Chloroethyl urea derivatives block tumour growth and thioredoxin-1 nuclear translocation

Aryl chloroethyl ureas (CEUs) are new protein alkylating agents exhibiting anticancer activity both in vitro and in vivo. We report herein that 14C-labeled CEU derivatives, designated CEU-025 and CEU-027, covalently bind to thioredoxin-1 (TRX1). Covalent binding of these molecules slightly decreases the disulfide-reducing activity of recombinant TRX1, when compared with the effect of strong thioalkylating agents such as N-ethylmaleimide. Moreover, site-directed mutagenesis and diamide competition assays demonstrated that TRX1 cysteinyl residues are not the prime targets of CEUs. CEU-025 abrogates the nuclear translocation of TRX1 in human cancer cells. In addition, we show that CEU-025 can block TRX1 nuclear translocation induced by cisplatin. Unexpectedly, pretreatment with sublethal CEU-025 concentrations that block TRX1 nuclear translocation protected the cells against cisplatin cytotoxicity. Overexpression of TRX1 in HT1080 fibrosarcoma cells attenuated CEU-025 cytotoxicity, while its suppression using TRX1-specific siRNA increased the effects of CEU-025, suggesting that loss of function of TRX1 is involved, at least in part, in the cytotoxic activity of CEU-025. These results suggest that CEU-025 and CEU-027 exhibit anticancer activity through a novel, unique mechanism of action. The importance of TRX1 and the dependence of the cytotoxicity of CEU-025 and CEU-027 on TRX1 intracellular localization are also discussed.     

Cycloalkyl-substituted aryl chloroethylureas inhibiting cell cycle progression in G0/G1 phase and thioredoxin-1 nuclear translocation

1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.     

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives

The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling.     

Calcium and cell cycle progression: possible effects of external perturbations on cell proliferation.

Exit from the phase of cellular division appears to be driven by a calcium signal that triggers a cascade of events leading to the completion of mitosis. Here we propose a model that relates the dynamics of cytosolic calcium to progression through mitosis, G1 and G2 phases of the cell cycle. To this end, the assumption has been made that the transient rise ir cytosolic calcium concentration during mitosis is induced by inositol(1,4,5)triphosphate (IP3), which in turn is released at high levels of mitosis-promoting factor (MPF). On this basis, a system of ordinary differential equations is proposed to simulate the evolution of ten cell-cycle-specific molecular species, including cyclins A and B, MPF, IP3, Ca2+, the CaMKII holoenzyme, and the ubiquitination complex. The influence on the cell proliferation capacity exerted by external perturbations, like calcium microinjections, depletion of intracellular calcium stores, electromagnetic fields, or stimulation/inhibition of different calcium currents through the plasma membrane, can be studied by appropriate modulation of the parameters involved in the signal transduction pathway.     

Regulation of apoptosis and cell cycle progression by MCL1. Differential role of proliferating cell nuclear antigen

MCL1 (ML1 myeloid cell leukemia 1), a Bcl-2 (B- cell lymphoma-leukemia 2) homologue, is known to function as an anti-apoptotic protein. Here we show in vitro and in vivo that MCL1 interacts with the cell cycle regulator, proliferating cell nuclear antigen (PCNA). This finding prompted us to investigate whether MCL1, in addition to its anti-apoptotic function, has an effect on cell cycle progression. A bromodeoxyuridine uptake assay showed that the overexpression of MCL1 significantly inhibited the cell cycle progression through the S-phase. The S-phase of the cell cycle is also known to be regulated by PCNA. A mutant of MCL1 that lacks PCNA binding (MCL1(Delta)(4A)) could not inhibit cell cycle progression as effectively as wild type MCL1. In contrast, MCL1(Delta)(4A) retained its anti-apoptotic function in HeLa cells when challenged by Etoposide. In addition, the intracellular localization of MCL1(Delta)(4A) was identical to that of wild type MCL1. An in vitro pull-down assay suggested that MCL1 is the only Bcl-2 family protein to interact with PCNA. In fact, MCL1, not other Bcl-2 family proteins, contained the PCNA-binding motif described previously. Taken together, MCL1 is a regulator of both apoptosis and cell cycle progression, and the cell cycle regulatory function of MCL1 is mediated through its interaction with PCNA.     

Fumonisin B1 influenced the effects of arachidonic acid, prostaglandins E2 and A2 on cell cycle progression, apoptosis induction, tyrosine- and CDC2-kinase activity in oesophageal cancer cells

In a previous study, we showed that, of a group of lipids including arachidonic acid (AA), prostaglandins E2 (PGE2) and A2 (PGA2), PGA2 had the most marked effect on the inhibition of cell growth, activation of tyrosine kinase activity, lowering of the number of G1-phase cells, and induction of p53 levels in oesophageal carcinoma (WHCO3) cells. No significant effects by the three lipids were seen in normal monkey kidney cells. In the present study, the effects of the inhibitor of ceramide synthesis, fumonisin B1 (FB1), a metabolite of Fusarium verticillioides (= F. moniliforme) which is implicated in the high incidence of oesophageal cancer, were determined on AA, PGE2 and PGA2 WHCO3 treated cells. In the presence of FB1, the lipid-enhanced tyrosine kinase activity was lowered. Flow cytometric and morphological studies showed that FB1 lowered the marked apoptosis induced by especially PGA2. FB1, however, in combination with AA, PGE2 or PGA2 increased the number of G2/M cells. AA>PGE2>PGA2 alone decreased CDC2-kinase activity, but, in the presence of FB1, CDC2-kinase activity was significantly increased. The PGA2- and AA-induced p53 levels were lowered in the presence of FB1. We concluded that FB1 diminished the cytotoxic effects of the lipids on oesophageal tumour cells.     

HsRec2/Rad51L1, a protein influencing cell cycle progression, has protein kinase activity

Human Rec2/Rad51L1 is a member of the Rad51 family of proteins. Although recombinase activity, typical of this family, could not be established, its overexpression in mammalian cells has been shown to cause a delay in G1. Moreover, since hsRec2/Rad51L1 has been found to be induced by both ionizing and UV irradiation, it is likely that hsRec2/Rad51L1 is elevated following any DNA damage and causes a G1 delay to allow time for DNA repair to occur. Limited homology with catalytic domains X and XI of protein kinase A suggested that kemptide, an artificial substrate containing one phosphorylatable residue, a serine, might serve as a substrate for hsRec2/Rad51L1. Here, we report that hsRec2/Rad51L1 can phosphorylate kemptide, as well as myelin basic protein, p53, cyclin E, and cdk2, but not a peptide substrate containing tyrosine only. The finding that hsRec2/Rad51L1 exhibits protein kinase activity is a first step toward identifying a mechanism whereby this protein affects the cell cycle.     

Design, microwave-assisted synthesis, and spasmolytic activity of 2-(alkyloxyaryl)-1H-benzimidazole derivatives as constrained stilbene bioisosteres

A simple, fast, and efficient method for the preparation of several 2-(alkyloxyaryl)-1H-benzimidazole derivatives is reported. Compounds were synthesized through a rapid one-pot three component reaction via microwave irradiation, starting from commercially available aldehydes and o-phenylenediamine, in the presence of Na(2)S(2)O(5) and solvent-free conditions. The design of these compounds explore the hypothesis that the stilbene framework could be mimicked with an appropriate 2-(Alkyloxyphenyl)benzimidazole scaffold. This framework has a similar structural motif as the 6-phenylnaphthalene and behaves like stilbene bioisosteres. The spasmolytic activity of these compounds was recorded using isolated rat ileum test. Compound 12 was the most active of the series, showing an IC(50) of 1.19 microM.     

