Mitochondrial Membrane Collapse Research Articles

Diffusion kurtosis imaging and diffusion weighted imaging comparison in diagnosis of early hypoxic–ischemic brain edema

BackgroundHypoxic–ischemic encephalopathy (HIE) refers to cerebral hypoxic–ischemic injury caused by asphyxia during perinatal period, which is one of the important causes of neonatal death and sequelae. Early and accurate diagnosis of HIE is of great significance for the prognostic evaluation of patients. The purpose of this study is to explore the efficacy of diffusion-kurtosis imaging (DKI) and diffusion-weighted imaging (DWI) in the diagnosis of early HIE.MethodsTwenty Yorkshire newborn piglets (3–5 days) were randomly divided into control group and experimental group. DWI and DKI scanning were performed at timepoints of 3, 6, 9, 12, 16, and 24 h after hypoxic–ischemic exposure. At each timepoint, the parameter values obtained by each group scan were measured, and the lesion area of the apparent diffusion coefficient (ADC) map and mean diffusion coefficient (MDC) map were measured. (For better interpretation of this study, we replaced the description of MD with MDC). Then, we completely removed the brain for pathological examination, and observed the state of cells and mitochondria in the ADC/MDC matching area (the actual area of the lesion), and the mismatch area (the area around the lesion).ResultsIn the experimental group, the ADC and MDC values decreased with time, but the MDC decreased more significantly and the change rate was higher. Both MDC and ADC values changed rapidly from 3 to 12 h and slowly from 12 to 24 h. The MDC and ADC images showed obvious lesions at 3 h for the first time. At this time, the area of ADC lesions was larger than that of MDC. As the lesions developed, the area of ADC maps was always larger than that of the MDC maps within 24 h. By observing the microstructure of the tissues by light microscopy, we found that the ADC and MDC matching area in the experimental group showed swelling of neurons, infiltration of inflammatory cells, and local necrotic lesions. Consistent with the observation under light microscope, pathological changes were observed in the matching ADC and MDC regions under electron microscopy as well, including collapse of mitochondrial membrane, fracture of partial mitochondrial ridge, and emergence of autophagosomes. In the mismatching region, the above pathological changes were not observed in the corresponding region of the ADC map.ConclusionsDKI’s characteristic parameter MDC is better than ADC (parameter of DWI) to reflect the real area of the lesion. Therefore, DKI is superior to DWI in diagnosing early HIE.

Protective Effects of Co-administration of Zinc and Selenium Against Streptozotocin-Induced Alzheimer's Disease: Behavioral, Mitochondrial Oxidative Stress, and GPR39 Expression Alterations in Rats.

Changes in the concentrations of trace metals such as zinc (Zn) and selenium (Se) can pathologically lead to neurodegenerative conditions such as the Alzheimer's disease (AD). Previous studies have shown that mitochondrial dysfunction plays an important role in the pathogenesis of AD. Several male Wistar rats were randomly divided into five groups: sham group, AD group that received 3mg/kg of streptozotocin (STZ) intracerebroventricularly, AD + Zn group that received 10mg/kg of Zn intraperitoneally (i.p.) for 1week, AD + Se group that received 0.1mg/kg of Se i.p. for 1week, and AD + Zn + Se group that received 10mg/kg of Zn i.p. plus 0.1mg/kg of Se i.p. for 1week. At end of the study, behavioral tests and mitochondrial oxidative stress and GPR39 gene expression evaluations were carried out. Co-administration of Zn and Se significantly decreased the potential collapse of mitochondrial membrane, reactive oxygen species levels, and lipid peroxidation levels while significantly increased cognitive performance, superoxide dismutase (SOD), glutathione peroxidase, and catalase activity in the brain mitochondria compared with the STZ group. In addition, no significant changes were observed in GPR39 expression in the co-treated group. Findings of the current study showed that ZnR/GPR39 receptor, mitochondrial dysfunction, and oxidative stress play important roles in the pathogenesis of AD. Co-treatment of Zn and Se improved the cognitive performance, mitochondrial dysfunction, and oxidative stress caused by STZ-induced AD. Therefore, therapeutic approaches to improve mitochondrial function could be effective in preventing the initiation and progression of AD.

Influence of the introduction of a triphenylphosphine group on the anticancer activity of a copper complex

Aiming at obtaining new copper complexes with good cytotoxicity against cancer cells, triphenylphosphine (TPP) was introduced to obtain insight into the influence of the co-ligands. In this paper, two copper complexes, Cu(2-pbmq)(CH3OH)Br2 (1) and [Cu(2-pbmq)(TPP)Br]2 (2) were designed, synthesized, and characterized by X-ray crystallography, 2-((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl))quinolone (2-pbmq), to investigate the influence of the TPP group on the anticancer activity of the metal complex. Although the presence of the TPP group diminished the intensity of the interaction properties of the complex with DNA, the in vitro anticancer activity and cellular uptake of the TPP-containing complex were markedly superior to those of its TPP-lacking counterpart. Detailed studies on the more potently cytotoxic complex 2 revealed that it accumulated in nucleus, arrested the cell cycle at the G0-G1 phase, causing mitochondrial dysfunction, involving the potential simultaneous mitochondrial membrane collapse, cellular ATP level depletion, and Ca2+ leakage, eventually inducing cell apoptosis. In summary, the introduction of a TPP group enhances the biological activity and cytotoxicity of the complex.

Adjuvant contributes Roundup's unexpected effects on A549 cells

Roundup® (RDP) is one of the most representative glyphosate-based herbicides (GBHs), which extensive use increases pressure on environmental safety and potential human health risk. The aim of this study was to investigate whether the adjuvant polyethoxylated tallow amine (POEA) or the herbicidal active ingredient glyphosate isopropylamine salt (GP) in formulation confers RDP cytotoxicity. We demonstrated that RDP and POEA could inhibit the proliferation of human lung A549 cells. Intracellular biochemical assay indicated that collapse of mitochondrial membrane, release of cytochrome c into cytosol, activation of caspase-9/-3, cleavage of poly (ADP-ribose) polymerase (PARP), oxidative DNA damage, DNA single-strand breaks and double-strand breaks are occurred in RDP and POEA treated A549 cells, not occurred in GP treated A549 cells. We conclude that the RDP's effect of apoptosis and DNA damage on human A549 cells is related to the presence of adjuvant POEA in formulation, independent of the herbicidal active ingredient GP. This study would enrich the theoretical basis of the RDP toxicity effects and attract attention on potential human health and environmental safety threat caused by adjuvant.

Mitochondrial Permeability Transition Pore Sealing Agents and Antioxidants Protect Oxidative Stress and Mitochondrial Dysfunction Induced by Naproxen, Diclofenac and Celecoxib

Nonsteroidal anti-inflammatory drugs (NSAIDs) like naproxen, diclofenac and celecoxib used to reduce pain. Many of these drugs have been associated with an increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which NSAIDs induce CVD up to now is unknown. We investigated the effects of naproxen, diclofenac and celecoxib with different structures and mechanism action on isolated rat heart mitochondria. All tested NSAIDs increased reactive oxygen species (ROS) formation, mitochondrial membrane collapse (MMP), mitochondrial swelling, lipid peroxidation, and glutathione and ATP depletion, which all of them play important roles in developing cardiotoxicity. We reported that mitochondrial permeability transition (MPT) pore sealing agents and antioxidants have the capacity to significantly prevent mitochondrial toxicity. Therefore, the inhibition of mitochondrial oxidative stress and mitochondrial dysfunction by MPT pore sealing agents and antioxidants can double confirm NSAID-induced cardiomyocytes toxicity is resulted from induction of apoptosis signaling trough ROS-mediated mitochondrial permeability transition.

Roundup® confers cytotoxicity through DNA damage and Mitochondria-Associated apoptosis induction

Glyphosate-based herbicides (GBH) are the most widely used pesticides in the world. The extensive use of them increases the potential human health risk, including the human inhalation toxicity risk. We studied the effect of the most famous GBH Roundup® (RDP) in the concentration range from 50 to 125 μg/mL on Mitochondria-Associated apoptosis and DNA damage in Human alveolar carcinoma cells (A549 cells). Alkaline comet assay, immunofluorescence assay and Flow Cytometric Analysis assay were employed to detect DNA damages and apoptosis of A549 cells. We found RDP caused concentration-dependent increases in DNA damages and proportion of apoptotic cells in A549 cells. RDP induced the DNA single-strand breaks and double-strand breaks; the collapse of mitochondrial membrane by increasing Bax/Bcl-2, resulting in the release of cytochrome c into cytosol and then activated caspase-9/-3, cleaved poly (ADP-ribose) polymerase (PARP) in human lung tissue cells. The results demonstrate that RDP can induce A549 cells cytotoxic effects in vitro at the concentration lower than the occupational exposures level of workers, which means RDP has a potential threat to human health.

PRIMA-1MET-induced neuroblastoma cell death is modulated by p53 and mycn through glutathione level

BackgroundNeuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1MET, in inducing neuroblastoma cell death.MethodsCellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential.ResultsNeuroblastoma cell lines were at least four times more susceptible to PRIMA-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET activated p53 transactivation activity, p53’s role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition.ConclusionsPRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.

Antioxidant potential and phenolic profile of blackberry anthocyanin extract followed by human gut microbiota fermentation

Beneficial properties attributed to the intake of blackberry fruit are associated with the presence of high content of anthocyanins. However, their low absorption and accumulation in the gut have generated the belief that gut metabolites of anthocyanins are probably reason for their protective effects. In this study, blackberry anthocyanins were prepared and subjected to in vitro human gut microbiota fermentation at different time intervals (0–48 h) to study their gut metabolites and antioxidant properties. The content of cyanidin-3-O-glucoside was found highest in blackberry and it degraded completely after 6 h fermentation. Gut metabolites of blackberry anthocyanins were found to improve the glucose consumption and glycogen content significantly in HepG2 cells. Furthermore, gut metabolites significantly ameliorated high glucose plus palmitic acid (HG + PA)-induced ROS, mitochondrial membrane collapse, and glutathione depletion in HepG2 cells. Overall, this study reveals that blackberry anthocyanins subjected to gut microbiota fermentation resulted in the formation of active metabolites with potential antioxidant activity against HG + PA-induced oxidative stress.

A polysaccharide from Enterobacter cloacae induces apoptosis of human osteosarcoma cells through the activation of p53 and mitochondrial intrinsic pathway

In the present study, a polysaccharide (ECP) from Enterobacter cloacae dose and time-dependently inhibited cell growth of human osteosarcoma U-2 OS cells via induction of apoptosis. ECP treatment was selectively toxic to U-2 OS cells whereas had no cytotoxic effect on normal human osteoblast cell line NHOst. ECP-induced apoptotic cell death was associated with collapse of mitochondrial membrane, cytochrome c release into the cytosol, activation of caspase-9 and-3, degradation of poly (ADP-ribose) polymerase (PARP), elevated the ratio of Bax/Bcl-2 protein and overexpression of p53, suggesting the involvement of the activation of p53 and mitochondrial intrinsic pathway in ECP-induced apoptosis. Likewise, ECP oral administration significantly inhibited the U-2 OS cancer growth in xenograft tumor model. All these first evidence indicated that ECP was a potential antitumor supplement for the treatment of human osteosarcoma.

Photobiomodulation Therapy Attenuates Hypoxic-Ischemic Injury in a Neonatal Rat Model

Photobiomodulation (PBM) has been demonstrated as a neuroprotective strategy, but its effect on perinatal hypoxic-ischemic encephalopathy is still unknown. The current study was designed to shed light on the potential beneficial effect of PBM on neonatal brain injury induced by hypoxia ischemia (HI) in a rat model. Postnatal rats were subjected to hypoxic-ischemic insult, followed by a 7-day PBM treatment via a continuous wave diode laser with a wavelength of 808nm. We demonstrated that PBM treatment significantly reduced HI-induced brain lesion in both the cortex and hippocampal CA1 subregions. Molecular studies indicated that PBM treatment profoundly restored mitochondrial dynamics by suppressing HI-induced mitochondrial fragmentation. Further investigation of mitochondrial function revealed that PBM treatment remarkably attenuated mitochondrial membrane collapse, accompanied with enhanced ATP synthesis in neonatal HI rats. In addition, PBM treatment led to robust inhibition of oxidative damage, manifested by significant reduction in the productions of 4-HNE, P-H2AX (S139), malondialdehyde (MDA), as well as protein carbonyls. Finally, PBM treatment suppressed the activation of mitochondria-dependent neuronal apoptosis in HI rats, as evidenced by decreased pro-apoptotic cascade 3/9 and TUNEL-positive neurons. Taken together, our findings demonstrated that PBM treatment contributed to a robust neuroprotection via the attenuation of mitochondrial dysfunction, oxidative stress, and final neuronal apoptosis in the neonatal HI brain.

Greensporone C, a Freshwater Fungal Secondary Metabolite Induces Mitochondrial-Mediated Apoptotic Cell Death in Leukemic Cell Lines.

Therapeutic agents used in the treatment of cancer are known to develop resistance against cancer cells. Hence, there is a continuing need to investigate novel agents for the treatment and management of cancer. Antitumor activity of greensporone C (GC), a new resorcylic acid lactone isolated from an organic extract of a culture of a Halenospora sp. freshwater fungus, was subjected for screening against a panel of leukemic cell lines (K562, U937, and AR320). In all the three cell lines, cell proliferation was inhibited in dose-dependent fashion. GC further arrested the cells in SubG0 phase in dose-dependent manner. Annexin V/PI dual staining data confirmed apoptotic death of treated K562 and U937 leukemic cells. Treatment with GC suppressed constitutively phosphorylated AKT and downregulated expression of inhibitor of apoptotic proteins XIAP, cIAP-1, and cIAP-2. In summation to this, GC-treated leukemic cells upregulated protein expression of pro-apoptotic proteins, Bax with concomitant decrease in expression of anti-apoptotic proteins including Bcl-2 and Bcl-xL. Upregulation of Bax was associated with cytochrome c release which was confirmed from the collapse of mitochondrial membrane. Released cytochrome c further activated caspase cascade which in turn initiated apoptosis process. Anticancer activity of this isolated fungal compound GC was potentiated via stimulating production of reactive oxygen species (ROS) along with depletion of reduced glutathione (GSH) levels in K562 and U937 leukemic cells. Pretreatment of these cells with N-acetyl cysteine prevented GC-induced depletion of reduced GSH level and mitochondrial-caspase-induced apoptosis. Altogether, our data show that GC modulates the apoptotic response of human leukemic cells and raises the possibility of its use as a novel therapeutic strategy for hematological malignancies.

Antioxidant and antidiabetic activity of blackberry after gastrointestinal digestion and human gut microbiota fermentation

Blackberry fruit contains high levels of polyphenols particularly anthocyanins which contribute to its biological activities. Bioavailability of polyphenols especially anthocyanins is generally low, it has been proposed that metabolites from polyphenol biotransformation under colonic fermentation are components that exert health benefits. In this study, blackberry was subjected to simulated gastrointestinal digestion and gut microbiota fermentation at different time intervals (0–48 h) to study the changes in bioactive components, its antioxidant and antidiabetic activities. Phenolic compounds, during digestion and fermentation were also analysed. Gut metabolites of blackberry significantly increased the glucose consumption and glycogen content in HepG2 cells. Furthermore, gut metabolites ameliorated high glucose plus palmitic acid-induced ROS overproduction, mitochondrial membrane collapse, and glutathione depletion in HepG2 cells. The mechanism of antidiabetic activity of blackberry was via its potent antioxidant activity. Therefore, our results suggest that blackberry could be recommended as a functional food due to potential antioxidant and antidiabetic activity.

The toxic effect of Vu-Defr, a defensin from Vigna unguiculata seeds, on Leishmania amazonensis is associated with reactive oxygen species production, mitochondrial dysfunction, and plasma membrane perturbation.

Plant defensins are plant antimicrobial peptides that present diverse biological activities in vitro, including the elimination of Leishmania amazonensis. Plant defensins are considered promising candidates for the development of new drugs. This protozoan genus has great epidemiological importance and the mechanism behind the protozoan death by defensins is unknown, thus, we chose L. amazonensis for this study. The aim of the work was to analyze the possible toxic mechanisms of Vu-Defr against L. amazonensis. For analyses, the antimicrobial assay was repeated as previously described, and after 24 h, an aliquot of the culture was tested for viability, membrane perturbation, mitochondrial membrane potential, reactive oxygen species (ROS) and nitric oxide (NO) inductions. The results of these analyses indicated that after interaction with L. amazonensis, the Vu-Defr causes elimination of promastigotes from culture, membrane perturbation, mitochondrial membrane collapse, and ROS induction. Our analysis demonstrated that NO is not produced after Vu-Defr and L. amazonensis interaction. In conclusion, our work strives to help to fill the gap relating to effects caused by plant defensins on protozoan and thus better understand the mechanism of action of this peptide against L. amazonensis.

Curcumin analog EF24 induces apoptosis via ROS-dependent mitochondrial dysfunction in human colorectal cancer cells.

Colorectal cancer is the most commonly diagnosed malignancy with high mortality rates worldwide. Improved therapeutic strategies with minimal adverse side effects are urgently needed. In this study, the anti-tumor effects of EF24, a novel analog of the natural compound curcumin, were evaluated in colorectal cancer cells. The anti-tumor activity of EF24 on human colon cancer lines (HCT-116, SW-620, and HT-29) was determined by measures of cell cycle arrest, apoptosis, and mitochondrial function. The contribution of ROS in the EF24-induced anti-tumor activity was evaluated by measures of H2O2 and pretreatment with an ROS scavenger, NAC. The findings indicated that EF24 treatment dose-dependently inhibited cell viability and caused cell cycle arrest at G2/M phase in all the tested colon cancer cell lines. Furthermore, we demonstrated that EF24 treatment induced apoptosis effectively via enhancing intracellular accumulation of ROS in both HCT-116 and SW-620 cells, but with moderate effects in HT-29 cells. We found that EF24 treatment decreased the mitochondrial membrane potential in the colon cancer cells, leading to the release of mitochondrial cytochrome c. Also, EF24 induced activation of caspases 9 and 3, causing decreased Bcl-2 protein expression and Bcl-2/Bax ratio. Pretreatment with NAC, a ROS scavenger, abrogated the EF24-induced cell death, apoptosis, cell cycle arrest, and mitochondrial dysfunction, suggesting an upstream ROS generation which was responsible for the anticancer effects of EF24. Our findings support an anticancer mechanism by which EF24 enhanced ROS accumulation in colon cancer cells, thereby resulting in mitochondrial membrane collapse and activated intrinsic apoptotic signaling. Thus, EF24 could be a potential candidate for therapeutic application of colon cancer.

Mitochondrial superoxide mediates mitochondrial and endoplasmic reticulum dysfunctions in TRAIL-induced apoptosis in Jurkat cells

Reactive oxygen species (ROS), such as superoxide (O2•-) and hydrogen peroxide (H2O2), have been reported to be important mediators of the apoptosis induced by death ligands, including Fas, tumor necrosis factor-α, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Conversely, there is evidence that H2O2 and prooxidative conditions are protective. Therefore, the roles of ROS in death ligand-induced apoptosis are a matter of debate. In this study, we attempted to define the oxidant species mediating TRAIL-induced apoptosis in human tumor cells. The generation of intracellular O2•-, but not H2O2, was correlated with apoptosis in the cells. TRAIL treatment resulted in increased mitochondrial O2•- generation and the oxidation of cardiolipin. The O2•--selective scavenger MnTBaP [Mn(III) tetrakis (4-benzoic acid) porphyrin chloride] specifically blocked TRAIL-induced apoptosis and proapoptotic events including mitochondrial membrane collapse and caspase-3/7 activation. TRAIL also induced endoplasmic reticulum (ER) stress responses including caspase-12 activation, while inhibition of caspase-12 prevented the apoptosis. In addition, increased mitochondrial O2•- generation by uncoupling of oxidative phosphorylation or inhibition of the electron transport chain amplified the TRAIL-induced apoptosis and proapoptotic events. This amplification was also significantly abolished by MnTBaP treatment. Our data indicate that mitochondrial O2•- mediates mitochondrial and ER dysfunctions during TRAIL-induced apoptosis in Jurkat cells. The present findings suggest that pharmacological agents increasing mitochondrial O2•- may serve as clinical drugs that amplify TRAIL effectiveness toward cancer cells.

LED Light-Activated Hypocrellin B Induces Mitochondrial Damage of Ovarian Cancer Cells

Objective. Hypocrellin is a natural photosensitizer from a traditional Chinese herb. In the present study, our aim is to investigate the effect of LED light-activated hypocrellin B on mitochondria of ovarian cancer cells.Material and Methods. Ovarian cancer HO-8910 cells were incubated with hypocrellin B at the concentration of 2.5 μM for 5 h and then irradiated by blue light from a novel LED source. Cell survival rate of HO-8910 cells was measured using MTT assay 24 h after photodynamic treatment of hypocrellin B. Mitochondrial morphology was observed using transmission electron microscopy (TEM). Mitochondrial membrane potential was measured using flow cytometry with JC-1 staining.Results. MTT assay showed that cell survival rate of HO-8910 cells in the photodynamic treatment group has significantly decreased down to27.22±1.26% (P<0.01). Light irradiation alone or hypocrellin B alone showed no significant impact. In our TEM mitochondria of the cells after photodynamic treatment of hypocrellin B showed severe damage with swollen mitochondria that had nearly nonexistant cristae. Mitochondrial membrane potential remarkably decreased after photodynamic action of hypocrellin B.Conclusion. The findings demonstrated that photodynamic action of hypocrellin B significantly decreased cell proliferation of ovarian cancer HO-8910 cells, caused severe damage to mitochondrial structure, and induced mitochondrial membrane collapse.

