Search

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/REF-1) is a multifunctional protein acting on cellular signaling pathways, including DNA repair and redox activities. APE1/REF-1 has emerged as a target for cancer therapy, and its role in breast cancer models would reveal new strategies for cancer therapy. APX2009 is a specific APE1/REF-1 redox inhibitor whose anticancer properties have not been described in breast cancer cells. Here, we investigated the effect of the APX2009 treatment in the breast cancer cell lines MDA-MB-231 and MCF-7. Breast cancer cell lines were cultured, and WST1 and colony formation assays were performed to evaluate cell proliferation. Annexin V-FITC/7-AAD and LDH-Glo™ assays were performed to evaluate cell death. The wound healing assay and Matrigel transwell assay were performed after APX2009 treatment to evaluate the cellular migration and invasion processes, respectively. Our findings demonstrated that APX2009 treatment decreased breast cancer cell proliferative, migratory, and invasive properties. Furthermore, it induced apoptosis in both cell lines. Our study is the first to show the effects of APX2009 treatment on apoptosis in a breast cancer cell. Therefore, this study suggested that APX2009 treatment is a promising anticancer molecule for breast cancer.

Since the reporting of Endre Mester's results, researchers have investigated the biological effects induced by non-ionizing radiation emitted from low-power lasers. Recently, owing to the use of light-emitting diodes (LEDs), the term photobiomodulation (PBM) has been used. However, the molecular, cellular, and systemic effects involved in PBM are still under investigation, and a better understanding of these effects could improve clinical safety and efficacy. Our aim was to review the molecular, cellular, and systemic effects involved in PBM to elucidate the levels of biological complexity. PBM occurs as a consequence of photon-photoacceptor interactions, which lead to the production of trigger molecules capable of inducing signaling, effector molecules, and transcription factors, which feature it at the molecular level. These molecules and factors are responsible for cellular effects, such as cell proliferation, migration, differentiation, and apoptosis, which feature PBM at the cellular level. Finally, molecular and cellular effects are responsible for systemic effects, such as modulation of the inflammatory process, promotion of tissue repair and wound healing, reduction of edema and pain, and improvement of muscle performance, which features PBM at the systemic level., (© 2023. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

We evaluated whether early-life protein restriction alters structural parameters that affect β-cell mass on the 15th day and 20th day of gestation in control pregnant (CP), control non-pregnant (CNP), low-protein pregnant (LPP) and low-protein non-pregnant (LPNP) rats from the fetal to the adult life stage as well as in protein-restricted rats that recovered after weaning (recovered pregnant (RP) and recovered non-pregnant). On the 15th day of gestation, the CNP group had a higher proportion of smaller islets, whereas the CP group exhibited a higher proportion of islets larger than the median. The β-cell mass was lower in the low-protein group than that in the recovered and control groups. Gestation increased the β-cell mass, β-cell proliferation frequency and neogenesis frequency independently of the nutritional status. The apoptosis frequency was increased in the recovered groups compared with that in the other groups. On the 20th day of gestation, a higher proportion of islets smaller than the median was observed in the non-pregnant groups, whereas a higher proportion of islets larger than the median was observed in the RP, LPP and CP groups. β-Cell mass was lower in the low-protein group than that in the recovered and control groups, regardless of the physiological status. The β-cell proliferation frequency was lower, whereas the apoptosis rate was higher in recovered rats compared with those in the low-protein and control rats. Thus, protein malnutrition early in life did not alter the mass of β-cells, especially in the first two-thirds of gestation, despite the increase in apoptosis.

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes' immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1β and nf-κb expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-κb and p65 . This study showed the ability of methionine surplus to improve leucocytes' response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Machado, Serra, Oliva-Teles and Costas.)

Photobiomodulation (PBM) has been used to modulate the inflammatory and immune responses, pain relief, and to promote wound healing. PBM is widely used in dental practice and its cellular effects should be investigated. The aim was to evaluate if PBM changes proteins cell death-related, such as caspase-6 and Bcl-2, in periodontal ligament cells. Eighteen mice were divided in three groups (n = 6), i.e., (I) control, (II) 3 J cm -2 , and (III) 30 J cm -2 . Low power infrared laser (830 nm) parameters were power at 10 mW, energy densities at 3 and 30 J cm -2 in continuous emission mode, exposure time of 15 and 150 s, respectively for 4 days in a row. Twenty-four hours after last irradiation, the animals were euthanized, and their jaws were fixed and decalcified. Caspase-6 and Bcl-2 were analyzed by real-time polymerase chain reaction and immunocytochemical techniques, and DNA fragmentation was evaluated by TUNEL. Statistical differences were not significant to caspase-6 mRNA relative levels in tissues from jaws at both energy densities, but a significant increase of Bcl-2 mRNA relative levels was obtained at 30 J cm -2 group. Also, 30 J cm -2 group showed caspase-6 positive-labeled cells decreased and Bcl-2 positive-labeled cells significantly increased. TUNEL-labeled cells demonstrated DNA fragmentation decreased at 30 J cm -2 . PBM can alter Bcl-2 mRNA relative level and both caspase-6 and Bcl-2 protein, modulating cell survival, as well as to reduce DNA fragmentation. More studies must be performed in order to obtain conclusive results about photobiostimulation effects using infrared low-level laser in apoptosis process as to achieve the optimum dosage.

Dengue virus (DENV) co-circulation in Brazil represents a challenge for treatment and vaccine development. Despite public health impact, the occurrence of coinfections with other viruses is a common event. Increased T cell activation and altered inflammatory response are found during DENV coinfection with Human Immunodeficiency Virus (HIV) impacting HIV-pathogenesis. Even with Antiretroviral therapy (ART), HIV- treated patients had chronic immune activation and lymphocyte apoptosis. However, apoptotic mechanisms have not been investigated during coinfection with DENV. Our attention was attracted to apoptotic cell markers expressions in PBMCs from DENV and DENV/HIV coinfected patients. We found CD4/CD8 ratio inversion in most coinfected patients. CD4 T and CD8 T-cell subsets from DENV and DENV/HIV groups expressed low levels of anti-apoptotic protein Bcl-2. Furthermore, CD8 CD95 double positive cells frequency expressing low levels of Bcl-2 were significantly higher in these patients. Additionally, the density of Bcl-2 on classical monocytes (CD14 ++ CD16 - ) was significantly lower during DENV infection. Upregulation of pro-apoptotic proteins and anti-apoptotic proteins were found in DENV and DENV/HIV, while catalase, an antioxidant protein, was upregulated mainly in DENV/HIV coinfection. These findings provide evidence of apoptosis triggering during DENV/HIV coinfection, which may contribute to knowledge of immunological response during DENV acute infection in HIV-patients treated with ART.

