Stanisław Szala

Publications of Stanisław Szala

• Evolving models of tumor origin and progression.

  Authors: Iwona Mitrus, Ewa Bryndza, Aleksander Sochanik, Stanisław Szala

  Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 04/2012;

  History of cancer disease models clearly illustrates the evolving nature of these concepts. Since such models undergo continual revisions and additions as a result of underlying medical research,History of cancer disease models clearly illustrates the evolving nature of these concepts. Since such models undergo continual revisions and additions as a result of underlying medical research, they also tend to reorganize knowledge and allow perceiving previously unseen relationships. Growth of medical thought has been influenced for many centuries by an ancient Hippocratic concept of disease seen as a disturbance in bodily "humors." True mechanisms of cell and tissue injury started to be elucidated only with the advent of postmortem pathological findings. Concerning cancer, when first disease-producing bacteria were identified in the nineteenth century, also neoplasms were treated as infectious diseases. Foreign organisms were thought to be present inside tumors. However, this hypothesis could not be confirmed by microscopic or histochemical studies. The latter suggested, instead, that tumors were rather formed by abnormal cells. Cancer was then started to be regarded as a disease of cells. This interpretation was radically altered by later developments in genetics which suggested that neoplasms can be treated as genetic diseases as pathologic cellular lesions are caused by mutations in specific genes. More recent models have compared carcinogenesis to evolutionary processes. Due to genetic instability, successive mutations, appearing in cells, lead to selection of cancer cells which feature specific phenotypic traits. The newest data indicate that there may be also a link between cancer and mutated stem cells. The review discusses main concepts of tumor origin forwarded since the beginnings of the nineteenth century.

• [Tumor blood vessels].

  Authors: Stanisław Szala, Magdalena Jarosz

  Postȩpy higieny i medycyny doświadczalnej (Online). 01/2011; 65:437-46.

  Growth of tumors usually depends on the development of the tumor’s own vasculature. Small avascular tumors (1–2 mm3) cannot continue growth provided an equilibrium between pro-angiogenic andGrowth of tumors usually depends on the development of the tumor’s own vasculature. Small avascular tumors (1–2 mm3) cannot continue growth provided an equilibrium between pro-angiogenic and anti-angiogenic factors is maintained within the tumor environment. Angiogenesis is not the only factor responsible for tumor blood vessels forming, as vasculogenic mimicry plays an equally substantial role in this process. Vessel-like structures formed during this process are made up from cancer cells, macrophages and mast cells. Certain neoplasms are capable of growing without developing their own vasculature; instead they secure growth via normal blood vessels of the host. Slowed-down blood flow through an abnormally built tumor vascular network is the main reason for cancer cells’ underoxygenation (hypoxia). Defective blood vessels, with hypoxia resulting, play a major role in tumor progression. Underoxygenation induces formation of novel vessels and these new defective vessels are in turn the principal reason for hypoxia. The latter increases cancer cells’ malignancy and invasiveness. A particular process, called transdifferentiation, takes place in tumor vasculature when hypoxia is present and involves neoplastic cells transforming into endothelial cells. Since growth of a tumor is dependent on its own blood supply, inhibition of such vascular network growth and/or damage to this network should exert a strong impact on tumor growth. Long-term administration of anti-angiogenic drugs, however, encounters unexpected problems. Anti-angiogenic drug resistance, together with paradoxical stimulation of invasiveness and metastasis by these drugs, has lately become a dominant issue in anticancer therapy.

• Can inhibition of angiogenesis and stimulation of immune response be combined into a more effective antitumor therapy?

  Authors: Stanisław Szala, Iwona Mitrus, Aleksander Sochanik

  Cancer immunology, immunotherapy : CII. 10/2010; 59(10):1449-55.

  Cancer initiation and progression is strongly influenced by the tumor microenvironment consisting of various types of host cells (inflammatory cells, vascular cells and fibroblasts), extracellularCancer initiation and progression is strongly influenced by the tumor microenvironment consisting of various types of host cells (inflammatory cells, vascular cells and fibroblasts), extracellular matrix and non-matrix molecules. Host cells play a defining role in two major processes crucial for tumor growth: angiogenesis and escape from immune surveillance. The interdependence of these processes resemble the principles of Yin and Yang, as the stimulation of tumor angiogenesis inhibits effective immune responses, while angiogenesis inhibition may have the opposite effect. These considerations may be useful in developing anticancer strategies based on the potentially synergistic combinations of antiangiogenic and immunostimulatory drugs.