Reactive Oxygen Species Production and Mitochondrial Dysfunction Contribute to Quercetin Induced Death in Leishmania amazonensis

BackgroundLeishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied.Methodology/Principal FindingsIn the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC50 for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential.Conclusions/SignificanceThe effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function.

Inhibition of Superoxide Generation upon T-Cell Receptor Engagement Rescues Mart-127-35–Reactive T Cells from Activation-Induced Cell Death

Cytotoxic T lymphocytes (CTL) may undergo massive expansion upon appropriate antigenic stimulation. Homeostasis is maintained by a subsequent "contraction" of these cells. Activation-induced cell death (AICD) and programmed cell death prevent the untoward side effects, arising from excessive numbers and prolonged persistence of activated CTL, that occur upon uncontrolled and/or continued expansion. However, effector cell persistence has been identified as a hallmark of successful T-cell-mediated adoptive immunotherapy. Thus, prevention of AICD may be critical to achieve more successful clinical results. We have previously shown that treatment with the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125 protects human melanoma epitope Mart-1(27-35)-reactive CTL from apoptotic death upon their reencounter with cognate antigen. However, inhibition of JNK also interferes with the functional ability of the CTL to secrete IFN-gamma. Here, we show that reactive oxygen species (ROS) inhibitors, such as the superoxide dismutase mimetic Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), efficiently protected Mart-1(27-35)-reactive primary CTL from AICD without impairing their functional capability. MnTBAP prevented the increase in intracellular ROS, mitochondrial membrane collapse, and DNA fragmentation observed in control-treated cells upon cognate antigen encounter. Furthermore, the mechanism of AICD prevention in primary CTL included blockade of JNK activation. Finally, tumor-reactive in vitro expanded tumor infiltrating lymphocytes, which are used clinically in cancer immunotherapy, also benefit from MnTBAP-mediated antioxidant treatment. Thus, modulation of the redox pathway might improve CTL persistence and lead to better clinical results for T cell-based immunotherapies.

Cytotoxic activity of violacein in human colon cancer cells

Several studies have shown that violacein, a purple pigment extracted from Chromobacterium violaceum, is capable to induce apoptosis in a variety of cancer cells, including those leukemia cell lines. Herein, we examined the effects of violacein on reactive oxygen species (ROS) production during the apoptotic colon cancer cell death. We demonstrate that violacein mediates ROS production followed by activation of Caspase-3, release of cytochrome c, and calcium release to citosol in Caco-2 cells. Moreover, presence of ROS scavengers such as N-acetyl-cysteine (NAC) diminishes ROS cytotoxicity induced by violacein in Caco-2 cells, indicating that violacein mediates cellular critical mechanisms in the triggering of apoptotic tumor cell death. These data also imply that violacein-induced ROS are collectively key mediators of mitochondrial membrane collapse, leading to cytochrome c release, and culminating in tumor apoptosis. Unlike in Caco-2 cells, violacein was incapable of increasing ROS levels in HT29 cells, suggesting the existence of violacein cell-type specific mechanisms. Those findings bring light to the violacein cytotoxic mechanism studies, indicating that oxidative stress play a role in the violacein-induced cytotoxicity.

Fascioscapulohumeral muscular dystrophy: A progressive degenerative disease that responds to diltiazem

The authors believe that with fascioscapulohumeral muscular dystrophy (FSHD), like Duchenne muscular dystrophy, there is Ca2+ dysregulation in the muscle cells. The dysregulated Ca2+ can cause cell death in various ways. One mechanism may be Ca2+ triggering abnormal levels of tumor necrosis factor (TNF-alpha). Another mechanism may involve excessive Ca2+ levels within the mitochondria which would cause this organelle's membrane to collapse ultimately inducing apoptosis and/or necrosis. With this in mind, it has been reported that in FSHD there is over expression of adenine nucleotide translocator-1 (ANT-1). This Ca2+ dependent protein, which is a component of the mitochondrial permeability transition pore, could be an important culprit in mitochondrial membrane collapse. Therefore, dysregulated Ca2+ as well as TNF-alpha, in addition to over-expression of ANT-1, may result in cell disruption ultimately causing the characteristic dystrophic muscle wasting. The present investigators have noted that some individuals with FSHD benefit from a regimen of diltiazem, a Ca2+ channel blocker. Initial results using diltiazem may represent the first beneficial treatment for a form of muscular dystrophy. Even if there is only a slowing of progression, this would be a positive first step. A combination of several different Ca2+ regulating agents and TNF-alpha inhibitors may be necessary to truly alter and/or reverse the deleterious effects of this form of muscular dystrophy.