Photobiomodulation (PBM) by low-level laser has demonstrated excellent results for inflammatory treatments, promoting repair of injured tissues. Knowledge regarding the molecular mechanisms involved in this process has been increasing, but its effect on cell death/survival-related gene expression after laser irradiation with different doses is not well understood. So, it is important to know these effects in order to guarantee the safety of therapeutic protocols based on PBM. This study aimed to investigate the mRNA levels of genes related to proteins involved in cell death/survival pathways of healthy tissues from talocrural joint of mice after PBM. Mice were divided into three groups: control, PBM at 3 J cm -2 , and PBM at 30 J cm -2 . Laser irradiation was performed on talocrural joint during four consecutive days. Morphological analyses, immunocytochemistry, FasL, Fas, Bax, Apaf1, Caspase9, Caspase3, Caspase6, Bcl2 mRNA levels, and DNA fragmentation were performed to verify cell death induction after laser irradiation. PBM can increase mRNA levels of almost genes pro-apoptotic. On the other hand, mRNA level of anti-apoptotic protein Bcl-2 gene was not significantly altered. Bcl-2/Bax ratio (indicator of protective molecular response) was decreased after PBM at 30 J cm -2 , trending to DNA fragmentation. Results obtained in this study indicate that PBM by low-level infrared laser alters mRNA relative levels of genes involved in cell death pathways. However, these molecular alterations were not able to cause DNA fragmentation in cells in talocrural joint tissues, indicating that infrared laser was not enough to cause cell death.

Xylan is one of the most abundant hemicellulose constituents in the plant kingdom. We extracted xylan from corn cobs (XCC) using ultrasound. The structure of XCC was determined by NMR analysis, which revealed a composition of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar percentage ratio of 48:21:16:10:2.5:2.5. XCC induced the proliferation of murine macrophage cells (RAW 264.7) and inhibited the proliferation of human lung adenocarcinoma epithelial cells (A549) by 20% and human cervical adenocarcinoma cells (HeLa) by 60%. Several cell death-associated morphological changes were observed after the exposure of HeLa cells to XCC for 24 h. In addition, by using fluorescent probes, we observed a larger number of irregular and pyknotic nuclei with condensed chromatin bodies (nuclear condensation). FACS analysis showed an increase in the number of annexin V-positive and propidium iodide (PI)-positive cells (37.0%) in the presence of XCC compared with that of the negative control cells (5.0%). XCC also increased the ratio of Bax:Bcl-2 (3.5:1) and the levels of cytochrome c, caspase 3, and apoptosis-inducing factor (AIF) in treated cells. Therefore, the results demonstrated the antiproliferative potential of XCC, which induced apoptosis in HeLa cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: BRN2 transcription factor is associated with the development of malignant melanoma. The cytotoxic activities and cell death mechanism against B16F10-Nex2 cells were determined with synthetic peptide R18H derived from the POU domain of the BRN2 transcription factor., Objective: To determine the cell death mechanisms and in vivo activity of peptide R18H derived from the POU domain of the BRN2 transcription factor against B16F10-Nex2 cells., Methods: Cell viability was determined by the MTT method. C57Bl/6 mice were challenged with B16F10-Nex2 cells and treated with R18H. To identify the type of cell death, we used TUNEL assay, Annexin V and PI, Hoechst, DHE, and determination of caspase activation and cytochrome c release. Transmission electron microscopy was performed to verify morphological alterations after peptide treatment., Results: Peptide R18H displayed antitumor activity in the first hours of treatment and the EC50% was calculated for 2 and 24h, being 0.76 ± 0.045 mM and 0.559 ± 0.053 mM, respectively. After 24h apoptosis was evident, based on DNA degradation, chromatin condensation, increase of superoxide anion production, phosphatidylserine translocation, activation of caspases 3 and 8, and release of extracellular cytochrome c in B16F10-Nex2 cells. The peptide cytotoxic activity was not affected by necroptosis inhibitors and treated cells did not release LDH in the extracellular medium. Moreover, in vivo antitumor activity was observed following treatment with peptide R18H., Conclusion: Peptide R18H from BRN2 transcription factor induced apoptosis in B16F10-Nex2 and displayed antitumor activity in vivo., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aneuploidy is associated with different human diseases including cancer. However, different cell types appear to respond differently to aneuploidy, either by promoting tumorigenesis or causing cell death. We set out to study the behavior of adult Drosophila melanogaster intestinal stem cells (ISCs) after induction of chromosome missegregation either by abrogation of the spindle assembly checkpoint or through kinetochore disruption or centrosome amplification. These conditions induce moderate levels of aneuploidy in ISCs, and we find no evidence of apoptosis. Instead, we observe a significant accumulation of ISCs associated with increased stem cell proliferation and an excess of enteroendocrine cells. Moreover, aneuploidy causes up-regulation of the JNK pathway throughout the posterior midgut, and specific inhibition of JNK signaling in ISCs is sufficient to prevent dysplasia. Our findings highlight the importance of understanding the behavior of different stem cell populations to aneuploidy and how these can act as reservoirs for genomic alterations that can lead to tissue pathologies., (© 2018 Resende et al.)

