Levels Of Cytosolic Cytochrome Research Articles

Abstract 17456: Inositol 1,4,5-Trisphosphate Receptors (ITP3Rs) and Activation of Cardiac Fibroblasts in Ischemia/Reperfusion Injury

Introduction: Inositol 1,4,5-trisphosphate receptors (ITP3Rs) are Ca 2+ release channels on the endoplasmic reticulum (ER). The role of ITP3Rs in cardiomyocytes has been previously investigated for hypertrophy and arrhythmias, however, the exact function in the activation of cardiac fibroblasts (FBs) is yet to be determined. The main goal of this project is to investigate the mechanisms linking FB ITP3Rs to post-ischemia/reperfusion (I/R) cardiac fibrosis. Hypothesis: We hypothesize that ITP3Rs activate FB via a direct transcriptional regulation of αSMA and other genes involved in myoFB activation. Indeed, myoFB differentiation and persistence is regulated by a balance between pro-and anti-apoptotic signaling pathways. Methods: We generated myoFB-specific ITP3Rs KO mice (ITP3R KO ) and isolated FBs as well as cardiomyocytes via collagenase digestion and performed immunoprecipitation (IP) assays showing that ITP3Rs bind p53 upregulated modulator of apoptosis (PUMA). Cytosolic levels of Cytochrome C (Cyt C) were assessed in myoFB isolated from areas remote from injury and scar areas 7 d after I/R. After having immunoprecipitated for ITP3R1 we blotted the isolated FBs for PUMA and, vice versa. PUMA levels were measured in cytosol, ER, and mitochondria of FBs isolated from regions remote from injury and scar areas 7 d post I/R. Results: Our data show reduced levels of cytosolic Cyt C following I/R, which was prevented by IP3Rs ablation, providing an explanation for the persistence of myoFBs in remote areas. We found that the mitochondrial expression of PUMA was significantly decreased - while ER levels were markedly increased - in myoFBs isolated from areas remote from I/R injury, indicating a sequestration of PUMA by ITP3Rs; this phenotype was rescued by ITP3Rs ablation. Instead, in myoFBs isolated from the scar areas, mitochondrial PUMA levels were increased both in ITP3R fl/fl and ITP3R KO mice, and no sequestration was observed, consistent with the absence of ITP3R upregulation. Conclusions: Using a specific Cre/lox KO model, we provide the first assessment of functional contribution of ITP3R to post-I/R activation of cardiac FBs and understanding the molecular mechanisms underlying excessive reactive fibrosis in the heart.

(D620N) VPS35 causes the impairment of Wnt/\u03b2-catenin signaling cascade and mitochondrial dysfunction in a PARK17 knockin mouse model

Patients with familial type 17 of Parkinson’s disease (PARK17) manifest autosomal dominant pattern and late-onset parkinsonian syndromes. Heterozygous (D620N) mutation of vacuolar protein sorting 35 (VPS35) is genetic cause of PARK17. We prepared heterozygous VPS35D620N/+ knockin mouse, which is an ideal animal model of (D620N) VPS35-induced autosomal dominant PARK17. Late-onset loss of substantia nigra pars compacta (SNpc) dopaminergic (DAergic) neurons and motor deficits of Parkinson’s disease were found in 16-month-old VPS35D620N/+ mice. Normal function of VPS35-containing retromer is needed for activity of Wnt/β-catenin cascade, which participates in protection and survival of SNpc DAergic neurons. It was hypothesized that (D620N) VPS35 mutation causes the malfunction of VPS35 and resulting impaired activity of Wnt/β-catenin pathway. Protein levels of Wnt1 and nuclear β-catenin were reduced in SN of 16-month-old VPS35D620N/+ knockin mice. Downregulated protein expression of survivin, which is a target gene of nuclear β-catenin, and upregulated protein levels of active caspase-8 and active caspase-9 were observed in SN of VPS35D620N/+ mice at age of 16 months. VPS35 is involved in controlling morphology and function of mitochondria. Impaired function of VPS35 caused by (D620N) mutation could lead to abnormal morphology and malfunction of mitochondria. A significant decrease in mitochondrial size and resulting mitochondrial fragmentation was found in tyrosine hydroxylase-positive and neuromelanin-positive SNpc DAergic neurons of 16-month-old VPS35D620N/+ mice. Mitochondrial complex I activity or complex IV activity was reduced in SN of 16-month-old VPS35D620N/+ mice. Increased level of mitochondrial ROS and oxidative stress were found in SN of 16-month-old VPS35D620N/+ mice. Levels of cytosolic cytochrome c and active caspase-3 were increased in SN of VPS35D620N/+ mice aged 16 months. Our results suggest that PARK17 mutant (D620N) VPS35 impairs activity of Wnt/β-catenin signaling pathway and causes abnormal morphology and dysfunction of mitochondria, which could lead to neurodegeneration of SNpc DAergic cells.

