Pro-apoptotic Bcl-2 Proteins Research Articles

Inhibition of chemokine-like factor 1 protects against focal cerebral ischemia through the promotion of energy metabolism and anti-apoptotic effect

Chemokine-like factor 1 (CKLF1) is a novel C-C chemokine, and plays important roles in immune response and brain development. In previous study, we have found that the expression of CKLF1 increased after focal cerebral ischemia and inhibition of CKLF1 using antagonist C19 peptide protected against cerebral ischemia. However, few studies have focused on the role of CKLF1 on neuronal apoptosis. The objective of present study is to investigate the role of CKLF1 on neuronal apoptosis by applying anti-CKLF1 antibodies in rat focal cerebral ischemia and reperfusion model. Antibodies against CKLF1 was applied to the right cerebral ventricle immediately after transient middle cerebral artery occlusion (MCAO), and infarct volume, neurological score, glucose metabolism and apoptosis-related protein were measured. Treatment with anti-CKLF1 antibody decreased infarct volume and neurological score, and inhibited neuronal apoptosis in a dose-dependent manner at 24h after reperfusion. Anti-CKLF1 antibody also reduced the level of phosphorylation of Akt (P-Akt), and led to decrease of pro-apoptotic protein Bcl-2 associated X protein (Bax) and increase of anti-apoptotic protein B cell lymphoma-2 protein (Bcl-2) and the ratio of Bcl-2/Bax, and inhibited caspase-3 at last. In addition, positron emission tomography (PET) indicated that anti-CKLF1 antibody increased glucose metabolism in ischemic hemisphere. These results suggest that CKLF1 is associated with neuronal apoptosis after cerebral ischemia and reperfusion. Neutralization of CKLF1 with antibodies shows neuroprotective effects against cerebral ischemia, which may be involved in inhibition of Akt pathway, regulation of apoptosis-related protein expression, and improvement glucose metabolism in ischemic hemisphere. Therefore, CKLF1 may be a novel target for the treatment of stroke.

Ox-LDL induces endothelial cell apoptosis via the LOX-1-dependent endoplasmic reticulum stress pathway

ObjectiveTo investigate the effect of lectin-like ox-LDL receptor-1 (LOX-1) on oxidized low-density lipoprotein (ox-LDL)-induced apoptosis and the involvement of the endoplasmic reticulum (ER) stress response pathway. Methods and resultsHuman umbilical vein endothelial cells were treated with 50, 100, or 200 μg/ml ox-LDL and cultured for 12, 24, or 48 h for concentration- and time-dependent studies. Cells were transfected with LOX-1 or Nox-4 shRNAs, and target proteins were inhibited with the corresponding antibodies for mechanistic studies. Active proteins and mRNAs were analyzed by Western blotting and RT-PCR, respectively. Cell apoptosis was analyzed by Annexin and Hoechst staining assays. Ox-LDL induced both apoptosis and protein expression of LOX-1 and Nox-4 through activation of ER stress sensors IRE1 and PERK, and nuclear translocation of ATF6 and their subsequent pathways were indicated by JNK, eukaryotic initiation factor 2 phosphorylation, XBP-1, and chaperone GRP78 expression; up-regulation of proapoptotic proteins CHOP and Bcl-2; and caspase-12 activity. LOX-1 gene silencing and treatment with an anti-LOX-1 antibody attenuated the effects of ox-LDL. Pretreatment with irestatin 9389, salubrinal, or AEBSF also blocked ox-LDL-induced expression of CHOP and Bcl-2 and activation of caspase-12 activity, leading to an attenuation of endothelial cell apoptosis. Furthermore, Nox-4 siRNA attenuated the up-regulated expression of GRP78, PERK, IRE1, and XBP-1 to reduce ox-LDL-induced endothelial cell apoptosis. ConclusionsLOX-1 plays a critical role in ox-LDL-induced endothelial cell apoptosis via the ER stress pathway.

Evaluation of pro-apoptotic BIM gene loss of function on lapatinib efficacy in patients with metastatic breast cancer (MBC) and in HER2-amplified cell lines.