Ketamine is a widely used pharmaceutical that has been detected in water sources worldwide. Zebrafish embryos were used in this study to investigate the oxidative stress and apoptotic signals following a 24h exposure to different ketamine concentrations (0, 50, 70 and 90 mg L -1 ). Early blastula embryos (∼2 h post fertilisation-hpf) were exposed for 24 h and analysed at 8 and 26 hpf. Reactive oxygen species and apoptotic cells were identified in vivo, at 26 hpf. Enzymatic activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE)), glutathione levels (oxidised (GSSG) and reduced (GSH)), oxidative damage (lipid peroxidation (LPO) and protein carbonyls (CO)) as well as oxidative stress (gclc, gstp1, sod1 and cat), apoptosis (casp3a, casp6, casp8, casp9, aifm1 and tp53) and cell proliferation (pcna) related-genes were evaluated at 8 and 26 hpf. Caspase (3 and 9) activity was also determined at both time-points by colorimetric methods. Superoxide dismutase (SOD), catalase (CAT), glutathione levels (GSSG), caspase-9 and reactive oxygen species (ROS) were shown to be affected by ketamine exposure while in vivo analysis showed no difference in ROS. A significant up-regulation of superoxide dismutase (sod1) and catalase (cat) genes expression was also perceived. Ketamine-induced apoptosis was observed in vivo and confirmed by the apoptotic-related genes up-regulation. The overall results suggest that ketamine induced oxidative stress and apoptosis through the involvement of p53-dependent pathways in zebrafish embryos which could be important for the evaluation of the overall risk of ketamine in aquatic environments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M] +• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25μg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC 50 ]=29.85 and 5.964μg/mL, respectively) but not NIH-3T3 cells (IC 50 =1579 and 911.5μg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25μg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC 50 ≥800μg/mL) and no hemolytic activity. LEG (400 and 800μg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway., (Copyright © 2017. Published by Elsevier Ltd.)

AIP56 (apoptosis-inducing protein of 56 kDa) is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), the causative agent of a septicaemia affecting warm water marine fish species. Phdp -associated pathology is triggered by AIP56, a short trip AB toxin with a metalloprotease A domain that cleaves the p65 subunit of NF-κB, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes and plays a central role in host responses to infection. During infection by Phdp , AIP56 is systemically disseminated and induces apoptosis of macrophages and neutrophils, compromising the host phagocytic defence and contributing to the genesis of pathology. Although it is well established that the secretion of AIP56 is crucial for Phdp pathogenicity, the protein secretion systems operating in Phdp and the mechanism responsible for the extracellular release of the toxin remain unknown. Here, we report that Phdp encodes a type II secretion system (T2SS) and show that mutation of the EpsL component of this system impairs AIP56 secretion. This work demonstrates that Phdp has a functional T2SS that mediates secretion of its key virulence factor AIP56., Competing Interests: The authors declare no conflict of interest.

The human microbiome consists of a multitude of bacterial genera and species which continuously interact with one another and their host establishing a metabolic equilibrium. The dysbiosis can lead to the development of pathology, such as inflammatory bowel diseases. Sulfide-producing prokaryotes, including sulphate-reducing bacteria (SRB) constituting different genera, including the Desulfovibrio, are commonly detected within the human microbiome recovered from fecal material or colonic biopsy samples. It has been proposed that SRB likely contribute to colonic pathology, for example ulcerative colitis (UC). The interaction of SRB with the human colon and intestinal epithelial cell lines has been investigated using Desulfovibrio indonesiensis as a model mono-culture and in a co-culture with E. coli isolate, and with SRB consortia from human biopsy samples. We find that D. indonesiensis, whether as a mono- or co-culture, was internalized and induced apoptosis in colon epithelial cells. This effect was enhanced in the presence of E. coli. The SRB combination obtained through enrichment of biopsies from UC patients induced apoptosis of a human intestinal epithelial cell line. This response was not observed in SRB enrichments from healthy (non-UC) controls. Importantly, apoptosis was detected in epithelial cells from UC patients and was not seen in epithelial cells of healthy donors. Furthermore, the antibody raised against exopolysaccharides (EPS) of D. indonesiensis cross reacted with the SRB population from UC patients but not with the SRB combination from non-UC controls. This study reinforces a correlation between the presence of sulphate-reducing bacteria and an inflammatory response in UC sufferers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

Anti-inflammatory property of low-level laser therapy (LLLT) has been widely described in literature, although action mechanisms are not always clarified. Thus, this study aimed to evaluate apoptosis mechanisms in the LLLT anti-inflammatory effects on the arthritis experimental model in vivo at two different energy densities (3 and 30 Jcm -2 ). Arthritis was induced in mice by zymosan solution, animals were distributed into five groups, and morphological analysis, immunocytochemistry and gene expressions for apoptotic proteins were performed. Data showed an anti-inflammatory effect, DNA fragmentation in polymorphonuclear (PMN) cells and alteration in gene expression of proteins related to apoptosis pathways after LLLT. p53 gene expression increased at both energy densities, Bcl2 gene expression increased at 3 Jcm -2 , and Bcl2 tissue expression decreased at 30 Jcm -2 . In addition, apoptosis was restricted to PMN cells. Results suggest that apoptosis in PMN cells comprise part of LLLT anti-inflammatory mechanisms by disbalance promotion between expression of pro-apoptotic (Bax and p53) and anti-apoptotic (Bcl-2) proteins, with pro-apoptotic gene expression selectively in PMN cells.

Mitochondria are deeply involved in cell fate decisions via their multiple roles in metabolism, cell growth, and cell death. In healthy cells, these functions are highly regulated to provide sufficient energy for cell function, maintain cell homeostasis, and avoid undesirable cell death. This is achieved by an orchestrated cooperation of cellular and molecular mechanisms such as mitochondrial mass control (mitophagy vs biogenesis), oxidative phosphorylation, redox and calcium homeostasis, and the balance between pro- and antiapoptotic proteins. In the 1990s, mitochondria have been demonstrated to directly control some forms of regulated cell death as well indirectly through energetic metabolism modulation. However, a large body of literature revealed that distinct cell death modalities can coexist in vivo as well as that mitochondria can be dispensable for certain forms of cell death. Likewise, unexpected interconnections between cell death pathways can lead to an amplification of lethality, as well as a defeat of cell death resistance mechanisms. This revealed a complexity of the control of cell fate and a crucial need to reevaluate the role of mitochondria. Here, we will review the various cell death pathways such as apoptosis and mitochondrial permeability transition-driven necrosis and discuss how mitochondrial proteins and mitophagy regulate them. Finally, the role of mitochondrial proteins in the triggering of cell death and mitophagy in pathological models, such as cardiac and brain pathologies, will be highlighted. This may help to define efficient cytoprotective therapeutic strategies based on the targeting of mitochondria., (© 2017 Elsevier Inc. All rights reserved.)