Nuclear translocation of phospholipase C-delta1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate

Nuclear phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, fluctuate throughout the cell cycle and are linked to proliferation and differentiation. Here we report that phospholipase C-delta(1) accumulates in the nucleus at the G(1)/S boundary and in G(0) phases of the cell cycle. Furthermore, as wild-type protein accumulated in the nucleus, nuclear phosphatidylinositol 4,5-bisphosphate levels were elevated 3-5-fold, whereas total levels were decreased compared with asynchronous cultures. To test whether phosphatidylinositol 4,5-bisphosphate binding is important during this process, we introduced a R40D point mutation within the pleckstrin homology domain of phospholipase C-delta(1), which disables high affinity phosphatidylinositol 4,5-bisphosphate binding, and found that nuclear translocation was significantly reduced at G(1)/S and in G(0). These results demonstrate a cell cycle-dependent compartmentalization of phospholipase C-delta(1) and support the idea that relative levels of phosphoinositides modulate the portioning of phosphoinositide-binding proteins between the nucleus and other compartments.     

New V1a receptor antagonist. Part 1. Synthesis and SAR development of urea derivatives

Solid preclinical evidence links vasopressin to social behavior in animals, so, extensive work has been initiated to find new vasopressin V1a receptor antagonists which can improve deteriorated social behavior in humans and can treat the core symptoms of autistic behavior, as well. Our aim was to identify new chemical entities with antagonizing effects on vasopressin V1a receptors. Starting from a moderately potent HTS hit (7), we identified a molecule (49) having nanomolar binding strength and functional activity, which is in the same range as the potency of clinically tested V1a antagonists.     

Biological activity of usnic acid and its derivatives: Part 2. effects on higher organisms. Molecular and physicochemical aspects

Usnic acid (UA) is a commercially available lichen metabolite. Its biological activity is diverse, and it is of interest for pharmacopoeia. The second part of the review is dedicated to the biological action of UA and its derivatives on higher organisms. Effects exhibited by UA at the cellular level and the molecular and physicochemical aspects of its biological activity are considered. Special attention is placed on the possibility of modifying the biological activity of UA by change of its bioavailability or modification of its molecular structure.     

Unconventional effects of UVA radiation on cell cycle progression in S. pombe

UVA radiation, the most abundant solar UV radiation reaching Earth’s surface, induces oxidative stress through formation of reactive oxygen species (ROS) that can damage different cell components. Because of the broad spectrum of the possible targets of ROS, the cellular response to this radiation is complex. While extensive studies have allowed dissecting the effects of UVB, UVC and gamma radiations on cell cycle progression, few studies have dealt with the effect of UVA so far. Here we use Schizosaccharomyces pombe as a model organism to study biological effects of UVA radiation in living organisms. Through analysis of cell cycle progression in different mutant backgrounds we demonstrate that UVA delays cell cycle progression in G2 cells in a dose dependent manner. However, despite Chk1 phosphorylation and in contrast to treatments with others genotoxic agents, this cell cycle delay is only partially dependent on DNA integrity checkpoint pathway. We also demonstrate that UVA irradiation of S phase cells slows down DNA replication in a checkpoint independent manner, activates Chk1 to prevent entry into abnormal mitosis and induces formation of Rad22 (homologue to human Rad52) foci. This indicates that DNA structure integrity is challenged. Furthermore, the cell cycle delay observed in checkpoint mutants exposed to UVA is not abolished when stress response pathway is inactivated or when down regulation of protein synthesis is prevented. In conclusion, fission yeast is a useful model to dissect the fundamental molecular mechanisms involved in UVA response that may contribute to skin cancer and aging.     

The novel 3,4-dihydropyrimidin-2(1H)-one urea derivatives of N-aryl urea: synthesis, anti-inflammatory, antibacterial and antifungal activity evaluation

A series of novel 3,4-dihydropyrimidin-2(1H)-one urea derivatives of biological interest were prepared by sequential Bigineli’s reaction, reduction followed by reaction of resulting amines with different arylisocynates. All the synthesized (1-23) compounds were screened against the pro-inflammatory cytokines (TNF-α and IL-6) and antimicrobial activity (antibacterial and antifungal). Biological activity evaluation study reveled that among all the compounds screened, compounds 12 and 17 found to have promising anti-inflammatory activity (68-62% TNF-α and 92-86% IL-6 inhibitory activity at 10 μM). Interestingly compounds 3, 4, 5, 6, 15, 22 and 23 revealed promising antimicrobial activity at MIC of 10-30 μg/mL against selected pathogenic bacteria and fungi.     

