IN HUMAN GLIOMA CELLS

Monika Sobczak, Elzbieta Pastwa

Department of Molecular Genetics, Medical University of Lodz, 92-215 Lodz, Mazowiecka 6/8, Poland.

            Gefitinib (ZD1839) is a derivative of chinazolin, which suppresses the activity of the tyrosine kinase of epidermal growth factor receptor (EGFR). Signal pathways of EGFR proteins are associated with unchecked proliferation and the resistance to radiotherapy of cells of the glioma. Activation of EGFR influences mitosis, mobility associated with metastases, as well as repair of DNA damage and angiogenesis. Since in gliomas, the EGFR genes often impact gene overexpression, DNA amplification and mutations, the inhibition of tyrosine kinase of EGFR may allow the use of lower doses of drugs to treat brain tumors.

The aim of our study was to determine if gefitinib, inhibitor of EGFR, can sensitize human glioma cells to DNA damaging agents: cisplatin and bleomycin.

We performed XTT growth inhibition assay in order to evaluate the effect of the EGFR inhibitor, gefitinib, on the response of two glioma cell lines – MO59K and MO59J to cisplatin and bleomycin, the latter cell line lacking the expression of the catalytic subunit of DNA-dependent protein kinase (DNA-PK_cs)_.

Our results demonstrated that gefitinib increases the cytotoxicity of bleomycin in MO59K and MO59J cells. Simultaneous incubation with 10 µM gefitinib potentiates two times (R=2) the growth inhibition of the drug in both cell lines. Additionally, MO59J cells are twice more sensitive than MO59K to the cytotoxic effect of bleomycin, what is manifesting itself in IC₅₀ values. In contrast, no sensitization to cisplatin was observed in MO59K and MO59J cells. We also found that cytotoxic effect of cisplatin and bleomycin in combination is higher than that of every drug individually (synergism, combination index CI<1).

In conclusion, our data suggest that the inhibition of EGFR activity may increase the effectiveness of bleomycin therapy in human glioma.

 

32. CYTOTOXICITY OF ETOPOSIDE IN CANCER CELL LINES IN VITRO AFTER SIMULTANEOUS BCL-2 AND C-RAF GENE SILENCING WITH ANTISENSE OLIGONUCLEOTIDES

Daniel Sypniewski_, Ilona Bednarek, Tomasz Loch_, Grzegorz Machnik, Sabina Gałka, Daria Błaszczyk, Dagna Sołtysik

Department of Biotechnology and Genetic Engineering, Medical University of Silesia, Katowice, Poland.

BCL-2 and C-RAF genes are overexpressed in most types of cancers. Although these genes are mediators in different molecular pathways, their main characteristic is the antiapoptotic activity thus, cells that overexpress either BCL-2 or C-RAF lose their ability to undergo apoptotic death being resistant to chemotherapeutic agents and/or physiologic mediators of cell death (e.g. TNF-α). Both anti-C-RAF, and anti-BCL-2 oligonucleotides were tested as chemosensitizers in cancer therapy.

            The aim of the study was to test the effects of simultaneous / combined use of antisense oligonucleotides (ASOs) targeting the BCL-2 and C-RAF transcripts on the cancer cell cultures in vitro exposed to etoposide. BCL-2 and C-RAF ASOs with phosphorothioate backbones were designed according to the general rules within the coding mRNA sequences. For BCL-2 a consensus sequence targeting both α-, and β- isoform was designed. Cells were transfected with BCL-2 or C-RAF ASOs or with combination of both daily for 5 h, either for 1 or 3 days. 3-day transfection was followed by a single treatment with 20 μM etoposide for 5 h. The following cancer cell lines were tested: A549, HeLa, and T24.

