Kinetics and Mechanism of In(III) Ions Electroreduction on Cyclically Renewable Liquid Silver Amalgam Film Electrode: Significance of the Active Complexes of In(III)-Acetazolamide.

The results of kinetic measurements revealed an accelerating effect of acetazolamide (ACT) on the multistep In(III) ions electroreduction in chlorates(VII) on a novel, cyclically renewable liquid silver amalgam film electrode (R-AgLAFE). The kinetic and thermodynamic parameters were determined by applying the DC polarography, square-wave (SWV) and cyclic voltammetry (CV), as well as electrochemical impedance spectroscopy (EIS). It was shown that ACT catalyzed the electrode reaction ("cap-pair" effect) by adsorbing on the surface of the R-AgLAFE electrode. The catalytic activity of ACT was explained as related to its ability to form active In(III)- acetazolamide complexes on the electrode surface, facilitating the electron transfer process. The active complexes constitute a substrate in the electroreduction process and their different structures and properties are responsible for differences in the catalytic activity. The determined values of the activation energy ΔH≠ point to the catalytic activity of ACT in the In(III) ions electroreduction process in chlorates(VII). Analysis of the standard entropy values ΔS0 confirm changes in the dynamics of the electrode process.

Long-lasting reduction in clonogenic potential of colorectal cancer cells by sequential treatments with 5-azanucleosides and topoisomerase inhibitors.

BACKGROUND: The currently approved therapies fail in a substantial number of colorectal cancer (CRC) patients due to the molecular heterogeneity of CRC, hence new efficient drug combinations are urgently needed. Emerging data indicate that 5-azanucleosides are able to sensitize cancer cells to the standard chemotherapeutic agents and contribute to overcoming intrinsic or acquired chemoresistance. METHODS: CRC cells with different genetic backgrounds (HCT116, DLD-1, HT-29) were sequentially treated with 5-azanucleosides and topoisomerase inhibitors. The combined effects of these two drug classes on cell viability, apoptosis, signaling pathways, and colony formation were investigated. RESULTS: Here, we demonstrate that pretreatment with DNA demethylating agents, 5-aza-2'-deoxycytidine and 5-azacytidine, sensitizes CRC cells to topoisomerase inhibitors (irinotecan, etoposide, doxorubicin, mitoxantrone), reducing cell viability and clonogenicity and increasing programmed cell death more effectively than individual compounds at the same or even higher concentrations. 5-Azanucleosides did not cause considerable immediate toxic effects as evaluated by analysis of cell viability, apoptosis, DNA damage (γH2A.X), and endoplasmic reticulum (ER) stress (CHOP). However, 5-azanucleosides exerted long-lasting effects, reducing cell viability, changing cell morphology, and affecting phosphoinositide 3-kinase (PI3-kinase)/Akt signaling pathway. We found that a single exposure to 5-azanucleosides is sufficient to induce long-lasting sensitization to topoisomerase inhibitors. The combinatorial, but not separate, treatment with low doses of 5-aza-2'-deoxycytidine (0.1 µM) and etoposide (0.5 µM) caused a long-lasting (almost 70 days) reduction in clonogenic/replating ability of DLD-1 cells. CONCLUSIONS: These results suggest that sequential treatments with DNA demethylating agents and topoisomerase inhibitors may exert clinically relevant anticancer effects.

The interplay between epigenetic silencing, oncogenic KRas and HIF-1 regulatory pathways in control of BNIP3 expression in human colorectal cancer cells.

Bcl-2/adenovirus E1B-19kDa-interacting protein 3 (BNIP3) is an important mediator of cell survival and a member of the Bcl-2 family of proteins that regulate programmed cell death and autophagy. We have previously established a link between the expression of oncogenic HRas and up-regulation of BNIP3 and the control of autophagy in cancer cells. However, in view of varied expression of BNIP3 in different tumor types and emerging uncertainties as to the role of epigenetic silencing, oncogenic regulation and the role of BNIP3 in cancer are still poorly understood. In the present study we describe profound effect of KRas on the expression of methylated BNIP3 in colorectal cancer cells and explore the interplay between HIF-1, hypoxia pathway and oncogenic KRas in this context. We observed that BNIP3 mRNA remains undetectable in aggressive DLD-1 cells harboring G13D mutant KRAS and HT-29 colorectal cancer cells unless the cells are exposed to demethylating agents such as 5-aza-2'-deoxycytidine. Following this treatment BNIP3 expression remains uniquely dependent on the Ras activity. We found that hypoxia or pharmacological activation of HIF-1 alone contributes to, but is not sufficient for efficient induction of BNIP3 mRNA transcription in cells lacking mutant KRas activity. The up-regulation of BNIP3 by KRas in this setting is mediated by the MAPK pathway, and is attenuated by the respective inhibitors (PD98059, U0126). Thus, we demonstrate the novel mechanism where activity of Ras is essential for 5-aza-2'-deoxycytidine-mediated BNIP3 expression. Moreover, we found that 5-aza-2'-deoxycytidine-mediated or enforced up-regulation of BNIP3 in DLD-1 cells results in KRas-dependent resistance to 5-Fluorouracil.