Mitochondrial Dysfunction Secondary to Endoplasmic Reticulum Stress in Acute Myocardial Ischemic Injury in Rats.

BackgroundThe relationship between endoplasmic reticulum and mitochondria during acute myocardial ischemic injury is still unclear. Our study aimed to define the dynamics of endoplasmic reticulum stress and mitochondrial dysfunction during acute ischemic injury.Material/MethodsA rat model of acute myocardial infarction and hypoxic cardiomyocytes were used in this study. Groups were set at 0 hours, 1 hour, 2 hours, 4 hours, and 6 hours after ischemic injury for both in vivo and in vitro studies. ATF6 and GRP-78 were examined to indicate endoplasmic reticulum stress. Cellular ATP and cytosolic levels of mitochondrial DNA and cytochrome c were detected to evaluate mitochondrial dysfunction. Caspase-3 was used for apoptosis analysis.ResultOur results showed that both mRNA and protein levels of ATF6 and GRP-78 were elevated from 1 hour after ischemic injury in vivo and in vitro (P<0.05). However, ATP levels were increased at 2 hours after ischemic injury and significantly decreased from 4 hours after ischemic injury in vivo, while ATP level of cultured cardiomyocytes decreased remarkably from 2 hours after ischemic injury (P<0.05). Cytosolic mitochondrial DNA levels began to increase from 2 hours after ischemic injury (P<0.05). Cytosolic levels of cytochrome c increased from 4 hours after ischemic injury. Additionally, both mRNA and protein expressions of caspase-3 started to significantly elevate at 6 hours after ischemic injury (P<0.05).ConclusionsThe present study suggested that mitochondrial dysfunction was secondary to endoplasmic reticulum stress, which provides a novel experimental foundation for further exploration of the detailed mechanism after ischemic injury, especially the interaction between endoplasmic reticulum and mitochondria.

The effects of sevoflurane on myocardial damage after cardiopulmonary resuscitation in rats

Objective To investigate the effects of sevoflurane on myocardial damage after cardiopulmonary resuscitation in rats. Methods The cardiac arrest model was established by asphyxia. Thirth-six male Wistar rats were assigned to three groups randomly(random number). Rats in the sham group were anesthetized with intubation, without induced cardiac arrest. Rats in the sevo group inhaled 1minimal alveolar concentration(MAC) of sevoflurane during CPR. Rats in the control group received the standard CPR. At 4 h and 24 h after ROSC , half the rats of every group received the echocardiography examination. At 4 h after ROSC, the rest of rats in every group were sacrificed with overdose anesthetics, the hearts were removed and mitochondria was isolated promptly. Determination of the status of mitochondrial permeability transition pore(MPTP) using the spectrophotometer. The level of cytosolic cytochrome C was measured by Western blot. Results After ROSC, the left ventricular posterior wall during diastolic(LVPWd) became thick and the cardiac output was decreased dramatically. At 4 h after ROSC, the mice of the sevo group had a slighter thickness of LVPWd (mm)(1.95±0.14)vs.(2.32±0.34), P<0.05 and an enhanced cardiac output(mL/min)(46.94±3.61)vs.(38.77±6.63), P<0.05 than the control group. The mitochondria swelling was triggered by the addition of CaCl2,the sevo group showed an slighter decrease in the absorbance of light compared with the control group(0.048±0.007)vs.(0.069±0.011), P<0.01. After ROSC, cytochrome C was released to cytosol, and the sevo group had a lower level of this protein than the control group(1.02±0.22)vs.(1.59±0.31), P<0.05. Conclusions After ROSC, sevoflurane can alleviate myocardial dysfunction, attenuate the MPTP opening and decrease the level of cytosolic cytochrome C. Key words: Cardiac arrest; Cardio-pulmonary resuscitation; Asphyxia; Sevoflurane; Post-resuscitation myocardial dysfunction; Mitochondrial permeability transition pore

Aeroallergen Der p 2 induces apoptosis of bronchial epithelial BEAS-2B cells via activation of both intrinsic and extrinsic pathway.

