Reduced Caspase-3 Activity Research Articles

Bradykinin Preconditioning Improves Therapeutic Potential of Human Endothelial Progenitor Cells in Infarcted Myocardium

ObjectivesStem cell preconditioning (PC) is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs) with bradykinin (BK) enhance cell survival, inhibit apoptosis and repair the infarcted myocardium.MethodsThe hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF) levels were assessed in hEPCs. For in vivo studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging.Results In vitro data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. In vivo data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively.ConclusionsThe activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function in vivo. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used.

UTX coordinates steroid hormone-mediated autophagy and cell death

Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. Here we show that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. We demonstrate that dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. We further show that salivary gland degradation requires dUTX catalytic activity. Our findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development.

P38MAPK-regulated induction of p62 and NBR1 after photodynamic therapy promotes autophagic clearance of ubiquitin aggregates and reduces reactive oxygen species levels by supporting Nrf2–antioxidant signaling

Emerging evidence indicates that oxidative stress instigates the formation of ubiquitin (Ub) aggregates, substrates of autophagy, through a process requiring the ubiquitin binding adaptors p62/SQSTM1 and NBR1. Here, we have investigated the role of p62 and NBR1 in cell survival after hypericin-mediated photodynamic therapy (Hyp-PDT), a procedure known to incite robust reactive oxygen species (ROS)-based endoplasmic reticulum stress and autophagy pathways. We found that Hyp-PDT stimulated the formation of p62- and NBR1-associated Ub aggregates in normal and cancer cells, which were ultimately removed by autophagy, through a mechanism partially regulated by p38MAPK. In line with this, genetic or pharmacological p38MAPK inhibition reduced p62 and NBR1 levels and aggregate formation and impaired Nrf2 activation, thus increasing photo-oxidative stress and cell death. p62-deficient cells, or cells lacking p62 and with reduced levels of NBR1 (through siRNA knockdown), also displayed reduced aggregate formation but exhibited attenuated ROS levels, reduced caspase activation, and improved survival after Hyp-PDT. The increased resistance to photo-oxidative stress exhibited by cells lacking p62 and/or NBR1 was overruled by the inhibition of p38MAPK, which restored cytotoxic ROS levels, thus indicating the relevance of this signal in the control of cell viability. Taken together these findings provide evidence that in photodynamically treated cells a p38MAPK-regulated pathway coordinates the p62/NBR1-mediated clearance of cytosolic aggregates and mitigates PDT-induced proteotoxicity. They also reveal that a functional p38MAPK–Nrf2 signal is required to keep ROS levels in check and protect against PDT-induced proteotoxicity, independent of aggregate formation.

Mitigation of ROS Insults by Streptomyces Secondary Metabolites in Primary Cortical Neurons

Oxidative stress is a common point in neurodegenerative diseases, widely connected with mitochondrial dysfunction. In this study, we screened seven natural products from Streptomyces sources against hydrogen peroxide insult in primary cortical neurons, an oxidative stress in vitro model. We showed the ability of these compounds to inhibit neuronal cytotoxicity and to reduce ROS release after 12 h treatment. Among the tested compounds, the quinone anhydroexfoliamycin and the red pyrrole-type pigment undecylprodigiosin stand out. These two compounds displayed the most complete protection against oxidative stress with mitochondrial function improvement, ROS production inhibition, and increase of antioxidant enzyme levels, glutathione and catalase. Further investigations confirmed that anhydroexfoliamycin acts over the Nrf2-ARE pathway, as a Nrf2 nuclear translocation inductor, and is able to strongly inhibit the effect of the mitochondrial uncoupler FCCP over cytosolic Ca(2+), pointing to mitochondria as a cellular target for this molecule. In addition, both compounds were able to reduce caspase-3 activity induced by the apoptotic enhancer staurosporine, but undecylprodigiosin failed to inhibit FCCP effects and it did not act over the Nrf2 pathway as was the case for anhydroexfoliamycin. These results show that Streptomyces metabolites could be useful for the development of new drugs for prevention of neurodegenerative disorders such as Parkinson's and Alzheimer's diseases and cerebral ischemia.

