Jan Šmarda - Academia.edu (original) (raw)

Papers by Jan Šmarda

Oncogene, 2017

Metastasis accounts for most of cancer-related deaths. Paracrine signaling between tumor cells an... more Metastasis accounts for most of cancer-related deaths. Paracrine signaling between tumor cells and the stroma induces changes in the tumor microenvironment required for metastasis. Transcription factor c-Myb was associated with breast cancer (BC) progression but its role in metastasis remains unclear. Here we show that increased c-Myb expression in BC cells inhibits spontaneous lung metastasis through impaired tumor cell extravasation. On contrary, BC cells with increased lung metastatic capacity exhibited low c-Myb levels. We identified a specific inflammatory signature, including Ccl2 chemokine, that was expressed in lung metastatic cells but was suppressed in tumor cells with higher c-Myb levels. Tumor cell-derived Ccl2 expression facilitated lung metastasis and rescued trans-endothelial migration of c-Myb overexpressing cells. Clinical data show that the identified inflammatory signature, together with a MYB expression, predicts lung metastasis relapse in BC patients. These results demonstrate that the c-Myb-regulated transcriptional program in BCs results in a blunted inflammatory response and consequently suppresses lung metastasis.

Molecular Cancer, 2012

Background The c-Myb transcription factor is essential for the maintenance of stem-progenitor cel... more Background The c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells. Results Ectopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP) 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed...

Biochemical and Biophysical Research Communications, 2007

c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor... more c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor that participates in regulation of proliferation, differentiation, and apoptosis. In this study, we explored functional interactions of the c-Jun protein with several regulators of the G1/S transition in serum-deprived v-myb-transformed chicken monoblasts BM2. We show that the c-Jun protein induces expression of cyclin A, thus up-regulating activity of cyclin A-associated cyclin-dependent kinase 2 (CDK2), and causing massive programmed cell death of starved BM2cJUN cells. Specific inhibition of CDK2 suppresses frequency of apoptosis of BM2cJUN cells. We conclude that up-regulation of cyclin A expression and CDK2 activity can represent important link between the c-Jun protein, cell cycle machinery, and programmed cell death pathway in leukemic cells.

Journal of Applied Biomedicine

Healthy cells strictly regulate gene transcription to control crucial cellular regulatory pathway... more Healthy cells strictly regulate gene transcription to control crucial cellular regulatory pathways. Members of the Jun protein family, c-Jun, JunB, and JunD are key subunits of the transcription factor AP-1 that controls transcription from various gene promoters. The genes targeted by Jun affect essential life processes, such as cell cycle progression, differentiation or programmed cell death. Therefore, the loss of proper Jun function is often associated with cancer. This review summarizes recent advances in understanding of function of the Jun proteins in healthy and cancer cells.

Metallomics : integrated biometal science, Jan 17, 2018

Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cance... more Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cancer agent. The underlying mechanisms of its action are not fully understood and appear to involve interplay with copper ions. Herein, we examined coordination and redox interactions of WL with Cu in phosphate buffer (pH 7), and in two breast cancer cell lines. EPR, UV-Vis and fluorescence spectroscopy showed that WL and Cu build a coordination complex with 2 : 1 stoichiometry and distorted tetrahedral geometry. WL showed strong fluorescence that was quenched by Cu. The sequestration of the intracellular copper pool with neocuproine led to a significant drop in the cytotoxic effects of WL, whereas the co-application of Cu and WL and the formation of an extracellular complex suppressed both the cytotoxic effects of WL and copper loading. Fluorescence microscopy showed that WL is mainly localized in the cytosol and significantly less in the nuclei. WL fluorescence was stronger in cells pretre...

Journal of The American Society for Mass Spectrometry

The practicality of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass... more The practicality of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) applied to molecular imaging of biological tissues is limited by the analysis speed. Typically, a relatively low speed of stop-and-go micromotion of XY stages is considered as a factor substantially reducing the rate with which fresh sample material can be supplied to the laser spot. The sample scan rate in our laboratory-built high-throughput imaging TOF mass spectrometer was significantly improved through the use of a galvanometer-based optical scanner performing fast laser spot repositioning on a target plate. The optical system incorporated into the ion source of our MALDI TOF mass spectrometer allowed focusing the laser beam via a modified grid into a 10-μm round spot. This permitted the acquisition of high-resolution MS images with a well-defined pixel size at acquisition rates exceeding 100 pixel/s. The influence of selected parameters on the total MS imaging time is discussed. The new scanning technique was employed to display the distribution of an antitumor agent in 3D colorectal adenocarcinoma cell aggregates; a single MS image comprising 100 × 100 pixels with 10-μm lateral resolution was recorded in approximately 70 s.

International journal of molecular sciences, Jan 29, 2017

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer ce... more Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytot...

European journal of nutrition, Jan 16, 2016

Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon ca... more Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon carcinogenesis have been implicated, the mechanisms of their action are not fully clear. Here, we investigated modulations of composition of individual phospholipid (PL) classes, with a particular emphasis on cardiolipins (CLs), in colon cells treated with DHA, sodium butyrate (NaBt), or their combination (DHA/NaBt), and we evaluated possible associations between lipid changes and cell fate after fatty acid treatment. In two distinct human colon cell models, foetal colon (FHC) and adenocarcinoma (HCT-116) cells, we compared patterns and composition of individual PL classes following the fatty acid treatment by HPLC-MS/MS. In parallel, we measured the parameters reflecting cell proliferation, differentiation and death. In FHC cells, NaBt induced primarily differentiation, while co-treatment with DHA shifted their response towards cell death. In contrast, NaBt induced apoptosis in HCT-116 ce...

Tumor Biology, 2016

The MYB gene codes for the c-Myb transcription factor maintaining proliferation of colon epitheli... more The MYB gene codes for the c-Myb transcription factor maintaining proliferation of colon epithelial progenitors, thus controlling colon development and homeostasis. This gene is overexpressed in early phases of colorectal cancer (CRC) tumorigenesis. The aim of this study was to examine the expression of c-Myb in CRC tissue samples both at the messenger RNA (mRNA) and protein levels and to evaluate their associations with clinicopathological characteristics in a group of 108 CRC patients. Statistically significant negative association was found between the frequency of the c-Myb-positive tumor cells assessed by immunohistochemistry and the presence of distant metastases (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01) but not tumor differentiation, tumor stage, lymph node involvement, vascular invasion, tumor localization, age, and gender of the patients. Although the c-Myb protein level in the tumor tissue correlated with its mRNA level, no significant association between MYB mRNA and any clinicopathological characteristics was observed. We conclude that albeit overexpression of c-Myb is considered as an important factor contributing to early phases of CRC tumorigenesis, it may later have negative effect on tumor cell dissemination as observed recently in breast cancer as well. Further studies are required to explain the role of c-Myb during formation of CRC distant metastases.

International journal of oncology, 2012

The p53 protein is a sequence-specific transcription factor controlling the expression of multipl... more The p53 protein is a sequence-specific transcription factor controlling the expression of multiple genes and protecting cells from oncogenic transformation. In many tumors, the p53 protein is completely or partially inactivated by mutations in the p53 gene. We analyzed the transactivating activity of nine human temperature-dependent (td) p53 mutants in yeast cells. Mutations in seven of them were localized in the β-sandwich-coding region of the p53 gene, eight p53 mutants were temperature-sensitive and the R283C mutant was cold-sensitive. Patterns of their transactivation abilities towards three different responsive elements, the extent of their temperature dependency as well as discriminativity, were considerably variable. Similarly, their capacity to become reactivated by amifostine varied from complete resistance to high sensitivity. Transactivation abilities and temperature dependency of six p53 td mutants were determined in transiently-transfected H1299 human cells and revealed...