• Experimental anticancer therapy with vascular-disruptive peptide and liposome-entrapped chemotherapeutic agent.

  Authors: Aleksander Sochanik, Iwona Mitrus, Ryszard Smolarczyk, Tomasz Cichoń, Mirosław Snietura, Maria Czaja, Stanisław Szala

  Archivum immunologiae et therapiae experimentalis. 04/2010; 58(3):235-45.

  Vasculature is essential for the sustained growth of solid tumors and metastases. Tumor cells surviving vascular-disruptive therapeutic intervention (especially those present at the tumor rim) canVasculature is essential for the sustained growth of solid tumors and metastases. Tumor cells surviving vascular-disruptive therapeutic intervention (especially those present at the tumor rim) can contribute to tumor regrowth. The aim was to strengthen, by carrier-mediated delivery of a chemotherapeutic, the curative effects of a bifunctional anti-vascular oligopeptide capable of inducing vascular shutdown and tumor shrinkage. For the in vitro experiments and animal therapy, ACDCRGDCFC-GG-(D)(KLAKLAK)(2) peptide (900 microM in D-PBSA, i.e. Dulbecco's PBS without Ca(2+) and Mg(2+)) and size-calibrated, passively or actively targeted liposomes based on distearoylphosphatidylcholine, cholesterol, and N-carbamoyl-methoxypolyethyleneglycol coupled to distearoylphosphatidylethanolamine (PEG-DSPE) and containing gradient-entrapped doxorubicin were used. The KB (human nasopharyngeal carcinoma) cell line overexpressing folate receptors was used in the fluorescence studies of liposomal uptake. The B16-F10 melanoma cell line was used for confirming, by flow cytometry and confocal microscopy, doxorubicin intracellular transfer as well as to induce experimental tumors in C57BL/6 mice. Animal therapy was achieved with injections of vascular-disrupting peptide, doxorubicin-loaded liposomes, or alternating combined therapy. The results (tumor growth inhibition and survival) were compared using the Mann-Whitney U test and the log-rank test. Necrosis in H&E-stained tumor sections was assessed microscopically by pathologists. Treatment of C57BL/6 mice bearing B16-F10 experimental tumors with a combination of vascular-disruptive peptide and doxorubicin-carrying pegylated liposomes (either passively targeted liposomes (PTL) or folate receptor targeted) gave better therapeutic effects when tumor development was re-challenged with a second cycle of combined therapy. Marked inhibition of tumor growth and a statistically significant extension of the lifespan of the treated mice were observed when the re-challenge involved the use of folate receptor-targeted liposomes (FTL). Anticancer therapy involving vascular-disruptive peptide and doxorubicin delivered via pegylated folate receptor-targeted liposomes is more effective than either monotherapy, especially when tumor growth is re-challenged with the therapeutic combination.

• Anticancer effects of CAMEL peptide.

  Authors: Ryszard Smolarczyk, Tomasz Cichoń, Wojciech Kamysz, Magdalena Głowala-Kosińska, Anna Szydło, Lukasz Szultka, Aleksander L Sieroń, Stanisław Szala

  Laboratory investigation; a journal of technical methods and pathology. 03/2010; 90(6):940-52.

  This study analyzed whether therapy with CAMEL, an antimicrobial peptide (KWKLFKKIGAVLKVL), possess anticancer benefits. Although the peptide was cytotoxic for all the cell lines tested, it did notThis study analyzed whether therapy with CAMEL, an antimicrobial peptide (KWKLFKKIGAVLKVL), possess anticancer benefits. Although the peptide was cytotoxic for all the cell lines tested, it did not cause hemolysis, which suggests that CAMEL does not damage cell membranes. After cellular internalization, CAMEL localized to mitochondria and lowered the mitochondrial potential, resulting in the organelles' swelling, a decrease in cellular ATP level and, finally, cellular breakdown. High mobility group box 1 (HMGB1) protein, a necrotic death marker, was shown to be released from cells treated with CAMEL. Growth of B16-F10 melanoma tumors was clearly restrained after injections with CAMEL and could be kept in check throughout the period of peptide administration. However, if therapy was stopped, tumors started to grow again 3-4 days later. To reduce tumor volume and block tumor relapse, a combined therapy was required involving CAMEL and plasmid DNA carrying the interleukin-12 (IL-12) gene. The two therapeutic agents used in combination (a series of CAMEL injections first, followed by daily administration of plasmid DNA) delayed tumor growth and extended survival of treated animals in a statistically significant manner. Complete tumor regression was found in 60% of cases.