e11590 Background: BIM is a pro-apoptotic Bcl-2 protein. Cellular BIM levels are upregulated by tyrosine kinase inhibitors (TKI) and BIM expression is correlated with TKI-induced cancer cell apoptosis. Limited studies suggest lapatinib response is correlated with BIM expression in HER2-amplified cell lines and in tumor samples from a small cohort of MBC patients. A common germline deletion polymorphism in BIM confers loss of function and has been reported to mediate resistance to TKI therapy in patients with NSCLC or CML. Methods: Using blood sample germline DNA collected from patients with HER2 positive MBC during the clinical study EGF104535 (NCT00281658), we investigated the effect of the BIM deletion polymorphism on response to lapatinib, when administered in combination with paclitaxel. In addition, the effect of BIM siRNA knockdown on lapatinib anti-proliferative activity was evaluated in three HER2-amplified breast cancer cell lines (BT474, SrBr3, and HCC1954). Results: DNA was collected from 279 of the 444 randomized patients. Consistent with published data, BIM deletion was only observed among the 238 Asian patients. The deletion polymorphism was identified in 13 patients (11 heterozygous, 2 homozygous) randomized to lapatinib plus paclitaxel (n=120) and in 20 patients (19 heterozygous, 1 homozygous) receiving paclitaxel alone (n=118), providing 80% power to identify moderate genetic effects (hazard ratio>2, one-sided test; p<0.05). There was a clear PFS response distinction between treatments (lapatinib plus paclitaxel being favorable), however, there was no PFS distinction between BIM deletion carriers versus non-carriers in either treatment arm. No differences were observed in lapatinib IC50 values when cell lines were exposed to BIM-specific or scrambled siRNA. Conclusions: In these limited studies, reductions in BIM function, conferred by the BIM deletion polymorphism in HER2 positive, lapatinib plus paclitaxel-treated MBC patients, or by BIM-specific siRNA treatment of HER2-amplified cell lines did not influence lapatinib efficacy. Further evaluation of the genetics and expression of BIM and other Bcl-2 proteins is warranted. Clinical trial information: NCT00281658.

JunB protects β-cells from lipotoxicity via the XBP1-AKT pathway.

Diets rich in saturated fats may contribute to the loss of pancreatic β-cells in type 2 diabetes. JunB, a member of the activating protein 1 (AP-1) transcription factor family, promotes β-cell survival and mediates part of the beneficial effects of GLP-1 agonists. In this study we interrogated the molecular mechanisms involved in JunB-mediated β-cell protection from lipotoxicity. The saturated fatty acid palmitate decreased JunB expression, and this loss may contribute to β-cell apoptosis, as overexpression of JunB protected cells from lipotoxicity. Array analysis of JunB-deficient β-cells identified a gene expression signature of a downregulated endoplasmic reticulum (ER) stress response and inhibited AKT signaling. JunB stimulates XBP1 expression via the transcription factor c/EBPδ during ER stress, and forced expression of XBP1s rescued the viability of JunB-deficient cells, constituting an important antiapoptotic mechanism. JunB silencing inhibited AKT activation and activated the proapoptotic Bcl-2 protein BAD via its dephosphorylation. BAD knockdown reversed lipotoxic β-cell death potentiated by JunB siRNA. Interestingly, XBP1s links JunB and AKT signaling as XBP1 knockdown also reduced AKT phosphorylation. GLP-1 agonists induced cAMP-dependent AKT phosphorylation leading to β-cell protection against palmitate-induced apoptosis. JunB and XBP1 knockdown or IRE1 inhibition decreased AKT activation by cAMP, leading to β-cell apoptosis. In conclusion, JunB modulates the β-cell ER stress response and AKT signaling via the induction of XBP1s. The activation of the JunB gene network and the crosstalk between the ER stress and AKT pathway constitute a crucial defense mechanism by which GLP-1 agonists protect against lipotoxic β-cell death. These findings elucidate novel β-cell-protective signal transduction in type 2 diabetes.

Unbiased transcriptomes of resting human CD4+CD45RO+T lymphocytes

Unbiased transcriptomes of resting human <scp>CD</scp>4⁺<scp>CD</scp>45<scp>RO</scp>⁺<scp>T</scp> lymphocytes

Bim Regulates Alloimmune-Mediated Vascular Injury Through Effects on T-Cell Activation and Death

Bim is a proapoptotic Bcl-2 protein known to downregulate immune responses and to also be required for antigen-induced T-cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T-cell responses in a model of vascular rejection. Bim was required for proliferation of CD4 and CD8 T cells, and for interleukin-2 production, in T cells stimulated with alloantigen in vitro. Moreover, a partial reduction in Bim expression was sufficient to attenuate T-cell activation, whereas a complete elimination of Bim was required to prevent CD4 T-cell death in response to cytokine withdrawl. When alloimmune-mediated vascular rejection was examined using an aortic interposition model, there was significantly less intimal thickening in Bim(+/-), but not Bim(-/-), graft recipients. T-cell proliferation in response to allograft arteries was significantly reduced in both Bim(+/-) and Bim(-/-) mice, but cell death was attenuated only in Bim(-/-) animals. Bim controls both T-cell activation and death in response to alloantigen stimulation. These processes act cooperatively to determine the outcome of immune responses in allograft arteries.

