The stabilization of hypoxia inducible factor modulates differentiation status and inhibits the proliferation of mouse embryonic stem cells.

Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O2 and 5% O2) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells.

Immunomodulatory Potency of Microcystin, an Important Water-Polluting Cyanobacterial Toxin.

Microcystins (MCs) are primarily hepatotoxins produced by cyanobacteria and are responsible for intoxication in humans and animals. There are many incidents of chronic exposure to MCs, which have been attributed to the inappropriate treatment of water supplies or contaminated food. Using RAW 264.7 macrophages, we showed the potency of microcystin-LR (MC-LR) to stimulate production of pro-inflammatory cytokines (tumor necrosis factor α and interleukin-6) as a consequence of fast nuclear factor κB and nitrogen-activated protein kinase activation. In contrast to other studies, the observed effects were not attributed to the intracellular inhibition of protein phosphatases 1/2A due to lack of specific transmembrane transporters for MCs. However, the MC-LR-induced activation of macrophages was effectively inhibited by a specific peptide that blocks signaling of receptors, which play a pivotal role in the innate immune responses. Taken together, we showed for the first time that MC-LR could interfere with macrophage receptors that are responsible for triggering the above-mentioned signaling pathways. These findings provide an interesting mechanistic explanation of some adverse health outcomes associated with toxic cyanobacteria and MCs.

Modulation of endothelial glycocalyx structure under inflammatory conditions.

The glycocalyx of the endothelium is an intravascular compartment that creates a barrier between circulating blood and the vessel wall. The glycocalyx is suggested to play an important role in numerous physiological processes including the regulation of vascular permeability, the prevention of the margination of blood cells to the vessel wall, and the transmission of shear stress. Various theoretical models and experimental approaches provide data about changes to the structure and functions of the glycocalyx under various types of inflammatory conditions. These alterations are suggested to promote inflammatory processes in vessels and contribute to the pathogenesis of number of diseases. In this review we summarize current knowledge about the modulation of the glycocalyx under inflammatory conditions and the consequences for the course of inflammation in vessels. The structure and functions of endothelial glycocalyx are briefly discussed in the context of methodological approaches regarding the determination of endothelial glycocalyx and the uncertainty and challenges involved in glycocalyx structure determination. In addition, the modulation of glycocalyx structure under inflammatory conditions and the possible consequences for pathogenesis of selected diseases and medical conditions (in particular, diabetes, atherosclerosis, ischemia/reperfusion, and sepsis) are summarized. Finally, therapeutic strategies to ameliorate glycocalyx dysfunction suggested by various authors are discussed.

The isolation and characterization of lipopolysaccharides from Microcystis aeruginosa, a prominent toxic water bloom forming cyanobacteria.

Massive toxic blooms of cyanobacteria represent a major threat to water supplies worldwide, yet serious gaps exist in understanding their complex toxic effects, including the role of lipopolysaccharides (LPS). The present comparative study focused on the levels and biological activities of LPS isolated from Microcystis aeruginosa, which is one of the most globally distributed toxic species. Using hot phenol extraction, LPS was isolated from 3 laboratory cultures and 11 natural water blooms. It formed 0.2-0.7% of the original dry biomass of the cyanobacteria, based on gravimetry. Additional analyses by commercial anti-LPS ELISA were correlated with gravimetry but showed concentrations that were about 7-times lower, which indicated either impurities in isolated LPS or the poor cross-reactivity of the antibodies used. LPS isolates from M. aeruginosa were potent pyrogens in the traditional Limulus amebocyte lysate (LAL)-test, but comparison with the PyroGene test demonstrated the limited selectivity of LAL with several interferences. The determined pyrogenicity (endotoxin units, EU) ranged from very low values in laboratory cultures (less than 0.003 up to 0.008-EU per 100 pg LPS) to higher values in complex bloom samples (0.01-0.078 EU per 100 pg of LPS), which suggested the role of bloom-associated bacteria in the overall effects. Potent pro-inflammatory effects of the studied LPS from both cultures and bloom samples were observed in a highly-relevant ex vivo human blood model by studying reactive oxygen species production in phagocytes as well as increased productions of interleukin 8, IL-8, and tumor necrosis factor α, TNF-α. LPS from M. aeruginosa seem to modulate several pathways involved in the regulation of both innate immunity and specific responses. In comparison to the standard pathogenic bacterial LPS (World Health Organization Escherichia coli O113:10 endotoxin; activity 1 EU per 100 pg), the studied cyanobacterial samples had pyrogenicity potencies that were at least 12-times lower. However, the health risks associated with LPS from M. aeruginosa should not be underestimated, especially with respect to diverse biological effects observed ex vivo and in the case of massive blooms in drinking water reservoirs, where the estimated pyrogenicity can reach up to 46,000 EU per mL of water.

LA-12 overcomes confluence-dependent resistance of HT-29 colon cancer cells to Pt (II) compounds.

BACKGROUND: LA-12 is a new platinum (IV) drug with promising cytotoxic effects in a wide range of cancer cell lines. Its confluence-dependent effects were compared with cisplatin (CDDP) and oxaliplatin (L-OHP) in HT-29 cells. MATERIALS AND METHODS: Cytotoxicity was determined by MTT test, eosin exclusion assay, and cell number quantification. The cell cycle was analysed using propidium iodide DNA staining (flow cytometry), apoptosis by phosphatidylserine externalisation (annexin-V assay), mitochondrial membrane potential by flow cytometry, nuclear morphology by means of fluorescence microscopy, and PARP cleavage by Western blotting. RESULTS: While L-OHP and CDDP were practically inactive in the subconfluent cell population, LA-12 showed a similar toxicity in both subconfluent and growing populations. All compounds induced apoptosis, although with different potentials. CONCLUSION: LA-12 was able to overcome confluence-dependent resistance of HT-29 cells observed for other platinum compounds, which may have potential therapeutic use in slowly growing tumours.

Concentrations of methylated naphthalenes, anthracenes, and phenanthrenes occurring in Czech river sediments and their effects on toxic events associated with carcinogenesis in rat liver cell lines.

Alkylated polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. In the present study, we determined levels of monomethylated naphthalenes (MeNap), phenanthrenes (MePhe), and anthracenes (MeAnt) in Czech river sediments. The levels of MePhe generally were lower than the concentrations of phenanthrene. In contrast, both MeNap and MeAnt were found at levels higher than their respective parent compounds in the majority of sampling sites. We then investigated their aryl hydrocarbon receptor (AhR)-mediated activity, accumulation of phosphorylated p53 protein, induction of expression of cytochrome P450 1A1 (CYP1A1), inhibition of gap junctional intercellular communication (GJIC), and effects on cell proliferation in rat liver cell models to evaluate the relative importance of these toxicity mechanisms of low-molecular-weight methylated PAHs. Methylated phenanthrene and anthracene compounds were weak inducers of AhR-mediated activity as determined both in a reporter gene assay system and by detection of the endogenous gene (Cyp1a1) induction. 2-Methylphenanthrene was the most potent AhR ligand. Contribution of MeAnt and MePhe to overall AhR-inducing potencies should be taken into account in PAH-contaminated environments. Nevertheless, their effects on AhR were not sufficient to modulate cell proliferation in a normal rat liver progenitor cell model system. These PAHs only had a marginal effect on p53 phosphorylation at high doses of 1-, 3-, and 9-MePhe as well as 1 MeAnt. On the other hand, both 2- and 9-MeAnt as well as all the MePhe under study were efficient inhibitors of GJIC, suggesting that these compounds might act as tumor promoters. In summary, inhibition of GJIC and partial activation of AhR seem to be the most prominent toxic effects of the methylated PAHs in the present study.

Different cell cycle modulation following treatment of human ovarian carcinoma cells with a new platinum(IV) complex vs cisplatin.

Platinum (IV) derivative with adamantylamine-LA-12-represents a new generation of highly efficient anti-cancer drug derived from cisplatin and is currently in the final stage of phase I clinical trials. Understanding the specific mechanisms of its effects on cell cycle is necessary for defining the mode of action of LA-12. In this study, we characterized the ability of LA-12 to induce cell cycle perturbations in ovarian cancer cell line A2780 as compared to equitoxic cisplatin treatment. LA-12 induced a permanent accumulation of A2780 cells in S phase while cisplatin caused G2/M arrest at 24-h time point, where we also detected an increased expression of Gadd45alpha protein. Although both derivatives induced a rapid increase of p53 expression, this was not associated with a down-regulation of Mdm2 protein. Increased expression of p21(Cip1/WAF1) protein and its association with cyclins A and B1 suggested that this cyclin-dependent kinase inhibitor might contribute significantly to the observed perturbations of cell cycle. The results of this study provide insight into the mechanism of action of platinum-based derivative with adamantylamine on cell cycle in ovarian cancer cells. The differences between effects of LA-12 and cisplatin suggest that more attention should be paid to elucidation of modes of action of novel platinum(IV) complexes at cellular level.