The Contrasting Delayed Effects of Transient Exposure of Colorectal Cancer Cells to Decitabine or Azacitidine.

(1) Background: Decitabine and azacitidine are cytosine analogues representing the class of drugs interfering with DNA methylation. Due to their molecular homology and similar clinical application, both drugs are often regarded as interchangeable. Despite their unique mechanism of action the studies designed for observation and comparison of the prolonged activity of these drugs are rare. (2) Methods: The short-time (20-72 h) and long-term (up to 20 days) anti-cancer activity of decitabine and azacitidine has been studied in colorectal cancer cells. We observe the impact on cell culture's viability, clonogenicity, proliferation, and expression of CDKN1A, CCND1, MDM2, MYC, CDKN2A, GLB1 genes, and activity of SA-ß-galactosidase. (3) Results: Decitabine has much stronger anti-clonogenic activity than azacitidine. We show that azacitidine, despite significant immediate toxicity, has negligible long-term effects. Contrary, decitabine, which does not exert initial toxicity, profoundly worsened the condition of the cells over time. On the 13th day after treatment, the viability of cells was decreased and proliferation inhibited. These functional changes were accompanied by up-regulation of expression CDKN1A, CCND1, and CDKN2A genes and increased activation of SA-ß-galactosidase, indicating cellular senescence. (4) Conclusions: Our head-to-head comparison revealed profound differences in the activities of decitabine and azacitidine important in their anti-cancer potential and clinical application. The effects of decitabine need relatively long time to develop. This property is crucial for proper design of studies and therapy concerning decitabine and undermines opinion about the similar therapeutic mechanism and interchangeability of these drugs.

Therapeutic effects of an azaphenothiazine derivative in mouse experimental colitis.

Phenothiazines represent a class of compounds of potential therapeutic utility. In this report we evaluated therapeutic value of an azaphenothiazine derivative, 6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine (QBT), given intragastrically, in the model of dextran sodium sulfate-induced colitis in C57BL/6 mice using 5-aminosalicylic acid (5-ASA) as a reference drug. Colitis symptoms such as body weight loss, diarrhea and hematochezia (blood in stool) were observed and registered and disease activity index (DAI) was calculated. In addition, weight and cell numbers in the lymphatic organs and histological parameters of the colon wall were analyzed. The effects of QBT on viability of colon epithelial cell lines were also determined. We showed that weight and cell number of draining mesenteric lymph nodes were lower in mice treated with QBT in comparison to their control counterparts. The number of thymocytes, drastically reduced in control mice, was elevated in mice treated with the compounds with a significant effect of 5-ASA. In addition, an abnormal composition of blood cell types was partially corrected in these groups. Histological analysis of the colon revealed that the pathological changes were partially normalized by QBT and even to a higher degree by 5-ASA. In conclusion we demonstrated a therapeutic efficacy of the compound in amelioration of local and systemic pathological changes associated with chemically-induced colitis in mice. A possible mechanism of action of the compound is discussed.

HDL-associated lysosphingolipids inhibit NAD(P)H oxidase-dependent monocyte chemoattractant protein-1 production.