BackgroundExcessive apoptosis of airway epithelium is reported to induce airway remodeling and inhibited airway epithelium repair is highly associated with development of asthma and chronic obstructive pulmonary disease. Der p 2 is a major allergen derived from Dermatophagoides pteronyssinus and commonly causes airway hypersensitiveness and asthma; however, the connection between Der p 2 and epithelial apoptosis remains unclear. This study was aimed to explore whether Der p 2 induces apoptosis of airway epithelial cells and the underlying mechanisms.ResultsOur results showed that recombinant Der p 2 (rDP2) inhibited cell growth and induced apoptosis of human bronchial epithelial cell BEAS-2B. Further investigation revealed that rDP2 increased intracellular reactive oxygen species, level of cytosolic cytochrome c and cleavage of caspase-9 and caspase-3. rDP2 also induced activation of p38 mitogen-activated protein kinase (P38) and c-Jun N-terminal kinase (JNK), and triggered proapoptotic signals including decrease of Bcl-2, increase of Bax and Bak, and upregulation of Fas and Fas ligand. In parallel, rDP2 inhibited glycogen synthase kinase 3beta and consequently enhanced degradation of cellular (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP). Involvement of toll-like receptor (TLR)2 in rDP2-induced apoptosis was also demonstrated using specific small inhibitory RNA.ConclusionsOur findings indicate that rDP2 suppresses cell growth and trigger apoptosis of BEAS-2B cells, which may attribute to induction of both intrinsic and extrinsic pathway via TLR2 and P38/JNK signaling and c-FLIP degradation. It suggests that Der p 2 may aggravate respiratory disorders through enhancement of apoptosis and the consequent airway injury.

A small molecule activator of AKT does not reduce ischemic injury of the rat heart.

BackgroundActivation of protein kinase AKT is required for cardioprotection by ischemic preconditioning, and transgenic overexpression of AKT protects the heart against ischemia. However, it is unknown whether acute pharmacological activation of AKT alone, using a therapeutically relevant strategy, induces cardioprotection. In this study we provide the first evidence to clarify this question.MethodsWe used a recently described specific activator of AKT, the small molecule SC79, to treat rat hearts submitted to ischemia and reperfusion. Initially, isolated rat hearts were perfused with increasing doses of SC79 to verify the magnitude of AKT activation. Low and high doses were determined and used to treat hearts submitted to ischemia (35 minutes) and reperfusion (60 minutes), in a randomized and blinded design. AKT activation was verified by western immunobloting. Metabolic profile was determined by cardiac ATP content and mitochondrial enzyme activity, while cytosolic levels of cytochrome C and caspase-3 activity were used as markers of apoptosis. Ischemic injury was assessed by quantification of infarct size and cardiac release of creatine kinase and lactate dehydrogenase.ResultsSC79 activated cardiac AKT within 30 minutes in a dose-dependent fashion. ATP content was largely reduced by ischemia, but was not rescued by SC79. Similarly, mitochondrial enzyme activity was not affected by SC79. SC79 administered before ischemia or at reperfusion did not prevent cytosolic accumulation of cytochrome C and overactivation of caspase-3. Finally, SC79 failed to reduce infarct size or release of cardiac injury biomarkers at reperfusion.ConclusionWe conclude that selective AKT activation by the synthetic molecule SC79 does not protect the rat heart against ischemic injury, indicating that acute pharmacological activation of AKT is not sufficient for cardioprotection.

VvpM, an extracellular metalloprotease of Vibrio vulnificus, induces apoptotic death of human cells.

A pathogenic bacterium, Vibrio vulnificus produces various extracellular proteases including the elastolytic metalloprotease, VvpE. In silico analysis of its genome revealed a VvpE-homologous protease, VvpM whose proteolytic activity was abolished by specific inhibitors against metalloproteases. To investigate whether this newly identified protease has pathogenic role in host interaction in addition to proteolytic role, human cell lines were incubated with recombinant VvpM (rVvpM). rVvpM-challenged cells showed typical morphological changes found in cells under apoptosis. Apoptotic cell death was further evidenced by estimating the Annexin V-stained cells, whose proportions were dependent upon the concentrations of rVvpM treated to human cells. To elucidate the signaling pathway for VvpM-induced apoptosis, three MAPKs were tested if their activation were mediated by rVvpM. ERK1/2 was phosphorylated by treatment of rVvpM and rVvpM-induced cell death was blocked by a specific inhibitor against ERK1/2. In rVvpM-treated cells, the cytosolic levels of cytochrome c were increased in a VvpM concentration-dependent manner, while the levels of cytochrome c in mitochondria were decreased. Cell deaths were accompanied by apparent cleavages of procaspases-9 and -3 to the active caspases-9 and -3, respectively. Therefore, this study demonstrates that an extracellular metalloprotease of V. vulnificus, VvpM induces apoptosis of human cells via a pathway consisting of ERK activation, cytochrome c release, and then activation of caspases-9 and -3.