The Journal of steroid biochemistry and molecular biology, Jan 28, 2015

Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate c... more Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at μM concentrations. In this study, however, a novel biological activity of nM dose of wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) α and β as demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either ERα or ERβ and by molecular docking of this coumestan into ligand binding pocket of both ERα and ERβ. In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be inhibited by pretreatment with pure ER antagonist ICI 182,780 and they ...

Klinicka onkologie, 2014

Apoptóza je typ programované buněčné smrti (typ I), který je nezbytný pro správný vývoj organizmu... more Apoptóza je typ programované buněčné smrti (typ I), který je nezbytný pro správný vývoj organizmu a tkáňovou homeostázu. Její průběh může být určen dvěma signálními drahami-vnější (receptorovou) dráhou řízenou receptory smrti a vnitřní (mitochondriální) apoptotickou dráhou, kde klíčovou roli plní mitochondrie. Mitochondrie jsou důležité buněčné organely s nepostradatelnými funkcemi pro život buňky, jako je např. tvorba energie ve formě molekul ATP (adenosintrifosfátu). Mitochondriální buněčná smrt je charakteristická změnou transmembránového potenciálu a permeabilizací vnější mitochondriální membrány. Mitochondrie jsou elektronegativní organely a depolarizace mitochondriální membrány je důležitá pro uvolnění proapoptotických signálů. Narušená regulace mitochondriální buněčné smrti se může podílet na patogenezi různých onemocnění, včetně rakoviny. Mitochondrie jsou také zdrojem reaktivních kyslíkových radikálů, iontů Ca 2+ a proteinů ovlivňujících procesy iniciace a progrese nádorů nezávisle na indukci apoptózy. Současné studie se zaměřují na výzkum mitochondriálního membránového potenciálu a kyslíkových radikálů, které modulují různé signální dráhy uvnitř buňky a vymezení jejich významu v kancerogenezi, případně v léčbě onkologických pacientů. Monitorování apoptotických markerů, jako je stav mitochondriálního membránového potenciálu a určení hladiny reaktivních kyslíkových radikálů ve vzorcích nádorových pacientů, má prediktivní hodnotu pro výstup léčebných protokolů. Klíčová slova mitochondrie-průtoková cytometrie-apoptóza-volné radikály-membránový potenciál mitochondrií Summary Apoptosis is type I programmed cell death, a process that is essential for development and tissue homeostasis. It is a prevalent form of cell death and it proceeds via two signaling pathways-external (receptor pathway) triggered by death receptors and intrinsic (mitochondrial) apoptotic pathway with major involvement of mitochondria. Mitochondria are important cellular organelles producing energy stored in molecules of adenosine triphosphate that are essential for cell survival. The mitochondrial cell death is characterized by permeabilization of the mitochondrial outer membrane and dissipation of the transmembrane potential. Mitochondria are electronegative organelles and depolarization of the mitochondrial membrane is important for the release of proapoptotic signals. Aberrant control of the mitochondrial cell death might contribute to several diseases including cancer. Mitochondria are also a source of reactive oxygen species, Ca 2+ ions and other proteins that aff ect processes important for the initiation and progression of tumors independently of apoptosis. Current studies focus on research of mitochondrial membrane potential and reactive oxygen species modulating various signaling pathways within the cell, their importance in carcinogenesis, and in treatment of oncological patients. Monitoring of the apoptotic markers, such as the mitochondrial membrane potential (MMP), and the level of reactive oxygen species in samples of oncological patients has a predictive value for the output of treatment protocols.

International Journal of Molecular Medicine, 2009

White blood cell (WBC) count is considered a prognostic risk factor in acute myeloid leukemia. As... more White blood cell (WBC) count is considered a prognostic risk factor in acute myeloid leukemia. As density of leukemic cells increases, the cytotoxic activity of certain anticancer drugs, such as vincristine and doxorubicin, progressively decreases. In this study, we investigated the cell density-dependent induction of apoptosis of human acute myeloid leukemia U937 and ML-1 cells by disulfiram (DSF), the dithiocarbamate drug recently proposed for treatment of human cancers. This effect is dependent on uptake of extracellular copper and its intracellular accumulation. Highdensity cells cannot uptake and accumulate this metal to a sufficient level that would allow induction of apoptosis due to progressive decrease of its extracellular concentration. Simple addition of copper can resume sensitivity of highdensity leukemic cells to DSF and improve efficiency of antileukemic therapies using this drug, thus providing benefit to patients with high WBC count.

Experimental and Therapeutic Medicine, 2010

Increased production of the pro-inflammatory enzyme cyclooxygenase-2 (Cox-2) and altered expressi... more Increased production of the pro-inflammatory enzyme cyclooxygenase-2 (Cox-2) and altered expression and activity of peroxisome proliferator-activated receptor γ (PPARγ) have been observed in many malignancies. Both the pparγ ligands and the Cox-2 inhibitors possess antiinflammatory and anti-neoplastic effects in vitro and have been assessed for their therapeutic potential in several pre-clinical and clinical studies. Recently, multiple interactions between pparγ and Cox-2 signaling pathways have been revealed. Understanding of the cross-talk between PPARγ and Cox-2 might provide important novel strategies for the effective treatment and/or prevention of cancer. This article summarizes recent achievements involving the functional interactions between the PPARγ and Cox-2 signaling pathways and discusses the implications of such interplay for clinical use. Contents 1. Introduction 2. Cox-2 and regulation of PPARγ 3. PPARγ ligands as Cox-2 activators 4. PPARγ ligands as Cox-2 suppressors 5. Cox-2 inhibitors and PPARγ ligands can act synergistically to suppress Cox-2 and activate PPARγ 6. Conclusion

Anti-Cancer Agents in Medicinal Chemistry, 2014

Natural products are often used in drug development due to their ability to form unique and diver... more Natural products are often used in drug development due to their ability to form unique and diverse chemical structures. Coumestans are polycyclic aromatic plant secondary metabolites containing a coumestan moiety, which consists of a benzoxole fused to a chromen-2-one to form 1-Benzoxolo[3,2-c]chromen-6-one. These natural compounds are known for large number of biological activities. Many of their biological effects can be attributed to their action as phytoestrogens and polyphenols. In the last decade, anticancer effects of these compounds have been described in vitro but there is only limited number of studies based on models in vivo. More information concerning their in vivo bioavailability, stability, metabolism, toxicity, estrogenicity, cellular targets and drug interactions is therefore needed to proceed further to clinical studies. This review focuses on coumestans exhibiting anticancer properties and summarizes mechanisms of their toxicity to cancer cells. Moreover, the possible role of coumestans in cancer prevention is discussed.

Journal of Hematotherapy & Stem Cell Research, 2003

BM2 cells are chicken monoblasts transformed by the v-myb oncogene of avian myeloblastosis virus.... more BM2 cells are chicken monoblasts transformed by the v-myb oncogene of avian myeloblastosis virus. The constitutively high v-myb expression interferes with the terminal differentiation of BM2 cells, but these cells can be induced to differentiate into macrophage-like cells by phorbol esters. Histone acetylation plays an important role in regulation of transcription and is particularly relevant to the regulation and pathology of hematopoiesis. In the present study, we examined the contribution of elevated histone acetylation to the differentiation of BM2 cells. Inhibition of the activity of endogenous histone deacetylases by trichostatin A (TSA) resulted in histone hyperacetylation causing cell cycle arrest and differentiation of BM2 cells into macrophage polykaryons. TSA did not affect the level of v-Myb protein in BM2 cells, but it downregulated its transcription activation capability. This suggests that chromatin remodeling can be significantly engaged in regulation of proliferation and differentiation of leukemic cells.