Clinical Significance of BIM Deletion Polymorphism in Non–Small-Cell Lung Cancer with Epidermal Growth Factor Receptor Mutation

Background:Germline alterations in the proapoptotic protein Bcl-2–like 11 (BIM) can have a crucial role in tumor response to treatment. To determine the clinical utility of detecting BIM deletion polymorphism in non–small-cell lung cancer positive for epidermal growth factor receptor (EGFR) mutation, we examined outcomes of patients with and without BIM alterations.Methods:We studied 70 patients with EGFR mutation-positive non–small-cell lung cancer who were treated with an EGFR tyrosine kinase inhibitor between January 2008 and January 2013. BIM deletion was analyzed by polymerase chain reaction in 58 samples of peripheral blood and 24 formalin-fixed paraffin-embedded slides of surgical specimens (20 of lung tissue and four of brain tissue); both blood and tissue specimens were available for 12 patients. We retrospectively analyzed clinical characteristics, response rate, toxicity, and outcomes among patients with and without BIM deletion.Results:BIM deletion was present in 13 of 70 patients (18.6%). There were no significant differences between patients with and without BIM deletion in clinical characteristics, rate of response to EGFR tyrosine kinase inhibitor, or incidence of adverse events. Patients with BIM deletion had significantly shorter progression-free survival (PFS) than those without BIM deletion (median, 227 versus 533 days; p < 0.001). Multivariate Cox regression analysis showed that BIM deletion was an independent indicator of shorter PFS (hazard ratio, 3.99; 95% confidence interval, 1.864–8.547; p < 0.001).Conclusions:Polymerase chain reaction successfully detected BIM deletion in samples of peripheral blood and formalin-fixed paraffin-embedded slides of surgical specimens. BIM deletion was the most important independent prognostic factor in shorter PFS.

Loss of the Proapoptotic BH3-Only Protein BCL-2 Modifying Factor Prolongs the Fertile Life Span in Female Mice1

The duration of the female fertile life span is influenced by the number of oocytes stored in the ovary as primordial follicles. Cell death, both during ovarian development in the embryo and in the postnatal ovary, plays a critical role in determining how many primordial follicles are established and maintained within the ovary. However, the roles of individual apoptotic regulators in mediating cell death within the ovary have not yet been characterized. In this study, gene targeted mice were used to investigate the role of BCL-2-modifying factor (BMF), a proapoptotic protein belonging to the BH3-only subgroup of the BCL-2 family, in determining the number of primordial follicles maintained in the adult ovary and the length of the fertile life span. Stereological analysis of ovaries showed that Bmf(-/-) mice had significantly more primordial follicles than wild-type (WT) control animals at Postnatal Days 100, 200, 300, and 400 but not at Day 20. No differences were observed between WT and Bmf(-/-) mice in the number of ova shed following ovulatory stimulation with exogenous gonadotropins. Bmf(-/-) females were fertile and produced the same number pups/litter as WT females, but Bmf(-/-) females produced litters more frequently and consequently more offspring than WT females over a 6-mo period. Furthermore, the fertile life span of Bmf(-/-) females was significantly extended compared to WT females. Our findings support an important role for BMF in determining the number of primordial follicles maintained in the ovary throughout adult reproductive life and thus indicate that the length of female fertility may be extended by increasing the number of primordial follicles maintained within the ovary through inhibition of BMF.

Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer.

Antioxidant Chinese yam polysaccharides and its pro-proliferative effect on endometrial epithelial cells

Chinese yam polysaccharide (CYP) was successfully isolated from the rhizoma of Dioscorea opposita and its chemical characteristics were determined. Antioxidant and pro-proliferative activity of CYP on human endometrial epithelial cells in vitro were investigated. Role of CYP on cell proliferation was also evaluated by examining pro-apoptotic and anti-apoptotic proteins Bax and Bcl-2 by Western blot analysis. Chemical composition analysis indicated that CYP was mainly composed of mannose, glucose, galactose and glucuronic acid in the ratio of 0.5:1.2:0.3:0.3. In vitro, CYP exhibited a potent scavenging activity on the DPPH radical, hydroxyl radical and superoxide radical. Furthermore, CYP apparently promoted the proliferation of human endometrial epithelial cells, especially beyond the concentration of 100μg/ml after 36h exposure. Western blot revealed that the anti-apoptotic protein Bcl-2 was upregulated after endometrial epithelial cells were treated with CYP, while the protein level of Bax was attenuated, thus leading to the downregulation of Bax/Bcl-2 ratio. Our findings provide the first evidence that CYP may prove to be a potential candidate of the natural antioxidants as a therapeutic agent for female infertility.