• Antitumoral effect of IL-12 gene transfected via liposomes into B16F0 cells.

  Authors: Lucía Speroni, Julieta Gasparri, Victoria de Los A Bustuoabad, Nadia Chiaramoni, Andrzej Smagur, Stanisław Szala, María Taira, Silvia Del V Alonso

  Acta biochimica Polonica. 06/2009;

  Murine melanoma B16FO cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greaterMurine melanoma B16FO cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greater transfection efficiency compared to DOTAP-containing liposomes. The lipid:DNA ratio was 2:1 (w/w). Control groups were mock transfected or transfected with an empty plasmid (pNeo). pNeo or IL-12 transfected cells and controls were inoculated intradermically into the dorsal region of the foot or the lateral flank of C57BL6 mice. Results showed that IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors developed in both anatomic sites, significantly retarding their growth and prolonging host survival.

• Oxidation of carbidopa by tyrosinase and its effect on murine melanoma.

  Authors: Beata Gąsowska-Bajger, Bożena Frąckowiak-Wojtasek, Sabina Koj, Tomasz Cichoń, Ryszard Smolarczyk, Stanisław Szala, Hubert Wojtasek

  Bioorganic & medicinal chemistry letters. 06/2009;

  Oxidation of the anti-Parkinsonian agent carbidopa by tyrosinase was investigated. The products of this reaction were identified as 3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid andOxidation of the anti-Parkinsonian agent carbidopa by tyrosinase was investigated. The products of this reaction were identified as 3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid and 6,7-dihydroxy-3-methylcinnoline. These results demonstrate that after oxidation of the catechol moiety to an o-quinone either a redox exchange with the hydrazine group or a cyclization reaction occur. The cyclization product underwent additional oxidation reactions leading to aromatization. The cyclization reaction is undesired in the case of hydrazine-containing anti-melanoma prodrugs and will have to be taken into account in designing such compounds. Carbidopa was tested against B16(F10) melanoma cells in culture and showed cytotoxicity significantly higher than either of its oxidation products and l-dopa. This effect, however, was not specific to this cell line.

• Chimeric protein ABRaA-VEGF(121) is cytotoxic towards VEGFR-2-expressing PAE cells and inhibits B16-F10 melanoma growth.

  Authors: Andrzej Smagur, Mariya Boyko, Nadiya Biront, Tomasz Cichoń, Stanisław Szala

  Acta biochimica Polonica. 03/2009;

  It has been known that VEGF(121) isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric proteinIt has been known that VEGF(121) isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF(121). Expression of the ABRaA-VEGF(121) chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF(121) preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (~84 kDa homodimer and ~42 kDa monomer). ABRaA-VEGF(121) is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF(121) inhibited protein biosynthesis in cell-free translation system. Preincubation of ABRaA-VEGF(121) with antibody neutralizing the biological activity of human VEGF abolishe the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF(121) inhibits growth of B16-F10 murine melanoma tumors.

• Combination of combretastatin A4 phosphate and doxorubicin-containing liposomes affects growth of B16-F10 tumors.

  Authors: Iwona Mitrus, Aleksander Sochanik, Tomasz Cichoń, Stanisław Szala

  Acta biochimica Polonica. 02/2009; 56(1):161-5.

  The study aimed to check the effectiveness of anticancer therapy combining a vascular-disruptive drug (combretastatin phosphate, CA4P) and a liposomal formulation of a chemotherapeutic (doxorubicin).The study aimed to check the effectiveness of anticancer therapy combining a vascular-disruptive drug (combretastatin phosphate, CA4P) and a liposomal formulation of a chemotherapeutic (doxorubicin). CA4P was synthesized in our laboratory according to a previously described procedure. The antivascular drug and long-circulating doxorubicin-loaded liposomes were used to treat B16-F10 murine melanoma experimental tumors. Seventy-four hours after drug administration, a decrease in the number of tumor blood vessels was apparent and necrotic areas within tumors were visible. Combination therapy consisting of alternate administrations of CA4P and liposomal doxorubicin yielded greater inhibition of tumor growth than monotherapies alone. The best therapeutic results were obtained with the antivascular drug administered intratumorally every second day at 50 mg/kg body mass. In the case of combined therapy, the best results were obtained when the vascular-disruptive agent (CA4P) and the antineoplastic agent (liposomal doxorubicin) were administered in alternation.

