Gallic acid sensitizes paclitaxel-resistant human ovarian carcinoma cells through an increase in reactive oxygen species and subsequent downregulation of ERK activation.

Paclitaxel (PTX) is currently used as a front-line chemotherapeutic agent for several types of cancer, including ovarian carcinoma; however, PTX-resistance frequently arises through multiple mechanisms. The development of new strategies using natural compounds and PTX in combination has been the aim of several prior studies, in order to enhance the efficacy of chemotherapy. In this study, we found the following: (i) gallic acid (GA), a phenolic compound, potentiated the capacity of PTX to decrease proliferation and to cause G2/M cycle arrest in the PTX-resistant A2780AD ovarian cancer cell line; (ii) GA exerted a pro-oxidant action by increasing the production of reactive oxygen species (ROS), and co-treatment with the antioxidant agent N­acetyl-L­cysteine (NAC) prevented GA+PTX-induced cell proliferation inhibition and G2/M phase arrest; (iii) PTX stimulated ERK phosphorylation/activation, and co-treatment with the MEK/ERK inhibitor PD98049 potentiated the proliferation inhibition and G2/M phase arrest; (iv) and finally, GA abrogated the PTX-induced stimulation of ERK phosphorylation, a response that was prevented by co-treatment with NAC. Taken together, these results indicate that GA sensitizes PTX-resistant ovarian carcinoma cells via the ROS­mediated inactivation of ERK, and suggest that GA could represent a useful co-adjuvant to PTX in ovarian carcinoma treatment.

Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response.

Endoglin is an auxiliary receptor for members of the TGF-ß superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-ß receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Eng(fl/fl)LysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Eng(fl/fl)LysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-ß1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients.

L718P mutation in the membrane-proximal cytoplasmic tail of beta 3 promotes abnormal alpha IIb beta 3 clustering and lipid microdomain coalescence, and associates with a thrombasthenia-like phenotype.

BACKGROUND: Support for the role of transmembrane and membrane-proximal domains of alpha IIb beta 3 integrin in the maintenance of receptor low affinity comes from mutational studies showing that activating mutations can induce constitutive bi-directional transmembrane signaling. DESIGN AND METHODS: We report the functional characterization of a mutant alpha IIb beta 3 integrin carrying the Leu718Pro mutation in the membrane-proximal region of the beta 3 cytoplasmic domain, identified in heterozygosis in a patient with a severe bleeding phenotype and defective platelet aggregation and adhesion. RESULTS: Transiently transfected cells expressed similar levels of normal and mutant alpha IIb beta 3, but surface expression of mutant alpha v beta 3 was reduced due to its retention in intracellular compartments. Cells stably expressing mutant alpha IIb beta 3 showed constitutive binding to soluble multivalent ligands as well as spontaneous fibrinogen-dependent aggregation, but their response to DTT was markedly reduced. Fibrinogen-adherent cells exhibited a peculiar spreading phenotype with long protrusions. Immunofluorescence analysis revealed the formation of alpha IIb beta 3 clusters underneath the entire cell body and the presence of atypical high-density patches of clustered alpha IIb beta 3 containing encircled areas devoid of integrin that showed decreased affinity for the fluorescent lipid analog DiIC(16) and were disrupted in cholesterol-depleted cells. CONCLUSIONS: These findings are consistent with an important role of the membrane-proximal region of beta 3 in modulating alpha IIb beta 3 clustering and lateral redistribution of membrane lipids. Since the beta 3 mutant was associated with a thrombasthenic phenotype in a patient carrying one normal beta 3 allele, these results support a dominant role of clustering in regulating integrin alpha IIb beta 3 functions in vivo.

Involvement of ERK1/2, p38 and PI3K in megakaryocytic differentiation of K562 cells.

Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3-kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12-myristate 13-acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA-treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbalpha expression, DNA endoreduplication, and formation of platelet-like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down-regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.

Possible role for cellular FXIII in monocyte-derived dendritic cell motility.

The A subunit of plasma factor XIII (FXIII-A) is thought to function as an intracellular transglutaminase (TG) in the monocyte/macrophage lineage to regulate certain intracellular processes involving cytoskeleton remodeling, but its precise role and the functional consequences of its absence remain poorly understood. In the present study, we show that cellular FXIII (cFXIII) expression is largely upregulated during in vitro differentiation of monocytes into dendritic cells (DCs). Monodansyl-cadaverine, a competitive substrate of TG activity, inhibited basal and CCL19-stimulated migration of mature DCs. In agreement, FXIII-A-deficient DCs showed a reduced chemotactic response to CCL19. Consistent with these findings, CHO cells stably expressing human FXIII-A showed enhanced motility in transwell and scratch-wound assays. These cells displayed increased formation of membrane blebs, dynamic cell protrusions implicated in cell movement that were also observed in DCs. The results provide evidence suggesting that upregulation of cFXIII in DCs has a role in regulating cell motility.