            Sequence-specific decrease in BCL-2 and C-RAF mRNA levels were confirmed by Real-Time RT-PC. After 1-day treatment mRNA levels decreased by 9%-42% of the normal expression in cells treated with 50-1200 nM ASOs. One day treatment also confirmed induction of cell death in all transfected cultures in a concentration-dependent manner (even at the lowest 50 nM concentrations) as was revealed by MTT assay and by microscopic analysis of cell morphology (Hoechst 33258 staining). To sustain high intracellular level of ASOs directed against BCL-2/C-RAF we used repeated (3-day period) ASOs transfection. Results showed that both ASOs effectively stimulated cell death and potentiated etoposide-induced cytotoxicity. We also confirmed that the cytotoxic effect was at least partially mediated by apoptosis induction which was evidenced by microscopic analysis Those effects depended on ASOs concentrations. Significant differences were evidenced in the response of certain cancer types (models) analyzed in that study. The strongest resistance to ASOs treatment was observed in HeLa cultures while the most promising results were obtained in A549 (lung cancer) cells. This observation suggests that lower concentrations may be used for this type of cancer to obtain high efficiency of enhancement in etoposide-induced cell death. Comparison of the control ASO (scrambled sequence) with sequence-specific anti-BCL-2 or anti-C-RAF ASOs indicates that at higher concentrations of ASOs (~ 1 μM) a significant proportion of the cytotoxic effects came from general oligonucleotide and/or lipofectamine toxicity. Nevertheless, simultaneous transfection with both ASOs targeting BCL-2 and C-RAF transcripts allowed us to use lower concentrations to obtain maximal results. These conclusions suggest that maximal attainable effects were gained and the rest of cell pools were resistant to etoposide and/or gene silencing by ASOs. Simultaneous use of two ASOs in 3-day treatment allows us to lower the concentration needed to obtain the same treatment results compared with single use of each ASO thus enabling to diminish sequence-unsepcific toxicitiy, although the combined treatment does not lead to higher efficiency overall.

The study was supported by the funds of Medical University of Silesia (KNW-1-117/09, KNW-2-198/08).

 

33. TNFa-INDCUCED ACTIVATION OF NFkB SIGNALING AFFECTS REGULATION OF P53-DEPENDENT PATHWAY IN UV-IRRADIATED HCT116 CELLS

Szołtysek K.¹, Janus P.¹, Kalinowska-Herok M.¹, Herok R.¹, Puszyński K.², Lipniacki T.², Kimmel M.², Widłak P.¹

¹Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland; ²Silesian University of Technology, Gliwice, Poland.

Signaling pathways that depend on p53 or NFkB transcription factors are essential components of cellular responses to stress. In general, p53 is involved in either activation of cell cycle arrest or induction of apoptosis, while NFkB exerts mostly anti-apoptotic functions. Both regulatory pathways apparently interfere with each other yet molecular details of such interactions remains to be elucidated. A mathematical model describing functional interactions between p53- and NFkB-dependent pathways has been recently published by Puszyński & Lipniacki, that suggested different output depending on the time sequence of each pathway activation.

Here we aimed to verify experimentally effects of NFkB pathway on activation of p53-dependent genes. Colon carcinoma HCT116 cell line was used, in two congenic variants either containing or lacking transcriptionaly competent p53. Cells were incubated with TNFa cytokine to induce NFkB, and/or treated with ultraviolet radiation to induce p53 pathway; both factors were used in several different combinations. Activation of NFkB and p53 pathways was monitored by Western-blotting, while levels of expression of selected genes were assessed by semi-quantitative RT-PCR or quantitative real-time Q-RT-PCR. We have observed that activation of p53-dependent genes was enhanced in cells exposed to TNFa cytokine after UV irradiation. In contrast, exposure to TNFa before irradiation reduced UV-induced activation of p53-dependent genes.

This work was supported by the Ministry of Science and Higher Education, Grant PBZ-MNiI-2/1/2005 and N514-411936.

 

34. COMPARATIVE STUDY OF DIFFERENT DIAGNOSTIC TECHNIQUES (AI, MLPA, FISH) IN BREAST CANCER PATIENTS WITH 1q25.3 ALTERATIONS

Wiechec E.¹, Overgaard J.², Kjeldsen E.^3*, Hansen L.L.^1*

¹Department of Human Genetics, University of Aarhus, The Bartholin Building, 8000 Aarhus C, Denmark;

²Department of Experimental Clinical Oncology, Aarhus University Hospital, 8000 Aarhus C, Denmark;

³Department of Clinical Genetics, Aarhus University Hospital, 8000 Aarhus C, Denmark.