Abstract 145: Mitophagy Protects Against Statin-Induced Cell Death in Muscle Cells

Statins are a family of cholesterol lowering drugs associated with undisputable cardiovascular benefits. However, their use is associated with the risk of developing skeletal muscle myopathy which remains a significant barrier to maximizing the efficacy of statin therapy. Many recognize that the instances of statin-associated myopathy reported grossly underestimate the actual frequency of these events. Statin myopathy is a major reason for why many patients are unable to comply with their prescribed statin treatments. Identifying and understanding the molecular events involved in the development of statin-induced myopathy will pave the way to mitigating the unwanted effect of statin-myopathy. We have identified that statins induce mitochondrial loss in both C2C12 myotubes, and in gastrocnemius muscle of mice evidenced by loss of mitochondrial markers Tom70 and Cox4 by western blot analysis. C2C12 myotubes were treated with 2μM simvastatin for 24 hours, and wild-type C57BL/6 mice were given a daily i.p. dose of 20mg/kg simvastatin for 2 weeks. Pretreatment of C2C12 cells with 100nM bafilomycin for 2 hours before statin treatment attenuated autophagic flux, and prevents the observed loss of mitochondria through Tom70 and Cox4 mitochondrial markers. Thus, the loss of mitochondria is attributable to autophagy (mitophagy). Statin treatment increases the expression of Bnip3 and p62/SQSTM1 (sequestosome 1) which have been identified as important facilitators of mitophagy. We found increased Bnip3 dimerization via western blot under non-reducing conditions which implies increased activity. Furthermore, we observed increased mitochondrial translocation of p62/SQSTM1. Based on our findings, we hypothesized that statin-induced mitophagy may play a key role in the development of muscle myopathy. To investigate the role of mitophagy in statin myopathy, we silenced p62/SQSTM1 via RNAi prior to statin treatment. Surprisingly, reducing mitophagic flux via p62/SQSTM1 silencing resulted in increased destruction of myotubes. This was associated with increased levels of cytosolic cytochrome c. Our data combined suggests that mitophagy protects against statin-mediated cell death by sequestering and eliminating decrepit mitochondria via autophagy.

Osteopontin stimulates apoptosis in adult cardiac myocytes via the involvement of CD44 receptors, mitochondrial death pathway, and endoplasmic reticulum stress

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify the receptor for OPN and get insight into the mechanism by which OPN induces cardiac myocyte apoptosis. Adult rat ventricular myocytes (ARVMs) and transgenic mice expressing OPN in a myocyte-specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral-mediated OPN expression induced apoptosis in ARVMs. OPN co-immunoprecipitated with CD44 receptors, not with β1 or β3 integrins. Proximity ligation assay confirmed interaction of OPN with CD44 receptors. Neutralizing anti-CD44 antibodies inhibited OPN-stimulated apoptosis. OPN activated JNKs and increased expression of Bax and levels of cytosolic cytochrome c, suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase-12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase-12 inhibitor significantly reduced OPN-stimulated apoptosis. Expression of OPN in adult mouse heart in myocyte-specific manner associated with decreased left ventricular function and increased myocyte apoptosis. In the heart, OPN expression increased JNKs and caspase-12 activities, and expression of Bax and Gadd153. Thus, OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of mitochondrial death pathway and ER stress.

Osteopontin Stimulates Cardiac Myocyte Apoptosis via the Involvement of ER Stress and Mitochondrial Death Pathway

Increased osteopontin (OPN; matricellular protein) expression associates with myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify OPN receptor, and understand the mechanism by which OPN induces myocyte apoptosis. Adult rat ventricular myocytes (ARVM) and transgenic mice expressing OPN in a myocyte‐specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral‐mediated OPN expression induced apoptosis in ARVM. OPN co‐immunoprecipitated with CD44 receptor. Neutralizing anti‐CD44 antibodies inhibited OPN‐stimulated apoptosis. OPN activated JNKs, and increased expression of Bax and levels of cytosolic cytochrome c suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase‐12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase‐12 inhibitor significantly reduced OPN‐stimulated apoptosis. Expression of OPN in adult mouse heart associated with increased myocyte apoptosis and LV dysfunction. In the heart, OPN expression increased JNKs and caspase‐12 activities, and expression of Bax and Gadd153. OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of ER stress and mitochondrial death pathway.

Cystamine attenuates lupus‐associated apoptosis of ventricular tissue by suppressing both intrinsic and extrinsic pathways

Cystamine, a disulphide metabolite, has been demonstrated to ameliorate various lupus-associated tissue damages by animal models. However, effects of cystamine on apoptosis of cardiac tissue, a main cardiac damage attributing to lupus, are less obvious. Therefore, we aimed to investigate whether or not cystamine possesses anti-apoptotic effects with emphasis on LV tissue of lupus-prone mice NZB/W-F1. Cystamine treatment was performed by daily intraperitoneal administration. Morphology and apoptotic status of ventricular tissues in the treated mice were assessed by microscopy and TUNEL assay, respectively. Levels of apoptotic biomarkers were determined using immunoblot. Our results revealed that cystamine significantly attenuated the apoptosis of LV tissues in NZB/W-F1 mice, whereas the morphology of the tissues was slightly altered. In addition, cystamine reduced level of Fas and inhibited activation of caspase-8. Cystamine also increased level of Bcl-2 and phosphorylation of Bad, and decreased level of Bad and truncated Bid (tBid). Moreover, level of cytosolic cytochrome c and Apaf-1, and activation of caspase-9 and caspase-3 were suppressed in response to cystamine treatment. In Balb/c mice, as normal control mice, changes in cell morphology and levels of the tested apoptotic components were found insignificant in the LV tissues. These findings indicate that cystamine treatment attenuates apoptosis of LV tissues of NZB/W-F1 mice through suppressing both intrinsic and extrinsic apoptotic pathways. Therefore, cystamine is considered beneficial to alleviating lupus-associated cardiac damages.

Taxol, a Microtubule Stabilizer, Improves Cardiac Functional Recovery during Postischemic Reperfusion in Rat in Vitro

Microtubule disruption contributes to cellular and organic dysfunction, and is implicated in ischemia/reperfusion (I/R) injury. The purpose of this study was to explore the effects of taxol, a microtubule stabilizer, on cardiac functional recovery during reperfusion. Left ventricular developed pressure, left ventricular end-diastolic pressure, maximal time derivatives of pressure and the severity of ventricular arrhythmias were analyzed in isolated rat heart. Microtubule structure was immunohistochemically measured. Apoptosis and necrosis was identified with TUNEL or TTC staining, respectively. Mitochondrial permeability transition pore (mPTP) mRNA expression was examined by real-time polymerase chain reactions. mPTP opening, reactive oxygen species (ROS), and oxidative enzyme activities were measured with fluorometric or spectrophotometric techniques. Intracellular calcium concentration ([Ca(2+) ](i) ) and Ca(2+) transients were examined by Fura-2-AM and Fluo-3-AM, respectively. Cytosolic cytochrome c, sarcoplasmic reticulum Ca(2+) -ATPase (SERCA2), ryanodine receptors (RyR), phospholamban (PLB), and PLB phosphorylation were analyzed by Western blot. Effective refractory period (ERP) and afterpotential-mediated activity were detected using microelectrode. Taxol improved the functional recovery of post-I/R. Taxol preserved the intact microtubule structure in reperfusion. mPTP mRNA expression was unchanged while the mPTP opening was reduced by taxol, and this effect was accompanied by the decreased ROS level caused by oxidative enzymes activities' changes. Taxol reduced apoptosis and the level of cytosolic cytochrome c in reperfusion. Taxol also promoted rapid recovery of [Ca(2+) ](i) , prevented reduction of the amplitude of Ca(2+) transients and shortened the decay time of Ca(2+) transients. The protein expression of SERCA2, RyR, and PLB remained unchanged in reperfusion. Taxol prevented the increase of Phospho-Thr17-PLB and Phospho-Ser16-PLB in reperfusion. In addition, taxol facilitated rapid recovery of ERP and counter-acted afterpotential-mediated activity. Taxol may effectively improve cardiac functional recovery during reperfusion via inhibiting mPTP opening, ameliorating abnormal calcium homeostasis, and reducing the substrates associated with arrhythmias.

Combination treatment with photodynamic therapy and curcumin induces mitochondria-dependent apoptosis in AMC-HN3 cells

Photodynamic therapy (PDT) is a treatment for the selective destruction of cancerous and non-neoplastic cells that involves the simultaneous presence of light, oxygen and a light-activatable chemical known as a photosensitizer. Curcumin is one of the most extensively investigated phytochemicals with chemopreventive potential and antitumor effects. In this study, the effect of a combination of PDT and curcumin on apoptotic cell death in AMC-HN3 cells and the molecular mechanism underlying apoptosis was examined to confirm the interaction between photofrin-induced PDT and curcumin during combined mortality. The combination treatment with curcumin and PDT inhibited approximately 70% of the cell viability after PDT, whereas the PDT and curcumin only groups showed a 50 and 10% decrease in cell viability, respectively. In addition, the combination treatment increased the apoptotic events, such as nuclear fragmentation and nuclear condensation. This combination group showed an increase in ROS generation that was higher than that observed after each single treatment. Compared to the single agent treatments, the combination therapy induced the enhanced loss of ∆ψm. Furthermore, the cytosolic levels of cytochromec were significantly elevated in the combination group. Caspases-9, -3 and PARP, which are apoptosis-related proteins induced by mitochondrial activation, were upregulated remarkably by the combination treatment. When co-treated with glutathione, a singlet oxygen quencher, the combination treatment-induced synergistic cytotoxic and apoptotic effects, enhanced the generation of ROS and suppressed the upregulation of caspase-3 and PARP. These results suggest that the combination modality with PDT and curcumin have a better treatment effect invitro. The induction of mitochondrial-dependent apoptosis due to the increased generation of ROS may be involved in this combination treatment.

Tetramethylphenylenediamine protects the isolated heart against ischaemia‐induced apoptosis and reperfusion‐induced necrosis

Cytochrome c when released from mitochondria into cytosol triggers assembly of the apoptosome resulting in caspase activation. Recent evidence suggests that reduced cytochrome c is unable to activate the caspase cascade. In this study, we investigated whether a chemical reductant of cytochrome c, N,N,N',N'-tetramethylphenylene-1,4-diamine (TMPD), which we have previously shown to block cytochrome c-induced caspase activation, could prevent ischaemia-induced apoptosis in the rat perfused heart. The Langendorff-perfused rat hearts were pretreated with TMPD and subjected to stop-flow ischaemia or ischaemia/reperfusion. The activation of caspases (measured as DEVD-p-nitroanilide-cleaving activity), nuclear apoptosis of cardiomyocytes (measured by dUTP nick end labelling assay), mitochondrial and cytosolic levels of cytochrome c (measured spectrophotometrically and by elisa), and reperfusion-induced necrosis (measured as the activity of creatine kinase released into perfusate) were assessed. We found that perfusion of the hearts with TMPD strongly inhibited ischaemia- or ischaemia/reperfusion-induced activation of caspases and partially prevented nuclear apoptosis in cardiomyocytes. TMPD did not prevent ischaemia- or ischaemia/reperfusion-induced release of cytochrome c from mitochondria into cytosol. TMPD also inhibited ischaemia/reperfusion-induced necrosis. These results suggest that TMPD or related molecules might be used to protect the heart against damage induced by ischaemia/reperfusion. The mechanism of this protective effect of TMPD probably involves electron reduction of cytochrome c (without decreasing its release) which then inhibits the activation of caspases.

Abstract 2655: Apoptotic peptide hybrid for treatment of chemotherapy-resistant cancers

Abstract Resistance to chemotherapy is one of the major obstacles to successful cancer treatment. Many cancer patients develop metastatic cancers after responding to initial treatment, which are often refractory to chemotherapy, and remain incurable. Inhibition of apoptotic pathways due to up-regulation of anti-apoptotic elements, such as Bcl-2 and IAPs (inhibitors of apoptosis proteins) has been reported to account for the resistance to chemotherapeutic agents. Thus, it is critical to restore the inhibited apoptosis to re-sensitize the cancer to chemotherapy. In this study, we investigated whether a pro-apoptotic peptide and a proteasome inhibitor peptide can synergize to induce apoptosis in doxorubicin-resistant cancer cells in vitro. The pro-apoptotic peptide is a cationic amphipathic peptide which depolarizes mitochondrial membranes to release apoptosis initiators such as, cytochrom c and Smac/DIABLO to cell cytosols, which activate caspases, apoptosis executors by inhibiting IAPs (inhibitors of apoptosis proteins). MG132 (Z-Leu-Leu-Leu-CHO) has been reported to inhibit anti-apoptotic proteins including NF-[[Unable to Display Character: &amp;#312;]]B and Bcl-2 family proteins to stimulate apoptosis. We hypothesized that when concurrently delivered to the same cells, KLA and MG132 would synergize to induce apoptosis in resistant cancer cells by acting on different stages of apoptotic pathways. To test this, we conjugated KLA and MG132 by forming acid-labile Schiff bases between ε-amino groups of lysines in KLA and the aldehyde group of MG132. We expected that the formed KLA-MG132 conjugates would be internalized into cells by pinocytosis then, dissociated in the acidic environment (pH 4 – 5) of lysosomes to release intact KLA and MG132. The conjugation efficiency was found to be ∼ 100%, as measured indirectly by quantifying amine groups of un-conjugated lysine residues by TNBS assay. The conjugation was confirmed further by determining the molecular masses of the formed conjugates by mass spectrometry after reducing the conjugates by NaBH4 to stabilize Schiff bases. The conjugates induced upto 50 % of cell death in doxorubicin-resistant human cancer cells at the concentration of 0.6 μM as determined by MTT assay meanwhile; un-conjugated MG132 and KLA were inactive at the same concentration. And, the levels of cytosolic cytochrome c were 2-fold greater in the cells treated with KLA-MG132 conjugates than in cells treated with MG132 alone. The levels of active caspase-3 were higher by up to 70 % in cells treated with KLA-MG132 conjugate than in cells treated with MG132 alone. In conclusion, we have observed that KLA and MG132 conjugated via Schiff base induced potent apoptosis in doxorubicin-resistant human cancer cell lines at sub-micromolar concentrations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2655.

Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis

In the past few years, there has been much concern about the adverse health effects of environmental contaminants in general and organochlorine in particular. Studies have shown the repro-toxic effects of long-term exposure to methoxychlor, a member of the organochlorine family. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. In the present study we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to methoxychlor. Experimental rats were divided into six groups of four each. Animals were orally administered with a single dose of methoxychlor (50 mg/kg body weight) and killed at 0, 3, 6, 12, 24, and 72 h post-treatment. The levels and time-course of induction of apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas–FasL and NF-κB were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay and flowcytometry. Administration of methoxychlor resulted in a significant increase in the levels of cytosolic cytochrome c and procaspase 9 as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL, pro- and cleaved caspase 3 were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells, and also by flowcytometry of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of methoxychlor.

Extracellular ubiquitin inhibits -AR-stimulated apoptosis in cardiac myocytes: role of GSK-3 and mitochondrial pathways

Beta-adrenergic receptor (beta-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVMs) via the activation of glycogen synthase kinase-3beta (GSK-3beta) and mitochondrial pathways. However, beta-AR stimulation induces apoptosis only in a fraction ( approximately 15-20%) of ARVMs. We hypothesized that ARVMs may secrete/release a survival factor(s) which protects 80-85% of cells from apoptosis. Using two-dimensional gel electrophoresis followed by MALDI TOF and MS/MS, we identified ubiquitin (Ub) in the conditioned media of ARVMs treated with beta-AR agonist (isoproterenol). Western blot analysis confirmed increased Ub levels in the conditioned media 3 and 6 h after beta-AR stimulation. Inhibition of beta1-AR and beta2-AR subtypes inhibited beta-AR-stimulated increases in extracellular levels of Ub, whereas activation of adenylyl cyclase using forskolin mimicked the effects of beta-AR stimulation. Incubation of cells with exogenous biotinylated Ub followed by western blot analysis of the cell lysates showed uptake of extracellular Ub into cells, which was found to be higher after beta-AR stimulation (1.9 +/- 0.4-fold; P < 0.05 vs. control, n = 6). Pre-treatment with Ub inhibited beta-AR-stimulated increases in apoptosis. Inhibition of phosphoinositide 3-kinase using wortmannin and LY-294002 prevented anti-apoptotic effects of extracellular Ub. Ub pre-treatment inhibited beta-AR-stimulated activation of GSK-3beta and c-Jun N-terminal kinase (JNK) and increases in the levels of cytosolic cytochrome c. The use of methylated Ub suggested that the anti-apoptotic effects of extracellular Ub are mediated via monoubiquitination. beta-AR stimulation increases levels of Ub in the conditioned media. Extracellular Ub plays a protective role in beta-AR-stimulated apoptosis, possibly via the inactivation of GSK-3beta/JNK and mitochondrial pathways.