Purpose: Ischemia and reperfusion injury may induce apoptosis and lead to sustained tissue damage and loss of function, especially in neuronal organs. While carbon monoxide is known to exert protective effects after various harmful events, the mechanism of carbon monoxide releasing molecules in neuronal tissue has not been investigated yet. We hypothesize that the carbon monoxide releasing molecule (CORM) ALF-186, administered after neuronal ischemia-reperfusion injury (IRI), counteracts retinal apoptosis and its involved signaling pathways and consecutively reduces neuronal tissue damage., Methods: IRI was performed in rat´s retinae for 1 hour. The water-soluble CORM ALF-186 (10 mg/kg) was administered intravenously via a tail vein after reperfusion. After 24 and 48 hours, retinal tissue was harvested to analyze mRNA and protein expression of Bcl-2, Bax, Caspase-3, ERK1/2, p38 and JNK. Densities of fluorogold pre-labeled retinal ganglion cells (RGC) were analyzed 7 days after IRI. Immunohistochemistry was performed on retinal cross sections., Results: ALF-186 significantly reduced IRI mediated loss of RGC. ALF-186 treatment differentially affected mitogen-activated protein kinases (MAPK) phosphorylation: ALF-186 activated p38 and suppressed ERK1/2 phosphorylation, while JNK remained unchanged. Furthermore, ALF-186 treatment affected mitochondrial apoptosis, decreasing pro-apoptotic Bax and Caspase-3-cleavage, but increasing anti-apoptotic Bcl-2. Inhibition of p38-MAPK using SB203580 reduced ALF-186 mediated anti-apoptotic effects., Conclusion: In this study, ALF-186 mediated substantial neuroprotection, affecting intracellular apoptotic signaling, mainly via MAPK p38. CORMs may thus represent a promising therapeutic alternative treating neuronal IRI., Competing Interests: Competing Interests: We have the following interests: The co-author Carlos C. Romão is a co-founder, administrator and scientific officer of the company Alfama Lda. where he has equity interests. Carlos C. Romão is an inventor of Patent-No.: US 2011/0038955 A1 2011, where ALF-186 exemplifies the protection of the stomach against NSAID generated ulcers by the administration of CORMs, and is therefore irrelevant for the matter of the present article. The drug profile of ALF-186 is published in detail in: Dalton Transactions DOI: 10.1039/c2dt32174b. "Alfama Ltd. provided support in supplying C.C.R. with the ALF-186 compound, which is not commercially available. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Treatment of metastatic melanoma still remains a challenge, since in advanced stage it is refractory to conventional treatments. Most patients with melanoma have either B-RAF or N-RAS mutations, and these oncogenes lead to activation of the RAS-RAF-MEK-ERK and AKT signal pathway, keeping active the proliferation and survival pathways in the cell. Therefore, the identification of small molecules that block metastatic cell proliferation and induce cell death is needed. Violacein, a pigment produced by Chromobacterium violaceum found in Amazon River, has been used by our group as a biotool for scrutinizing signaling pathways associated with proliferation, survival, aggressiveness, and resistance of cancer cells. In the present study, we demonstrate that violacein diminished the viability of RAS- and RAF-mutated melanoma cells (IC 50 value ∼500 nM), and more important, this effect was not abolished after treatment medium removal. Furthermore, violacein was able to reduce significantly the invasion capacity of metastatic melanoma cells in 3D culture. In the molecular context, we have shown for the first time that violacein causes a strong drop on histone deacetylase 6 expression, a proliferating activator, in melanoma cells. Besides, an inhibition of AXL and AKT was detected. All these molecular events propitiate an inhibition of autophagy, and consequently, melanoma cell death by apoptosis.

Objective: This study aimed to evaluate apoptosis by assessing cleaved caspase-3 immunoexpression in hyperplastic, potentially malignant disorder (PMD), and malignant tumors in intraoral and lower lip sites., Material and Methods: A retrospective study using paraffin blocks with tissues from patients with inflammatory fibrous hyperplasia (IFH), actinic cheilitis, oral leukoplakia, lower lip and intraoral squamous cell carcinoma (SCC) was performed. The tissues were evaluated by immunohistochemical analysis with anti-cleaved caspase-3 antibody. Apoptotic area index was then correlated with lesion type., Results: From 120 lesions assessed, 55 (46%) were cleaved caspase-3-positive. The SCC samples (n=40) had the highest apoptotic area indices (n=35; 87.5%). Significant differences were detected between SCCs and PMDs (p=0.0003), as well as SCCs and IFHs (p=0.001), regarding caspase-3 immunopositivity. Carcinomas of the lower lip had lower apoptotic area indices than intraoral cancer (p=0.0015)., Conclusions: Cleaved caspase-3 immunoexpression showed differences in oral SCCs and PMDs and demonstrated a distinct role of apoptosis in carcinogenesis of intraoral and lower lip cancer. In future, the expression of cleaved caspase-3 with other target molecules in oral cancer may be helpful in delineating the prognosis and treatment of these tumors.

Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

Some chemotherapeutic agents used for breast cancer (BC) treatment can induce severe side effects in the ovarian tissue. The combination of cyclophosphamide and docetaxel (TC) is widely used for BC treatment; however, its late effects in the ovary are not completely understood. The main purpose of this study was to evaluate the structural and ultrastructural alterations in the ovarian stroma induced by TC treatment. Wistar rats were divided into two groups: a control group and a TC group. They were euthanized 5 months after the end of treatment, and their plasma and ovaries were collected. Important alterations were noted. The serum estradiol level was significantly reduced in the TC group compared with the control group. Additionally, the number of apoptotic nuclei was higher in the TC group. The role of the inflammatory response in the development of ovarian damage was investigated, and we found an increased number of mast cells and increased expression of TNF-α in the TC group. The involvement of fibrosis was also investigated. The results showed that the TC group had increased expression levels of TGF-β1, collagen type I (col-I) and collagen type III (col-III) compared with the control group. Ultrastructural analysis revealed the presence of collagen fibrils in the treated group and illustrated that the ovarian tissue architecture was more disorganized in this group than in the control group. The results from this study are important in the study of chemotherapy-induced ovarian failure and provide further insight into the mechanisms involved in the development of this disease., (© 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.)

Streptococcus agalactiae (Group B Streptococcus; GBS) is an important pathogen and is associated with pneumonia, sepsis and meningitis in neonates and adults. GBS infections induce cytotoxicity of respiratory epithelial cells (A549) with generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (ψm). The apoptosis of A549 cells by GBS was dependent on the activation of caspase-3 and caspase-9 with increased pro-apoptotic Bim and Bax molecules and decreased Bcl-2 pro-survival protein. Treatment of infected A549 cells with ROS inhibitors (diphenyleniodonium chloride or apocynin) prevented intracellular ROS production and apoptosis. Consequently, oxidative stress is included among the cellular events leading to apoptosis during GBS human invasive infections.

The Optic atrophy 1 protein (OPA1) is a key element in the dynamics and morphology of mitochondria. We demonstrated that the absence of IκB kinase-α, which is a key element of the nonclassical NF-κB pathway, has an impact on the mitochondrial network morphology and OPA1 expression. In contrast, the absence of NF-κB essential modulator (NEMO) or IκB kinase-β, both of which are essential for the canonical NF-κB pathway, has no impact on mitochondrial dynamics. Whereas Parkin has been reported to positively regulate the expression of OPA1 through NEMO, herein we found that PARK2 overexpression did not modify the expression of OPA1. PARK2 expression reduced the levels of Bax, and it prevented stress-induced cell death only in Bak-deficient mouse embryonic fibroblast cells. Collectively, our results point out a role of the nonclassical NF-κB pathway in the regulation of mitochondrial dynamics and OPA1 expression.

Streptomyces are aerobic Gram-positive bacteria characterized by a complex life cycle that includes hyphae differentiation and spore formation. Morphological differentiation is triggered by stressful conditions and takes place in a pro-oxidant environment, which sets the basis for an involvement of the oxidative stress response in this cellular process. Characterization of the phenotypic traits of Streptomyces natalensis ΔkatA1 (mono-functional catalase) and ΔcatR (Fur-like repressor of katA1 expression) strains in solid medium revealed that both mutants had an impaired morphological development process. The sub-lethal oxidative stress caused by the absence of KatA1 resulted in the formation of a highly proliferative and undifferentiated vegetative mycelium, whereas de-repression of CatR regulon, from which KatA1 is the only known representative, resulted in the formation of scarce aerial mycelium. Both mutant strains had the transcription of genes associated with aerial mycelium formation and biosynthesis of the hyphae hydrophobic layer down-regulated. The first round of the programmed cell death (PCD) was inhibited in both strains which caused the prevalence of the transient primary mycelium (MI) over secondary mycelium (MII). Our data shows that the first round of PCD and morphological differentiation in S. natalensis is dependent on oxidative stress in the right amount at the right time.

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2 μM). Nek5 silenced cells as well as cells expressing a "kinase dead" version of Nek5, displayed an increase in ROS formation after 4 h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor (a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.

Introduction: A balanced proliferation, apoptosis and differentiation in trophoblast cells of the human placenta is crucial for a proper placental development. Alteration in trophoblast apoptosis and differentiation are associated with gestational-related complications, such as preeclampsia, intrauterine growth restriction or miscarriages. The endocannabinoids (eCBs) have been recognized as new interveners in pregnancy events such as implantation and decidualization. However, their importance in placentation is poorly understood. We hypothesise that these novel lipid mediators may intervene in cytotrophoblast apoptosis and, concomitantly, have a role during placental development., Methods: primary human cytotrophoblasts (hCTs) and the human trophoblast-like choriocarcinoma cell line BeWo cells were exposed to Anandamide (AEA). It was investigated the cellular pathways involved in cell death, by the assessment of cell morphology, caspases activity, mitochondrial membrane potential (Δψm), reactive oxygen/nitrogen species (ROS/RNS) and western blot of cleaved Poly (ADP-ribose) polymerase 1 (PARP-1), truncated Bid (t-Bid) and IκB-α., Results: AEA decreased hCTs viability and induced morphological features of apoptosis (chromatin condensation and fragmentation), caspase-3/7 activation and PARP-1 cleavage. In BeWo, AEA also increased the activities of caspase-3/7 and 9, induced loss in Δψm and production of ROS/RNS. These effects were reversed by either CB1 or CB2 antagonists, whereas the increase in caspase-3/7 activity was only reversed with CB2 blockage. AEA-treated cells showed increased caspase-8 activation and formation of t-Bid, suggesting the interplay between intrinsic and extrinsic apoptotic pathways. AEA also increased IκB-α expression, a NF-κB regulatory protein., Conclusion: Our results highlight the importance of eCBs in cytotrophoblast cell apoptosis and indicate that a crosstalk between intrinsic and extrinsic apoptotic pathways is involved in AEA-induced effects., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We pinpoint CZT-1 (cell death-activated zinc cluster transcription factor) as a novel transcription factor involved in tolerance to cell death induced by the protein kinase inhibitor staurosporine in Neurospora crassa. Transcriptional profiling of staurosporine-treated wild-type cells by RNA-sequencing showed that genes encoding the machinery for protein synthesis are enriched among the genes repressed by the drug. Functional category enrichment analyses also show that genes encoding components of the mitochondrial respiratory chain are downregulated by staurosporine, whereas genes involved in endoplasmic reticulum activities are upregulated. In contrast, a staurosporine-treated Δczt-1 deletion strain is unable to repress the genes for the respiratory chain and to induce the genes related to the endoplasmic reticulum, indicating a role for CZT-1 in the regulation of activity of these organelles. The Δczt-1 mutant strain displays increased reactive oxygen species accumulation on insult with staurosporine. A genome-wide association study of a wild population of N. crassa isolates pointed out genes associated with a cell death role of CZT-1, including catalase-1 (cat-1) and apoptosis-inducing factor-homologous mitochondrion-associated inducer of death 2 (amid-2). Importantly, differences in the expression of czt-1 correlates with resistance to staurosporine among wild isolate strains. Our results reveal a novel transcription factor that regulates drug resistance and cell death in response to staurosporine in laboratory strains as well as in wild isolates of N. crassa., (Copyright © 2014 Gonçalves et al.)

