Synthesis and antiplatelet, antiinflammatory, and antiallergic activities of substituted 3-chloro-5,8-dimethoxy-1,4-naphthoquinone and related compounds

2-Amino (6), 2-alkylamino (7-8), 2-methoxy (9), 2-acetamido (10), and 5,8-diacetoxy (11) derivatives of the lead compound 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (4) were synthesized, together with 6,7-dichloro-5,8dimethoxy-1,4-naphthoquinone (5), a positional isomer of 4. Antiplatelet, antiinflammatory, and antiallergic activities were evaluated, and most compounds were quite potent in all assays. Compounds 5 and 9-11 were especially active; however, 5 was ineffective against neutrophil superoxide formation, and 10 was ineffective against mast cell degranulation.     

Vesicular anthracycline accumulation in doxorubicin-selected U-937 cells: participation of lysosomes

The U-A10 cell line, a doxorubicin-selected variant of human U-937 myeloid leukemia cells, exhibits a redistribution of anthracyclines into a expanded vesicular compartment. The acidic nature of this compartment was confirmed by vital staining with a pH sensitive dye, LysoSensor yellow/blue DND-160. Identification of the vesicular compartment was performed by immunofluorescence analysis. Staining for the LAMP-1 and LAMP-2 antigens showed that the vesicles are enlarged lysosomes that are eccentrically placed near the nucleus of U-A10 cells. By contrast, the expression of the multidrug resistance-associated protein and the P-glycoprotein was observed predominately on the plasma membrane of the drug-resistant cells. The accumulation of daunorubicin into cellular compartments was quantified using radiolabeled drug. Exposing cells to 3[H]-daunorubicin and then isolating intact nuclei showed that nuclei from U-A10 cells accumulated twofold to threefold less anthracycline than nuclei from U-937 cells. However, when nuclei were isolated first and then exposed to 3[H]-daunorubicin, little difference in net nuclear drug accumulation was detected. Cytoplasts prepared from U-A10 and U-937 cells were exposed to 3[H]-daunorubicin to measure cytoplasmic drug accumulation. At external daunorubicin concentrations of 100 ng/mL or higher, cytoplasts from U-A10 cells accumulated significantly more daunorubicin than cytoplasts from U-937 cells. Moreover, studies with the lysosomotropic agent chloroquine showed that U-A10 cells accumulated twofold more chloroquine and showed twofold enhanced sensitivity to this agent as compared with parental U-937 cells. Fluorescence microscopy showed that chloroquine affects vesicular anthracycline sequestration in U-A10 cells with an associated increase in daunorubicin nuclear fluorescence. Although chloroquine did not alter anthracycline cytotoxicity in parental cells, it restored daunorubicin and doxorubicin sensitivity to U-A10 cells. Taken together, these studies demonstrate that U-A10 cells exhibit a redistribution of the lysosomal compartment. The trapping of drug into an expanded acidic vesicular compartment results in decreased nuclear drug accumulation and decreased cytotoxicity. Lysosomotropic agents, such as chloroquine, warrant further study as modulators of this acquired drug-resistance phenotype.     

Shedding of CD44 from PMA-differentiated U-937 cells is enhanced by treatment with mineral particles

In this report, we show that enhanced shedding of CD44 might contribute to the down-regulation of this receptor observed after phagocytosis of MnO2 particles by PMA-differentiated U-937. The apparent Mr of the soluble CD44 detected in culture supernatants was slightly lower than that of the membrane form suggesting that shedding resulted from proteolytic cleavage. Increased shedding of CD44 was also noted with other mineral particles (chrysotile and DQ12) but to a lower extent whereas some (TiO2 and amosite) had no effect on this process. These results indicate that shedding enhancement was particle-specific rather than a general consequence of phagocytosis. The ability of the particles to enhance CD44 shedding was not directly dependent on their cytotoxic potency. Different patterns of reactivity were noted with CD11b, suggesting that the underlying mechanisms are specific.     

