Zdeněk Knejzlík

Differences in antitumor effects of various statins on human pancreatic cancer

Statins are widely used for the treatment of hypercholesterolemia. However, their inhibitory action on HMG‐CoA reductase also results in the depletion of intermediate biosynthetic products, which importantly contribute to cell proliferation. The aim of the present study was to compare the effects of the individual commercially available statins on experimental pancreatic cancer. The in vitro effects of individual statins (pravastatin, atorvastatin, simvastatin, lovastatin, cerivastatin, rosuvastatin and fluvastatin) on the viability of human pancreatic cancer were evaluated in CAPAN‐2, BxPc‐3 and MiaPaCa‐2 cell lines. The in vivo experiments were performed on nude mice xenotransplanted with CAPAN‐2 cells. The mice received oral treatments either with a placebo, or with the statins mentioned earlier in a daily dose corresponding to a hypocholesterolemic dose in humans. The effect of these statins on the intracellular Ras protein, trafficking in MiaPaCa‐2 transfected cells, was also investigated. Substantial differences in the tumor‐suppressive effects of all statins were detected in both in vitro and in vivo experiments. While simvastatin exerted the highest tumor‐suppressive effects in vitro, rosuvastatin (p = 0.002), cerivastatin (p = 0.002) and fluvastatin (p = 0.009) were the most potent compounds in an animal model. All statins (except pravastatin) inhibited intracellular Ras protein translocation. In summary, substantial tumor‐suppressive effects of various statins on the progression of experimental pancreatic adenocarcinoma were demonstrated, with marked differences among individual statins. These results support greatly the potential of statins for the chemoadjuvant treatment of pancreatic cancer.

D-retrovirus morphogenetic switch driven by the targeting signal accessibility to Tctex-1 of dynein.

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason–Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.

Antiproliferative effects of carbon monoxide on pancreatic cancer

BACKGROUND Carbon monoxide, the gaseous product of heme oxygenase, is a signalling molecule with a broad spectrum of biological activities. The aim of this study was to investigate the effects of carbon monoxide on proliferation of human pancreatic cancer. METHODS In vitro studies were performed on human pancreatic cancer cells (CAPAN-2, BxPc3, and PaTu-8902) treated with a carbon monoxide-releasing molecule or its inactive counterpart, or exposed to carbon monoxide gas (500 ppm/24h). For in vivo studies, pancreatic cancer cells (CAPAN-2/PaTu-8902) were xenotransplanted subcutaneously into athymic mice, subsequently treated with carbon monoxide-releasing molecule (35 mg/kg b.w. i.p./day), or exposed to safe doses of carbon monoxide (500 ppm 1h/day; n = 6 in each group). RESULTS Both carbon monoxide-releasing molecule and carbon monoxide exposure significantly inhibited proliferation of human pancreatic cancer cells (p<0.05). A substantial decrease in Akt phosphorylation was observed in carbon monoxide-releasing molecule compared with inactive carbon monoxide-releasing molecule treated cancer cells (by 30-50%, p<0.05). Simultaneously, carbon monoxide-releasing molecule and carbon monoxide exposure inhibited tumour proliferation and microvascular density of xenotransplanted tumours (p<0.01), and doubled the survival rates (p<0.005). Exposure of mice to carbon monoxide led to an almost 3-fold increase in carbon monoxide content in tumour tissues (p=0.006). CONCLUSION These data suggest a new biological function for carbon monoxide in carcinogenesis, and point to the potential chemotherapeutic/chemoadjuvant use of carbon monoxide in pancreatic cancer.

SUMO-2/3 conjugates accumulating under heat shock or MG132 treatment result largely from new protein synthesis☆

Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.

Reversible buckling and diffusion properties of silica-coated hydrogel particles.

The structure and diffusion properties of composite particles consisting of a calcium alginate hydrogel core and a thin SiO(2) surface layer have been investigated. The composite particles were formed by depositing a silica layer onto calcium alginate cores using a sol-gel process starting from alkoxysilane precursors. The composite particles were found to have a remarkable ability to reversibly rehydrate and return to their original size and shape after partial drying. The organo-silica skin was able to sustain large local deformations (such as complete folding) without the formation of cracks or defects. Such mechanical properties are uncharacteristic of pure silica and they can be attributed to the specific microstructure of the alginate-silica composite. The structure and composition of the alginate-silica particles were characterised by SEM, X-ray micro-tomography, Laser Scanning Confocal Microscopy and Thermo-gravimetry. In order to quantify the effect of the organo-silica layer on the diffusional transport into and out of the alginate particles, the uptake and release rates of several test molecules with increasing molecular weight were measured for both un-coated and silica-coated particles. While the diffusion rate of small and medium-size molecules (water, vitamin B12) was essentially unaffected by the presence of the silica layer, the diffusion rate of a larger biomolecule (lysozyme) was found to be slowed down by the presence of the surface layer. The flexibility of the organo-silica layer combined with the ability of even large biomolecules to diffuse through it indicate that the silica layer is macroporous, formed by individual SiO(2) nanoparticles dispersed and immobilised in the surface layer of the alginate hydrogel.

Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization.

The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibers is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.

Effect of surface functionalisation on the interaction of iron oxide nanoparticles with polymerase chain reaction

The combination of nanoparticles with the polymerase chain reaction (PCR) can have benefits such as easier sample handling or higher sensitivity, but also drawbacks such as loss of colloidal stability or inhibition of the PCR. The present work systematically investigates the interaction of magnetic iron oxide nanoparticles (MIONs) with the PCR in terms of colloidal stability and potential PCR inhibition due to interaction between the PCR components and the nanoparticle surface. Several types of MIONs with and without surface functionalisation by sodium citrate, dextran and 3-aminopropyl-triethoxysilane (APTES) were prepared and characterised by Transmission Electron Microscopy (TEM), dynamic light scattering (DLS) and Fourier Transform Infrared (FT-IR) spectroscopy. Colloidal stability in the presence of the PCR components was investigated both at room temperature and under PCR thermo-cycling. Dextran-stabilized MIONs show the best colloidal stability in the PCR mix at both room and elevated temperatures. Citrate- and APTES-stabilised as well as uncoated MIONs show a comparable PCR inhibition near the concentration 0.1mgml-1 while the inhibition of dextran stabilized MIONs became apparent near 0.5mgml-1. It was demonstrated that the PCR could be effectively carried out even in the presence of elevated concentration of MIONs up to 2mgml-1 by choosing the right coating approach and supplementing the reaction mix by critical components, Taq DNA polymerase and Mg2+ ions.

Human UBL5 protein interacts with coilin and meets the Cajal bodies.

UBL5 protein, a structural homologue of ubiquitin, was shown to be involved in pre-mRNA splicing and transcription regulation in yeast and Caenorhabditis elegans, respectively. However, role of the UBL5 human orthologue is still elusive. In our study, we observed that endogenous human UBL5 that was localized in the nucleus, partially associates with Cajal bodies (CBs), nuclear domains where spliceosomal components are assembled. Simultaneous expression of exogenous UBL5 and coilin resulted in their nuclear colocalization in HeLa cells. The ability of UBL5 to interact with coilin was proved by GST pull-down assay using coilin that was either in vitro translated or extracted from HEK293T cells. Further, our results showed that the UBL5-coilin interaction was not influenced by coilin phosphorylation. These results suggest that UBL5 could be targeted to CBs via its interaction with coilin. Relation between human UBL5 protein and CBs is in the agreement with current observations about yeast orthologue Hub1 playing important role in alternative splicing.