• [Peptides: a new class of anticancer drugs]

  Authors: Ryszard Smolarczyk, Tomasz Cichoń, Stanisław Szala

  Postȩpy higieny i medycyny doświadczalnej (Online). 02/2009; 63:360-8.

  Peptides are a novel class of anticancer agents embracing two distinct categories: natural antibacterial peptides, which are preferentially bound by cancer cells, and chemically synthesized peptides,Peptides are a novel class of anticancer agents embracing two distinct categories: natural antibacterial peptides, which are preferentially bound by cancer cells, and chemically synthesized peptides, which bind specifically to precise molecular targets located on the surface of tumor cells. Antibacterial peptides bind to both cell and mitochondrial membranes. Some of these peptides attach to the cell membrane, resulting in its disorganization. Other antibacterial peptides penetrate cancer cells without causing cell membrane damage, but they disrupt mitochondrial membranes. Thanks to phage and aptamer libraries, it has become possible to obtain synthetic peptides blocking or activating some target proteins found in cancer cells as well as in cells forming the tumor environment. These synthetic peptides can feature anti-angiogenic properties, block enzymes indispensable for sustained tumor growth, and reduce tumor ability to metastasize. In this review the properties of peptides belonging to both categories are discussed and attempts of their application for therapeutic purposes are outlined.

• [Angiogenesis and immune supression: yin and yang of tumor progression?]

  Authors: Stanisław Szala

  Postȩpy higieny i medycyny doświadczalnej (Online). 01/2009; 63:598-612.

  Podczas progresji nowotworowej powstaja swoiste warianty komórek nowotworowych, które maja zdolność rekrutowania ze szpiku i z krwiobiegu niektórych komórek hematopoetycznych i mezenchymalnych. WśródPodczas progresji nowotworowej powstaja swoiste warianty komórek nowotworowych, które maja zdolność rekrutowania ze szpiku i z krwiobiegu niektórych komórek hematopoetycznych i mezenchymalnych. Wśród rekrutowanych komórek hematopoetycznych sa m.in. monocyty, makrofagi, granulocyty, komórki tuczne, komórki dendrytyczne i mieloblastyczne komórki supresorowe. Komórki nowotworowe rekrutuja takze wywodzace sie z komórek mezenchymalnych komórki fibroblastów. Pod wpływem komórek nowotworowych niektóre z rekrutowanych komórek (zwłaszcza makrofagi i fibroblasty) podlegaja fenotypowemu przeprogramowaniu: swoistej "edukacji". W wyniku takiej "edukacji" powstaja charakterystyczne dla nowotworów makrofagi (TAM) oraz fibroblasty (CAF). Komórki TAM, CAF, pozostałe komórki zrekrutowane oraz macierz pozakomórkowa (ECM) tworza swoiste mikrośrodowisko nowotworowe. Komórki mikrośrodowiska wraz z komórkami nowotworowymi biora udział w dwóch, ściśle ze soba powiazanych, nierozerwalnych procesach: angiogenezie i immunosupresji. Powstajace podczas angiogenezy niesprawne naczynia krwionośne i zwiazany z nimi zmienny przepływ krwi powoduja powstawanie niedotlenienia, które ma istotny wpływ na metaboliczny profil komórek nowotworowych (np. niskie zuzycie tlenu). W niedotlenowanych komórkach zachodza takze inne procesy korzystne dla dalszej progresji: np. wzmozona, nasilona angiogeneza, przejście epitelialno-mezenchymalne, dzieki któremu komórki nowotworowe nabywaja zdolności samodzielnego przemieszczania sie. Niedotlenowanie wpływa takze na wzrost genetycznej niestabilności komórek nowotworowych. Komórki mikrośrodowiska maja takze swój udział w powstaniu środowiska immunosupresyjnego, umozliwiajacego ucieczke komórek nowotworowych spod nadzoru immunologicznego. Tworzac wysoce swoiste otoczenie selekcjonujace odpowiednie komórki nowotworowe (majace fenotyp angiogenny i immunosupresyjny) komórki mikrośrodowiska maja istotny wpływ na progresje komórek nowotworowych. Zahamowanie angiogenezy umozliwia powstanie odpowiedzi odpornościowej (zarówno swoistej jak i nieswoistej). Spostrzezenia te moga posłuzyć do zaprojektowania nowych rozwiazań terapeutycznych, w których leki antyangiogenne beda kojarzone z lekami immunomodulacyjnymi.

