The effect of the Fanconi anemia polypeptide, FAC, upon p53 induction and G2 checkpoint regulation

Fanconi anemia (FA) is an autosomal recessive disease marked by developmental defects, bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA cross-linking and alkylating agents and accumulate in the G2 phase of the cell cycle in response to these agents. FA cells also display genomic instability, suggesting a possible defect in the p53 pathway. To test the effect of heterologous expression of FAC cDNA on drug-induced cytotoxicity, G2 accumulation, and p53 induction in FA cells, we compared two isogenic FA cell lines: HSC536N (mock), a FA type C cell line sensitive to mitomycin C (MMC), and the same cell line transfected (corrected) with wild-type FAC cDNA (HSC536N [+FAC]). HSC536N (+FAC) cells showed a 30-fold increase in resistance to MMC concentration. Similarly, increases in resistance were observed following exposure to cisplatin, carboplatin, and cyclophosphamide. In addition, HSC536N (+FAC) cells showed a twofold lower G2 accumulation following MMC treatment. To analyze the possible interaction of FAC with the p53 pathway, we analyzed p53 induction in mock and corrected cell lines following exposure to MMC. HSC536N (mock) cells induced p53 at lower MMC concentrations than HSC536N (corrected). Caffeine, a known G2 checkpoint inhibitor, not only inhibited G2 accumulation seen in both cell lines but also caused the resistant HSC536N (+FAC) to become as sensitive to MMC as HSC536N (mock) cell line. We conclude that the FAC protein has a specific cytoprotective effect and may function as a cell cycle regulator of the G2 phase of the cell cycle.     

Soluble CD4, soluble CD8, soluble CD25, lymphopoieitic recovery, and endogenous cytokines after high-dose chemotherapy and blood stem cell transplantation

Mononuclear cell preparations from peripheral blood after mobilization with hematopoietic growth factors have been shown to induce accelerated neutrophil and platelet recovery as compared with that induced by autologous bone marrow transplantation after myeloablative chemotherapy. Because these mononuclear cell products contain many immunocompetent cells other than hematopoietic progenitors, these accessory cells might contribute to the rapid immunohematopoietic reconstitution. We have monitored the concentrations of soluble CD4 (sCD4), sCD8, and sCD25; the recovery of the lymphocyte subsets and of natural killer (NK) cells; and the endogenous levels of granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), IL-6, and granulocyte-macrophage-CSF (GM-CSF) in 12 patients who underwent high- dose chemotherapy supported by blood stem cells that were obtained by mobilization with chemotherapy and GM-CSF. The concentrations of both G- CSF and IL-6 peaked at 7 days after reinfusion of stem cells, and this transient elevation preceded the increase in the white blood cell count by approximately 5 to 7 days. The levels of sCD4 and sCD8 increased to a maximum on day 21, and the time to peak levels coincided with the maximum increase in white blood cell count, absolute neutrophil count, or lymphocytes. The levels of sCD25 were found to be elevated from day 7 to day 21. Statistically, the increases in sCD4, sCD8, sCD25, G-CSF, and IL-6 were highly significant, whereas there were no significant changes in IL-3 and GM-CSF. A rapid recovery of the NK activity was found in all 8 of the patients who could be monitored for this assay. Therefore, our study suggests that recovery of CD4+ cells, CD8+ cells, and NK activity coincided with that of neutrophils, which is preceded by a marked, but transient, elevation of IL-6 and G-CSF.     

Clostridium perfringens enterotoxin C‐terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy‐resistant ovarian cancer

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin‐3‐ and ‐4‐overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy‐naïve and chemotherapy‐resistant human ovarian cancer xenografts or patient‐derived xenografts (PDXs) were treated with the carboxyl‐terminal binding domain of the Clostridium perfringens enterotoxin (c‐CPE) conjugated to FITC (FITC‐c‐CPE) or the near‐infrared (NIR) fluorescent tag IRDye CW800 (CW800‐c‐CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC‐c‐CPE and the CW800‐c‐CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800‐c‐CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor‐to‐background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c‐CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c‐CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real‐time detection of micrometastatic ovarian disease overexpressing the Claudin‐3 and ‐4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.     

Aqueous Extract of Rosmarinus officinalis L. Inhibits Neutrophil Influx and Cytokine Secretion

Rosmarinus officinalis L. phenolic compounds have attracted considerable attention because of their antioxidant and antimicrobial properties, including its ability to treat inflammatory disorders. In this work, we investigated the in vivo and in vitro effects of R. officinalis aqueous extract on neutrophil trafficking from the blood into an inflamed tissue, on cell‐derived secretion of chemical mediators, and on oxidative stress. Anti‐inflammatory activity was investigated using carrageenan‐induced inflammation in the subcutaneous tissue of male Wistar rats orally treated with the R. officinalis extract (100, 200, or 400 mg/kg). The leukocyte influx (optical microscopy), secretion of chemical mediators (prostaglandin E2 (PGE2), TNF‐α, interleukin 6 (IL‐6), leukotriene B4 (LTB4), and cytokine‐induced neutrophil chemoattractant 1 by enzyme‐linked immunosorbent assay), and the anti‐oxidative profile (super oxide dismutase (SOD), glutathione peroxidase, and thiobarbituric acid reactive substance (TBARS) spectrophotometry) were quantified in the inflamed exudate. N‐Formyl‐methionine‐leucine‐phenylalanine‐induced chemotaxis, lipopolysaccharide‐induced NO₂^? production (Greiss reaction), and adhesion molecule expression (flow cytometry) were in vitro quantified using oyster glycogen recruited peritoneal neutrophils previous treated with the extract (1, 10, or 100 µg/mL). Animals orally treated with phosphate‐buffered saline and neutrophils incubated with Hank's balanced salt solution were used as control. R. officinalis extract oral treatment caused a dose‐dependent reduction in the neutrophil migration as well as decreased SOD, TBARS, LTB4, PGE2, IL‐6, and TNF‐α levels in the inflamed exudate. In vitro treatment with R. officinalis decreased neutrophil chemotaxis, NO₂^? production, and shedding of L‐selectin and β2 integrin expressions. Results here presented show that R. officinalis aqueous extract displays important in vivo and in vitro anti‐inflammatory actions by blocking pathways of neutrophil migration and secretion, suggesting its therapeutic application to acute inflammatory reactions. Copyright © 2014 John Wiley & Sons, Ltd.     

Uterine serous carcinoma: Molecular features,clinical management,and new and future therapies

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer. Multimodality treatment with surgery, radiotherapy, and chemotherapy is commonly used, given its propensity for extrauterine spread, distant recurrences, and poor prognosis. However, the use of molecularly-based therapy is expanding. Here, we review key molecular features of USC, discuss current management, and assess the landscape of novel therapies and combinations.     

Synthesis, anti-inflammatory activity and molecular docking studies of 2,5-diarylfuran amino acid derivatives

A series of 2,5-diaryl substituted furans functionalized with several amino acids were synthesized and evaluated as the cyclooxygenases COX-1 and COX-2 enzymes inhibitors. The proline-substituted compound inhibited PGE(2) secretion by LPS-stimulated neutrophils, suggesting selectivity for COX-2. Molecular docking studies in the binding site of COX-2 were performed.     

Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa

Because free factor VIIa is inactivated only very slowly by a plasma concentration of antithrombin III (AT III) even in the presence of heparin, it has been assumed that AT III plays no significant role in regulating the initiation of tissue factor-dependent blood coagulation. However, in the present study, we present evidence that factor VIIa bound to tissue factor, unlike free factor VIIa, is readily inactivated by AT III in the presence of heparin. In a reaction mixture containing calcium ions and approximately equimolar concentrations of relipidated tissue factor (8.9 nmol/L) and factor VIIa (10 nmol/L), AT III (100 micrograms/mL) plus heparin (1 U/mL) inhibited 50% of the factor VIIa coagulant activity of the reaction mixture within 5 minutes. AT III/heparin was also shown to inhibit the catalytic activity towards factor X of factor VIIa/tissue factor complexes formed on monolayers of an ovarian carcinoma cell line (OC-2008) that constitutively expresses surface membrane tissue factor. AT III, even in the absence of exogenously added heparin, substantially inhibited the functional activity of factor VIIa/cell surface tissue factor complexes on intact monolayers. AT III alone and AT III/heparin, to a greater extent, also inhibited factor VIIa on "nonfunctional" factor VIIa/tissue factor complexes on intact monolayers, with resultant inhibition of their expression of factor VIIa/tissue factor catalytic activity toward factor X after cell lysis. The potential physiologic significance of these findings is discussed.