Oligonucleotide biological activity: Relationship to the cell cycle and nuclear transport

Previous studies suggest that oligodeoxynucleotide (ODN) cellular uptake is cell cycle-dependent which may have important implications in cancer cell targeting. To further our understanding of ODN transport and activity, this study examines the relationships between the cell cycle, ODN cellular uptake, intracellular transport, and activity. An antisense c-myc ODN 21-mer was used to study ODN cellular uptake in Rauscher erythroleukemia cells synchronized by either chemical methods or flow cytometry. ODN uptake was examined using subcellular fractionation and confocal fluorescence microscopy. Western blot analysis was used to measure ODN-mediated decreases in c-myc protein levels. Intracellular ODN distribution and extent of uptake was influenced by the phase of the cell cycle, but the mechanism of uptake was not. The relative activity of the antisense ODN was positively correlated to ODN distribution to the cytosol, but negatively correlated to total cellular uptake. Although ODN total cellular uptake is positively influenced by the cell cycle, retention of the ODN in the cytosol (presumably extra-vesicularly) appeared to be relevant in determining the activity of an antisense ODN. Novel methods to target cytosol-acting drugs to the cytoplasm may therefore be warrented.     

Radiosensitization of glioma cells by TP53-induced glycolysis and apoptosis regulator knockdown is dependent on thioredoxin-1 nuclear translocation

TP53-induced glycolysis and apoptosis regulator (TIGAR) knockdown is proven to radiosensitize glioma cells, but the mechanisms are not fully understood. Thioredoxin-1 (TRX1) is a redox-sensitive oxidoreductase, which plays critical roles in DNA damage signal transduction via nuclear translocation in irradiated cells. Because the TRX1-dependent DNA damage signaling pathway relies on NADPH to maintain the reduced state of TRX1, and TIGAR functions to increase NADPH generation under oxidative stress, in this study, the role of TRX1 in TIGAR abrogation-induced radiosensitization was investigated. It was demonstrated that ionizing radiation (IR)-induced nuclear translocation of TRX1 was significantly inhibited by TIGAR interference and reversed by wild-type (WT)-TRX1 overexpression. In addition, WT-TRX1 overexpression could accelerate the process of DNA damage repair postponed by TIGAR knockdown in irradiated glioma cells. The reduction process of IR-oxidized TRX1 was also delayed by TIGAR knockdown but restored by WT-TRX1 overexpression. Therefore, we conclude that TIGAR knockdown-induced radiosensitization of glioma cells may be dependent on the inhibition of TRX1 nuclear translocation.     

Resveratrol inhibits MAPK activity and nuclear translocation in coronary artery smooth muscle: reversal of endothelin-1 stimulatory effects.

In porcine coronary arteries, short-term treatment with resveratrol (RSVL) substantially inhibited MAPK activity (IC50 = 37 microM); and immunoblot analyses revealed consistent reduction in the phosphorylation of ERK-1/-2, JNK-1 and p38, at active sites. Endothelin-1 (ET-1), a primary antecedent in coronary heart diseases, enhanced MAPK activity, phosphorylation and nuclear translocation in a concentration-responsive but RSVL-sensitive manner. RSVL had no effect on basal or forskolin-stimulated cAMP levels, a known downregulator of the MAPK cascade. Likewise, inhibition of MAPK by RSVL was not reversed by the estrogen receptor blockers tamoxifen and ICI-182,780. Conversely, RSVL remarkably attenuated basal and ET-1-evoked protein tyrosine phosphorylation. Because MAPKs promote smooth muscle proliferation and contraction, their current inhibition may contribute to the putative protection by RSVL against coronary heart diseases. These effects apparently do not involve interaction with estrogen receptors.