• Combination of vasostatin and cyclophosphamide in the therapy of murine melanoma tumors.

  Authors: Joanna Jazowiecka-Rakus, Magdalena Jarosz, Dorota Kozłowska, Aleksander Sochanik, Stanisław Szala

  Acta biochimica Polonica. 02/2007; 54(1):125-33.

  Growth of tumors is strongly dependent upon supply of nutrients and oxygen by de novo formed blood vessels. Inhibiting angiogenesis suppresses growth of primary tumors as well and affects developmentGrowth of tumors is strongly dependent upon supply of nutrients and oxygen by de novo formed blood vessels. Inhibiting angiogenesis suppresses growth of primary tumors as well and affects development of metastases. We demonstrate that recombinant MBP/vasostatin fusion protein inhibits proliferation of endothelial cells in vitro. The therapeutic usefulness of such intratumorally delivered recombinant protein was then assessed by investigating its ability to inhibit growth of experimental murine melanomas. In the model of B16-F10 melanoma the MBP/vasostatin construct significantly delayed tumor growth and prolonged survival of treated mice. A combination therapy involving MBP/vasostatin construct and cyclophosphamide was even more effective and led to further inhibition of the tumor growth and extended survival. We show that such combination might be useful in the clinical setting, especially to treat tumors which have already formed microvessel networks.

• Antitumor effect of RGD-4C-GG-D(KLAKLAK)2 peptide in mouse B16(F10) melanoma model.

  Authors: Ryszard Smolarczyk, Tomasz Cichoń, Klaudyna Graja, Joanna Hucz, Aleksander Sochanik, Stanisław Szala

  Acta biochimica Polonica. 02/2006; 53(4):801-5.

  Vasculature targeting agents have been tested as cancer therapeutics for the past few years. Such therapy could be accomplished using, for example, bifunctional (two-domain) peptides.Vasculature targeting agents have been tested as cancer therapeutics for the past few years. Such therapy could be accomplished using, for example, bifunctional (two-domain) peptides. RGD-4C-GG-D(KLAKLAK)2, a peptide designed by Ellerby and coworkers (1999) (full sequence: ACDCRGDCFCGGKLAKLAKKLAKLAK), binds selectively to alphaVbeta3 integrin receptors expressed in tumor neovasculature and, after internalization, effectively induces apoptosis of endothelial cells. The aim of this study was to examine if RGD-4C-GG-D(KLAKLAK)2 would efficiently target cells, among them B16(F10), that overexpress alphaVbeta3 receptors, and whether it would be suitable for therapeutic treatment of primary B16(F10) murine melanoma tumors. Thus, the peptide would target two distinct tumor compartments: that formed by endothelium of blood vessels and that made up of neoplastic cells. The therapeutic peptide was recognized and did induce apoptosis in B16(F10) cell line. Tumor growth inhibition was observed following direct intratumoral administration. However, cessation of peptide administration led to rapid tumor growth and death of the animals.

• Combination of IL-12 gene therapy and CTX chemotherapy inhibits growth of primary B16(F10) melanoma tumors in mice.

  Authors: Iwona Mitrus, Klaudia Delić, Natalia Wróbel, Ewa Missol-Kolka, Stanisław Szala

  Acta biochimica Polonica. 02/2006; 53(2):357-60.

  We investigated suppression of murine B16(F10) melanoma tumor growth following a therapy which involved concomitant administration of cyclophosphamide and plasmid DNA bearing interleukin-12 gene.We investigated suppression of murine B16(F10) melanoma tumor growth following a therapy which involved concomitant administration of cyclophosphamide and plasmid DNA bearing interleukin-12 gene. Since both therapeutic factors display antiangiogenic capabilities, we assumed that their use in blocking the formation of new blood vessels would result in augmented inhibition of tumor growth. This combined therapy regimen indeed resulted in a considerable suppression of tumor growth. We observed a statistically significant extension of treated animals' lifespan. Interestingly, the therapeutic effect was also obtained using a plasmid without an interleukin gene insert. This observation suggests that plasmid DNA, which has been widely applied for treating neoplastic tumors, contains element(s) that elicit immune response in mice.