Modulation of heat-shock protein 70 (HSP70) gene expression by sodium butyrate in U-937 promonocytic cells: relationships with differentiation and apoptosis

The administration of sodium butyrate at 0.75 mM induced the functional differentiation of U-937 human promonocytic leukemia cells with negligible cell mortality. However, the drug rapidly caused cell death with characteristics of apoptosis when used at concentrations of 5 mM and above. In addition, butyrate stimulated the expression of the stress-responsive heat-shock protein 70 (HSP70) gene when applied at both differentiation-inducing and apoptosis-inducing concentrations. The induction of HSP70 by butyrate was inhibited by the simultaneous addition of cAMP-increasing agents (dibutyryl cAMP or the combination of forskolin plus theophylline). However, these agents did not prevent differentiation and only partially reduced apoptosis. Moreover, the DNA topoisomerase II inhibitor etoposide, which provoked U-937 cell differentiation and apoptosis with the same or greater efficiency than butyrate, failed to stimulate HSP70 expression. Finally, it was observed that cAMP-increasing agents also abrogated the induction of HSP70 and reduced the apoptosis caused by cadmium chloride, a typical inducer of the stress response. Taken together, these results indicate that HSP70 expression is not required for differentiation of promonocytic cells, as earlier proposed, and that butyrate probably triggers the stress response in these cells.     

Inhibition of human immunodeficiency type 1 virus replication in monocytic U-937 cells by superinfection with Chlamydia trachomatis

Status epilepticus is a neurological emergency associated with substantial morbidity and mortality. Experimental and clinical investigations suggest that prolonged seizure activity is associated with injury to vulnerable neurons. Compounds with neuroprotective properties may minimize such injury. Existing methods of inducing experimental status epilepticus result in seizure activity of variable duration and neuronal injury of variable degree. To minimize such variability, status epilepticus may be stopped with anticonvulsants, but this limits the ability to screen for independent neuroprotective properties. We have developed a simple and reliable non-pharmacological model of limbic status epilepticus in which the duration of status epilepticus is under direct experimental control. Status epilepticus is induced by continuous, unilateral hippocampal stimulation. Using this model, the degree of hippocampal pyramidal cell injury varies in direct proportion to status epilepticus duration across a range of 15-140 min. A progressive sequence of EEG changes unfolds with increasing status epilepticus duration, resembling that seen in other models. This model may serve as a reference against which the effects of potential neuroprotective compounds can be studied.     

Epinephrine-induced reduction in insulin receptor mRNA level and stability in U-937 human promonocytic cells

Abscess of the psoas muscle (PA) is every more frequently observed in recent years. The PA diagnosed in the authors' center over a period of 91 months are presented, analyzing the main clinical features, microbiologic causal agents, risk factors, treatment and the differences between primary and secondary PA. A total of 19 cases of which 14 were secondary PA (73.7%) and 5 primary PA (26.3%) were diagnosed. The main foci of infection of the former were the bone and the genitourinary tract, with intestinal infection being rare. The most frequent clinical data were lumbar pain with possible irradiation to the lower limb, fever, and leucocytosis with neutrophilia. Gram negative and enteric anaerobes were the bacteria most often identified, followed by Staphylococcus aureus and Mycobacterium tuberculosis. In a high percentage of patients (57.8%) a history of immunodeficiency was reported. In regard to treatment, surgical drainage was performed in 5 cases (26.3%), while ten cases (52.6%) were treated by DPCT. Four patients (21%) were exclusively treated with antibiotics. Recurrence was observed in three cases (15.3%) of the DPCT group requiring new drainage. Of all the cases, 18 were cured while one death occurred, being attributed to the underlying tumoral disease of advanced stage. The authors believe DPCT to be a good therapeutic option in both primary and secondary PA, thereby avoiding the risks of major surgery. In the cases with no underlying immunodeficiency the existence of secondary PA should be discarded as occurred in 7 out of 8 cases with no history of immunodeficiency in this series of patients.     