* these authors share senior authorship.

Breast cancer is the most frequently diagnosed malignancy among women and is the leading cause of cancer related death worldwide. Molecular genetic studies have revealed many subgroups of breast cancer within which the genomic alterations affecting chromosome arm 1q are considered to be an early event in breast carcinogenesis and are correlated with good prognosis for the patients. We have found a high percentage of concordance between Allelic Imbalance (AI) and Multiplex Ligation-dependent Probe Amplification (MLPA) assay pointing towards gain of the 1q25.3 region that includes Regulators of G protein Signaling (RGS) genes in breast cancer (1).

Our main objective was to compare the sensitivity and specificity of AI and MLPA with other cytogenetic method (FISH) in breast cancer patients with or without previously confirmed alterations within 1q25.3.

FISH was performed on formalin-fixed paraffin-embedded tumour material in order to verify previous findings and assess the level of genetic alterations of 1q25.3 in breast cancer. The 1q25.3 test and 1p32 reference probes labeled with different fluorochromes were utilized for analysis.

A total of 70 nuclei from each breast cancer case were examined and scored for the percentage of 1q25.3 alterations. The non-tumourigenic nuclei obtained from healthy individuals served as adequate cut-off for the 1q25.3-specific changes. The overall FISH results are consistent with results obtained from previous analysis in majority of analysed cases. Futhermore, FISH resolved the level of 1q25.3 alterations in few cases that were uncertain by AI and MLPA analysis.

This study shows that both AI and MLPA assays are able to map regions of copy number changes in cancer genome and are in concordance with molecular cytogenetics. This study shows that these three techniques complement each other and are able to map regions of copy number changes in the cancer genome and proved to be an efficient tool for diagnosis of 1q25.3 alterations in breast cancer.

References:

[1]. Wiechec, E., Overgaard., J., Hansen., L.L. 2008. A new fragile site within the HPC1 region at 1q25.3 affecting RGS16, RGSL2 and RGSL1 in human breast carcinomas. Genes, Chromosomes and Cancer, 47:766-80.

 

35. D-METHIONINE IN NOISE INDUCED HEARING LOSS

Wiktorek-Smagur A., Pawełczyk M., Rajkowska E. , Politański P., Śliwińska-Kowalska M.

Nofer Institute of Occupational Medicine, 8 Teresy street, 91-348 Lodz, Poland.

Both ROS and reactive nitrogen species can attack a wide range of biological targets, leading to oxidative cellular injury. ROS have been shown to play a toxic role in the cochlea both in vitro and in vivo, such as noise-induced cochlear injury in animal studies. D-Methionine, a sulfur containing amino acid, functioning as an ROS and/or reactive nitrogen species scavenger, may thus prevent noise-induced cochlear dysfunction.

C57BL/6 mice were used for this study. The animals were divided for three group: I – non-exposed control, II – exposed to noise, III – exposed to noise and given injections with D-Met i.p. D‑methionine was administrated 1 h before and 1 h after noise exposure. One additional dose each was given on days 1, 2 and 3 after noise exposure. The control and II groups received equivalent volume of saline ate the same intervals as the D-Met.

In result, D-methionine treatment significantly decreased threshold shift 1, 7 and 14 days after noise exposure. A significantly lower growth in LPO level was observed after  day 1 and  day 14 after noise exposure in the mice group exposed to noise and treated with D-Met in comparison with the mice group exposed to noise, only. In the mice group exposed to noise and treated with D‑methionine a significant decreased was observed in catalase activity after day 14 and SOD activity after day 1 noise exposure as compared to the group exposed to noise alone.

In summary, the administration of D-methionine seems to attenuate the noise-induced changes. It results in decreased hearing threshold shift and influence on oxidative stress process.