• Combination of vasostatin gene therapy with cyclophosphamide inhibits growth of B16(F10) melanoma tumours.

  Authors: Joanna Jazowiecka-Rakus, Magdalena Jarosz, Stanisław Szala

  Acta biochimica Polonica. 02/2006; 53(1):199-202.

  Angiogenesis, i.e. formation of new blood vessels out of pre-existing capillaries, is essential to the development of tumour vasculature. The discovery of specific antiangiogenic inhibitors hasAngiogenesis, i.e. formation of new blood vessels out of pre-existing capillaries, is essential to the development of tumour vasculature. The discovery of specific antiangiogenic inhibitors has important therapeutic implications for the development of novel cancer treatments. Vasostatin, the N-terminal domain of calreticulin, is a potent endogenous inhibitor of angiogenesis and tumour growth. In our study, using B16(F10) murine melanoma model and electroporation we attempted intramuscular transfer of human vasostatin gene. The gene therapy was combined with antiangiogenic drug dosing schedule of a known chemotherapeutic (cyclophosphamide). The combination of vasostatin gene therapy and cyclophosphamide administration improved therapeutic effects in melanoma tumours. We observed both significant inhibition of tumour growth and extended survival of treated mice. To our knowledge, this is one of the first reports showing antitumour efficacy of electroporation-mediated vasostatin gene therapy combined with antiangiogenic chemotherapy.

• Gene expression profile of B 16(F10) murine melanoma cells exposed to hypoxic conditions in vitro.

  Authors: Magdalena Olbryt, Michał Jarzab, Joanna Jazowiecka-Rakus, Krzysztof Simek, Stanisław Szala, Aleksander Sochanik

  Gene expression. 01/2006; 13(3):191-203.

  Hypoxia is an important feature of tumor microenvironment, exerting far-reaching effects on cells and contributing to cancer progression. Previous studies have established substantial differences inHypoxia is an important feature of tumor microenvironment, exerting far-reaching effects on cells and contributing to cancer progression. Previous studies have established substantial differences in hypoxia response between various cell lines. Investigating this phenomenon in melanoma cells contributes to a better understanding of cell lineage-specific hypoxia response and could point out novel hypoxia-regulated genes. We investigated transcriptional activity of B 16(F10) murine melanoma cells cultured for 24 h under hypoxic (nominal 1% O2, 15 samples including controls) and hypoxia-mimicking conditions (cobalt chloride, 100 or 200 microM, 6 samples including controls). Gene expression profiles were analyzed using MG-U74Av2 oligonucleotide microarrays. Data analysis revealed 2541 probesets (FDR <5%) for 1% oxygen experiment and 364 probesets (FDR <5%) for cobalt chloride, which showed differences in expression levels. Analysis of hypoxia-regulated genes (true hypoxia, 1% O2) by stringent Family-Wise Error Rate estimation indicated 454 significantly changed transcripts (p < 0.05). The most upregulated genes were Lgals3, Selenbpl, Nppb (more than ten-fold increase). We observed significant differences in expression levels of genes regulating glycolysis (Pfkp, Hk2, Aldo3, Eno2), apoptosis (Bnip3, Bnip31, Cdknla), transcription (Bhlhb2, Sap30, Atf3, Mxil), angiogenesis (Vegfa, Adm, Anxa2, Ctgf), adhesion (Pkp2, Itga4, Mcam), migration (Cnn2, Tmsb4x), and other processes. Both true hypoxia and hypoxia mimicry induced HIF-1-regulated genes. However, unsupervised analysis (Singular Value Decomposition) revealed distinct differences in gene expression between these two experimental conditions. Contrary to hypoxia, cobalt chloride caused suppression of gene expression rather than stimulation, especially concerning transcripts related to proliferation, immune response, DNA repair, and melanin biosynthesis.

• Negligible induction of IFN-gamma, IL-12 and TNF-alpha by DNA-PEI 750 kDa/albumin complexes.

  Authors: Ryszard Smolarczyk, Tomasz Cichoń, Aleksander Sochanik, Stanisław Szala

  Cytokine. 04/2005; 29(6):283-7.