Bone Marrow Stromal Cell-Derived Exosomes Facilitate Multiple Myeloma Cell Survival Through Inhibition Of The JNK Pathway

Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a crucial role in MM pathogenesis by secreting growth factors, cytokines, and other functional components. Exosomes are 30-100nm diameter membranous vesicles constitutively released by several cell types including reticulocytes, cytotoxic T lymphocytes, B lymphocytes, epithelial and endothelial cells. Exosomes mediate local cell-cell communication by transferring mRNAs, miRNAs and proteins. Due to their ability to transfer functional components, exosomes play multiple roles by stimulating target cells, transferring membrane receptors, delivering proteins, and inducing epigenetic changes in recipient cells. Although the promotion of MM growth and survival induced by BMSCs has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here, we investigated the effect and mechanisms of BMSC-derived exosomes on the proliferation and survival of MM cells using the murine 5T33MM model. This model mimics the human disease closely and of this model two lines exist: the 5T33MMvv model which is propagated in vivo and the 5T33MMvt line which is derived from 5T33MMvv cells but which can grow stroma-independently.Exosomes were isolated from conditioned medium using the ExoQuick-TC Exosome Precipitation Solution (System Biosciences) after culture of primary BMSCs obtained from naïve C57BL/KaLwRij mice or 5T33MM diseased mice. The size of exosomes derived from naïve BMSCs, 5T33 BMSCs and 5T33MMvt cells were confirmed using a NanoSight LM10. Several exosomal markers such as CD63, Flotillin-1, heat shock protein 90 (HSP90), and HSP70 were detected using Western blot. We co-cultured the BMSCs or MM cells with fluorescent dye-labeled exosomes to examine whether exosomes could be transferred into cells. The results showed that both naïve and 5T33 BMSC-derived exosomes could fuse with 5T33MMvt cells and that the uptake of 5T33MMvt cell-derived exosomes by BMSCs was also observed. As several cytokines were found to be present in BMSC- and MMvt cell-derived exosomes, this suggests that BMSCs and MM cells could exchange cytokines with each other through exosomes secretion and uptake. Furthermore, the cytokine composition of 5T33BMSC-derived exosomes compared to naïve BMSC-derived exosomes was different.We next performed luminescent cell viability assays, BrdU cell proliferation assays and 7-AAD/annexin-V stainings to examine the effects of BMSC-derived exosomes on MM cell viability, proliferation and survival, respectively. Both naïve and 5T33 BMSC-derived exosomes increased 5T33MMvt and MMvv cell viability in a dose- and time-dependently manner. BrdU uptake in 5T33MMvt and MMvv cells was also increased after treatment with BMSC-derived exosomes. Significantly reduced apoptosis of 5T33 MMvt and MMvv cells was observed when they were treated with BMSC-derived exosomes as judged by 7-AAD/annexin-V staining. 5T33MMvt and MMvv cells were treated with different amounts of BMSC-derived exosomes and apoptosis-related proteins Bcl-2, Bax, and caspase-3 were determined using western blot. Bcl-2 was increased slightly and activated (cleaved) caspase-3 was reduced after co-culture with exosomes, coinciding with the results of 7-AAD/annexin-V staining. To elucidate the mechanisms responsible for exosome-induced MM cell survival, we examined the activation of several proteins involved. Reduced phosphorylation of p53, p38MAPK and JNK were detected when 5T33MMvt were treated with naïve-BMSC-derived exosomes for 24h, whereas phosphorylated Erk1/2, Akt, and IGF1Rβ were not changed. Surprisingly, activation of p53 and p38MAPK were not changed after the treatment with 5T33 BMSC-derived exosomes. 5T33 BMSC-derived exosomes further decreased the activation of JNK, Bim expression and phosphorylated Bim compared to naïve BMSC-derived exosomes. As Bim is a pro-apoptosis protein, mainly regulated by the JNK pathway; promotion of MM cell survival likely results from the inhibition of the JNK pathway by BMSC-derived exosomes.In summary, our results demonstrate a positive role for BMSC-derived exosomes in induction of MM cell proliferation and survival. BMSC-derived exosomes could inhibit the JNK pathway, thereby reducing caspase-3 activation and protecting MM cells from apoptosis. Disclosures:No relevant conflicts of interest to declare.

Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins

Virulent Clostridium difficile strains produce toxin A and/or toxin B that are the etiological agents of diarrhea and pseudomembranous colitis. Treatment of C. difficile infections (CDI) has been hampered by resistance to multiple antibiotics, sporulation, emergence of strains with increased virulence, recurrence of the infection, and the lack of drugs that preserve or restore the colonic bacterial flora. As a result, there is new interest in non-antibiotic CDI treatments. The human conjugated bile salt taurocholate was previously shown in our laboratory to inhibit C. difficile toxin A and B activities in an in vitro assay. Here we demonstrate for the first time in an ex vivo assay that taurocholate can protect Caco-2 colonic epithelial cells from the damaging effects of the C. difficile toxins. Using caspase-3 and lactate dehydrogenase assays, we have demonstrated that taurocholate reduced the extent of toxin B-induced apoptosis and cell membrane damage. Confluent Caco-2 cells cultured with toxin B induced elevated caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively. Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only. Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production. These findings open up a new avenue for the development of non-antibiotic therapeutics for CDI treatment.

Baicalin inhibits colistin sulfate-induced apoptosis of PC12 cells.

Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress co-listin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate (62.5–500 μg/mL) for 24 hours resulted in PC12 cell apoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin (25, 50 and 100 μg/mL), and exposed to 125 μg/mL colistin sulfate. Cell morphology markedly changed, and cell viability increased. Moreover, caspase-3 activity, tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhi-bited colistin sulfate-induced PC12 cell apoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.

Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

To investigate the mechanisms underlying the protective effects of sodium tanshinone IIA sulfonate (STS) in an ischemia-reperfusion (I/R)-induced rat myocardial injury model. Male SD rats were iv injected with STS, STS+LY294002 or saline (NS) for 15 d. Then the hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. Cardiac function, infarction size and area at risk were assessed. Cell apoptosis was evaluated with TUNEL staining, DNA laddering and measuring caspase-3 activity. In addition, isolated cardiomyocytes of neonatal rats were pretreated with the above drugs, then exposed to H2O2 (200 mol/L) for 1 h. Cell apoptosis was detected using flow cytometric assay. The levels of p-Akt, p-FOXO3A and Bim were examined with immunoblotting. Compared to NS group, administration of STS (20 mg/kg) significantly reduced myocardial infarct size (40.28%±5.36% in STS group vs 59.52%±7.28% in NS group), and improved the myocardial function as demonstrated by the increased values of dp/dtmax, LVDP and coronary flow at different reperfusion time stages. Furthermore, STS significantly decreased the rate of apoptotic cells (15.11%±3.71% in STS group vs 38.21%±7.83% in NS group), and reduced caspase-3 activity to nearly a quarter of that in NS group. Moreover, STS significantly increased the phosphorylation of Akt and its downstream target FOXO3A, and decreased the expression of pro-apoptotic gene Bim. Co-treatment with the PI3K inhibitor LY294002 (40 mg/kg) partially countered the protective effects induced by STS treatment. In isolated cardiomyocytes, STS exerted similar protective effects as shown in the ex vivo I/R model. STS pretreatment reduces infarct size and improves cardiac function in an I/R-induced rat myocardial injury model via activation of Akt/FOXO3A/Bim-mediated signal pathway.