beta-Naphthoflavone and 3'-methoxy-4'-nitroflavone exert ambiguous effects on Ah receptor-dependent cell proliferation and gene expression in rat liver 'stem-like' cells.

Both natural and synthetic flavonoids are known to interact with the aryl hydrocarbon receptor (AhR); however, their agonist/antagonist properties in vitro have been so far studied mostly in the context of cytochrome P450 1A1 gene (Cyp1a1) regulation. We investigated effects of two synthetic flavones known either as AhR agonist (beta-naphthoflavone; BNF) or antagonist (3'-methoxy-4'-nitroflavone; 3M4NF), using an in vitro model of liver 'stem-like' cells, on expression of various AhR target genes and AhR-dependent cell proliferation. We found that the presumed antagonist 3M4NF induces a partial nuclear translocation and activation of AhR. Although inhibiting the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced Cyp1a1 expression, 3M4NF alone induced a minor increase of CYP1A1 mRNA and protein. However, 3M4NF did not induce AhR binding to synthetic dioxin response elements (DRE). In contrast to Cyp1a1, 3M4NF induced a marked expression of other AhR-regulated genes, such as Cyp1b1 and Nqo1, as well as transcriptional repression of Cdh13 gene, confirming that its effects may be promoter-context specific. Like BNF, 3M4NF induced AhR-dependent cell proliferation of contact-inhibited rat liver 'stem-like' WB-F344 cells, associated with a marked upregulation of Cyclin A, as well as the downregulation of proteins involved in formation of cell-cell contacts. Based on these experimental findings, we conclude that partial agonists/antagonists of AhR can increase cell proliferation rate and AhR-dependent genes expression in both cell type- and gene-specific manner. The specificity of effects of flavones on diverse AhR targets should be taken into account, when studying AhR signaling using presumed AhR antagonists.

The aryl hydrocarbon receptor-dependent deregulation of cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells.

Disruption of cell proliferation control by polycyclic aromatic hydrocarbons (PAHs) may contribute to their carcinogenicity. We investigated role of the aryl hydrocarbon receptor (AhR) in disruption of contact inhibition in rat liver epithelial WB-F344 'stem-like' cells, induced by the weakly mutagenic benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and by the strongly mutagenic benzo[a]pyrene (BaP). There were significant differences between the effects of BaA and BbF, and those of the strongly genotoxic BaP. Both BaA and BbF increased percentage of cells entering S-phase and cell numbers, associated with an increased expression of Cyclin A and Cyclin A/cdk2 complex activity. Their effects were significantly reduced in cells expressing a dominant-negative AhR mutant (dnAhR). Roscovitine, a chemical inhibitor of cdk2, abolished the induction of cell proliferation by BbF. However, neither BaA nor BbF modulated expression of the principal cdk inhibitor involved in maintenance of contact inhibition, p27(Kip1), or pRb phosphorylation. The strongly mutagenic BaP induced apoptosis, a decrease in total cell numbers and significantly higher percentage of cells entering S-phase than either BaA or BbF. Given that BaP induced high levels of Cyclin A/cdk2 activity, downregulation of p27(Kip1) and hyperphosphorylation of pRb, the accumulation of cells in S-phase was probably due to cell proliferation, although S-phase arrest due to blocked replication forks can not be excluded. Both types of effects of BaP were significantly attenuated in dnAhR cells. Transfection of WB-F344 cells with siRNA targeted against AhR decreased induction of Cyclin A induced by BbF or BaP, further supporting the role of AhR in proliferative effects of PAHs. This suggest that activation of AhR plays a significant role both in disruption of contact inhibition by weakly mutagenic PAHs and in genotoxic effects of BaP possibly leading to enhanced cell proliferation. Thus, PAHs may increase proliferative rate and the likelihood of fixation of mutations.

7H-Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial 'stem-like' cells.

Immature liver progenitor cells have been suggested to be an important target of hepatotoxins and hepatocarcinogens. The goal of the present study was to assess the impact of 7H-dibenzo[c,g]carbazole (DBC) and its tissue-specific carcinogenic N-methyl (N-MeDBC) and 5,9-dimethyl (DiMeDBC) derivatives on rat liver epithelial WB-F344 cells, in vitro model of liver progenitor cells. We investigated the cellular events associated with both tumor initiation and promotion, such as activation of aryl hydrocarbon receptor (AhR), changes in expression of enzymes involved in metabolic activation of DBC and its derivatives, effects on cell cycle, cell proliferation/apoptosis and inhibition of gap junctional intercellular communication (GJIC). N-MeDBC, a tissue-specific sarcomagen, was only a weak inhibitor of GJIC or inducer of AhR-mediated activity, and it did not affect either cell proliferation or apoptosis. DBC was efficient GJIC inhibitor, while DiMeDBC manifested the strongest AhR inducing activity. Accordingly, DiMeDBC was also the most potent inducer of cytochrome P450 1A1 (CYP1A1) and CYP1A2 expression among the three compounds tested. Both DBC and DiMeDBC induced expression of CYP1B1 and aldo-keto reductase 1C9 (AKR1C9). N-MeDBC failed to significantly upregulate CYP1A1/2 and it only moderately increased CYP1B1 or AKR1C9. Only the potent liver carcinogens, DBC and DiMeDBC, caused a significant increase of p53 phosphorylation at Ser15, an increased accumulation of cells in S-phase and apoptosis at micromolar concentrations. In addition, DiMeDBC was found to stimulate cell proliferation of contact-inhibited WB-F344 cells at 1 microM concentration, which is a mode of action that might further contribute to its hepatocarcinogenicity. The present data seem to suggest that the AhR activation, induction of enzymes involved in metabolic activation, inhibition of GJIC or stimulation of cell proliferation might all contribute to the hepatocarcinogenic effects of DBC and DiMeDBC.

Platinum(IV) complex with adamantylamine overcomes intrinsic resistance to cisplatin in ovarian cancer cells.

OBJECTIVES: The resistance of tumor cells to cisplatin remains a major cause of treatment failure in cancer patients. In this study, the ability of Pt(IV) complex with adamantylamine-LA-12 and its reduced counterpart with lower oxidation state Pt(II)-LA-9 to overcome intrinsic cisplatin resistance was investigated. METHODS: The ovarian adenocarcinoma SK-OV-3 cells were exposed to cisplatin, LA-9, or LA-12 for 72 h and the effects of drug concentrations that caused 10% or 50% inhibition of cell proliferation were determined. After 24-72 h of sustained exposure viability, apoptosis and inhibition of proliferation were analyzed. DNA synthesis and cell cycle analysis were performed simultaneously in order to determine the modulation of cell cycle after platinum complexes treatment. RESULTS: Lung Resistance-related Protein (LRP/MVP) was detected in SK-OV-3 cells but not in the other two ovarian cancer lines with different sensitivity to cisplatin. LRP/MVP overexpression may be an important factor contributing to intrinsic cisplatin resistance. Interestingly, Pt(IV) complex-LA-12 had approximately 2.7-fold lower IC(50) concentration than LA-9 or cisplatin in SK-OV-3 cells. Moreover, LA-12 caused persistent accumulation of cells in S-phase of the cell cycle while LA-9 and cisplatin treatment-induced S-phase arrest was transient and shifted to G(2)/M-phase at later intervals. Apoptosis seemed to be not the dominant type of cell death caused by such the derivatives, but it was the most intensive after LA-12 treatment. CONCLUSIONS: We found strong differences between effects of Pt(IV) complex-LA-12 and Pt(II) derivatives-LA-9 and cisplatin on cytokinetic parameters. Overall, LA-12 but not its reduced Pt(II) counterpart LA-9 is the compound effective in p53 null human ovarian cancer cells and it is able to overcome intrinsic cisplatin resistance in these cells.

High effectiveness of platinum(IV) complex with adamantylamine in overcoming resistance to cisplatin and suppressing proliferation of ovarian cancer cells in vitro.

[(OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand - adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC(50) and IC(90) concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC(50) or IC(90)) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G(2)/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC(50) or IC(90) of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied.