Direct and Indirect Effect of Air Particles Exposure Induce Nrf2-Dependent Cardiomyocyte Cellular Response In Vitro.

Air particulate matter has been associated with adverse effects in the cardiorespiratory system leading to cytotoxic and pro-inflammatory effects. Particulate matter-associated cardiac effects may be direct or indirect. While direct interactions may occur when inhaled ultrafine particles and/or particle components cross the air-blood barrier reaching the cardiac tissue, indirect interactions may occur as the result of pulmonary inflammation and consequently the release of inflammatory and oxidative mediators into the blood circulation. The aim of the study is to investigate the direct or indirectly the effect of Urban Air particles from downtown Buenos Aires (UAP-BA) and residual oil fly ash (ROFA), a surrogate of ambient air pollution, on cardiomyocytes (HL-1 cells). HL-1 cultured cells were directly exposed to particulate matter [UAP-BA (10-200 µg/ml), ROFA (1-100 µg/ml)] or indirectly exposed to conditioned media (CM) from particle-exposed alveolar macrophages (AM). Metabolic activity, reactive oxygen species (ROS), and Nrf2 expression were assessed by MTT, DHR 123, and immunocytochemistry techniques, respectively. We found that direct exposure of cardiomyocytes to UAP-BA or ROFA increased ROS generation but the oxidative damage did not alter metabolic activity likely by a concomitant increase in the cytoplasmic and nuclear Nrf2 expression. However, indirect exposure through CM caused a marked reduction on cardiac metabolic activity probably due to the rise in ROS generation without Nrf2 translocation into the cell nuclei. In this in vitro model, our results indicate both direct and indirect PM effects on cardiomyocytes cells in culture. Our findings employing lung and cardiomyocytes cells provide support to the hypothesis that particle-induced cardiac alteration may possibly involve lung-derived mediators.

Role of apoptosis-related miRNAs in resveratrol-induced breast cancer cell death.

Breast cancer is the most frequently diagnosed cancer in women, and one of the leading causes of cancer-related deaths worldwide. Recent evidences indicate that dietary agents such as resveratrol may inhibit cancer progression through modulation of microRNAs (miRNAs). We demonstrate that resveratrol regulates apoptotic and cell cycle machinery in breast cancer cells by modulating key tumor-suppressive miRNAs including miR-125b-5p, miR-200c-3p, miR-409-3p, miR-122-5p and miR-542-3p. Resveratrol-mediated miRNA modulation regulates key anti-apoptotic and cell cycle proteins including Bcl-2, X-linked inhibitor of apoptosis protein and CDKs, which are critical for its activity. Modulating miRNAs with mimics or inhibitors further validated a key role for miR-542-3p in MCF-7 and miR-122-5p in MDA-MB-231 breast cancer cell death in response to resveratrol. In conclusion, this study reveals novel miRNAs modulated by resveratrol that have a key role in breast cancer cell death.

Particulate matter cytotoxicity in cultured SH-SY5Y cells is modulated by simvastatin: Toxicological assessment for oxidative damage.

Epidemiological studies have shown a positive correlation between environmental particulate matter and adverse health effects. In particular, residual oil fly ash (ROFA) induces inflammation and reactive oxygen species (ROS), exerting not only local, but also systemic adverse effects. Previously, in an experimental animal model, we found that simvastatin (Sv) pretreatment was effective in preventing ROFA induced lung inflammation. Herein, using the human neuroblastoma SH-SY5Y cell line as a neurotoxicity in vitro model, we studied the potential Sv protective effect on ROFA cytotoxicity. We evaluated cell viability by the MTT assay, superoxide anion generation by NBT test, Nrf2 activation by immunofluorescence, apoptosis by cleaved-PARP and active-caspase 3 expressions, and senescence by ß-galactosidase activity. SH-SY5Y cells exposed to ROFA (10 and 50µg/ml) for 24h showed decreased cell viability, increased superoxide anion generation, apoptosis and senescence. Pretreatment with Sv (1µM) for 6 days, restored cell viability to basal levels, reduced ROFA-induced O2(-) generation as well as the number of apoptotic and senescent cells. Sv pretreatment stimulated the basal and ROFA-induced levels of Nrf2 nuclear translocation suggesting that activation of the cellular antioxidant defense system prevented particle-induced oxidative stress. In parallel, rescue experiments with mevalonate did not modify the effects of SV pretreatment in any of the parameters evaluated in this study. We conclude that simvastatin may provide neuroprotection against air particulate matter-induced neurotoxicity independently of its ability to inhibit cholesterol synthesis.

Direct and indirect air particle cytotoxicity in human alveolar epithelial cells.

Air particulate matter has been associated with adverse impact on the respiratory system leading to cytotoxic and proinflammatory effects. The biological mechanisms behind these associations may be initiated by inhaled small size particles, particle components (soluble fraction) and/or mediators released by particle-exposed cells (conditioned media). The effect of Urban Air Particles from Buenos Aires (UAP-BA) and Residual Oil Fly Ash (ROFA) a surrogate of ambient air pollution, their Soluble Fractions (SF) and Conditioned Media (CM) on A549 lung epithelial cells was examined. After 24 h exposure to TP (10 and 100 µg/ml), SF or CM, several biological parameters were assayed on cultured A549 cells. We tested cell viability by MTT, superoxide anion (O2(-)) generation by NBT and proinflammatory cytokine (TNFα, IL-6 and IL-8) production by ELISA. UAP-BA particles or its SF (direct effect) did not modify cell viability and generation of O2(-) for any of the doses tested. On the contrary, UAP-BA CM (indirect effect) reduced cell viability and increased both generation of O2(-) and IL-8 production. Exposure to ROFA particles, SF or ROFA CM reduced proliferation and O2(-) but, stimulated IL-8. It is worth to note that UAP-BA and ROFA depicted distinct effects on particle-exposed A549 cells implicating morphochemical dependence. These in vitro findings support the hypothesis that particle-induced lung inflammation and disease may involve lung-derived mediators.

The stemness phenotype model.

The identification of a fraction of cancer stem cells (CSCs) associated with resistance to chemotherapy in most solid tumors leads to the dogma that eliminating this fraction will cure cancer. Experimental data has challenged this simplistic and optimistic model. Opposite to the classical cancer stem cell model, we introduced the stemness phenotype model (SPM), which proposed that all glioma cells possess stem cell properties and that the stemness is modulated by the microenvironment. A key prediction of the SPM is that to cure gliomas all gliomas cells (CSCs and non-CSCs) should be eliminated at once. Other theories closely resembling the SPM and its predictions have recently been proposed, suggesting that the SPM may be a useful model for other type of tumors. Here, we review data from other tumors that strongly support the concepts of the SPM applied to gliomas. We include data related to: (1) the presence of a rare but constant fraction of CSCs in established cancer cell lines, (2) the clonal origin of cancer, (3) the symmetrical division, (4) the ability of "non-CSCs" to generate "CSCs," and (5) the effect of the microenvironment on cancer stemness. The aforenamed issues that decisively supported the SPM proposed for gliomas can also be applied to breast, lung, prostate cancer, and melanoma and perhaps other tumors in general. If the glioma SPM is correct and can be extrapolated to other types of cancer, it will have profound implications in the development of novel modalities for cancer treatment.

Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome.

During macronuclear development the Tetrahymena thermophila ribosomal RNA gene is excised from micronuclear chromosome 1 by site-specific cleavage at chromosome breakage sequence (Cbs) elements, rearranged into a 'palindromic' 21 kb minichromosome and extensively amplified. Gene amplification initiates from origins in the 5' non-transcribed spacer, and forks moving toward the center of the palindrome arrest at a developmentally regulated replication fork barrier (RFB). The RFB is inactive during vegetative cell divisions, suggesting a role in the formation or amplification of macronuclear rDNA. Using micronuclear (germline) transformation, we show that the RFB region facilitates Cbs-mediated excision. Deletion of the RFB inhibits chromosome breakage in a sub-population of developing macronuclei and promotes alternative processing by a Cbs-independent mechanism. Remarkably, the RFB region prevents spontaneous breakage of chromosome 1 in the diploid micronucleus. Strains heterozygous for DeltaRFB and wild-type rDNA lose the DeltaRFB allele and distal left arm of chromosome 1 during vegetative propagation. The wild-type chromosome is subsequently fragmented near the rDNA locus, and both homologs are progressively eroded, suggesting that broken micronuclear chromosomes are not 'healed' by telomerase. Deletion of this 363 bp segment effectively creates a fragile site in the micronuclear genome, providing the first evidence for a non-telomere cis-acting determinant that functions to maintain the structural integrity of a mitotic eukaryotic chromosome.

Disulfiram is a potent in vitro inhibitor of DNA topoisomerases.

The drug disulfiram is a thiol-reacting drug that is relatively nontoxic when used alone and has been used in the therapy of alcohol abuse for more than 40 years. Several effects of this drug have been reported for DNA synthesis and cell proliferation. In this study, the inhibitory effect of disulfiram on topoisomerase I and II activity was investigated by measuring the relaxation of superhelical plasmid pBR322 DNA. Disulfiram (1-100 microM) inhibited topoisomerase I and II in a concentration-dependent manner (IC(50) congruent with 42 +/- 8 and 30 +/- 9 microM, respectively). Consistent with the assumption that a thiol residue is involved, dithiothreitol (1 mM) markedly prevented the inhibitory effect of disulfiram on the activity of both classes of topoisomerases. These findings might explain certain aspects of disulfiram toxicity and encourage new studies to determine the usefulness of this drug and its analogues as antineoplastic agent.

Inhibition of DNA synthesis in human gliomas by roscovitine.

The early effect of 1-100 microM roscovitine, a purine analogue and cyclin-dependent kinase inhibitor, was studied on tissue specimens from eight human malignant gliomas. The tissue was incubated immediately after resection with DMEM containing [3H]methylthymidine plus vehicle alone or the proper concentration of roscovitine for 30-90 min. The DNA synthesis rate was assessed by measurement of [3H]methylthymidine incorporation into trichloroacetic acid insoluble material/mg protein/min. In all gliomas, 100 microM roscovitine inhibited DNA synthesis by 71-97% (average 89 +/- 8%, p<0.0001). This inhibitory effect of roscovitine appeared within 30 min of incubation and was concentration dependent.