 

36. TGFβ auxiliary receptors in endometrial cancer

Piotr K. Zakrzewski^1*, Adam I. Cygankiewicz¹, Jacek Mokrosiński¹, Andrzej Semczuk², Tomasz Rechberger², Wanda M. Krajewska¹

¹University of Lodz, Department of Cytobiochemistry, Poland;

²Lublin Medical University, Second Department of Gynaecology, Poland.

^*pkzak@biol.uni.lodz.pl

            Transforming growth factor β (TGFβ) signalling pathway regulates such divergent processes as cell differentiation and proliferation, apoptosis, embryogenesis, angiogenesis, immunological response. It has been shown that TGFβ plays an important role during neoplastic transformation, tumour progression and metastasis. Up till now, three TGFβ auxiliary receptors that differ in TGFβ isoforms binding specificity have been identified: betaglycan (TGFβ receptor type III), endoglin (CD105 antigen) and CD109 antigen. These molecules do not posses enzymatic activity, however they modulate presentation of TGFβ ligands to canonical TGFβ receptors type I and II.

            In this study, betaglycan, endoglin and CD109 expression was compared with TGFβ1, TGFβ2 and TGFβRII status in endometrial cancer and normal tissue. Quantitative PCR method was employed to asses mRNA levels, whereas ELISA assay was performed in order to measure protein expression.

            The results have shown betaglycan, endoglin and CD109 mRNA down-regulation and protein level up-regulation in tumour cases. Observed alterations in TGFβ auxiliary receptors expression were found to correlate with changes in expression level of other studied members of TGFβ signalling pathway. Equivocal expression of TGFβ accessory receptors at mRNA and protein level suggests complexity of TGFβ signalling regulation.

 

37. ANTI-PROLIFERATIVE PROPERTIES OF OLIGO(3-HYDROXY-BUTANOATE) CONJUGATES WITH IBUPROFEN

B. Zawidlak-Węgrzyńska¹, M. Kawalec¹, M. Juzwa¹, A. Rusin², P. Filipczak², M. Głowala-Kosińska², K. Wolańska², Z. Krawczyk², P. Kurcok^1,3

¹Centre of Polymer and Carbon Materials, Polish Academy of Sciences, 34, M. Curie-Skłodowska St., 41-819 Zabrze, Poland;

²Department of Tumor Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 15, Armii Krajowej,44-101 Gliwice, Poland;

³Institute of Chemistry and Environment Protection, Jan Długosz Academy, 13/15 Armii Krajowej Ave., 42-200 Częstochowa, Poland..

            Ibuprofen [2-(4-isobutylphenyl)propanoic acid] is a non-steroidal anti-inflammatory drug (NSAID) used widely in rheumatoid arthritis, osteoarthritis and a number of other painful conditions. Besides its positive effects on inflammation and pain, ibuprofen could also present potential use for cancer prevention of colorectal cancer and cancer treatment of disease such as cardiovascular disease[1,2].

            The therapeutic use of non-steroidal anti-inflammatory drugs (NSAIDs) is often restricted by the necessity to deliver the drug to specific sites of target organ or tissue. The use of this kind of drugs is also limited by their irritant side effects on the gastro-enteric mucus. Many drug-polymer delivery systems have been used offering improved efficacy, reduced toxicity and better patient compliance compared to conventional oral dosage forms of pure drugs. Looking for biodegradable and biocompatible non-toxic polymers for drug delivery systems we performed the synthesis of non-toxic biocompatible poly(3-hydroxybutyrate) [3-4].

            This communication shows that anionic ring-opening polymerization of (R,S)-β-butyrolactone initiated with an alkali metal salt of (S)-(+)-2-(4-isobutylphenyl) propionic acid (ibuprofen) may constitute a convenient conjugation methods of ibuprofen with biodegradable OHB. Furthermore using the MTT cell proliferation assay we demonstrated that ibuprofen conjugated with OHB exhibited significantly increased, as compared to free ibuprofen, potential to inhibit proliferation of HT-29 and HCT 116 colon cancer cells. Although the mechanism of antiproliferative activity of ibuprofen-OHB conjugates (Ibu-OHB) has to be established, we suggest that partially it can be related to more effective cellular uptake of the conjugate than the free drug. This assumption is based on the observation of much more efficient accumulation of a marker compound - OHB conjugated with fluorescein, in contrast to fluorescein sodium salt, which entered cells inefficiently.

