Microbial growth-promotion activity of 3-hydroxymonoazine- and N-hydroxydiazine-type heterocycles.
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Three 3-hydroxymonoazine- and three N-hydroxydiazine-type heterocycles were tested whether they act as artificial siderophores toward Aureobacterium flavescens JG-9 (ATCC No. 25091). Among them, 1-hydroxy-3,5,6-trimethyl-2(1H)-pyrazinone (3) showed the highest growth-promotion activity comparable to desferrioxamine B (DFB), a natural trihydroxamate siderophore, at 48.5 microM or above, followed by 1-hydroxy-5,6-dimethyl-2(1H)-pyrazinone (2), 1-hydroxy-4,6-dimethyl-2(1H)-pyrimidinone (1), and 3-hydroxy-2-methyl-1-phenyl-4(1H)-pyridinone (6), while 3-hydroxy-1,2-dimethyl-4(1H)-pyridinone (5) did not show the bioactivity. These results are the first examples of N-hydroxydiazine-type heterocycles acting as artificial siderophores for A. flavescens JG-9. (+info)
Identification of Src transformation fingerprint in human colon cancer.
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We used a classical rodent model of transformation to understand the transcriptional processes, and hence the molecular and cellular events a given cell undergoes when progressing from a normal to a transformed phenotype. Src activation is evident in 80% of human colon cancer, yet the myriad of cellular processes effected at the level of gene expression has yet to be fully documented. We identified a Src 'transformation fingerprint' within the gene expression profiles of Src-transformed rat 3Y1 fibroblasts demonstrating a progression in transformation characteristics. To evaluate the role of this gene set in human cancer development and progression, we extracted the orthologous genes present on the Affymetrix Hu95A GeneChip (12k named genes) and compared expression profiles between the Src-induced rodent cell line model of transformation and staged colon tumors where Src is known to be activated. A similar gene expression pattern between the cell line model and staged colon tumors for components of the cell cycle, cytoskeletal associated proteins, transcription factors and lysosomal proteins suggests the need for co-regulation of several cellular processes in the progression of cancer. Genes not previously implicated in tumorigenesis were detected, as well as a set of 14 novel, highly conserved genes with here-to-fore unknown function. These studies define a set of transformation associated genes whose up-regulation has implications for understanding Src mediated transformation and strengthens the role of Src in the development and progression of human colon cancer. Supportive Supplemental Data can be viewed at http://pga.tigr.org/PGApubs.shtml. (+info)
Roles of activated Src and Stat3 signaling in melanoma tumor cell growth.
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Activation of protein tyrosine kinases is prevalent in human cancers and previous studies have demonstrated that Stat3 signaling is a point of convergence for many of these tyrosine kinases. Moreover, a critical role for constitutive activation of Stat3 in tumor cell proliferation and survival has been established in diverse cancers. However, the oncogenic signaling pathways in melanoma cells remain to be fully defined. In this study, we demonstrate that Stat3 is constitutively activated in a majority of human melanoma cell lines and tumor specimens examined. Blocking Src tyrosine kinase activity, but not EGF receptor or JAK family kinases, leads to inhibition of Stat3 signaling in melanoma cell lines. Consistent with a role of Src in the pathogenesis of melanoma, we show that c-Src tyrosine kinase is activated in melanoma cell lines. Significantly, melanoma cells undergo apoptosis when either Src kinase activity or Stat3 signaling is inhibited. Blockade of Src or Stat3 is also accompanied by down-regulation of expression of the anti-apoptotic genes, Bcl-x(L) and Mcl-1. These findings demonstrate that Src-activated Stat3 signaling is important for the growth and survival of melanoma tumor cells. (+info)
Molecular characterization and sensitivity of STI-571 (imatinib mesylate, Gleevec)-resistant, Bcr-Abl-positive, human acute leukemia cells to SRC kinase inhibitor PD180970 and 17-allylamino-17-demethoxygeldanamycin.
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Using human acute leukemia HL-60/Bcr-Abl (with ectopic expression of p185 Bcr-Abl) and K562 cells (with endogenous expression of p210 Bcr-Abl) subjected to a continuous selection pressure of up to 1.0 micro M Gleevec (imatinib mesylate, STI-571), we have isolated Gleevec-resistant K562 R (+Bcr-Abl), K562 R (-Bcr-Abl), and HL-60/Bcr-Abl R cells, which display disparate level and activity of Bcr-Abl tyrosine kinase (TK). As compared with their sensitive counterparts, Gleevec-resistant cell types were >/=5-fold resistant to Gleevec-induced apoptosis. Bcr-Abl protein levels were significantly increased in HL-60/Bcr-Abl R and K562 R (+Bcr-Abl) cells, but K562 R (-Bcr-Abl) cells showed a marked decline in the mRNA and protein levels and activity of Bcr-Abl. Bcr-Abl TK level and activity corresponded to the signal transducers and activators of transcription-5 DNA binding activity and up-regulation of heat shock protein 70 levels. The decline in Bcr-Abl expression and TK activity in K562 R (-Bcr-Abl) cells was associated with reduced AKT kinase and signal transducers and activators of transcription-5 DNA binding activities and increased sensitivity to the death ligand Apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand and 1-beta-D-arabinofuranosylcytosine-induced apoptosis. All Gleevec-resistant cell types were sensitive to 17-allylamino-17-demethoxygeldanamycin (17-AAG)- and PD180970 (a SRC and Bcr-Abl TK inhibitor)-induced apoptosis. Treatment with 17-AAG or PD180970 also induced apoptosis of CD34+ leukemic cells from three patients with chronic myeloid leukemia in blast crisis who had progressive leukemia while receiving Gleevec therapy. Taken together, these findings indicate that in addition to overexpression or mutations in Bcr-Abl, resistance to Gleevec may also develop due to a loss of Bcr-Abl expression. These findings also support the rationale to test the in vivo efficacy of 17-AAG and PD180970 against STI-571-resistant Bcr-Abl-positive acute leukemias. (+info)
Effect of imipramine on 1-methyl-4-phenylpyridinium ion-induced hydroxyl radical generation in rat striatum.