Dual inhibition of MEK1/2 and EGFR synergistically induces caspase-3-dependent apoptosis in EGFR inhibitor-resistant lung cancer cells via BIM upregulation

Epidermal growth factor receptor (EGFR) gene mutations activate the KRAS-RAF-MEK-ERK pathway in lung cancer cells. EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib induce apoptosis of cancer cells, but prolonged treatment is often associated with acquired resistance. Here, we identified a novel MEK1/2 inhibitor, CZ0775, and compared its cytotoxic effects to those of AZD6244 (selumetinib) in non-small cell lung cancer (NSCLC) cell lines harboring EGFR mutations. The lapatinib-sensitive HCC827 and PC9 and lapatinib-resistant H1650 and H1975 cell lines showed poor responses to CZ0775 and AZD6244 monotherapy with an IC50 > 10 μM. By contrast, combination treatment with lapatinib and CZ0775 inhibited cell proliferation and produced a 2-fold higher number of annexin V-labeled cells than lapatinib alone in H1975 cells. Furthermore, combination treatment decreased phosphorylated extracellular signal related kinase (p-ERK) and survivin levels and upregulated the expression of the pro-apoptotic protein BIM. siRNA-mediated BIM depletion reduced caspase-3 activity (~40%) in lapatinib and CZ0775 treated H1975 cells. An in vitro ERK activity assay showed that p-ERK levels were approximately a 3-fold lower in H1975 cells treated with CZ0775 and lapatinib combination than in cells treated with lapatinib alone. CZ0775 was more cytotoxic than AZD6244 when used in combination with lapatinib. Our results suggest that combination treatment with CZ0774 and EGFR inhibitors is a promising therapeutic approach for the treatment of EGFR-TKI-resistant lung cancers and its effect is mediated by the inhibition of ERK and the induction of BIM.

Excitotoxin-induced caspase-3 activation and microtubule disintegration in axons is inhibited by taxol

BackgroundAxon degeneration, a key pathological event in many neurodegenerative diseases and injury, can be induced by somatodendritic excitotoxin exposure. It is currently unclear, however, whether excitotoxin-induced axon degeneration is mechanistically similar to Wallerian degeneration, which occurs following axon transection, but does not involve axonal caspase activation.ResultsWe have used mouse primary cortical neurons at 9 days in vitro, in a compartmented culture model that allows separation of the axon from the soma, to examine the pathological cascade of excitotoxin-induced axon degeneration. Excitotoxicity induced by chronic exposure to kainic acid, resulted in axonal fragmentation, which was associated with activation of caspase-3 in the axonal compartment. To examine the role of microtubules in these events, the microtubule-stabilizing agent, taxol, was added to either the axonal or somatodendritic compartment. Our results demonstrated that microtubule stabilization of axons resulted in a significant reduction in the number of fragmented axons following excitotoxin exposure. Interestingly, taxol exposure to either the somatodendritic or axonal compartment resulted in reduced caspase-3 activation in axons, suggesting that caspase activation is a downstream event of microtubule destabilization and involves signalling from the cell soma.ConclusionThese data suggest that excitotoxin-induced axon degeneration shows some mechanistic differences to Wallerian degeneration, and that microtubule stabilization may assist in protecting nerve cells from excitotoxic effects.

Protective Effect of N-Acetylserotonin against Acute Hepatic Ischemia-Reperfusion Injury in Mice

The purpose of this study was to investigate the possible protective effect of N-acetylserotonin (NAS) against acute hepatic ischemia-reperfusion (I/R) injury in mice. Adult male mice were randomly divided into three groups: sham, I/R, and I/R + NAS. The hepatic I/R injury model was generated by clamping the hepatic artery, portal vein, and common bile duct with a microvascular bulldog clamp for 30 min, and then removing the clamp and allowing reperfusion for 6 h. Morphologic changes and hepatocyte apoptosis were evaluated by hematoxylin-eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Activated caspase-3 expression was evaluated by immunohistochemistry and Western blot. The activation of aspartate aminotransferase (AST), malondialdehyde (MDA), and superoxide dismutase (SOD) was evaluated by enzyme-linked immunosorbent assay (ELISA). The data show that NAS rescued hepatocyte morphological damage and dysfunction, decreased the number of apoptotic hepatocytes, and reduced caspase-3 activation. Our work demonstrates that NAS ameliorates hepatic IR injury.

