Gallic acid ester derivatives induce apoptosis and cell adhesion inhibition in melanoma cells: The relationship between free radical generation, glutathione depletion and cell death

Malignant melanoma is a lethal disease, and the incidence and mortality associated with it are increasing worldwide. It has a significant tendency to develop both metastasis and resistance to chemotherapy. The tumor cells show abnormal redox regulation, and although the molecular mechanisms involved are not well characterized, they seem to be related to oxidative stress. In a previous study, we showed the antitumoral properties of gallic acid ester derivatives in leukemia cells. Here, we show the effect of octyl, decyl, dodecyl and tetradecyl gallates on B16F10 cells, a melanoma cell line. All compounds induced cytotoxic effects, and the IC₅₀ values obtained were between 7 μM and 17 μM after 48 h of incubation. Cell death occurred through apoptosis, as demonstrated by the genomic DNA fragmentation pattern. The gallates were able to induce significant production of free radicals, deplete both glutathione and ATP, activate NF-κB and promote the inhibition of cell adhesion under the experimental conditions. The glutathione depletion induced by these compounds was related to the inhibition of γ-glutamylcysteine synthase activity. These results suggest that gallates induce tumoral cell death through apoptosis as a consequence of oxidative stress, though they use different mechanisms to do so. These findings are important since melanoma cells are resistant to death because of their high level of antioxidant defense, adhesion capability and propensity to metastasize.     

Diabetes-induced changes in glucose synthesis,intracellular glutathione status and hydroxyl free radical generation in rabbit kidney-cortex tubules

Diabetes-induced changes in glucose formation, intracellular and mitochondrial glutathione redox states as well as hydroxyl free radicals (HFR) generation have been investigated in rabbit kidney-cortex tubules. In contrast to renal tubules of control animals, diabetes-evoked increase in glucose formation in the presence of either aspartate + glycerol + octanoate or malate as gluconeogenic precursors (for about 50%) was accompanied by a diminished intracellular glutathione reduced form (GSH)/glutathione oxidised one (GSSG) ratio by about 30–40%, while the mitochondrial GSH/GSSG ratio was not altered. However, a relationship between the rate of gluconeogenesis and the intracellular glutathione redox state was maintained in renal tubules of both control and diabetic rabbits, as concluded from measurements in the presence of various gluconeogenic precursors. Moreover, diabetes resulted in both elevation of the glutathione reductase activity in rabbit kidney-cortex and acceleration of renal HFR generation (by about 2-fold). On the addition of melatonin, the hormone exhibiting antioxidative properties, the control values of HFR production were restored, suggesting that this compound might be beneficial during diabetes therapy. In view of the data, it seems likely that diabetes-induced increase in HFR formation in renal tubules might be responsible for a diminished intracellular glutathione redox state despite elevated glutathione reductase activity and accelerated rate of gluconeogenesis, providing glucose-6-phosphate for NADPH generation via pentose phosphate pathway. (Mol Cell Biochem 261: 91–98, 2004)     

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO(*)) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.     

Habitual physical activity facilitates stress-induced HSP72 induction in brain,peripheral, and immune tissues

The mechanism(s) for how physically active organisms are resistant to many damaging effects of acute stressor exposure is unknown. Cellular induction of heat-shock proteins (e.g., HSP72) is one successful strategy used by the cell to survive the damaging effects of stress. It is possible, therefore, that the stress-buffering effect of physical activity may be due to an improved HSP72 response to stress. Thus the purpose of the current study was to determine whether prior voluntary freewheel running facilitates the stress-induced induction of HSP72 in central (brain), peripheral, and immune tissues. Adult male Fischer 344 rats were housed with either a mobile running wheel (Active) or a locked, immobile wheel [sedentary (Sed)] for 8 wk before stressor exposure. Rats were exposed to either inescapable tail-shock stress (IS; 100 1.6-mA tail shocks, 5-s duration, 60-s intertrial interval), exhaustive exercise stress (EXS; treadmill running to exhaustion), or no stress (controls). Blood, brain, and peripheral tissues were collected 2 h after stressor termination. The kinetics of HSP72 induction after IS was determined in cultured mesenteric lymph node cells. Activation of the stress response was verified by measuring serum corticosterone (RIA). Tissue and cellular HSP72 content were measured using HSP72 ELISA in cell lysates. Both Active and Sed rats had elevated levels of serum corticosterone after stress. In contrast, Active but not Sed rats exposed to IS and/or EXS had elevated HSP72 in dorsal vagal complex, frontal cortex, hippocampus, pituitary, adrenal, liver, spleen, mesenteric lymph nodes, and heart. In addition, Active rats exposed to IS demonstrated a faster induction of lymphocyte HSP72 compared with Sed rats. Thus Active rats responded to stress with both greater and faster HSP72 responses compared with Sed rats. These results indicate that previous physical activity potentiates HSP72 expression after a wide range of stressors. Facilitated induction of HSP72 may contribute to the increased stress resistance previously reported in physically active organisms.     

In vivo reduction of chromium (VI) and its related free radical generation

Chromium (VI) compounds are widely recognized as human carcinogens. Extensive studies in vitro and in model systems indicate that the reactive intermediate, Cr (V), generated by cellular reduction of Cr (VI), is likely the candidate for the ultimate carcinogenic form of chromium compounds. Here we review our current understanding of the in vivo reduction of Cr (VI) and its related free radical generation. Our results demonstrate that Cr (V) is indeed generated from the reduction of Cr (VI) in vivo, and that Cr (V) thus formed can mediate the generation of free radicals. Cr (V) and its related free radicals are very likely to be involved in the mechanism of Cr (VI)induced toxicity and carcinogenesis. These studies also illustrate that in vivo EPR spectroscopy and magnetic resonance imaging can be very useful and powerful tools for studying paramagnetic metal ions in chemical and biochemical reactions occurring in intact animals.     

Exposure to nickel, chromium, or cadmium causes distinct changes in the gene expression patterns of a rat liver derived cell line

Many heavy metals, including nickel (Ni), cadmium (Cd), and chromium (Cr) are toxic industrial chemicals with an exposure risk in both occupational and environmental settings that may cause harmful outcomes. While these substances are known to produce adverse health effects leading to disease or health problems, the detailed mechanisms remain unclear. To elucidate the processes involved in the toxicity of nickel, cadmium, and chromium at the molecular level and to perform a comparative analysis, H4-II-E-C3 rat liver-derived cell lines were treated with soluble salts of each metal using concentrations derived from viability assays, and gene expression patterns were determined with DNA microarrays. We identified both common and unique biological responses to exposure to the three metals. Nickel, cadmium, chromium all induced oxidative stress with both similar and unique genes and pathways responding to this stress. Although all three metals are known to be genotoxic, evidence for DNA damage in our study only exists in response to chromium. Nickel induced a hypoxic response as well as inducing genes involved in chromatin structure, perhaps by replacing iron in key proteins. Cadmium distinctly perturbed genes related to endoplasmic reticulum stress and invoked the unfolded protein response leading to apoptosis. With these studies, we have completed the first gene expression comparative analysis of nickel, cadmium, and chromium in H4-II-E-C3 cells.     

Effect of temperature and duration of hyperthermia on HSP72 induction in rat tissues

The aim of the present study was to determine whether heat shock protein 72 (HSP72) is induced in a heated rat model at rectal temperatures below 42 degrees C. Rats were divided into a control group and six groups (n = 6) heated to different rectal temperatures: 39 degrees C for 1 h (39), 40.0 degrees C for either 15 min (40S) or 1 h (40L), 41.0 degrees C for either 15 min (41S) or 1 h (41L) and 42.0 degrees C for 15 min (42). Tissues were sampled 4 h after heating. Following 1 h at 40.0 degrees C, HSP72 was significantly elevated in heart (p < 0.005), but not in gut or liver tissue. In all three tissues, HSP72 was significantly elevated under the conditions 41L and 42 compared to control tissue (p < 0.005). Marked differences were found in the amount of HSP72 induced in different tissues in response to the same heat stress. Duration of heating was important in modulating HSP72 induction, with a significantly greater induction of HSP72 following 1 h compared to 15 min at 41 degrees C in all three tissues (p < 0.02). A correlation was found between thermal load and HSP72 content in liver, heart (both p < 0.01) and gut (p < 0.001) for the rats heated to 41 and 42 degrees C. These data show that HSP72 is induced at temperatures below 42 degrees C, with striking differences between tissues.     

In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) increase

Adding the membrane-permeant oxidant tert-butylhydroperoxide (t-BOOH) to the incubation medium, in SH-SY5Y human neuroblastoma cells, induced a marked and progressive concentration-dependent (300, 500 and 1000 microM) increase of free radical production, as evaluated by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and of the intracellular Ca(2+) ion concentrations [Ca(2+)](i). The removal of extracellular Ca(2+) ions did not prevent t-BOOH-induced [Ca(2+)](i) elevation, whereas the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy) ethane-N,N, N',N'-tetraacetic acid (BAPTA) (10 microM) was shown to be effective. Both t-BOOH-induced free radical formation and the [Ca(2+)](i) increase were completely prevented by the peroxyl scavenger alpha-tocopherol (50 microM). t-BOOH induced a time-dependent SH-SY5Y cell injury, monitored by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (approximately 25% at 1 h, 50% at 3 h, 80% at 5 h) and by fluorescein diacetate (FDA)-propidium iodide (PI) fluorescent staining. The entity of t-BOOH-induced cell damage was the same both in the absence and in the presence of the intracellular Ca(2+) ion chelator BAPTA. By contrast, the peroxyl scavenger alpha-tocopherol (50 microM) completely prevented cell injury due to oxidative stress. Finally, superoxide dismutase (SOD) (500 ng/ml) caused a 30% reduction of t-BOOH-induced 2', 7'-dichlorofluorescein (DCF) fluorescence, whereas it did not modify the extent of cell injury produced by the oxidant. Collectively, the results of the present study demonstrated that in SH-SY5Y human neuroblastoma cells, the rise of [Ca(2+)](i) which occurs during oxidative stress is not involved in cell injury. Therefore, oxidative stress-induced cell death may be exclusively attributed to free radical overproduction.     

The carcinogen safrole increases intracellular free Ca2+ levels and causes death in MDCK cells

The effect of the carcinogen safrole on intracellular Ca2+ movement in renal tubular cells has not been explored previously. The present study examined whether safrole could alter Ca2+ handling in Madin-Darby canine kidney (MDCK) cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 33 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 400 microM. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+, but was not affected by nifedipine, verapamil, or diltiazem. Addition of Ca2+ after safrole had depleted intracellular Ca(2+)-induced dramatic Ca2+ influx, suggesting that safrole caused store-operated Ca2+ entry. In Ca(2+)-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca 2+. Inhibition of phospholipase C with 2 microM U73122 did not affect safrole-induced Ca2+ release. Trypan blue exclusion assays revealed that incubation with 650 microM safrole for 30 min did not kill cells, but killed 70% of cells after incubation for 60 min. Collectively, the data suggest that in MDCK cells, safrole induced a [Ca2+] increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent fashion, and by inducing Ca2+ influx via store-operated Ca2+ entry. Furthermore, safrole can cause acute toxicity to MDCK cells.     

Survival and cell death in cells constitutively unable to synthesize glutathione

We examined the role of GSH in survival and cell death using GCS-2 cells that are deficient in glutamate cysteine ligase (gamma-glutamyl cysteine synthetase, gammaGCS), an enzyme essential for GSH synthesis. Cells maintained in 2.5 mM GSH have GSH levels that are approximately 2% of wild type and grow indefinitely; however, they express both pro- and anti-apoptotic Bcl-2 family members and have detectable levels of cytoplasmic cytochrome C. Withdrawal of GSH from the medium results in a fall in intracellular GSH to undetectable levels, decreased mitochondrial dehydrogenase activity, decreased anti-apoptotic factor RNAs, increased pro-apoptotic factor RNAs, additional cytochrome C release, and a fall in ATP levels; however, cells continue to grow for another 24h. At 48 h, these trends continue with the exception that mitochondrial membrane potential and ATP levels rise; DNA fragmentation begins at 48 h. Thus, severe reduction of GSH to 2% of wild type produces a metastable state compatible with survival, but complete absence of GSH triggers apoptosis.     

Hypoxia-mediated prior induction of monocyte-expressed HSP72 and HSP32 provides protection to the disturbances to redox balance associated with human sub-maximal aerobic exercise

HSP72 is rapidly expressed in response to a variety of stressors in vitro and in vivo (including hypoxia). This project sought a hypoxic stimulus to elicit increases in HSP72 and HSP32 in attempts to confer protection to the sub-maximal aerobic exercise-induced disturbances to redox balance. Eight healthy recreationally active male subjects were exposed to five consecutive days of once-daily hypoxia (2,980?m, 75?min). Seven days prior to the hypoxic acclimation period, subjects performed 60?min of cycling on a cycle ergometer (exercise bout 1—EXB1), and this exercise bout was repeated 1?day post-cessation of the hypoxic period (exercise bout 2—EXB2). Blood samples were taken immediately pre- and post-exercise and 1, 4 and 8?h post-exercise for HSP72 and immediately pre, post and 1?h post-exercise for HSP32, TBARS and glutathione [reduced (GSH), oxidised (GSSG) and total (TGSH)], with additional blood samples obtained immediately pre-day 1 and post-day 5 of the hypoxic acclimation period for the same indices. Monocyte-expressed HSP32 and HSP72 were analysed by flow cytometry, with measures of oxidative stress accessed by commercially available kits. There were significant increases in HSP72 (P?<?0.001), HSP32 (P?=?0.03), GSSG (t?=?9.5, P?<?0.001) and TBARS (t?=?5.6, P?=?0.001) in response to the 5-day hypoxic intervention, whereas no significant changes were observed for GSH (P?=?0.22) and TGSH (P?=?0.25). Exercise-induced significant increases in HSP72 (P?<?0.001) and HSP32 (P?=?0.003) post-exercise in EXB1; this response was absent for HSP72 (P?≥?0.79) and HSP32 (P?≥?0.99) post-EXB2. The hypoxia-mediated increased bio-available HSP32 and HSP72 and favourable alterations in glutathione redox, prior to exercise commencing in EXB2 compared to EXB1, may acquiesce the disturbances to redox balance encountered during the second physiologically identical exercise bout.     

Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage

Iron storage proteins, ferritin and haemosiderin, release iron to a range of chelators and reducing agents, including citrate, acetate and ascorbate. Released iron promotes both hydroxyl radical formation in the presence of hydrogen peroxide and lipid peroxidation in liposomes. Ferritin protein is modified in such reactions, both by free radical cleavage and addition reactions with aldehyde products of lipid peroxidation.     

HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90''s previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.Necroptosis is an inflammatory, caspase-independent form of regulated cell death characterised by loss of cellular membrane integrity and release of cytoplasmic contents.¹ It is believed to have evolved as a defence mechanism against viruses;^{2, 3} however, there is increasing evidence that deregulated necroptosis has a role in the pathogenesis of a range of inflammatory, autoimmune and neurodegenerative diseases.^{4, 5, 6, 7, 8} Reduced capacity to undergo necroptosis has been correlated to increased aggressiveness of cancers;^{9, 10} and therapeutic initiation of necroptosis is currently being investigated as a cancer therapy.^{11, 12} Additionally, there is emerging evidence that the necroptotic signalling pathway has a general role in the modulation of inflammation.^{13, 14, 15, 16, 17} As such, unravelling the molecular events governing necroptosis, and potential avenues for therapeutic intervention, is of enormous interest.Necroptosis is initiated through activation of death receptors, such as Tumour Necrosis Factor Receptor 1 (TNFR1), or through microbial activation of pattern recognition receptors, such as Toll-like receptors or intracellular viral DNA sensors.^{3, 18, 19, 20} Receptor ligation initiates a signalling cascade, whereby Receptor Interacting Protein Kinase (RIPK)-3 oligomerises and is phosphorylated, a process known to be regulated by association with other effectors, such as the protein kinase RIPK1, TIR-domain-containing adapter-inducing IFN-β (TRIF), or DNA-dependent activator of IFN regulatory factors (DAI), via their RIP Homotypic Interaction Motifs (RHIMs).^{2, 21, 22} Once activated, RIPK3 phosphorylates the pseudokinase domain of Mixed Lineage Kinase domain-Like (MLKL), the most downstream known obligate effector of the necroptotic signalling pathway, to induce its activation.^{23, 24} MLKL phosphorylation is thought to trigger a molecular switch,^{25, 26, 27} leading to the unleashing of the N-terminal executioner four-helix bundle (4HB) domain,²⁸ MLKL oligomerisation and translocation to cellular membranes where cell death occurs via an incompletely-understood mechanism.^{28, 29, 30}Molecular chaperones have an integral role in modulating both the structure and function of proteins. One such chaperone is heat-shock protein 90 (HSP90), which interacts with a diverse group of protein ‘clients'', the largest group comprising the kinases and pseudokinases, with 50% of the human kinome estimated to interact with HSP90.³¹ These interactions are dependent on the recognition of the kinase or pseudokinase domain by the HSP90 co-chaperone Cdc37, which enables HSP90 to confer protein stabilisation, assist in late-stage folding and conformational modifications, and mediate intracellular transport.^{32, 33, 34, 35}It has already been demonstrated that the necroptotic pathway is subject to modulation by HSP90. RIPK1 is well established as an HSP90 client protein, with a number of studies finding HSP90 inhibition affects both the stability and function of RIPK1 and promotes an apoptotic phenotype.^{36, 37, 38, 39, 40, 41} More recently, RIPK3 was also identified as an HSP90 client.^{2, 42, 43} Surprisingly, HSP90 inhibition did not markedly impact RIPK3 abundance or stability, but rather was essential for RIPK3''s necroptotic functions, such as phosphorylation of MLKL.⁴² However, whether MLKL itself is a client of HSP90 has not been investigated.In this study, using a phenotypic screen for small-molecule inhibitors of MLKL-driven cell death, we identified HSP90 as a modulator of necroptosis that functions on, or downstream of, the terminal effector, MLKL. HSP90 inhibition did not markedly reduce levels of MLKL in human U937 or mouse dermal fibroblasts, suggesting instead that HSP90 has an active role in governing MLKL-mediated cell death. This idea is supported by our finding that cell death driven by the S345D activated mutant of MLKL in Ripk3-deficient fibroblasts in the absence of necroptotic stimuli was suppressed by three distinct chemical classes of HSP90 inhibitor, but MLKL abundance was not impacted by HSP90 inhibition. Although our data indicate that MLKL binds HSP90 weakly or transiently, HSP90 activity was essential for the assembly of MLKL into high molecular weight complexes and the membrane translocation known to precede cell death. These findings suggest an expanded role for HSP90 in regulating necroptosis, and further our understanding of the mechanisms controlling MLKL-mediated cell death.     

Divergent effects of resveratrol, a polyphenolic phytostilbene, on free radical levels and type of cell death induced by the histone deacetylase inhibitors butyrate and trichostatin A

We investigated the effects of the polyphenolic phytostilbene resveratrol on the steady-state free radical (FR) concentration and mode of cell death induced by the histone deacetylase inhibitors butyrate and trichostatin A. (i) There was no correlation between cell death induction by butyrate or trichostatin A (TSA) and FR levels. (ii) Treatment with resveratrol or N-acetyl-l-cystein (NAC) of cells, in which the FR concentration was high, resulted in an almost complete reduction of FR levels. (iii) When, however, the cellular FR concentration was marginal, resveratrol caused a minor, and NAC a marked increase of FRs as well as of the extent of cell death. Thus, resveratrol and NAC acted as antioxidants only when the cellular FR levels were high, and acted as pro-oxidants when facing a low FR concentration. (iv) Since resveratrol and the antioxidant NAC exhibited analogous effects, it is concluded that the observed actions of resveratrol are due to polyphenolic redox reactions and not related to the stilbene moiety of the molecule. (v) The results indicate that the redox status of a given cell type plays an important role in determining whether resveratrol and other antioxidants promote cell death or protect cells from it.     

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.     

Multiple roles of cadmium in cell death and survival

Cadmium is a toxic metal with no known biological function. It is increasingly important as an environmental hazard to both humans and wildlife, and it exemplifies the double edged nature of many toxic substances. Thus, on the one hand cadmium can act as a mitogen, stimulate cell proliferation, inhibit apoptosis, inhibit DNA repair, and promote cancer in a number of tissues. On the other hand, it causes tissue damage, notably in the kidney, by inducing cell death. At low and moderate concentrations in cell culture systems (e.g., 0.1–10 μM) cadmium primarily causes apoptosis, and at higher concentrations (>50 μM) necrosis becomes evident. This generalization appears to hold in vivo. There is also evidence of cadmium-induced autophagy, although whether this is a direct cause of cell death remains uncertain. After discussing these generalities, this review considers the details of apoptotic death, and its inhibition, in renal mesangial cells. We also present evidence for the effect of environmental exposure to cadmium in affecting renal function, and in particular review the evidence for the role of the mesangial cell in cadmium nephrotoxicity.     

N-acyl dopamines induce cell death in PC12 cell line via induction of nitric oxide generation and oxidative stress

It was shown that dopamine amides of arachidonic, oleic, and docosahexaenoic acids exhibit toxicity with respect to PC12 pheochromocytoma cell line. The mechanism of realization of the cytotoxic effect of acyl dopamines is the induction of oxidative stress. This event is preceded by triggering the synthesis of nitric oxide.     

Electron-spin-resonance evidence for enzymic reduction of oxygen to a free radical, the superoxide ion

1. An electron-spin-resonance signal with g( parallel)2.08 and g( perpendicular)2.00 is observed by the rapid-freezing technique during the oxidation of substrates by molecular oxygen catalysed by xanthine oxidase at pH10. 2. The intensity of this signal is shown to depend on oxygen rather than on enzyme concentration, indicating that it is due to an oxygen free radical and not to the enzyme. 3. The same species is shown to be produced in the reaction at pH10 between hydrogen peroxide and periodate ions. Studies with this system have facilitated comparison of the properties of the oxygen radical with data in the literature on the products of pulse radiolysis of oxygenated water over a wide pH range. 4. It is concluded that the species observed is the superoxide ion, O(2) (-), and that the stability of this ion is greatly increased in alkaline solution. A mechanism explaining the alkaline stability is proposed. 5. The importance of O(2) (-) in the enzymic reaction is discussed.     

The effect of hyperthermia on the induction of cell death in brain, testis, and thymus of the adult and developing rat

Stressful stimuli can elicit 2 distinct reactive cellular responses, the heat shock (stress) response and the activation of cell death pathways. Most studies on the effects of hyperthermia on the mammalian nervous system have focused on the heat shock response, characterized by the transient induction of Hsps, which play roles in repair and protective mechanisms. This study examines the effect of hyperthermia on the induction of cell death via apoptosis, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and active caspase 3 cytochemistry, in the adult rat brain, testis, and thymus. Results show that a fever-like increase in temperature triggered apoptosis in dividing cell populations of testis and thymus, but not in mature, postmitotic cells of the adult cerebellum. These differential apoptotic responses did not correlate with whole-tissue levels of Hsp70 induction. We further investigated whether dividing neural cells were more sensitive to heat-induced apoptosis by examining the external granule cell layer of the cerebellum at postnatal day 7 and the neuroepithelial layers of the neocortex and tectum at embryonic day 17. These proliferative neural regions were highly susceptible to hyperthermia-induced apoptosis, suggesting that actively dividing cell populations are more prone to cell death induced by hyperthermia than fully differentiated postmitotic neural cells.     

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl¹-NH-Aryl² scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.