Characterization of progressive periodontal lesions in chronic periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells.

BACKGROUND AND AIMS: Periodontitis is an infection with an episodic nature of tissue support destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells in periodontal sites characterized by active periodontal connective tissue destruction. MATERIAL AND METHOD: Fifty-six patients with moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth > or =5 mm, clinical attachment level > or =3 mm and radiographic bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for nuclear factor kappa B-ligand (RANK-L), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor-alpha (TNF-alpha), IL-1beta, MMP-13, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was performed using the Stata 7.0 software. Data were expressed as mean+/-SD and paired samples t-test and chi(2) tests were used. RESULTS: Higher RANK-L, IL-1beta and MMP-13 activity levels were observed in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the number of CD4(+) T were higher in active than in inactive sites (p>0.05). CONCLUSION: The detection of periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the destruction of the supporting tissues of the tooth.

Type II Glanzmann thrombasthenia in a compound heterozygote for the alpha IIb gene. A novel missense mutation in exon 27.

BACKGROUND AND OBJECTIVES: Glanzmann thrombasthenia is an autosomal recessive bleeding disorder characterized by a life-long hemorrhagic tendency and absent or severely reduced platelet aggregation in response to agonists, caused by quantitative or qualitative abnormalities in the platelet fibrinogen receptor, integrin alphaIIb beta3. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a patient with type II Glanzmann thrombasthenia. DESIGN AND METHODS: The expression of platelet alphaIIb beta3 was determined by flow cytometry and western blotting. Mutations were identified by sequencing both cDNA and genomic DNA. Functional characterization was assessed by exontrap and transient transfection analysis. RESULTS: Flow cytometry and western blot analysis revealed markedly reduced levels of platelet alphaIIb beta3, which may account for the residual fibrinogen binding detected upon platelet activation. Sequencing of genomic DNA revealed the presence of two mutations in the alphaIIb gene: a C1750T transition in the last codon of exon 17 changing Arg553 to STOP, and a C2829T transition in exon 27 that changes Pro912 to Leu. Sequence analysis of reversely transcribed alphaIIb mRNA did not detect cDNA from the C1750T mutant allele, and revealed a significant increase of the physiological splicing out of exon 28 in the cDNA carrying the C2829T mutation. Transient expression of [912Leu]alphaIIb in CHO-b3 cells showed a marked reduction in the rate of surface expression of alphaIIb beta3. INTERPRETATION AND CONCLUSIONS: The results suggest that the thrombasthenic phenotype is the result of reduced availability of alphaIIb-mRNA, enhanced expression of exon 28-deleted transcripts, and defective processing of [912Leu]alphaIIb.

Effect of metamizol on promyelocytic and terminally differentiated granulocytic cells. Comparative analysis with acetylsalicylic acid and diclofenac.

Metamizol is an analgesic and antipyretic agent that can induce agranulocytosis in certain patients. However, its effects on granulocyte viability and differentiation have been poorly evaluated. Here we analysed the effects of metamizol and its active metabolite, 4-methylaminoantipyrine (MAA), on the viability of HL60 promyelocytes and their dimethyl sulphoxide-induced differentiated granulocytes. Metamizol and MAA at 75 microM (above the peak of plasmatic concentration after 2g intake) did not alter granulocytic differentiation of HL60 cells. Only at concentrations above 100 microM, well over the pharmacological range, metamizol-induced apoptosis in about 30% of the HL60 promyelocytes, while HL60-granulocytic terminally differentiated cells were more resistant to this apoptotic action. When the effects of metamizol were compared with those of acetylsalicylic acid (ASA) and diclofenac on cell viability, at equivalent concentrations used in analgesic and antipyretic therapy (75 microM for metamizol, and ASA and 3 microM for diclofenac) their apoptotic effects were similar. Again, the HL60 promyelocytes were more sensitive to apoptosis than granulocytic differentiated cells, as measured by the percentage of sub-G(1) cells detected by flow cytometry and by determination of caspase activity as a function of poly(ADP-ribose) polymerase cleavage. Furthermore, when human blood-derived granulocytes were treated with metamizol, MAA, and ASA at 75 microM or diclofenac at 3 microM, less than 10% of apoptotic granulocytes were detected, whereas at toxicological/suprapharmacological concentrations (10mM), about 90% of granulocytes were apoptotic. These results demonstrate that metamizol, MAA, ASA, and diclofenac, at pharmacological concentrations, neither affect the granulocytic differentiation process nor induce relevant apoptosis on terminally differentiated granulocytes.