References

[1]. J. L. Williams, S. Borgo, I. Hasan, E. Castillo, F. Traganos, B. Rigas, Cancer Res. 2001, 61, 3285.

[2]. R.E. Harris, J. Beebe-Donk, H. Doss, D. Burr-Doss, Oncol Rep. 2005, 13, 559.

[3]. Z. Jedlinski, P. Kurcok, R. W. Lenz,  Macromolecules 1998, 31, 6718.

[4]. V. Piddubnyak, P. Kurcok,  A. Matuszowicz, M. Głowala, A. Fiszer-Kierzkowska, Z. Jedliński, M. Juzwa, Z. Krawczyk,  Biomaterials 2004, 25, 5271.

 

Addendum

 

38. WO SUBTYPES OF SEROUS OVARIAN CANCER WITH DIFFERENT SURVIVAL

Katarzyna Lisowska¹, Magdalena Olbryt¹, Michał Jarzab^1,2, Krzysztof Simek³, Elżbieta Nowara², Jolanta Kupryjańczyk⁴

¹Department of Tumor Biology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland;

²Clinical Oncology Department, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland;

³Department of Automatic Control, Silesian University of Technology, Gliwice, Poland; ⁴Department of Molecular Pathology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Warsaw, Poland.

Ovarian cancer is characterized by asymptomatic development until its advanced stages. Cancer cells spread by migration inside the peritoneal cavity rather than via lymph- or bloodstream. As the disease is diagnosed late, the standard treatment option is surgery and adjuvant chemotherapy. The aim of our study is to better understand the molecular mechanisms of ovarian cancer tumorigenicity and chemotherapy resistance. The long range aim is to select potential prognostic or predictive molecular markers and/or potential new therapeutic targets for serous ovarian cancer.

We studied 99 ovarian cancer specimens by DNA microarray technique. Unsupervised analysis revealed the two subtypes of serous ovarian cancers that differ significantly by gene expression pattern. This division did not overlap with the differences defined by most of the clinical or molecular features (FIGO, grade, etc.), however it appeared to be associated with the overall survival time (OS). The majority of genes that are differentially expressed in the two subtypes of serous cancer code for proteins engaged in cell adhesion and communication, development, metabolism, and immune response. We selected 6 genes to be analyzed by real time RT-PCR and all were positively validated. Expression values obtained by quantitative PCR were used to build a 6-gene predictor of the OS by Cox regression method. This model was able to predict successfully the subtype of a tumor and survival time of the patients (ROC 0.92). We conclude that among ovarian cancer samples of serous histology two molecular subtypes are present that differ by their biological properties and are associated with different survival of the patients. Our current project is aimed to evaluate the role of selected genes differentially expressed in the two subtypes, in particular, their impact on cancer cell tumorigenicity and sensitivity to chemotherapy.

This work was partially supported from the grant 1/0-PBZ-MNiI-2/1/2005 from Ministry of Science and Higher Education.

 

39. Expression of genes connected with glYcolYsis Process in colorectal ADenocarcinoma

Grażyna Janikowska¹, Marta Plato², Celina Kruszniewska – Rajs², Tomasz Janikowski², Jacek Olender², Szymon Jonczyk², Urszula Mazurek²

¹Division of Analytical Chemistry, Medical University of Silesia, Katowice;

²Department and Division of Molecular Biology, Medical University of Silesia, Katowice.

Glycolysis is the oldest evolutionary process which can raise energy (in the form of NADH and ATP)  from glucose, fructose, galactose, mannose and glycerol as a result of enzymatic degradation of these compounds to pyruvate. This process may have occurred both in aerobic and anaerobic condition, what gives great opportunities the cells, in which began running process or a malignancy. It is known that glycolysis is the primary source of energy for cancer cells. The Warburg effect (a reduction in respiration and growth of glycolysis), and hypoxia are commonly present in human cancer cells.

The aim of this study was to evaluate expression of genes closely connected with  glycolysis process in the cells of colorectal adenocarcinoma, comparing the expression of these genes in segments which found to be macroscopically intact with segments changed by cancer obtained from patients in different clinical stage of disease.

The total RNA extracted from segments of normal colon and adenocarcinoma using TRIzol reagent (Invitrogen). Then subjected to RNA purification, and about 8 micrograms of total RNA was used to synthesize double-stranded cDNA, then synthesized cRNA labeled biotin,  fragmentated and hybridizated with microarrays test3 and HGU 133A (Affymetrix), and labeled with streptavidin-phycoerythrin complex (according to the manufacturer's protocol). The fluorescence intensity of the transcripts were analyzed using a microarray scanner GeneArray Scanner G2500A (Agilent). Quantity and quality of total RNA, cDNA and cRNA received in subsequent degrees of transciptome determining, assessed spectrophotometrically and by agarose electrophoresis. After reading the fluorescence signal from the microarray for 22,283 mRNAs, before proceeding to further analysis of results, performed the statistical classification of microarray using Affymetrix's internal programs and other statistical programs, then the final were normalizing data using RMAExpress (Robust Multichip Analysis) based on the logarithmic function (log₂). From obtained transcripts identified 49 genes closely connected with glycolysis process and subjected them to analysis which allowing comparison of results between the studied groups: control and adenocarcinoma with different clinical stage.

The differentiating genes of these groups are determined by the analysis of linear regression using the Statistica 7.1. and the SAM (Significance Analisys of Microarrays) programs.

From obtained comparative analysis the expression of genes connected with glycolysis process in control samples and samples varying of stage of disease that PKM2 (PYRUVATE Kinase 2) and ENO2 (Enolase 2) in the fourth, LDH (Lactate dehydrogenase) only in the second and ALDOB (Fructose-1 ,6-bisphosphate aldolase) in both the second and first clinical stage of disease (CS) were overexpressed. But for PFKL (Phosphofructokinase; IICS) and ENO1 (Enolase 1, II and IV CS) and HK2 (Hexokinase-2, III and IVCS) related to the control were significantly silenced.

 

40. OXIDATIVELY DAMAGED DNA  AND ACTIVITY OF THE PROTEINS OF NF-кB PATHWAY IN LIVER AND KIDNEY OF SUPEROXIDE DISMUTASE KNOCK-OUT MICE

Siomek A.¹, Brzoska K.², Sochanowicz B.², Gackowski D.¹, Rozalski R.¹, Foksinski M.¹, Zarakowska E.¹, Szpila A.¹, Guz J.¹, Dziaman T.¹, Kalinowski B.¹, Bartlomiejczyk T.², Kruszewski M.², Bialkowski K.¹, Olinski R.¹

¹Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Departament of Clinical Biochemistry, ul.Karlowicza 24, 85-092 Bydgoszcz ,e-mail: asiomek@cm.umk.pl, Poland;

²Institute of Nuclear Chemistry and Technology, Centre of Radiobiology and Biological Dosimetry, Dorodna 16, 03-195 Warsaw, e-mail: kamil.brzoska@ichtj.waw.pl, Poland.

To counteract the deleterious effect of oxidative stress/oxidatively damaged DNA,  all organisms developed several strategies.  Important factors,  in antioxidant defense are antioxidant enzymes like superoxide dismutases (SODs). Deficiency in various forms of SOD may result in higher production of free radicals and subsequently in oxidative stress.  Moreover, the reactive oxygen species are considered as a second messengers leading to NF-кB pathway activation.

In our work the level of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a marker of oxidative stress, and the NF-кB pathway activity in the Cu,Zn SOD knock-out and wild type mice were examined. The level of 8-oxodG in DNA as well as the activity of the p50 (NF-κB1) and p65 (RelA) proteins were determined in liver, brain and kidney.