It has been established that sphingolipids are engaged in the regulation of apoptosis both as direct executors and as signalling molecules. However, the peculiarities of this class of bioactive lipids, namely the interconnectivity of their metabolic pathways, the specific subcellular localization where they are generated and the transport mechanisms involved, introduce a considerably high level of complexity in deciphering their role in the signalling and regulation of programmed cell death. Although yeast is undeniably a simple model, the conservation of the sphingolipid metabolism and of the core machinery engaged in regulated cell death has already provided valuable clues to the understanding of metabolic pathways involved in distinct cellular processes, including apoptosis. It can be anticipated that studies using this model system will further unravel mechanisms underlying the regulation of apoptosis by sphingolipids and contribute to novel therapeutic strategies against serious human diseases associated with dysfunction of sphingolipid-dependent cell death programmes., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

We explored Group B Streptococcus (GBS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC) and the role of phosphoramidon, a zinc metalloprotease inhibitor, in this process. GBS 90186 strain (serotype V, a blood isolate) and concentrated supernatant (CS) were used to investigate the viability and morphological alterations in HUVEC by Trypan blue uptake, electrophoresis in 2 % agarose gel and scanning electron microscopy assays. Apoptosis before and after phosphoramidon-treatment were verified by flow cytometry using annexin V-FITC labeling. Differences were considered significant when P < 0.05 using unpaired Student's t test. GBS and CS induced HUVEC death by apoptosis (76.5 and 32 %, respectively) with an increasing pro-apoptotic Bax expression and decreasing anti-apoptotic Bcl-2 expression. Caspase-3 was activated during GBS-induced endothelial apoptosis. Phosphoramidon reduced 89.3 and 100 % of GBS and CS cell death by apoptosis, respectively. Some GBS strains may induce cell death by apoptosis with involvement of metalloproteases and signaling through the intrinsic pathway of apoptosis, which may contribute to GBS survival during sepsis of adults and neonates.

In a previous study, we demonstrated that staurosporine (STS) induces programmed cell death (PCD) in the fungus Neurospora crassa and that glutathione has the capability of inhibiting both STS-induced reactive oxygen species (ROS) formation and cell death. Here, we further investigated the role of glutathione in STS-induced PCD in N. crassa and observed an efflux of reduced glutathione (GSH) together with a change in the cell internal redox state to a more oxidative environment. This event was also observed with another PCD inducer, phytosphingosine (PHS), although externally added GSH did not prevent PHS-induced PCD. The nature of ROS, detected under the experimental conditions at which GSH export occurred, is also different in the two systems, predominantly superoxide in the case of STS and hydrogen peroxide in the case of PHS. In both cases, GSH export preceded the alterations in the plasma membrane that lead to selective dye permeation. We conclude that glutathione export in the context of PCD is not exclusive of certain mammalian cells and can be extended to Fungi, being an early PCD event in N. crassa. In addition, STS and PHS induce different PCD pathways in this fungus and the role of GSH export in each of them is likely different.

We investigated the consequences of mild maternal malnutrition in rat dams, in terms of thymocyte responses and the putative role of leptin. The young progeny of dams submitted to protein deprivation (PD) during lactation showed at 30 days of age lower body and thymus weights, significant alterations in CD4/CD8-defined T cell subsets without modifications in total thymocyte number as well as in proliferative response. Despite, the rats from PD group did not present alterations in leptin circulating levels, the expression of leptin receptor ObRb was enhanced in their thymocytes. This change was accompanied by an increase in leptin signaling response of thymocytes from PD rats, with an increase in JAK2 and STAT3 phosphorylation after leptin stimulation. Thymocytes from PD rats also presented a decreased rate of spontaneous apoptosis when compared to controls. Accordingly, higher expression of anti-apoptotic protein Bcl-2, and lower of pro-apoptotic protein Bax, with no change of pro-apoptotic Bad, and higher pro-caspase 3 content were detected in PD thymocytes. Moreover, thymocytes from PD group exhibited a constitutive higher nuclear content of p65 NF-kB associated to a lower IkB content in the cytoplasm. Finally, although there was no change in ob gene expression in PD thymocytes, a higher mRNA expression for the Ob gene was observed in the thymic microenvironment from PD animals. Taken together, the results show that mild maternal protein deprivation during lactation affects thymic homeostasis, enhancing leptin activity, which in turn protects thymocytes from apoptosis in the young progeny, with possible consequences upon the immune response of these animals in adult life.

Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor.