  A 750 kDa polyethylenimine (PEI 750 kDa) combined with albumin has been found to mediate in vivo a highly efficient transfection of small amounts of plasmid DNA. Using this exceptional carrier systemA 750 kDa polyethylenimine (PEI 750 kDa) combined with albumin has been found to mediate in vivo a highly efficient transfection of small amounts of plasmid DNA. Using this exceptional carrier system we evaluated the inflammatory responses triggered by CpG sequences found in plasmid DNA. Using as little as 1 mug DNA transferred in vivo caused an almost negligible response from pro-inflammatory cytokines (IFN-gamma, IL-12 and TNF-alpha), as assessed in serum with a commercially available kit. Administering 750 kDa PEI/albumin/plasmid DNA complexes every three days assured a high and prolonged in vivo expression of a reporter protein. A further increase in the level of such protein was obtained by administering the investigated complexes concurrently with dexamethasone. High gene transfer capability and a relatively low pro-inflammatory response of 750 kDa PEI/albumin/DNA complexes can be exploited for recurrent gene transfer into lungs to treat (via inhalation or instillation) cancer or genetic disorders such as cystic fibrosis.

• Recombinant angioarrestin secreted from mouse melanoma cells inhibits growth of primary tumours.

  Authors: Andrzej Smagur, Jarosław Szary, Stanisław Szala

  Acta biochimica Polonica. 02/2005; 52(4):875-9.

  Angioarrestin is a recently described anti-angiogenic protein whose expression is down-regulated in solid tumours of various origins. It has a sequence identical to angiopoietin related protein-1. InAngioarrestin is a recently described anti-angiogenic protein whose expression is down-regulated in solid tumours of various origins. It has a sequence identical to angiopoietin related protein-1. In this study we investigated anti-tumour properties of angioarrestin in B16 (F10) melanoma tumour model. We constructed an expression vector encoding human angioarrestin under the control of EF-1alpha promoter. This vector was transferred to B16 (F10) cells and recombinant angioarrestin secreted from the transfected cells was tested for anti-angiogenic activity using endothelial cell proliferation assay. Finally, mice were injected subcutaneously with cells that had been transfected with either angioarrestin-encoding vector or empty vector and tumor growth was compared. The obtained recombinant angioarrestin inhibited proliferation of bovine aortic endothelial cells. Tumours derived from an angioarrestin-secreting B16 (F10) cell clone grew in vivo more slowly than tumours derived from a cell clone transfected with empty vector. These data show, to our knowledge for the first time, that angioarrestin can inhibit primary melanoma tumour growth.

• [Immunostimulatory properties of CpG sequences in bacterial DNA]

  Authors: Ryszard Smolarczyk, Stanisław Szala

  Postepy biochemii. 02/2004; 50(4):296-303.

• CEA-negative glioblastoma and melanoma cells are sensitive to cytosine deaminase/5-fluorocytosine therapy directed by the carcinoembryonic antigen promoter.

  Authors: Anna Dabrowska, Jarosław Szary, Małgorzata Kowalczuk, Stanisław Szala, Maciej Ugorski

  Acta biochimica Polonica. 02/2004; 51(3):723-32.

  Recent studies have suggested that carcinoembryonic antigen (CEA)-promoter sequences are active only in CEA-positive cells, filing in the criteria for tumor specific targeting of suicide genes.Recent studies have suggested that carcinoembryonic antigen (CEA)-promoter sequences are active only in CEA-positive cells, filing in the criteria for tumor specific targeting of suicide genes. However, the present study on gene therapy of colon cancer and cell-specificity of CEA promoter, provide evidence that CEA-positive and CEA-negative cells transfected with E. coli cytosine deaminase (CD) gene under the control of CEA promotor sequence are sensitive to enzyme/pro-drug therapy with 5-fluorocytosine (5-FC). Individual clones derived from the CEA-negative cell lines: melanoma Hs294T and glioblastoma T98G after transfection with CD differed profoundly in their sensitivity to 5-FC. The IC50 values for several clones of the CEA-negative cells were almost the same as for CEA-positive colon cancer cells. Such 5-FC-sensitive clones derived from the population of CEA-negative cells, present even in small number, because of the very effective bystender effect of this enzyme/pro-drug system can cause severe problems during therapy by efficiently killing surrounding normal cells. Safety is the major issue in gene therapy. Our data suggest that the safety of gene-directed enzyme pro-drug therapy (GDEPT) with CEA promoter driven expression of therapeutic genes is not so obvious as it has originally been claimed.