BIMELis a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

sBackgroundArsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death.MethodsHL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression.ResultsHL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO.ConclusionsThe enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.

Chapter Three - The Structural Biology of BH3-Only Proteins

B-cell lymphoma-2 (Bcl-2) homology-3 (BH3)-only proteins are considered members of the Bcl-2 family, though they bear little sequence or structural identity with the multi-BH motif prosurvival or proapoptotic Bcl-2 proteins like Bcl-2 or Bax. They are better considered a separate phylogenetic entity. In combination, results from biophysical, biochemical, cell biological, and animal studies in conjunction with structural investigations have elucidated the function and mechanism of action of these proteins. Either by antagonizing prosurvival Bcl-2 proteins or directly activating proapoptotic Bcl-2 proteins (Bax or Bak) they initiate apoptosis. BH3-only proteins are intrinsically disordered and fold and bind into a groove provided by their cognate receptor Bcl-2 family proteins. Our detailed molecular understanding of BH3-only protein action has aided the development of novel chemical entities that initiate cell death by mimicking the properties of BH3-only proteins.

Proteasome inhibition leads to altered signaling in the proteome of cisplatin-resistant human ovarian carcinoma cell line

To address a precise view into molecular mechanisms of apoptotic signaling pathways after single- or combinatory treatments with specific NF-κB- or proteasome inhibitors and/or cisplatin (CDDP), flow cytometry and western blotting of the cell proteome in human ovarian chemosensitive- and CDDP-resistant cell lines were used. We report here that proteasome inhibition (but not NF-κB inhibition) caused marked alterations in the cell proliferation and cell cycle, as well as in the levels of signaling anti- and pro-apoptotic proteins PARP, NF-κB, IκB-α, Bcl-2, Bax, and lysosome-associated LAMP-1 and ATP-7B molecules in particular proteome fractions. These findings refer to the possibility of regulation of CDDP resistance, inclusive the capacity of lysosomes to export CDDP in certain human ovarian cancer cells by proteasome inhibition.

Cryptotanshinone Ameliorates Hepatic Normothermic Ischemia and Reperfusion Injury in Rats by Anti-mitochondrial Apoptosis.

Cryptotanshinone (CT), isolated from the dried roots of Salvia militorrhiza, has been reported to have protective effects on myocardial and cerebral ischemia/reperfusion (I/R) injury both in vitro and in vivo. However, its effects and underlying mechanism on hepatic I/R injury remain unclear. To investigate its effects on hepatic I/R injury, thirty male Sprague-Dawley rats were randomized into 3 groups: a sham group, a vehicle-treated hepatic I/R group and a CT-treated (50 mg/kg) group. The hepatic I/R and CT-treated groups were subjected to 60 min of normothermic ischemia of the left lateral lobe of the liver, followed by 4 h of reperfusion. The animals were then sacrificed to collect the serum and the left liver lobe for assay. Hepatic function was protected, as evidenced by significantly reduced alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) levels in the CT-treated group as compared with I/R group. The terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) demonstrated significantly decreased apoptosis in the CT-administration animals. Western blotting demonstrated upregulation of the proapoptotic protein Bcl-2, as well as decreased levels of the activated form of caspase-3 and the cleaved form of its substrate, poly(ADP-ribose) polymerase (PARP) in the CT-treated group compared with those of the I/R group. In addition, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) was inhibited by CT. Our data suggest that CT attenuates hepatic I/R injury by inhibiting the intrinsic pathway of apoptosis, mediated partly through the inhibition of JNK and p38 MAPK phosporylation.

Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B.

BackgroundPrevious screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).ResultsIn vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3′ position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1′ position from I29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5′ leader sequence (5′UTR) of BNIP-2.ConclusionsDespite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.