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

We have examined the relationship between chemotherapy-induced mitotic catastrophe and cell death by apoptosis in both wild-type and p53(-/-) HCT116 human colon carcinoma cells treated with nanomolar concentrations of paclitaxel (PTX), a drug that acts on tubulin altering the normal development of mitosis. After treatment, HCT116 cells entered mitosis regardless of the presence of functional p53, which resulted in changes in the distribution of cells in the different phases of the cell cycle, and in cell death. In the presence of PTX, the percentage of polyploid cells observed was higher in p53-deficient cells, indicating that mitotic slippage was favored compared to wild-type cells, with the presence of large multinucleate cells. PTX caused mitotic catastrophe and about 50-60% cells that were entering an aberrant mitosis died through an apoptotic-like pathway characterized by the presence of phosphatidylserine in the outer cell membrane, which occurred in the absence of significant activation of caspases. Lack of p53 facilitated endoreduplication and polyploidy in PTX-treated cells, but cells were still killed with similar efficacy through the same apoptotic-like mechanism in the absence of caspase activity., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Apoptotic death and apoptotic mimicry are defined respectively as a non-accidental death and as the mimicking of an apoptotic-cell phenotype, usually by phosphatidylserine (PS) exposure. In the case of the murine infection by Leishmania spp, apoptotic death has been described in promastigotes and apoptotic mimicry in amastigotes. In both situations they are important events of the experimental murine infection by this parasite. In the present review we discuss what features we need to consider if we want to establish if a behavior shown by Leishmania is altruistic or not: does the behavior increases the fitness of organisms other than the one showing it? Does this behavior have a cost for the actor? If we manage to show that a given behavior is costly for the actor and beneficial for the recipient of the action, we will be able to establish it as altruistic. From this perspective, we can argue that apoptotic-like death and apoptotic mimicry are both altruistic with the latter representing a weaker altruistic behavior than the former.

Modulation of cerebral cell metabolism for improving the outcome of hypoxia-ischemia and reperfusion is a strategy yet to be explored. Because carbon monoxide (CO) is known to prevent cerebral cell death; herein the role of CO in the modulation of astrocytic metabolism, in particular, at the level of mitochondria was investigated. Low concentrations of CO partially inhibited oxidative stress-induced apoptosis in astrocytes, by preventing caspase-3 activation, mitochondrial potential depolarization, and plasmatic membrane permeability. CO exposure enhanced intracellular ATP generation, which was accompanied by an increase on specific oxygen consumption, a decrease on lactate production, and a reduction of glucose use, indicating an improvement of oxidative phosphorylation. Accordingly, CO increased cytochrome c oxidase (COX) enzymatic specific activity and stimulated mitochondrial biogenesis. In astrocytes, COX interacts with Bcl-2, which was verified by immunoprecipitation; this interaction is superior after 24 h of CO treatment. Furthermore, CO enhanced Bcl-2 expression in astrocytes. By silencing Bcl-2 expression with siRNA transfection, CO effects in astrocytes were prevented, namely: (i) inhibition of apoptosis, (ii) increase on ATP generation, (iii) stimulation of COX activity, and (iv) mitochondrial biogenesis. Thus, Bcl-2 expression is crucial for CO modulation of oxidative metabolism and for conferring cytoprotection. In conclusion, CO protects astrocytes against oxidative stress-induced apoptosis by improving metabolism functioning, particularly mitochondrial oxidative phosphorylation.

The genome from Neurospora crassa presented three open reading frames homologous to the genes coding for human AIF and AMID proteins, which are flavoproteins with oxidoreductase activities implicated in caspase-independent apoptosis. To investigate the role of these proteins, namely within the mitochondrial respiratory chain, we studied their cellular localization and characterized the respective null mutant strains. Efficiency of the respiratory chain was analyzed by oxygen consumption studies and supramolecular organization of the OXPHOS system was assessed through BN-PAGE analysis in the respective null mutant strains. The results demonstrate that, unlike in mammalian systems, disruption of AIF in Neurospora does not affect either complex I assembly or function. Furthermore, the mitochondrial respiratory chain complexes of the mutant strains display a similar supramolecular organization to that observed in the wild type strain. Further characterization revealed that N. crassa AIF appears localized to both the mitochondria and the cytoplasm, whereas AMID was found exclusively in the cytoplasm. AMID2 was detected in both mitochondria and cytoplasm of the amid mutant strain, but was barely discernible in wild type extracts, suggesting overlapping functions for the two proteins.

Aims: Oncogenic RET/PTC1 chromosomal rearrangements are hallmarks of thyroid papillary carcinoma. The resulting protein, mainly through tyrosine 451, is responsible for the activation of pathways controlling cell survival, including the PI3K/Akt/mTOR cascade. Vanadium compounds were shown to have anti-neoplastic potential. However, reports about their mechanism of action are contradictory, particularly in what concerns the signaling mediators that are involved. Here, the aim was to characterize the effects of orthovanadate in thyroid cancer cells harboring RET/PTC1., Main Methods: Growth behavior of orthovanadate-treated cells was evaluated by the sulphorhodamine B assay, cell cycle analysis and Terminal Transferase dUTP Nick End Labeling (TUNEL). Mitochondrial parameters such as the transmembrane potential and production of reactive oxygen species (ROS) were also assessed. Western blot was used to study cellular signaling., Key Findings: Low doses of the compound induce a pro-proliferative response. In contrast, treatment with inhibitory concentrations of orthovanadate results in increased phosphorylation of tyrosine 451 of RET/PTC1 and activation of the mTOR/S6R branch of the PI3K/Akt signaling pathway. These concentrations of the drug also induce typical features of apoptosis including DNA fragmentation, loss of mitochondrial membrane potential, production of ROS and activation of caspase-3. Addition of the glial cell line-derived neutrophic factor, a pro-survival stimulator that acts through RET, could not completely block orthovanadate-induced growth inhibition and cell death., Significance: In this model, orthovanadate induces caspase-dependent apoptosis and interferes with the PI3K/Akt/mTOR cascade. This work provides characterization of the effects of orthovanadate and underlines the possibility of its usefulness as a cell death modulator., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Treatment of human breast epithelial cells MCF-10F with 17-β-estradiol has been reported to result in E2-transformed cells which have given rise to highly invasive C5 cells that in turn generate tumors in SCID mice. From these tumors, various cell lines, among which C5-A6-T6 and C5-A8-T8, were obtained. Although different phases of the tumorigenesis process in this model have been studied in molecular biology and image analysis assays, no cytological data on apoptotic ratios and mitotic abnormalities have been established to accompany the various steps leading to 17-β-estradiol-treated MCF-10F cells to tumorigenesis. Here we detected that the apoptotic ratio decreases with the transformation and tumorigenesis progress, except for the tumor cell line C5-A8-T8, probably on account of its more intense proliferation rate and a more rapid culture medium consumption. Increased frequency of mitotic abnormalities contributed by triple- and tetrapolar metaphases, and by lagging chromosomes and chromosome bridges observed at the anaphase found by transformation and tumorigenesis progress. However, no difference was found under these terms when the C5-A6-T6 and C5-A8-T8 tumor cell lines were compared to each other. Present findings are in agreement with the nuclear instability and enrichment of dysregulated genes in the apoptotic process promoted by transformation and tumorigenesis in 17-β-estradiol-treated MCF-10F cells.