Early effects of protein kinase modulators on DNA synthesis in rat cerebral cortex.

By using tissue miniunits, protein kinase modulators, and topoisomerase inhibitors in short-term incubation (0-90 min) we studied (1) the role of protein phosphorylation in the immediate control of DNA replication in the developing rat cerebral cortex and (2) the mechanism of action for genistein-mediated DNA synthesis inhibition. Genistein decreased the DNA synthesis within less than 30 min. None of the other protein kinase inhibitors examined (herbimycin A, staurosporine, calphostin-C) or the protein phosphatase inhibitor sodium orthovanadate inhibited DNA synthesis and they did not affect the genistein-mediated inhibition. The selective topoisomerase inhibitors camptothecin and etoposide decreased the DNA synthesis to an extent similar to that of genistein and within less than 30 min. In addition, the effects of these substances on topoisomerase I and II were studied. Etoposide and genistein but not herbimycin A, staurosporine, or calphostin-C strongly inhibited the activity of topoisomerase II. Our results (1) strongly suggest that the net rate of DNA replication during the S phase of the cell cycle is independent of protein phosphorylation and (2) indicate that the early inhibitory effect of genistein on DNA synthesis is mediated by topoisomerase II inhibition rather than protein tyrosine kinase inhibition.

Fast and sensitive method for simultaneous measurement of cell proliferation rate and drug sensitivity in rat cerebral cortex.

A proliferation assay based on the production of mini-units of tissue was adopted and modified for the simultaneous determination of cell proliferation rate and the effect of genistein in rat cerebral cortex. Mini-units of tissue were produced from rat cerebral cortex immediately after killing the animal and incubated with culture medium containing 3H-methyl-thymidine during 90 min. The proliferation rate was assessed by measurement of 3H-methyl-thymidine incorporation into trichloroacetic acid insoluble material/mg of protein/min. The mini-unit method preserves the neural-cell topological relation existing in vivo and, in addition, has several additional advantages: (1) the short incubation time required limits the metabolic changes, (2) the sensitivity to drugs can be assessed simultaneously with the cell proliferation rate, (3) the complete procedure can be performed within 4-6 h, and (4) many experiments can be performed with the tissue from one animal. Genistein in doses from 10 to 100 microM inhibited cell proliferation in a concentration-dependent manner. The percentage of inhibition was highest in young animals and decreased with increasing age. This method is a powerful tool for the study of drugs with short-time onset mechanisms of action and can be useful for the screening of new drugs.

Early inhibition of DNA synthesis in the developing rat cerebral cortex by the purine analogues olomoucine and roscovitine.

The effects of the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine on DNA synthesis were studied using short time incubation (30-90 minutes). Both purine analogues at concentrations from 1-100 microM decreased the DNA synthesis of rat brain cortex in a dose-dependent manner and the maximum effect occurred within 30 min of incubation. Staurosporine, another potent CDK inhibitor did not affect the DNA synthesis in the concentration range 1-250 nM. These results indicate that olomoucine and roscovitine block DNA synthesis by a mechanism independent of CDK inhibition. We propose that the cellular effects of olomuocine and roscovitine on the cell cycle are at least in part due to this early inhibitory effect on DNA synthesis.

Growth inhibition of herpes simplex virus-type 1 in calphostin C-treated astrocytes.

The aim was to evaluate the effects of calphostin C (CC), a protein kinase C inhibitor, on lytic herpes simplex virus-type 1 infection of cultured rat astrocytes. At 24 h postinjection, the cell culture receiving CC treatment at 50 nM concentration showed decreased cell detachment and retraction versus untreated infected controls; likewise, the infective virus yield was significantly lower in a dose-dependent manner. In contrast, image analysis failed to disclose differences in viral antigen immunolabeling at low drug concentrations thus suggesting that CC-induced inhibition of cytopathic effects and infectivity taken place through mechanisms not involving viral protein synthesis. Given the low dose required and the apparent lack of cytotoxic effects, present findings encourage additional studies on CC antiviral potential in the whole organism.

Examination of the natural protein substrates affected by staurosporine in the developing cerebral cortex.

The protein substrates affected by staurosporine (SP), the most potent inhibitor of protein kinases yet described, are unknown. In order to approach this problem we incubated cerebral cortex tissue with 0, 20, 50 and 100 nM of SP using [32P]orthophosphate as radioactive precursor. The analysis of the phosphoproteins were made with a modified high resolution two dimensional gel electrophoresis, followed by autoradiography. We detected several proteins affected by SP. Specially noticeable was an approximately 55 kDa protein which strikingly diminished the intensity of phosphorylation. However, the reverse phenomenon was also observed. To the best of our knowledge this is the first examination of protein substrates affected by SP in intact tissue.