The level of 8-oxodG was higher in the liver and kidney of knock-out mice as compared to the wild type. We have found that activity of the p50 protein was also higher in kidneys, but surprisingly not in livers of SOD1-deficient mice, whereas p65 activity did not show any changes. Our results indicate the mice deficient in Cu,Zn-SOD may develop  oxidative stress in some tissues, and ROS-induced NF-кB activation could be cell/organ type depended. 

This work was supported by a grant of Ministry of Science and Higher Education N301 2052 33 and in part by  ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility) a network of excellence operating within the European Union 6th Framework Program, Priority 5: "Food Quality and Safety" (Contract No 513943).

 

Polymorphic variants of XPD gene - functional significance by in vitro studies.

Agnieszka Gdowicz-Kłosok¹, Wojciech Pigłowski³ , Joanna Rzeszowska-Wolny^1,2

¹Department of Experimental and Clinical Radiobiology, Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology,  Branch Gliwice, Poland;

²System Engineering Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland;

³Department of  Tumor Biology and Department of Pathology, Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology,  Branch Gliwice, Poland.

XPD gene is the main component of Nucleotide Excision Repair (NER) pathway. The XPD protein encoded by this gene, is a subunit of the transcription factor II H (TFIIH) and  functions as an ATP-dependent helicase which unwinds DNA around the lesion in the first step of  repair.

Even minute changes in the nucleotide sequence of this gene may affect its activity and thus contribute to disturbance of processes in which XPD participates. Previous studies conducted at the Department of Experimental and Clinical Radiobiology highlighted correlation between XPD polymorphism and the dynamics of  DNA repair, as well as indicated the effect of this polymorphism upon morbidity risk in various cancers. In order to better understand the ways in which XPD affects these processes, cell lines overexpressing XPD Asp/Asp gene and  XPD Asn/Asn were isolated and studied.

MATERIALS AND METHODS:  To obtain stable cell lines overexpressing XPD gene the pLNCX2 retroviral vector with an inserted gene (XPDAsp/Asp or XPDAsn/Asn) and PT67 cell line were employed. For the experiments HCT116 cell line was used (HCT116 control, HCT116 pLNCX2-transfected, HCT116 pLNCX2-XPDAsp/Asp-transfected and HCT116 pLNCX2-XPDAsn/Asn-transfected, respectively). XPD gene expression, cell cycle and DNA repair dynamics were analyzed after cell cultures’ exposure to ionizing radiation.

Experiments performed thus far indicate that exogenous XPD affects cell survival, cell cycle and the dynamics of repair of DNA lesions induced by ionizing radiation.

 

Synthesis of  potential to PDT photosesitizers with alkyl chains  .

Anna Jarczyk, Violetta Kozik, Krystyna Jarzembek, Marcin Rojkiewicz, Grzegorz Ziemba, Piotr Kuś

Department of Synthesis Chemistry, Institute of Chemistry, University of Silesia, Szkolna 9, 40-006 Katowice,

               The objective of the proposed synthesis and further chemical transformation of a porphyrin derivative was to obtain long alkyl chains terminated by the ester or carboxylic group, respectively.

The discussed series of compounds show biological activity as PDT and PDD agents, therefore might be used in current attempts of  therapy.

Figure 1. a) 4-O-[5-(4-phenyleno)-10,15,20-tritolliloporphiryn]-5-oksypentanan ethyl, b) 4-O-[5-(4-phenyleno)-10,15,20-tritolliloporphirya]-6-oksyheksanin ethyl.

Figure 2 3 a) 4-O-[5-(4-phenyleno)-10,15,20-tritolliloporphirya]-5-oksypentan acid, b)  4-O-[5-(4-phenyleno)-10,15,20-tritolliloporphiryn]-6-oksyheksan acid.

               The synthesized derivatives are able to penetrate the semipermeable membranes what makes them especially promising for the application in anti-cancer therapy. Encouraging results of biological tests enable us to apply these macromolecules in PDT and PDD.