Although group B Streptococcus (GBS) has been classically described as an exclusively extracellular pathogen, growing evidence suggests that it may be internalized by epithelial cells. However, the fates of intracellular GBS and of infected respiratory epithelial cells remain unclear. Little is known about the bacterial components involved in these processes. The present study investigated the bacterial internalization by A549 cells and the apoptosis/necrosis of the infected human epithelial cells. The morphological changes in A549 cells observed from 2 h post-infection with GBS included vacuolization and the formation of apoptotic bodies. Flow cytometry revealed that 81.2% of apoptotic A549 cells were infected with GBS serotype III 90356-liquor. Moreover, a double-staining assay using propidium iodide (PI)/Annexin V (AV) gave information about the numbers of viable (PI-/AV-) (18.27%) vs. early apoptotic (PI-/AV+) (73.83%) and late apoptotic cells (PI+/AV+) (7.37%) during infection of A549 cells with GBS III 90356-liquor. In addition, 37% necrotic cells were observed in A549 cells infected with GBS serotype V 90186-blood. In conclusion, GBS serotypes III and V induce apoptosis of epithelial cells in the early stages of GBS infection, resulting in tissue destruction, bacterial spreading and, in consequence, invasive disease or systemic infection.

It has been previously shown that the exotoxin of the important fish pathogen Photobacterium damselae ssp. piscicida is a key pathogenicity factor and is responsible for the extensive systemic apoptosis of macrophages and neutrophils seen in acute fish photobacteriosis. The focus of the present study was to further characterize the AIP56-induced apoptosis of sea bass professional phagocytes by assessing the involvement of caspases, mitochondria and oxidative stress. The resulting data indicate that the apoptotic response in peritoneal macrophages and neutrophils treated ex vivo with AIP56 involves activation of caspase-8, -9 and -3, and mitochondria as shown by loss of mitochondrial membrane potential, release of cytochrome c and over-production of ROS. These results together with previous data from this laboratory suggest that both the extrinsic and intrinsic apoptotic pathways are involved in the AIP56-induced phagocyte apoptosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Deregulation of cell proliferation and apoptosis is linked to malignant cell development. Leukemia is the most frequent cancer in children, and plants are important sources for new potential anti-cancer agents. Although anti-tumoral effects have been shown for Pterodon pubescens extracts, the mechanisms are still obscure. This study describes in Pterodon pubescens a furane diterpene only reported in Pterodon polygalaeflorus, the methyl-6α-acetoxy-7β-hydroxyvouacapan-17β-oate, indicated by HRMS and 13C-NMR analysis, and demonstrates some mechanisms of the anti-leukemia action of its terpene subfraction SF5. SF5 induced cytotoxic and anti-proliferative effects on K562 cells. Increased sub-G1 nuclei and Annexin V+-FITC cells confirmed apoptosis of leukemic cells by treatment of these cells with SF5. Down-regulation of DNMT1 gene transcription and over-expression of Apaf-1 mRNA suggested that SF5 may be inducing apoptosis of K562 cells by epigenetic up-regulation of pro-apoptotic proteins involved in the mitochondrial intrinsic pathway.

The predominant definition of apoptosis considers that the elimination of the apoptosing cell is by heterolytic degradation following phagocytosis by an assisting scavenger (efferocytosis). However, an alternative and largely underestimated outcome of apoptosis is secondary necrosis, an autolytic process of cell disintegration with release of cell components that occurs when there is no intervention of scavengers and the full apoptotic program is completed. Secondary necrosis is the typical outcome of apoptosis in unicellular eukaryotes but, importantly, it may also occur in multicellular animals and has been implicated in the genesis of important human pathologies. Secondary necrosis is a mode of cell elimination with specific molecular and morphological features and should be considered the natural outcome of the complete apoptotic program., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this work, the sea bass counterpart of mammalian caspase-8 was sequenced and characterized, and its involvement in the apoptogenic activity of a toxin from a fish pathogen was assessed. A 2472 bp cDNA of sea bass caspase-8 was obtained, consisting of 1455 bp open reading frame coding for 484 amino acids and with a predicted molecular weight of 55.2 kDa. The sea bass caspase-8 gene has 6639 bp and is organized in 11 introns and 12 exons. Several distinctive features of sea bass caspase-8 were identified, which include two death effector domains, the caspase family domains p20 and p10, the caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. The sea bass caspase-8 sequence revealed a significant degree of similarity to corresponding sequences from several vertebrate taxonomic groups. A low expression of sea bass caspase-8 was detected in various tissues of non-stimulated sea bass. Furthermore, it is shown that stimulation of sea bass with mid-exponential phase culture supernatants from Photobacterium damselae ssp. piscicida (Phdp), known to induce selective apoptosis of macrophages and neutrophils, resulted in an increased expression of caspase-8 in the spleen, one of the main affected organs by Phdp infection., (2010 Elsevier Ltd. All rights reserved.)

Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. alpha(9)beta(1) integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how alpha(9)beta(1) integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by alpha(9)beta(1) integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to alpha(9)beta(1) integrin in neutrophils, we demonstrate that alpha(9)beta(1) integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/alpha(9)beta(1) interaction. Moreover we show that VLO5/alpha(9)beta(1) engagement induces NF-kappaB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-x(L). VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of alpha(9)beta(1) integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role., (2010 Elsevier B.V. All rights reserved.)