Non-steroidal anti-inflammatory drugs decrease E2F1 expression and inhibit cell growth in ovarian cancer cells.

Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs) drugs is associated with a reduced risk of various cancers. In addition, in vitro and experiments in mouse models have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. However, there are limited preclinical studies investigating the effects of NSAIDs in ovarian cancer. Here, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in ovarian cancer cell lines and in a xenograft mouse model. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and apoptosis. In addition, diclofenac and indomethacin reduced tumor volume in a xenograft model of ovarian cancer. To identify possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, we performed microarray analysis of ovarian cancer cells treated with indomethacin or diclofenac. Interestingly, several of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1. E2F1 was downregulated at the mRNA and protein level upon treatment with diclofenac and indomethacin, and overexpression of E2F1 rescued cells from the growth inhibitory effects of diclofenac and indomethacin. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer in vitro and in vivo and the effects of NSAIDs may be mediated, in part, by downregulation of E2F1

Genome Analysis of F. nucleatum sub spp vincentii and Its Comparison With the Genome of F. nucleatum ATCC 25586

We present the draft genome sequence and its analysis for Fusobacterium nucleatum sub spp. vincentii (FNV), and compare that genome with F. nucleatum ATCC 25586 (FN). A total of 441 FNV open reading frames (ORFs) with no orthologs in FN have been identified. Of these, 118 ORFs have no known function and are unique to FNV, whereas 323 ORFs have functional orthologs in other organisms. In addition to the excretion of butyrate, H(2)S and ammonia-like FN, FNV has the additional capability to excrete lactate and aminobutyrate. Unlike FN, FNV is likely to incorporate galactopyranose, galacturonate, and sialic acid into its O-antigen. It appears to transport ferrous iron by an anaerobic ferrous transporter. Genes for eukaryotic type serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A are found in FNV but not in FN. Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in FNV. ORFs for ethanolamine utilization, thermostable carboxypeptidase, γ glutamyl-transpeptidase, and deblocking aminopeptidases are absent from FNV. FNV, like FN, lacks the classical catalase-peroxidase system, but thioredoxin/glutaredoxin enzymes might alleviate oxidative stress. Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present. These capabilities allow Fusobacteria to survive in a mixed culture in the mouth

NSAIDs decrease the viability of ovarian cancer cells in a dose-dependent manner.

<p>A) HEY, OVCAR5 or UCI-101 cells were treated for 48 hours with 300 µM diclofenac or indomethacin. Cells were then incubated with MTS reagents and absorbance at 490 nm was measured after 1 hr incubation at 37°C. The average of 3 experiments is shown. Statistical significance is represented as follows: * p<0.05 and *** p<0.001. B) Cells were treated for 24 hours with the indicated concentrations of diclofenac or indomethacin. Cell viability was measured using an MTS assay. The average of 3 experiments is shown. Statistical significance is represented as follows: * p<0.05 and *** p<0.001. C) Cells were treated for 48 hours with 300 µM diclofenac or indomethacin, washed and allowed to grow for a total of 7 to 14 days. Cells were then stained with crystal violet. Image shown is representative of 3 experiments.</p

Diclofenac and Indomethacin promote cell cycle arrest and apoptosis in ovarian cancer cells.

<p>A) HEY, OVCAR5 and UCI-101 cells were treated for 24 hours with 300 µM diclofenac or indomethacin. Cells were labeled with propidium iodide and analyzed in a FACS Caliber Flow Cytometer. Image shown is representative of 4 experiments. B) Cells were treated for 48 or 96 hours with 300 µM diclofenac or indomethacin. Apoptosis and DNA content were assayed by BrdU and propidium iodide staining using Apo-BrdU kit. Cells in upper quadrant box (P3 gate) represent the apoptotic population.</p

NSAIDs reduce the growth of HEY xenografts in nude mice.

<p>HEY cells (1.5×10⁶) were injected subcutaneously into both flanks of nude mice. A) Mice were randomized into 2 groups: control or diclofenac (18 mg/kg). Treatment was started three days after cell injection and was administered intraperitoneally twice a week for 4 weeks. Tumors were measured twice a week, starting at week 1. The control group (only PBS) consisted of 6 mice that developed 12 tumors in total; the diclofenac group (18 mg/kg) consisted of 6 mice that developed a total of 11 tumors. Values represent means ± standard errors. Statistical significance is represented as * p<0.05. B) Mice were randomized into 2 groups: control or indomethacin (2.5 mg/kg). Treatment was started the day after cell injection and was given daily in drinking water for 6 weeks. Tumors were measured twice a week, starting at week 3. The control group (only water) consisted of 5 mice that developed 9 tumors in total; the indomethacin group (2.5 mg/kg) consisted of 5 mice that developed a total of 5 tumors. Values represent means ± standard errors. Statistical significance is represented as * p<0.05.</p

E2F1 and target genes are downregulated in cells treated with NSAIDs.

<p>HEY, OVCAR5 and UCI-101 cells were treated for 24 hours with 300 µM diclofenac or indomethacin. A) RNA was analyzed by Illumina microarrays. The number of mRNAs that were differentially expressed 2-fold or more in all three cell lines is indicated for the diclofenac and indomethacin treatments (Shown in bold are the overexpressed mRNAs; shown in regular font are the downregulated mRNAs). B) RNA from the indicated cell lines treated with diclofenac or indomethacin was analyzed by Real-time RT-PCR. E2F1 and E2F4 mRNA levels are expressed as fold change in treated samples as compared to the untreated, after normalization with GAPDH. The average of 3 experiments is shown. Statistical significance is represented as *** p<0.001. C) Cell lysates of the indicated cell lines that were treated with diclofenac or indomethacin were analyzed by immunoblotting with antibodies specific for the indicated proteins. GAPDH was used as a protein loading control.</p

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1,381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified