Effects of lanthanum chloride on glutamate level, intracellular calcium concentration and caspases expression in the rat hippocampus

Lanthanum chloride (LaCl₃) can affect neurobehavioral development and impair cognitive abilities. The mechanism underlying LaCl₃-induced neurotoxic effects is still unknown. The purpose of this research was to investigate the neuronal impairment induced by LaCl₃ and discuss the possible mechanism from the aspects of the alteration of glutamate level, intracellular calcium concentration ([Ca²⁺]_i), Bax, Bcl-2 and caspases expression in the hippocampus. Lactational rats were exposed to 0, 0.25, 0.50 and 1.0 % LaCl₃ in drinking water, respectively. Their offspring were exposed to LaCl₃ by parental lactation and then administrated with 0, 0.25, 0.50 and 1.0 % LaCl₃ in drinking water for 1 month. The results showed that 0.25, 0.50 and 1.0 % LaCl₃ exposure induced neuronal impairment in the hippocampus of young rat. Hippocampal glutamate level, [Ca²⁺]_i and ratio of Bax and Bcl-2 expression increased significantly after LaCl₃ exposure. Besides, LaCl₃ exposure increased GRP78, GRP94, GADD153 and p-JNK expression, promoted the activation of caspase-3, caspase-9 and caspase-12, induced PARP cleavage and caused excessive apoptosis. These results indicate that LaCl₃ increases glutamate level, [Ca²⁺]_i and ratio of Bax and Bcl-2 expression, which cause excessive apoptosis by the mitochondrial and endoplasmic reticulum stress-induced pathway, and thus neuronal damages in the hippocampus.     

Endoplasmic Reticulum Calcium Release Engages Bax Translocation in Cortical Astrocytes

Apoptosis is a highly complex form of cell death that can be triggered by alterations in Ca²⁺ homeostasis. Members of the Bcl-2 family may regulate apoptosis and modulate Ca²⁺ distribution within intracellular compartments. Bax, a proapoptotic member of the family, is constitutively expressed and soluble in the cytosol and, under apoptotic induction, translocates to mitochondrial membranes. However, it is not clear if the intracellular Ca²⁺ stores and selective Ca²⁺ releases can modulate or control Bax translocation. The aim of this study was to investigate the relation of intracellular Ca²⁺ stores with Bax translocation in rat cortical astrocytes. Results show that the classical apoptotic inducer, staurosporine, caused high elevations of cytosolic Ca²⁺ that precede Bax translocation. On the other hand, agents that mobilize Ca²⁺ from endoplasmic reticulum such as noradrenaline or thapsigargin, induced Bax translocation, while mitochondrial Ca²⁺ release evoked by carbonyl cyanide-p-(trifluoromethoxyphenyl) hydrazone was not able to cause Bax punctation. In addition, microinjection of inositol 1,4,5- trisphosphate induced Bax translocation. Taken together, our results show that in Bax overexpressing cortical astrocytes, endoplasmic reticulum-Ca²⁺ release may induce Bax transactivation and specifically control apoptosis.     

High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced apoptosis via calcineurin pathways

It has been previously shown that Walker 256 tumor cells express a high content of the anti-apoptotic protein Bcl-2 which protects mitochondria against the damaging effects of Ca²⁺. In the present study, we analyze H₂O₂-induced apoptotic death in two different types of tumor cells: Walker 256 and SCC-25. Treatment with H₂O₂ (4mM) increased reactive oxygen species generation and the concentration of cytosolic free Ca²⁺. These alterations preceded apoptosis in both cell lines. In Walker cells, which show a high Bcl-2/Bax ratio, apoptosis was dependent on calcineurin activation and independent of changes in mitochondrial membrane potential (Δ < eqid1 > _m), as well as cytochrome c release. In contrast, in SCC-25 cells, which show a lower Bcl-2/Bax ratio, apoptosis was preceded by a decrease in Δ < eqid2 > _m, mitochondrial permeability transition, and cytochrome c release. Caspase-3 activation occurred in both cell lines. The data suggest that although the high Bcl-2/Bax ratio protected the mitochondria of Walker cells from oxidative stress, it was not sufficient to prevent apoptosis through calcineurin pathways.     

Modulation of Ca2+ signaling by antiapoptotic Bcl-2 versus Bcl-xL: From molecular mechanisms to relevance for cancer cell survival

Members of the Bcl-2-protein family are key controllers of apoptotic cell death. The family is divided into antiapoptotic (including Bcl-2 itself, Bcl-xL, Mcl-1, etc.) and proapoptotic members (Bax, Bak, Bim, Bim, Puma, Noxa, Bad, etc.). These proteins are well known for their canonical role in the mitochondria, where they control mitochondrial outer membrane permeabilization and subsequent apoptosis. However, several proteins are recognized as modulators of intracellular Ca²⁺ signals that originate from the endoplasmic reticulum (ER), the major intracellular Ca²⁺-storage organelle. More than 25 years ago, Bcl-2, the founding member of the family, was reported to control apoptosis through Ca²⁺ signaling. Further work elucidated that Bcl-2 directly targets and inhibits inositol 1,4,5-trisphosphate receptors (IP₃Rs), thereby suppressing proapoptotic Ca²⁺ signaling. In addition to Bcl-2, Bcl-xL was also shown to impact cell survival by sensitizing IP₃R function, thereby promoting prosurvival oscillatory Ca²⁺ release. However, new work challenges this model and demonstrates that Bcl-2 and Bcl-xL can both function as inhibitors of IP₃Rs. This suggests that, depending on the cell context, Bcl-xL could support very distinct Ca²⁺ patterns. This not only raises several questions but also opens new possibilities for the treatment of Bcl-xL-dependent cancers. In this review, we will discuss the similarities and divergences between Bcl-2 and Bcl-xL regarding Ca²⁺ homeostasis and IP₃R modulation from both a molecular and a functional point of view, with particular emphasis on cancer cell death resistance mechanisms.     

The BH4 Domain of Anti-apoptotic Bcl-XL,but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Excessive Ca²⁺ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca²⁺-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca²⁺-dependent apoptosis by binding and inhibiting IP₃Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP₃Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca²⁺ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1^−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca²⁺ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca²⁺ release in the cytosol or to prevent apoptosis, as done instead by an IP₃R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca²⁺ uptake into the mitochondria.     

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca²⁺) signals. Since the proper supply of Ca²⁺ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca²⁺ signaling. The endoplasmic reticulum (ER) is the main Ca²⁺ storage for the cell and Bcl-2 family proteins competitively regulate its Ca²⁺ concentration. Bcl-2 family proteins also regulate the flux of Ca²⁺ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP₃Rs) to mediate their opening. Type 1 IP₃Rs reside at the bulk ER to coordinate cytosolic Ca²⁺ signals, while type 3 IP₃Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca²⁺ uptake. In healthy cells, mitochondrial Ca²⁺ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca²⁺ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca²⁺ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca²⁺ signaling and how mitochondrial Ca²⁺ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.     

Protective effects of a wheat germ peptide (RVF) against H2O2-induced oxidative stress in human neuroblastoma cells

RVF (Arg-Val-Phe), a peptide derived from wheat germ, shows antioxidant properties. Here, the neuroprotective efficacies of RVF were investigated in human neuroblastoma cells (SH-SY5Y) that were pretreated with RVF (150–250 μM, 4 h) and exposed to H₂O₂ (200 μM). RVF increased viable cell numbers by 37 % and reduced the release of lactate dehydrogenase. Pretreatment with RVF also inhibited H₂O₂-induced accumulation of reactive oxygen species and maintained the mitochondrial transmembrane potential as well as preventing intracellular Ca²⁺ dysregulation during H₂O₂ exposure. Furthermore, pretreatment with RVF increased the Bcl-2/Bax ratio and blocked cleavage poly(ADP-ribose) polymerase by inhibiting caspase-3 activation, thus decreasing apoptosis.     

Lanthanum chloride suppresses oxysterol-induced ECV-304 cell apoptosis via inhibition of intracellular Ca<Superscript>2+</Superscript> concentration elevation,oxidative stress,and activation of ERK and NF-κB signaling pathways

Experimental studies have demonstrated that oral administration of lanthanum chloride (LaCl₃) inhibits the development of atherosclerosis, but the related mechanism has not been fully elucidated. Oxysterols are toxic to the vascular endothelial cells which are important in preventing the formation and progression of atheromatous plaque. In this study, we examined the effect of LaCl₃ on oxysterol cholestane-3β,5α,6β-triol (Triol)-induced apoptosis and the related mechanisms in ECV-304 cells, a presumptive endothelial cell line. Incubation with Triol resulted in apoptosis of ECV-304 cells, as determined by Hoechst 33342 staining, fluorescein isothiocyanate labeled annexin V/propidium iodide double staining, and the loss of mitochondrial membrane potential. Triol activated extracellular-signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), and inhibition of Triol-activated ERK and NF-κB signaling by specific inhibitors attenuated apoptosis induction by Triol in ECV-304 cells. Pretreatment with LaCl₃ (1 μM) for 12 h before exposure to Triol decreased Triol-mediated apoptosis as well as activation of ERK and NF-κB. In addition, Triol induced oxidative stress in ECV-304 cells, manifested by the increase of intracellular reactive oxygen species generation and malondialdehyde level, and the reduction of the content of total protein thiols and the activity of antioxidant glutathione peroxidases; LaCl₃ pretreatment significantly reversed these effects. Finally, LaCl₃ pretreatment significantly inhibited the increases of intracellular Ca²⁺ concentration induced by Triol. Our study suggests that Triol induced ECV-304 cell apoptosis, and LaCl₃ could suppress this effect probably by inhibiting intracellular Ca²⁺ concentration elevation, oxidative stress, as well as activation of ERK and NF-κB signaling pathways.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Lanthanum-containing bioparticles are associated with the influence of lanthanum on high phosphate mediated bone marrow stromal cells viability

Lanthanum (La)-based binder appears effective in reducing serum inorganic phosphate (Pi) levels among chronic dialysis patients, yet concern remains about La accumulation in bone during long-term oral administration. In this study, the effect of lanthanum chloride (LaCl₃) on bone marrow stromal cells (BMSCs) viability was investigated under high Pi situation. We found low concentration (10^?9 M) of LaCl₃ increased BMSCs viability, while high concentration (10^?5 M) of LaCl₃ not only inhibited BMSCs viability but also stimulated high Pi induced cell apoptosis. In addition, La-containing calcium phosphate (CaP) particles can be detected on cell surfaces and inside cells. We also found that inhibition of CaP formation by adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, abrogated LaCl₃ induced the BMSCs viability. For isolated La-containing CaP particles, the particle size increased and crystal phase switched with elevated concentration of LaCl₃. These results demonstrated that La-containing CaP particles were associated with the process of LaCl₃ mediated BMSCs viability and the physicochemical properties of these particles played an important role in modulating BMSCs function.     

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP‐1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l ‐Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP‐1 activity using overexpression of the dominant negative DRP‐1^K38A slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild‐type DRP‐1. In contrast, responses to l ‐Glu appeared DRP‐1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca²⁺ uptake or the ability of l ‐Glu to stimulate excitotoxic Ca²⁺ overload. Finally, apoptosis‐induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:335–341, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

Mfn2 Affects Embryo Development via Mitochondrial Dysfunction and Apoptosis

BackgroundGrowth factors, energy sources, and mitochondrial function strongly affect embryo growth and development in vitro. The biological role and prospective significance of the mitofusin gene Mfn2 in the development of preimplantation embryos remain poorly understood. Our goal is to profile the role of Mfn2 in mouse embryos and determine the underlying mechanism of Mfn2 function in embryo development.MethodsWe transfected Mfn2-siRNA into 2-cell fertilized eggs and then examined the expression of Mfn2, the anti-apoptotic protein Bcl-2, and the apoptosis-promoting protein Bax by Western blot. Additionally, we determined the blastocyst formation rate and measured ATP levels, mtDNA levels, mitochondrial membrane potential (ΔΨm), and apoptosis in all of the embryos.ResultsThe results indicate that the Mfn2 and Bcl-2 levels were markedly decreased, whereas Bax levels were increased in the T group (embryos transfected with Mfn2-siRNA) compared with the C group (embryos transfected with control-siRNA). The blastocyst formation rate was significantly decreased in the T group. The ATP content and the relative amounts of mtDNA and cDNA in the T group were significantly reduced compared with the C group. In the T group, ΔΨm and Ca²⁺ levels were reduced, and the number of apoptotic cells was increased.ConclusionLow in vitro expression of Mfn2 attenuates the blastocyst formation rate and cleavage speed in mouse zygotes and causes mitochondrial dysfunction, as confirmed by the ATP and mtDNA levels and mitochondrial membrane potential. Mfn2 deficiency induced apoptosis through the Bcl-2/Bax and Ca²⁺ pathways. These findings indicate that Mfn2 could affect preimplantation embryo development through mitochondrial function and cellular apoptosis.     

Baicalein-Induced Apoptosis via Endoplasmic Reticulum Stress Through Elevations of Reactive Oxygen Species and Mitochondria Dependent Pathway in Mouse–Rat Hybrid Retina Ganglion Cells (N18)

Studies were designed to investigate the effects of baicalein on mouse–rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, reactive oxygen species (ROS), cytoplasmic Ca²⁺, mitochondrial membrane potential (MMP), apoptosis induction, and caspases-3 activity were examined by flow cytometric assay. Apoptosis-associated proteins such as p53, Bax, Bcl-2, cytochrome c, and caspase-3 were examined by Western blot. We demonstrated the increase in the levels of p53, Bax, and cytochrome c and decrease in the level of Bcl-2, which are associated with the induction of apoptotic cell death after 24 h treatment with baicalein in N18 cells. Baicalein induced an increase in the cytoplasmic levels of ROS and Ca²⁺ in 1 h and reached their peak at 3 h, and thereafter a loss of MMP by flow cytometry. We also demonstrated a release of the cytochrome c from mitochondria into cytosol and an activation of caspase-3, which led to the occurrence of apoptosis in N18 cells treated with baicalein by Western blot. Pretreatment was conducted with BAPTA (intracellular calcium chelator) in baicalein-treated cells, the decline of MMP was recovered, and the increase in the level of cytoplasmic Ca²⁺ was suppressed, and the proportion of apoptosis was also markedly diminished. In conclusion, our data suggests that oxidative stress and cellular Ca²⁺ modulates the baicalein-induced cell death via a Ca²⁺-dependent mitochondrial death pathway in N18 cells.     

Guattegaumerine Protects Primary Cultured Cortical Neurons Against Oxidative Stress Injury Induced by Hydrogen Peroxide Concomitant with Serum Deprivation

Guattegaumerine is a natural product with characteristics of being lipophilic and reaching high concentration in the brain, but its function in the central nervous system has not yet been observed. This study was designed to evaluate the neuroprotective effects of guattegaumerine on rat primary cultured cortical neurons. Following a 24-h exposure of the cells to combined serum-starvation and hydrogen peroxide, a significant augment in neuron damage as determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release were observed. Preincubation of guattegaumerine dramatically improved the cell viability and inhibited LDH release. Preincubation of guattegaumerine also dramatically inhibited malondialhehyde (MDA) production and elevated the decreased total antioxidative capacity in cells caused by the combined injury. Results of flow cytometry and immunohistochemistry showed that pre-addition of guattegaumerine interrupted the apoptosis of the neurons, reversed the up regulation of the pro-apoptotic gene (Bax) and the down regulation of the anti-apoptotic gene (Bcl-2). Furthermore, guattegaumerine suppressed the increase of intracellular calcium ([Ca²⁺]_i) stimulated by either H₂O₂ or KCl in Ca²⁺-containing extracellular solutions, and high concentration of 2.5 μM guattegaumerine also suppressed the increase of [Ca²⁺]_i induced by H₂O₂ in Ca²⁺-free solution. These observations suggested that guattegaumerine may possess potential protection against oxidative stress injury, which might be beneficial for neurons.     

Effect of Hyperoxia on Cortical Neuronal Nuclear Function and Programmed Cell Death Mechanisms

There is growing concern over detrimental neurologic effects to human newborns caused by increased inspired oxygen concentrations. We hypothesize that hyperoxia (FiO₂ > 0.95) results in increased high-affinity Ca²⁺-ATPase activity, Ca²⁺-influx, and proapoptotic protein expression in cortical neuronal nuclei of newborn piglets. Neuronal cerebral energy metabolism was documented by determining ATP and phosphocreatine levels. Neuronal nuclear conjugated dienes and fluorescent compounds were measured as indices of lipid peroxidation. High-affinity Ca²⁺-ATPase activity and ATP-dependent Ca²⁺-influx were determined to document neuronal nuclear membrane function. Hyperoxia resulted in increases in lipid peroxidation, high-affinity Ca²⁺-ATPase activity, ATP-dependent Ca²⁺-influx, and Bax/Bcl-2 ratio in the cortical neuronal nuclei of newborn piglets. We conclude that hyperoxia results in modification of neuronal nuclear membrane function leading to increased nuclear Ca²⁺-influx, and propose that hyperoxia-induced increases in intranuclear Ca²⁺ activates the Ca²⁺/calmodulin-dependent protein kinase pathway, triggering increased CREB protein-mediated apoptotic protein expression in hyperoxic neurons.     

Concomitant Inhibition of MDM2 and Bcl-2 Protein Function Synergistically Induce Mitochondrial Apoptosis in AML

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance ofantiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acutemyeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus,functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects ofMdm2 inhibition. We here investigate the potential therapeutic utility of combinedtargeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors ofprotein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational changeand mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3ainduced p53-mediated apoptosis predominantly in S and G₂/M cells, while cells in G₁ were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G₁ , the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser⁷⁰ was absent in G₁ but detectable in G₂/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G₁ cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.     

The timing of re-institution of good blood glucose control affects apoptosis and expression of Bax and Bcl-2 in the retina of diabetic rats

Hyperglycemia initiates a sequence of events that leads to the development of diabetic retinopathy. We explored the effect of re-institution of good blood glucose control on apoptosis and apoptosis related genes (Bax and Bcl-2) in the retina of diabetic rats. Fifty male Wistar rats randomly divided into five groups : normal control group (CON), diabetic rats with high blood glucose levels for 8 months group (DM) ,diabetic rats with good blood glucose control for 8 months group (DM₁),diabetic rats with poor blood glucose control for 2 month followed by good blood glucose control for six additional months group (DM₂), rats with poor blood glucose control for 4 months followed by good blood glucose levels for four additional months group (DM₃). Expression of Bax and Bcl-2 in the retina was studied by immunohistochemistry and the apoptotic cells were stained using the TUNEL method. The apoptotic cell, expression of Bax and Bcl-2 and the ratio of Bax to Bcl-2 in the retina was increased in DM group compared with normal rats’ (P < 0.01). There was no significant difference in apoptotic cells and the ratio of Bax to Bcl-2 between DM₁ group and CON group. The number of TUNEL positive cells and Bax to Bcl-2 ratio was partially reversed in DM₂ group. But glucose control had no effect on the apoptotic cells and the expression of Bax and Bcl-2 in DM₃ group. There was a positive correlation between apoptotic cells and Bax/Bcl-2 ratio in the retina (r = 0.808, P < 0.01). Good blood glucose control at early stage can decrease the number of apoptotic cells in the retina; the decreased apoptosis is correlated with the down-regulation of Bax to Bcl-2 ratio.     

The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver

Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na₂SeO₃, 2 mg/kg), Cd (CdCl₂, 150 mg/kg), or Se + Cd (150 mg/kg of CdCl₂ and 2 mg/kg of Na₂SeO₃) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.     

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.