Melatonin‐enhanced autophagy protects against neural apoptosis via a mitochondrial pathway in early brain injury following a subarachnoid hemorrhage

Melatonin is a strong antioxidant that has beneficial effects against early brain injury (EBI) following a subarachnoid hemorrhage (SAH) in rats; protection includes reduced mortality and brain water content. The molecular mechanisms underlying these clinical effects in the SAH model, however, have not been clearly identified. This study was undertaken to determine the influence of melatonin on neural apoptosis and the potential mechanism of these effects in EBI following SAH using the filament perforation model of SAH in male Sprague Dawley rats. Melatonin (150 mg/kg) or vehicle was given via an intraperitoneal injection 2 hr after SAH induction. Brain samples were extracted 24 hr after SAH. The results show that melatonin treatment markedly reduced caspase-3 activity and the number of TUNEL-positive cells, while the treatment increased the LC3-II/LC3-I, an autophagy marker, which indicated that melatonin-enhanced autophagy ameliorated apoptotic cell death in rats subjected to SAH. To further identify the mechanism of autophagy protection, we demonstrated that melatonin administration reduced Bax translocation to the mitochondria and the release of cytochrome c into the cytosol. Taken together, this report demonstrates that melatonin improved the neurological outcome in rats by protecting against neural apoptosis after the induction of filament perforation SAH; moreover, the mechanism of these antiapoptosis effects was related to the enhancement of autophagy, which ameliorated cell apoptosis via a mitochondrial pathway.

The Effects of Intraperitoneal Pentoxifylline Treatment in Rat Pups With Hypoxic-Ischemic Encephalopathy

The aim of this study was to evaluate the effects of postischemic treatment with pentoxifylline on the cytokine gene expressions and neuronal apoptosis in neonatal rat model of hypoxic-ischemic encephalopathy. Seven-day-old Wistar rat pups (n = 40) of either sex, delivered spontaneously, were used in this experimental study. Control group (n = 8): after median neck incision was made, neither ligation nor hypoxia was performed, ischemia group (n = 16): 0.5 mL of saline was injected intraperitoneally immediately after hypoxia. Pentoxifylline and ischemia groups (n = 16): the rat pups were administered intraperitoneally 60 mg/kg of pentoxifylline immediately after hypoxia. Eight rats from ischemia and pentoxifylline + ischemia groups were sacrificed 4 and 24 hours after drug administration. Control group mice were decapitated 4 hours after hypoxia. Caspase-3 activity, interleukin-1β, and tumor necrosis factor-α messenger RNA expression levels were studied in the left half of the brain. Induction of cerebral ischemia increased tumor necrosis factor-α and interleukin-1β messenger RNA expression levels significantly at 4 hours and 24 hours following ischemia in the left ischemic hemispheres in the ischemia group as compared with the control group. Systemic administration of pentoxifylline immediately after hypoxic-ischemic encephalopathy significantly reduced the tumor necrosis factor-α and interleukin-1β messenger RNA expression levels in ischemic tissue as compared with the ischemia group. Caspase-3 activities in the left half of the brains of ischemia group were found to be increased significantly as compared with control group. Caspase-3 activities in the brains of pentoxifylline + ischemia groups were significantly lower than in that of ischemia group. Based on the significantly lower interleukin-1β and tumor necrosis factor-α gene expression measured after 4 and 24 hours and significantly reduced caspase-3 activity measured colorimetrically in the animals treated with pentoxifylline, our findings suggest that pentoxifylline may reduce brain damage due to hypoxic-ischemic injury.

Novel IRES-based lentivirus co-expressing IGF1 and BMP2 enhances both cardiomyogenesis and cytoprotection of bone marrow-derived mesenchymal stem cells

Purpose: The in vivo effects of bone marrow mesenchymal stem cells (BM-MSC) in repairing ischemic hearts are limited by poor engraftment, low differentiation rate and, in elderly patients, by reduced paracrine properties. To overcome these limitations, we genetically engineered BM-MSC with a novel bicistronic lentivirus co-expressing IGF1 and BMP2 (IB), two factors involved in both cardiac differentiation and cytoprotection. Methods: We cloned an IRES-based expression cassette into a novel lentiviral vector. Rat BM-MSC were transduced with a control (GFP-MSC) or IB (IB-MSC) virus. Cardiac differentiation was evaluated by RT-qPCR, western blot, and immunocytochemistry for cardiac specific markers such as GATA4, Nkx2.5, Tbx5, Mef2c, Cx43, cTnT, a-MHC, a-SA and a-SR1. Resistance to hypoxia-induced apoptosis was measured after 24hrs of hypoxia. To study the cytoprotective paracrine properties, H9c2 cells were deprived of oxygen and glucose (6hrs), and then reoxygenated (18hrs) in the presence of control medium (CTRL-M) or medium conditioned by either GFP-MSC (GFP-CM) or IB-MSC (IB-CM). Viability was measured by MTS assay, apoptosis by caspase-3 activation, cytotoxicity by LDH release. Transcriptional levels of known pro- and anti-apoptotic genes were measured by RT-PCR. Activation of I and B associated pathways (Akt, ERK1/2, and SMAD1/5/8) was assessed by western blot. Results: The transduction efficiency with our novel vector was extremely high (>90% with one cycle of infection). Compared with GFP-MSC, IB-MSC triggered expression of cardiac markers both at RNA (Tbx5 +3.8, p<0.01; Mef2c +1.7, p<0.05; GATA4 +3.1, p<0.05; Cx43 +7.5, p<0.001; cTnT +6.4, p<0.01; a-SA +4.8, p<0.001) and protein level and favoured the nuclear translocation of cardiac-specific transcription factors. Furthermore, IB-MSC showed increased viability (+26.8%, p<0.01), and marked reduction of apoptosis (-62.9%, p<0.001) and cytotoxicity (-22.9%, p<0.05) vs GFP-MSC after hypoxia. IB-CM also increased H9c2 viability (+44.2% vs CTRL-M, p<0.001; +21.4% vs GFP-CM, p<0.01) and reduced caspase-3 activation (-68% vs CTRL-M, p<0.001; -60.4% vs GFP-MSC, p<0.001) and LDH release (-34% vs CTRL-M, p<0.001; -20% vs GFP-MSC, p<0.05). These cytoprotective effects were matched by a strong activation of Akt, ERK1/2 and SMAD1/5/8 anti-apoptotic pathways, by enhanced expression of Bcl-2, Stat3 and Hsp20 pro-survival genes and by inhibition of FasL and TNFa pro-apoptotic genes. Conclusions: IB overexpression commits BM-MSC toward the cardiac lineage, increases their resistance to hypoxia and also improves their cytoprotective paracrine effects.

GW24-e3646 Adiponectin regulates SR Ca2+ cycling following ischaemia/reperfusion through sphingosine 1-phosphate-CaMKII signalling in mice

ObjectivesThe circulating, adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. Recent study demonstrated that APN induces a marked Ca2+ influx in skeletal muscle. However, whether...

Basal caspase-3 activity promotes migration, invasion, and vasculogenic mimicry formation of melanoma cells

Melanoma is the least common but most serious form of skin cancer. The leading cause of death in melanoma patients is widespread metastasis caused by increased cell motility and a rich blood supply for tumor cells. A unique form of microcirculation called vasculogenic mimicry, which efficiently supplies blood to tumor cells, has been reported recently. Apoptosis-related protein performs a nonapoptotic function to promote migration and invasion of tumor cells. This study focuses on the nonapoptotic role of caspase-3 in melanoma and its effects on the migration, invasion, and vasculogenic mimicry formation of melanoma cells. Human melanoma samples were used to detect active caspase-3 expression and determine its relationship with clinicopathologic parameters. In addition, a human melanoma A375 cell line was used to determine the role of caspase-3 in migration and invasion using z-DEVD-fmk, a selective caspase-3 inhibitor, to inhibit caspase-3 activity. The findings suggest that active caspase-3 is expressed in nonapoptotic melanoma cells and is related to metastasis and vasculogenic mimicry formation in patients with melanoma. Low doses of caspase-3 inhibitor reduced caspase-3 activity without affecting cell apoptosis. Inhibition of caspase-3 activity using low-dose z-DEVD-fmk decreased the migration, invasion, and vasculogenic mimicry formation of melanoma cells in vitro. Similarly, downregulation of caspase-3 by specific small interfering RNA also inhibited the migratory, invasive, and tube-forming potential of melanoma cells. The caspase-3-mediated promotion of melanoma cell motility may be because of the cleavage of matrix metalloproteinase-2.

