Protective<Emphasis Type="Italic">in vivo</Emphasis> effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays

Curcumin is a phytochemical with antiinflammatory, antioxidant and anticarcinogenic activities. Apparently, curcumin is not genotoxic in vivo, but in vitro copper and curcumin interactions induce genetic damage. The aim of this study was to test if in vivo copper excess induces DNA damage measured by comet and micronucleus assays in the presence of curcumin. We tested 0.2% curcumin in Balb-C mice at normal (13 ppm) and high (65, 130 and 390 ppm) copper ion concentrations. The comet and micronucleus assays were performed 48 hr after chemical application. Comet tail length in animals treated with 0.2% curcumin was not significantly different from the control. Animals exposed to copper cations (up to 390 ppm) exhibited higher oxidative DNA damage. Curcumin reduced the DNA damage induced by 390 ppm copper. We observed statistically significant increase in damage in individuals exposed to 390 ppm copper versus the control or curcumin groups, which was lowered by the presence of curcumin. Qualitative data on comets evidenced that cells from individuals exposed to 390 ppm copper had longer tails (categories 3 and 4) than in 390 ppm copper + curcumin. A statistically significant increase in frequency of micronucleated erythrocytes (MNE/10000TE) was observed only in 390 ppm copper versus the control and curcumin alone. Also cytotoxicity measured as the frequency of polychromatic erythrocytes (PE/1000TE) was attributable to 390 ppm copper. The lowest cytotoxic effect observed was attributed to curcumin. In vivo exposure to 0.2% curcumin for 48 hr did not cause genomic damage, while 390 ppm copper was genotoxic, but DNA damage induced by 390 ppm copper was diminished by curcumin. Curcumin seems to exert a genoprotective effect against DNA damage induced by high concentrations of copper cations. The comet and micronucleus assays prove to be suitable tools to detect DNA damage by copper in the presence of curcumin.     

Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine

We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.     

Antibody responses to NY-ESO-1 in primary breast cancer identify a subtype target for immunotherapy

The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)⁻ invasive ductal carcinomas of high grade, including both HER2⁻ and HER2⁺ tumors. In line with these results, we detected ESO expression in 20% of primary HR⁻ BC, including both ESO Ab⁺ and Ab⁻ patients, but not in HR⁺ BC. Interestingly, whereas expression levels in ESO⁺ BC were not significantly different between ESO Ab⁺ and Ab⁻ patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR⁻ (HER2⁻ or HER2⁺) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy.     

Homeostasis of cell composition during prolonged darkness

The chemical composition of organisms in relation to their environmental resource availability is an area of intense research activity. We studied the changes in cell composition of the cyanobacterium Phormidium autumnale in response to prolonged darkness. Cells allocated their internal resources in a homeostatic manner, oxidizing all the three major cellular constituents in a proportional way. This resulted in constant C/N and carbohydrates, lipids and proteins ratios that remained unaltered throughout the whole incubation period. We propose the maintenance of balanced cell composition (homeostasis) as an evolutionary strategy related to environmental transitory changes.     

Comparison of different cationic probes for electron microscopic visualization of bacterial polyanionic capsular material

We have isolated from the ovine rumen eight bacterial strains belonging to the speciesButyrivibrio fibrisolvens. DNA hybridization studies showed that the eight strains could be divided into four homology groups, of which none was closely related to the type strain ATCC 19171. Measurement of cross-hybridization between selected pairs of bacterial strains showed that DNA types which produced low, but significant, cross-hybridization on dot-blots were able to form heteroduplexes with between 8.4% and 32.9% of the efficiency of homoduplex formation. Thermal denaturation of the same heteroduplexes resulted in Tm values 6.4–7.5°C lower than those of the homologous duplexes formed under the same conditions. In some cases, hybridization between strains was below the level of reliable measurement. Similar experiments with ten recently isolated strains ofBacteroides ruminicola sub-sp.brevis revealed a similar degree of genetic divergence between isolates.     

The crystal structure of the carboxy-terminal domain of human translation initiation factor eIF5

The carboxy-terminal domain (CTD) of eukaryotic initiation factor 5 (eIF5) plays a central role in the formation of the multifactor complex (MFC), an important intermediate for the 43 S pre-initiation complex assembly. The IF5-CTD interacts directly with the translation initiation factors eIF1, eIF2-beta, and eIF3c, thus forming together with eIF2 bound Met-tRNA(i)(Met) the MFC. In this work we present the high resolution crystal structure of eIF5-CTD. This domain of the protein is exclusively composed out of alpha-helices and is homologous to the carboxy-terminal domain of eIF2B-epsilon (eIF2Bepsilon-CTD). The most striking difference in the two structures is an additional carboxy-terminal helix in eIF5. The binding sites of eIF2-beta, eIF3 and eIF1 were mapped onto the structure. eIF2-beta and eIF3 bind to non-overlapping patches of negative and positive electrostatic potential, respectively.