High molecular weight FGF-2 promotes postconditioning-like cardioprotection linked to activation of the protein kinase C isoforms Akt and p70 S6 kinaseThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Fibroblast growth factor 2 (FGF-2) is a multifunctional protein translated as high and low molecular weight isoforms (hi- and lo-FGF-2, respectively). Although the postconditioning cardioprotective effect of lo-FGF-2 (18 kDa) has been documented, hi-FGF-2 is less well studied. We used an isolated perfused rat heart model of ischemia-reperfusion to study the effects of postischemic (during reperfusion) administration of hi-FGF-2 on recovery of contractile function and tissue salvage, as indicated by decreased cytosolic cytochrome c levels. Compared with the vehicle-treated group, hi-FGF-2-treated hearts had significantly improved recovery of systolic pressure, developed pressure, rates of contraction and relaxation, and coronary flow, as well as decreased relative levels of cytosolic cytochrome c. The effects of hi-FGF-2 on functional recovery and cytosolic cytochrome c were indistinguishable from those induced by lo-FGF-2. Both hi- and lo-FGF-2 upregulated relative levels of phosphorylated (activated) Akt and p70 S6 kinase, and they both promoted translocation of alpha, epsilon, and zeta isoforms of protein kinase C (PKC) to the particulate fraction of reperfused hearts. The magnitude of the effect on PKCzeta and p70 S6 kinases, however, was significantly more potent in the hi-FGF-2 than in the lo-FGF-2 group. We conclude that acute postischemic cardioprotection by hi- or lo-FGF-2 is isoform nonspecific and likely to be mediated by PKC and Akt. Nevertheless, isoform-specific functions are suggested by the augmented sensitivity of p70 S6 and PKCzeta to hi-FGF-2.

Activity-dependent neuroprotective protein–derived peptide, NAP, preventing alcohol-induced apoptosis in fetal brain of C57BL/6 mouse

Possible prevention of the effects of prenatal alcohol exposure has been investigated using peptides that were previously shown to be involved in neuroprotection both in vitro and in vivo. I focused in this study on investigating the neuroprotective effects of one of these peptides with regard to the determination of the downstream signaling pathways involved in neuroprotection. This peptide with the sequence NAPVSIPQ, known as NAP, a fragment of activity-dependent neuroprotective protein, demonstrated a potent protective effect against oxidative stress associated with alcohol exposure. On embryonic day 7 (E7), weight-matched C57BL/6 pregnant females were assigned the following groups: (1) Ethanol liquid diet group (ALC) 25% (4.49%, v/v) ethano-derived calories, (2) Pair-fed (PF) control group (3) Chow control group, (4) treatment groups with alcohol alongside i.p. injections of d-NAP (ALC/d-NAP, 20 or 30 μg/20 g body weight), (5) PF/d-NAP control group. On E13, fetal brains were collected and assayed for TdT-mediated dUTP nick end labeling (TUNEL) staining, caspase-3 colorimetric assay and ELISA for cytochrome c detection. My results show that NAP significantly prevented alcohol-induced weight reduction of the fetal brain. Apoptosis was determined by TUNEL staining; NAP administration significantly prevented alcohol-induced increases in TUNEL-positive cells in primordium cingulate cortex and basal ganglia eminence. The investigation of downstream signaling pathways involving NAP neuroprotection revealed that this peptide significantly prevented alcohol-induced increase in the concentrations of caspase-3 in E13 fetal brains. Moreover, ELISA for cytochrome c shows that NAP significantly prevented both alcohol-induced increases in the level of cytosolic cytochrome c and alcohol-induced decreases in the level of mitochondrial cytochrome c. These data provide an understanding of NAP intracellular target, and the downstream mechanisms of action that will pave a path toward potential therapeutics against alcohol intoxication during prenatal stages.

Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas–FasL and mitochondria-dependent pathways

Lindane, an organochlorine pesticide, is known to impair testicular functions and fertility. To elucidate the mechanism(s) underpinning the gonadal effects of lindane, we sought to investigate the levels of apoptosis-related proteins, namely cytochrome c, caspase-3 and-9, Fas and FasL in the testis of adult rats. Furthermore, the study aims to delineate whether nuclear factor kappa B (NF-κB) is involved in meditating the testicular effects of lindane. Animals were administered with a single dose of lindane (5 mg/kg body weight) and sacrificed at specific post-treatment intervals (0, 3, 6, 12, 24 and 72 h). Significant elevations in the levels of cytosolic cytochrome c with a parallel increase in pro-caspase-9 were observed as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL and caspase-3 were observed. Immunofluorescence studies revealed increased colocalization of Fas and caspase-3 in peritubular germ cells. FasL levels were increased in Sertoli and peritubular germ cells. The cytoplasmic levels of NF-κB p65 decreased from 3 h following exposure with a maximal decline at 12 and 24 h. Changes in the localization of NF-κB were observed with maximal nuclear translocation in germ cells at 12 and 24 h. Terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL) assay revealed a time-dependent increase in the number of apoptotic cells. Taken together, the data illustrate induction of testicular apoptosis in adult rats following exposure to a single dose of lindane. Early activation of NF-κB in contrast to late increase in Fas expression suggests a pro-apoptotic role of NF-κB in testicular response to lindane.