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We examined the effect of imipramine (a tricyclic antidepressant drug) on hydroxyl radical (.OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP(+)) in extracellular fluid of rat striatum, using a microdialysis technique. Imipramine enhanced the formation of.OH trapped as 2,3-dihydroxybenzoic acid (DHBA) induced by MPP(+) (5 mM). Introduction of imipramine (0.1, 0.5 and 1.0 mM) dose-dependently increased the level of dopamine (DA) release. Concomitantly, imipramine enhanced DA efflux and the level of DHBA induced by MPP(+), as compared with MPP(+)-treated control. When corresponding experiments were performed with reserpinized rats, there were small increases in the levels of DA and nonsignificant increase in the formation of DHBA. When iron (II) was administered to imipramine (1 mM)-treated animals, a marked elevation of DHBA was observed, compared with MPP(+)-only treated animals. A positive linear correlation was observed between iron (II) and DHBA (R(2)=0.985) in the dialysate. These results indicate that imipramine enhances generation of.OH induced by MPP(+) during enhanced DA overflow. (+info)
5-ethoxymethyl-7-fluoro-3-oxo-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2a]pyridine-4 -N-(2-fluorophenyl)carboxamide (RWJ-51204), a new nonbenzodiazepine anxiolytic.
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5-ethoxymethyl-7-fluoro-3-oxo-1,2,3,5-tetrahydrobenzo[4,5] imidazo[1,2a]pyridine-4-N-(2-fluorophenyl)carboxamide) (RWJ-51204) binds selectively and with high affinity (K(i) = 0.2-2 nM) to the benzodiazepine site on GABA(A) receptors. Considering the GABA shift, the intrinsic modulatory activity of RWJ-51204 is lower than that of full agonist anxiolytics (lorazepam, diazepam, alprazolam, and clonazepam) but similar to partial agonists (bretazenil, abecarnil, panadiplon, and imidazenil). RWJ-51204 was orally active in anxiolytic efficacy tests; pentylenetetrazole induced seizure inhibition in mice (ED(50) = 0.04 mg/kg), Vogel conflict in rats (ED(50) = 0.36 mg/kg), elevated plus-maze in rats (minimal effective dose = 0.1 mg/kg), and conflict in squirrel monkeys (ED(50) = 0.49 mg/kg). RWJ-51204 attenuated chlordiazepoxide-induced motor impairment in mice. Usually, RWJ-51204 was more potent than reference anxiolytics in rodent efficacy tests but less potent in monkey conflict. Usually, the slope of the dose-response lines for RWJ-51204 was more shallow than the full agonist anxiolytics but steeper than partial agonists in efficacy tests but typically shallow in tests for central nervous system side effects. In monkeys only mild or moderate sedation was observed at doses equivalent to 20 or 40 times the anxiolytic ED(50). RWJ-51204 fits into the partial agonist class of GABA(A) receptor modulators. In conclusion, RWJ-51204 exhibits a profile in in vitro experiments and in animal models, in mice and monkeys (but not in rats), suggesting that it has a profile of anxiolytic activity associated with less sedation, motor impairment, or muscle relaxation than currently available GABA(A) receptor modulators, i.e., the benzodiazepines. (+info)
Src activation regulates anoikis in human colon tumor cell lines.
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Src is a non-receptor protein tyrosine kinase, the expression and activity of which is increased in >80% of human colon cancers with respect to normal colonic epithelium. Previous studies from this and other laboratories have demonstrated that Src activity contributes to tumorigenicity of established colon adenocarcinoma cell lines. Src participates in the regulation of many signal transduction pathways, among which are those leading to cellular survival. In this study, we addressed the potential role of Src activation to a specific aspect of tumor cell survival, resistance to detachment-induced apoptosis (anoikis). Using five colon tumor cell lines with different biologic properties and genetic alterations, we demonstrate that expression and activity of Src corresponds with resistance to anoikis. Enforced expression of activated Src in subclones of SW480 cells (of low intrinsic Src expression and activity) increases resistance to anoikis; whereas decreased Src expression in HT29 cells (of high Src expression and activity) by transfection with anti-sense Src expression vectors increases susceptibility to anoikis. In contrast, increasing or decreasing Src expression had no effect on susceptibility to staurosporine-induced apoptosis in attached cells. PD173955, a Src family-specific tyrosine kinase inhibitor, increases the susceptibility of HT29 cells to anoikis in a dose- and time-dependent manner. Increasing Src expression and activity led to increased phosphorylation of Akt, a mediator of cellular survival pathways, whereas decreasing Src activity led to decreased Akt phosphorylation. In colon tumor cells with high Src activity, the PI3 kinase inhibitor LY 294002 sensitized cells to anoikis. These results suggest that Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis. (+info)
Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.
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Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC(50) of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML. (+info)