Acute and delayed protective effects of pharmacologically induced hypothermia in an intracerebral hemorrhage stroke model of mice

Hemorrhagic stroke, including intracerebral hemorrhage (ICH), is a devastating subtype of stroke; yet, effective clinical treatment is very limited. Accumulating evidence has shown that mild to moderate hypothermia is a promising intervention for ischemic stroke and ICH. Current physical cooling methods, however, are less efficient and often impractical for acute ICH patients. The present investigation tested pharmacologically induced hypothermia (PIH) using the second-generation neurotensin receptor (NTR) agonist HPI-201 (formerly known as ABS-201) in an adult mouse model with ICH. Acute or delayed administrations of HPI-201 (2mg/kg bolus injection followed by 2 injections of 1mg/kg, i.p.) were initiated at 1 or 24h after ICH. HPI-201 induced mild hypothermia within 30min and body and brain temperatures were maintained at 32.7±0.4°C for at least 6h without causing observable shivering. With the 1-h delayed treatment, HPI-201-induced PIH significantly reduced ICH-induced cell death and brain edema compared to saline-treated ICH animals. When HPI-201-induced hypothermia was initiated 24h after the onset of ICH, it still significantly attenuated brain edema, cell death and blood–brain barrier breakdown. HPI-201 significantly decreased the expression of matrix metallopeptidase-9 (MMP-9), reduced caspase-3 activation, and increased Bcl-2 expression in the ICH brain. Moreover, ICH mice received 1-h delayed HPI-201 treatment performed significantly better in the neurological behavior test 48h after ICH. All together, these data suggest that systemic injection of HPI-201 is an effective hypothermic strategy that protects the brain from ICH injury with a wide therapeutic window. The protective effect of this PIH therapy is partially mediated through the alleviation of apoptosis and neurovascular damage. We suggest that pharmacological hypothermia using the newly developed neurotensin analogs is a promising therapeutic treatment for ICH.

Suppression of granzyme B activity and caspase-3 activation in leukaemia cells constitutively expressing the protease inhibitor 9

Immune surveillance against malignant cells is mediated by cytotoxic T-lymphocytes and NK-cells (CTL/NK) that induce apoptosis through the granzyme-B-dependent pathway. The serine protease inhibitor serpinB9/protease inhibitor-9 (PI-9) is a known inhibitor of granzyme B. Ectopic expression of PI-9 in tumour cells has been reported. However, the impact of PI-9 on granzyme-B-induced apoptosis in tumour cells remains unclear. The aim of this study was to investigate the influence of constitutive PI-9 expression in leukaemia cell lines on the activity of granzyme B and apoptosis induction. PI-9 negative (lymphoblastic Jurkat cells; myeloblastic U937 cells) and PI-9-expressing cell lines (myeloblastic K562 cells, EBV-transformed LCL-1 and LCL-2 B-cells, lymphoblastic Daudi cells, AML-R cells f leukaemia and the U937 subclone U937PI-9(+)). For accurate granzyme B activity determination a quantitative substrate (Ac-IEPD-pNA) cleavage assay was established and caspase-3 activation measured for apoptosis assessment. Cells were treated with a cytotoxic granule isolate that has previously been shown to induce apoptosis through granzyme B signalling. We found a robust correlation between constitutive PI-9 expression levels and the suppression of granzyme B activity. Further, inhibition of granzyme B translated into reduced caspase-3 activation. We conclude, suppression of granzyme B initiated apoptosis in PI-9-expressing cells could contribute to immune evasion and the measurement of granzyme B activity with our assay might be a useful predictive marker in immune-therapeutic approaches against cancer.