OBJECTIVE: High-density lipoprotein (HDL) levels are inversely proportional to the risk of atherosclerosis, but mechanisms of HDL atheroprotection remain unclear. Monocyte chemoatractant protein-1 (MCP-1) constitutes an early component of inflammatory response in atherosclerosis. Here we investigated the influence of HDL on MCP-1 production in vascular smooth muscle cells (VSMCs) and rat aortic explants. METHODS AND RESULTS: HDL inhibited the thrombin-induced production of MCP-1 in a concentration-dependent manner. The HDL-dependent inhibition of MCP-1 production was accompanied by the suppression of reactive oxygen species (ROS), which regulate the MCP-1 production in VSMCs. HDL inhibited NAD(P)H oxidase, the preponderant source of ROS in the vasculature, and prevented the activation of Rac1, which precedes NAD(P)H-oxidase activation. The HDL capacity to inhibit MCP-1 production, ROS generation, and NAD(P)H-oxidase activation was emulated by sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), two lysosphingolipids present in HDL, but not by apolipoprotein A-I. HDL-, S1P-, and SPC-induced inhibition of MCP-1 production was attenuated in VSMCs pretreated with VPC23019, an antagonist of lysosphingolipid receptors S1P(1) and S1P(3), but not by JTE013, an antagonist of S1P(2). In addition, HDL, S1P, and SPC failed to inhibit MCP1 production and ROS generation in aortas from S1P(3)- and SR-B1-deficient mice. CONCLUSIONS: HDL-associated lysosphingolipids inhibit NAD(P)H oxidase-dependent ROS generation and MCP-1 production in a process that requires coordinate signaling through S1P(3) and SR-B1 receptors.

Topically applied azaphenothiazines inhibit experimental psoriasis in mice.

The therapeutic efficacy of topically applied azaphenothiazine derivatives: 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5) in the amelioration of inflammatory symptoms of imiquimod-induced psoriasis in mice was investigated. Clobederm®, containing clobetasol propioniate, served as a reference drug. The application of the compounds led to thinning of the epidermis and reduction of the cell layers. The suppressive actions of the compounds were even stronger with regard to pathological changes of the dermis. The compounds also exerted generalized, anti-inflammatory effects by decreasing the number of circulating leukocytes, lowering subiliac lymph node weight and partially normalizing an altered blood cell composition. The changes in the composition of main cell types in the epidermis and dermis were less affected by the compounds. In addition, both compounds inhibited to a similar degree production of tumor necrosis factor α (TNF α) in human whole blood cell culture. Whereas compound 5 strongly inhibited IL-8 and CXCL10 chemokines in human keratinocytes - KERTr cell line, transfected with poly(I:C), the suppressive action of compound 4 in this model was weak. In addition, compound 5, but not compound 4, exhibited at low doses proapoptotic properties with regard to colonic cell lines. In summary, we demonstrated the therapeutic potential of two selected azaphenotiazines in the amelioration of the skin pathology elicited in a mouse experimental model of psoriasis.

Non-genomic effects of the NR4A1/Nur77/TR3/NGFIB orphan nuclear receptor.

The orphan nuclear receptor NR4A1/Nur77/TR3/NGFIB acts primarily as a transcription factor to regulate the expression of multiple genes. However, increasing research attention has recently been given to non-genomic activities of NR4A1. The first description of a non-genomic action of NR4A1 referred to the conversion of anti-apoptotic Bcl-2 into a pro-apoptotic protein by direct interaction with NR4A1. In response to certain apoptotic stimuli, NR4A1 translocates from the nucleus to the mitochondrial outer membrane (MOM) where it associates with Bcl-2 and thereby causes apoptosis. Afterwards, it appeared that NR4A1 could also bind and convert other anti-apoptotic Bcl-2 family members. The latest studies indicate a significant role of NR4A1 in the process of autophagy. For example, a new NR4A1-mediated pathway specific for melanoma cells has been described where NR4A1 interacts with the adenine nucleotide translocase 1 (ANT1) on the mitochondrial inner membrane (MIM) leading to induction of the autophagy pathway. Moreover, NR4A1 interaction with cytoplasmic p53 may also contribute to the induction of autophagy. In addition to mitochondria, NR4A1 could be translocated to the outer membrane of the endoplasmic reticulum (ER) and associate with Bcl-2 or translocon-associated protein subunit γ (TRAPγ) causing ER stress-induced apoptosis. NR4A1 also contributes to the proteasomal degradation of ß-catenin in colon cancer cells in vitro and in vivo, as well as to the stabilization of hypoxia-inducible factor-1α (HIF-1α) under non-hypoxic conditions. This review summarizes research findings on non-genomic effects of NR4A1 in normal and cancer cells.