Prostaglandins & Other Lipid Mediators, 2003

Inhibitors of arachidonic acid (AA) conversion were described as suppressors of proliferation and... more Inhibitors of arachidonic acid (AA) conversion were described as suppressors of proliferation and inducers of differentiation of various leukemic cells. Certain AA metabolites have been shown to cooperate with Jun proteins that are important factors controlling cell proliferation, differentiation and apoptosis. Using lipoxygenase (LOX) inhibitors of various specifity we studied possible participation of lipoxygenase pathway in regulation of proliferation and apoptosis of v-myb-transformed chicken monoblasts BM2 and its functional interaction with Jun proteins. We found that nordihydroguaiaretic acid (NDGA) and esculetin (Esc) negatively regulate proliferation of BM2 cells causing accumulation in either G0/G1-phase (nordihydroguaiaretic acid) or S-phase (esculetin) of the cell cycle. BM2 cells can be also induced to undergo growth arrest and partial differentiation by ectopic expression of Jun proteins. We demonstrated that lipoxygenase inhibitors further enforce tumor suppressive capabilities of Jun proteins by inducing either more efficient cell cycle block and/or apoptosis in BM2 cells. This suggests that there is a cross-talk between the lipoxygenase- and Jun-directed pathways in regulation of differentiation and proliferation of monoblastic cells. Thus pharmacologic agents that specifically block lipoxygenase-catalyzed activity and enforce the effects of differentiation-inducers may be important components in anti-tumor therapies.

Oncology Reports, 1994

Burkitt´s lymphomas (BL) are aggressive fast growing tumors typified by high c-myc expression res... more Burkitt´s lymphomas (BL) are aggressive fast growing tumors typified by high c-myc expression resulting from t(8;14)(q24;q32), t(2;8)(p12;q24) or t(8;22)(q24;q11) translocations. Alterations of the p53 tumor suppressor are also relatively frequent in BL. Several approaches have been adopted for detection of the p53 aberrations: immunohistochemical analyses, immunoblotting, DNA sequencing, fluorescence in situ hybridization (FISH), and functional assays. We used these methods to characterize the p53 mutation in tumor cells of the fifty three year old male suffering from Burkitt´s lymphoma. By immunohistochemical analyses, we detected high level of the p53 protein in the tumor tissue. Immunoblotting analysis showed higher molecular weight of the p53 protein overexpressed in tumor tissues than that of the standard p53 protein. Similarly, the molecular weight of the PCR product prepared by amplification of the tumor p53 cDNA was higher than the PCR product prepared from standard p53 cDNA. Functional analyses of separated alleles in yeast clearly revealed that the tumor p53 protein was transcriptionally nonfunctional. The yeast colonies expressing this p53 variant possessed unique phenotype: they were red with many white spots on their surface. Sequencing of the tumor cDNA revealed duplication of the 30 bp region of the p53 gene (g.12155_12184dup30) leading to repeat of ten amino acids (Pro-77 to Ala-86) in the p53 protein. Further analyses showed that the mutation was unstable in yeast cells. The FISH analyses did not confer loss of the p53-specific locus 17p13.

Nutrition and Cancer, 2013

Growth of tumor cells depends on sufficient supply of fermentable substrate, such as glucose. Thi... more Growth of tumor cells depends on sufficient supply of fermentable substrate, such as glucose. This provokes development of new anticancer therapies based on dietary restrictions. However, some tumor cells can lower their glucose dependency and activate processes of ATP formation/saving to retain viability even in limited glucose supply. In addition, tumor cells often lose sensitivity to many conventional anticancer drugs in the low-glucose conditions. Thus, development of the drugs effectively killing the tumor cells in nutrient-limited conditions is necessary. In this study, we show an enhanced cytotoxicity of tetrathiomolybdate, the drug exhibiting antiangiogenic and tumor-suppressing effects, to neuroblastoma SH-SY5Y and SK-N-BE(2) cells in the low-glucose conditions. This preference results from the tetrathiomolybdate-induced upregulation of cell dependency on glucose. The cells treated with tetrathiomolybdate increase the uptake of glucose, production of lactate, activate the Akt- and AMPK-signaling pathways and downregulate COX IV. In cells growing in the low-glucose conditions, these events result in significant decrease of the intracellular ATP supply and apoptosis. We propose tetrathiomolybdate as suitable agent to be used in combination with dietary restrictions in therapy of neuroblastoma.