C-Terminal Peptide of γ-Enolase Impairs Amyloid-β-Induced Apoptosis Through p75NTR Signaling

γ-Enolase acts as a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. It is shown in this study to exert a protective effect against amyloid-β-peptide (Aβ)-induced neurotoxicity in rat pheochromocytoma PC12 cells. Aβ-induced toxicity was abolished in the presence of the active C-terminal peptide of γ-enolase (γ-Eno) as measured by cell viability, lactate dehydrogenase release, sub-G1 cell population, intracellular reactive oxygen species, mitochondrial functions and apoptotic morphology. γ-Eno caused downregulation of the pro-apoptotic protein Bax and upregulation of the anti-apoptotic protein Bcl-2, as well as reduced caspase-3 activation. Exposure to Aβ increased surface expression of p75 neurotrophin receptor (p75(NTR)), and the increase was abolished in the presence of γ-Eno peptide. Further, pretreatment with γ-Eno suppressed the activation of mitogen-activated protein kinases p38 and Jun-N-terminal kinase, which are p75(NTR) downstream effectors in apoptotic signaling. Moreover, Aβ triggered γ-enolase co-immunoprecipitation with p75(NTR) as well as their strong association in the perimembrane region as shown by confocal microscopy, which further supports the interaction between these two proteins in cells insulted by Aβ peptide. Our results indicate the possible use of γ-enolase C-terminal peptide for treating or preventing Alzheimer's disease.

Protein Kinase C Modulates Aurora-kinase Inhibition Induced by CCT129202 in HMC-1560,816 Cell Line

The human mast cell line HMC-1⁵⁶⁰,⁸¹⁶ carries activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit receptor activation. The compound CCT129202 is a new and selective inhibitor of Aurora kinase A and B that decreases the viability of a variety of human tumor cell lines. The effect of Aurora kinase inhibition was assessed in the HMC-1⁵⁶⁰,⁸¹⁶ line in order to find a suitable tool for mastocytosis treatment. CCT129202 treatment induces a significant decrease in cell viability in HMC-1⁵⁶⁰,⁸¹⁶ cells after 48 hours of treatment. Moreover, caspase-3 and caspase-8 activation was induced after incubation of HMC-1⁵⁶⁰,⁸¹⁶ cells in the presence of CCT129202. It has been demonstrated that Protein Kinase C (PKC) plays a crucial role in mast cell activation as well as cell migration, adhesion and apoptotic cell death. Co-treatment of Ca²⁺-independent PKCs (δ ε and θ) inhibitor GF109203X with CCT129202, reduces caspase-3 activation which controls cell levels. In contrast, Go6976, an inhibitor of Ca²⁺-dependent PKCs, increases caspase-3 activation. Oppositely, GF109203X does not modify CCT129202-induced apoptosis through the caspase-8 pathway whereas Go6976 treatment abolishes the increase on caspase-8 activity due to CCT129202. This implies that Ca²⁺-independent PKC isoforms seems to be related with CCT129202-induced apoptosis through the caspase- 3 pathway, whereas Ca²⁺-dependent PKC isoforms are related with the CCT129202 effect on the caspase-8 pathway. Interestingly, CCT129202 cytotoxic effect remains even though Ca²⁺-dependent PKCs are inhibited, which shows that the Aurora kinase inhibitor effect is acting through the caspase-3 pathway. On the other hand, Ca²⁺-independent PKCs inhibition does not affect the final apoptotic CCT129202 effect because this seems to be mediated by the caspase-8 pathway. Moreover, CCT129202 does not affect PKCδ and Ca²⁺-dependent PKC translocation, which indicates that PKC translocation pivots on its activation. This demonstrates that Aurora kinase inhibition is not related to this process. Finally, when PKC is silenced in HMC-1⁵⁶⁰,⁸¹⁶ cells, the effect of CCT129202 on the caspase-3 pathway disappears, which indicates that the CCT129202 effect is clearly PKC-dependent.