Induction of differentiation in mouse neuroblastoma cells

In the presence of 1--2% dimethyl sulfoxide (DMSO), mouse neuroblastoma cells are induced to differentiate morphologically as well as electrically. In addition, treatment of neurolbastoma cells with 2% DMSO results in a marked increase in the veratridine-activated K+ or Rb+ efflux. At 4% DMSO, neurite outgrowth is completely repressed and electrical activity is poorly developed. However, at this concentration, the cells have a relatively high resting potential which suggested that membrane components determining passive and active permeability properties are not necessarily under coordinated control. Induction of differentiation by 2% DMSO is also accompanied by an increase in a heavier molecular form of acetylcholinesterase sedimenting at 10.5S. The effect of other agents on the growth and differentiation of neuroblastoma cells is also presented.     

Induction of NG108-15 cells differentiation by human bone marrow stromal cells

The survival and differentiation of neuronal cells is dependent on factors such as neurotrophins, cytokines and components of extracellular matrix. Bone marrow stromal cells have been shown to support the growth and differentiation of neuroblastoma cells. In an attempt to study the effects of bone marrow stromal cells on neuronal differentiation, we have co-cultured neuroblastoma x glioma hybrid NG108-15 cells with human bone marrow stromal cells. After co-culturing, clones exhibiting morphological differentiated phenotype and high level of neurofilament expression were isolated. Interestingly, these clones maintain their ability to proliferate in contrast to differentiated NG108-15 cells induced by dibutyryladenosine 3',5'-cyclic monophosphate. These results suggested that bone marrow stromal cells can induce partial differentiation of NG108-15 cells.     

Induction of osteoblast differentiation in human adipose derived stem cells by lanthanum ions

Adipose derived stem cells represent a readily available source of adult stem cells for various biomedical applications. In this study, the proliferation and osteogenic differentiation potential of lanthanum nitrate（La3＋） on human adipose derived mesenchymal stem cells（hADSCs） were investigated for the first time and compared with that of dexamethasone（Dex）. Our results provided evidence that La3＋ at 50 μmol/L concentration promoted proliferation of hADSCs upto 2.4 fold when treated for 21 d in DMEM medium. Treatment of hADSCs with La3＋ containing osteogenic induction medium（α-MEM with ascorbic acid and β-glycerophosphate） for 7 d resulted in higher calcium deposition than that in the presence of Dex（0.1 μmol/L） as shown by Alizarin red S and von Kossa staining. Scanning electron micrographs also showed more extracellular matrix mineralization in the presence of La3＋. After 7 d of treatment with La3＋（10 μmol/L） the expression of RunX2, osteopontin（OP） and osteocalcin（OC） increased 3.4, 5.5 and 2.7 fold respectively. Our results provided evidence that in the presence of La3＋ osteogenic differentiation occurred earlier than that in the presence of Dex.     

Proteasome (prosome) subunit variations during the differentiation of myeloid U937 cells

20S proteasomes (prosomes/multicatalytic proteinase) are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol-myristate-acetate or retinoic acid plus 1,25-dihydroxy-cholecalciferol by western blot, flow cytometry and immuno-fluoresence. p25K (C3), p27K (IOTA) and p30/33K (C2) subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA + VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno-fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA + VD-induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA-induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non-induced cells; these membrane proteins disappeared when treated with RA + VD, whereas some increased on PMA-induced cells. The differential changes in the distribution and type of proteasomes in RA + VD and PMA-induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.     

Induction of apoptosis in leukemia U937 cells by 5'-deoxy-5'-methylthioadenosine, a potent inhibitor of protein carboxylmethyltransferase

We found dramatic changes in leukemia U937 cells treated with 5'-deoxy-5'-methylthioadenosine (MTA), a potent inhibitor of protein carboxylmethyltransferase (protein methylase II). Initiation of cell death was observed by 1 day after MTA treatment, and it was induced in a dose- and time-dependent manner. However, cell viability measured by trypan blue exclusion was not consistent with the actual percentage of cell death. These results indirectly indicated that the type of cell death is apoptosis rather than necrosis. Nuclear fragmentation and DNA condensation of MTA-treated U937 cells were analyzed by both fluorescent and electron microscopy. MTA-treated cells first began to arrest in the M phase of the cell cycle, and they then exhibited a mitotic-like nuclear fragmentation process with partially membraneless chromatin. Furthermore, agarose gel electrophoresis of DNA extracted from cells treated with MTA showed DNA laddering with production of fragments of approximately 200 bp multiples. These studies indicated that cell death induced by MTA has the characteristics of apoptosis, although nuclear fragmentation is atypical. It seems likely that the process of apoptosis in U937 cells induced by MTA correlates with incomplete assembly of the nuclear envelope, since MTA itself could inhibit the carboxylmethylation of nuclear lamin B and delayed incorporation of lamin B into the nuclear envelope.     

Induction of apoptosis and potentiation of TNF- and Fas-mediated apoptosis in U937 cells by the xanthogenate compound D609

The purpose of Study 1 was to examine the effect of dietary soy on the progression of MDA-MB-435 human breast cancer cell solid tumors in nude mice. When toasted soy chips were fed at levels of 5%, 10%, or 20% (wt/wt) in a high-fat, linoleic acid-rich diet for 12 weeks, there was a trend for larger mammary fat pad tumors to occur with increasing soy intake. However, compared with the controls the severity of macroscopic lung metastasis was reduced significantly in the groups fed 10% and 20% soy. Study 2 compared the effects of diets containing 23% corn oil (CO), 18% menhaden oil (MO) + 5% CO, 18% MO + 5% CO + 10% soy chips, and MO or soy-supplemented diets + indomethacin treatment in the same animal model. Feeding the 18% MO diet without soy or indomethacin reduced primary tumor growth; statistically significant effects were not observed in any of the other groups. All three of the groups with MO supplementation showed a reduction in the occurrence and severity of macroscopic lung metastases, together with the expected decreases in tumor prostaglandin E levels. These effects were most pronounced when MO was combined with indomethacin treatment. When indomethacin was given with dietary soy, the previously reported suppressive effect of the cyclooxygenase inhibitor on MDA-MB-435 cell tumor progression was lost, despite reductions in tumor prostaglandin E concentrations.     

Induction of pulmonary allergic responses by antigen-specific Th2 cells

The development of pulmonary allergic responses was examined in mice following pulmonary transfer of Ag (conalbumin)-specific Th2 cells. The levels of serum-specific IgE, cellular infiltrates, airway mucus goblet cells, and airway responsiveness were analyzed and compared with those in Ag-sensitized and -challenged mice. Pulmonary transfer of the conalbumin-specific Th2 clone (D10) induced, in an Ag-specific manner, high levels of the Th2 cytokines IL-4 and IL-5 in the bronchoalveolar lavage fluids and mucosal eosinophils, concomitant with an increase in airway responsiveness. The D10 cell-induced responses were seen in the absence of serum specific IgE. In the presence of Ag, the transferred D10 cells not only remained in the lungs, but also increased in number 72 h post-cell transfer. Although significantly higher levels of IL-4 and IL-5 in the bronchoalveolar lavage fluids were found in D10-transferred mice, the levels of pulmonary eosinophilia, mucus goblet cells, and airway responsiveness were significantly lower than those in Ag-sensitized and -challenged mice. These results demonstrate that although Ag-specific activation of Th2 cells at mucosal sites is able to mediate the recruitment of eosinophils and the subsequent induction of airway hyper-responsiveness, the more severe pulmonary allergic responses were observed only in mice sensitized and challenged with Ag.