Anti- and Pro-Apoptotic Bcl2 Proteins Distribution and Metabolic Profile in Human Aorta Endothelial Cells Before and After Hyp-PDT

Deregulation of apoptosis can contribute to diverse pathologic processes. Understanding its regulatory mechanisms in endothelial cells (ECs) has great importance for the development of novel therapy strategies for cancer and cardiovascular pathologies. An oxidative stress with the generation of radical oxygen species (ROS) is a common mechanism causing ECs' dysfunction and apoptosis. The generation of ROS can be triggered by various stimuli including photodynamic therapy (PDT). The molecular mechanisms underlying PDT, and specifically Hypericin PDT (HypPDT), are not completely understood, although it has been shown that the sub-cellular Hyp localization and distribution determine which signaling pathway will lead to cell death. Cell responses to HypPDT are highly dependent on the Hyp intracellular localization and accumulation. The mechanisms by which ROS cause or regulate ECs apoptosis typically include receptor activation, caspase activation, Bcl-2 family proteins, and mitochondrial dysfunction. We were particularly interested in Bcl-2 family proteins and investigated their role in apoptosis of ECs triggered by HypPDT. In the present work, we show that the presence of Hyp itself has an effect on the distribution of Bcl2 family members. Presence of Hyp triggers translocation of Bax into mitochondria, and translocation of Bax and Bcl2 into nuclei in ECs. Further, HypPDT results in oxidative stress via mitochondrial superoxide production and primarily in necrotic type of death in HAEC cells. HAEC cell respiratory control is similar in control cells and in cells after 24 hrs incubation with Hyp without irradiation, suggesting that even though Hyp presence resulted in an increased number of apoptotic cells, the mitochondria function was not affected. However, HypPDT resulted in diminished OCR, indicating that HypPDT oxidative stress abolished mitochondria function in intact cells.

The key role of sphingosine kinases in the molecular mechanism of neuronal cell survival and death in an experimental model of Parkinson's disease.

Sphingosine kinases (Sphk1/2 EC 2.7.1.91) are responsible for synthesis of sphingosine-1-phosphate (S1P) and for regulation of the bioactive sphingolipids homeostasis. Sphingosine-1-phosphate can act as a potent messenger in an autocrine/paracrine manner through five specific G protein-coupled receptors (GPCR) S1P1-5. This sphingolipid is involved in the mechanism of transcription, mitochondrial function, neuronal viability and degeneration. Until now the involvement of Sphk1/2 and sphingolipid alterations in Parkinson's disease (PD) remains unknown. Recent studies have indicated the role of sphingolipids in the regulation of alpha-synuclein (ASN) in the PD brain. Our latest data demonstrated significant inhibition of Sphk1 gene expression and activity in an in vitro PD model, induced by 1-methyl-4-phenylpyridinium (MPP+). The aim of this study was to investigate the role of Sphks inhibition in ASN secretion and in the molecular mechanism of neuronal death in the PD model. Our study was carried out using neuronal dopaminergic SH-SY5Y control cells, transfected with the human gene for ASN or with an empty vector. These cells were treated with MPP+ (1-3 mM), which represents an experimental PD model, or with the Sphks inhibitor (1-5 µM SKI II) for 3-24 h. Our data indicated that MPP+ (3 mM) induced significant alterations of Sphks and S1P lyase (SPL) gene expression. Reduced activity of Sphk1 and Sphk2 in the cytosolic fraction and in the crude nuclear fraction, respectively, was observed. Sphks inhibition evoked enhancement of ASN secretion, suppression of PI3K/Akt phosphorylation and activation of gene expression for the pro-apoptotic Bcl-2 proteins Bax and BH3-only protein Harakiri. Moreover, a lower level of cytochrome c in the mitochondrial fraction and caspase-dependent degradation of DNA-bound enzyme poly(ADP-ribose) polymerase (PARP-1) were observed. The caspase inhibitor (20 µM Z-VAD-FMK) significantly enhanced neuronal cell viability in MPP+ oxidative stress. However, exogenous S1P (1 µM) exerted a more efficient neuroprotective effect as compared to Z-VAD-FMK. In summary, these data indicated that Sphk1 inhibition plays an important role in caspase-dependent apoptotic neuronal death in an experimental PD model.

Nickel-induced oxidative stress and apoptosis in Carassius auratus liver by JNK pathway

Nickel (Ni) is ubiquitous in the biosphere and is a common component of natural fresh waters. When present in high concentrations, it becomes toxic to aquatic organisms. It is known that Ni toxicity may induce oxidative stress and apoptosis. However, the precise mechanism and the pathways that are activated in fish are still unclear. Thus, this study aimed to assess which apoptotic pathways are triggered by Ni in Carassius auratus liver, the main target of waterborne pollutants. Fish were exposed to 10, 25, 50 and 100mg/L of nickel sulfate for 96h. Our data showed that Ni exposure caused fish weight loss (by 10–12%) and decreased locomotory activity (by 1–25%). Ni exposure significantly decreased the relative lymphocyte count (by 1–24%) and increased the relative count of monocytes (by 25–111%) and neutrophils (by 10–322%) as compared to controls. Ni induced oxidative stress, as evidenced by increasing of lipid peroxidation level (29–91%) and depleting of the glutathione levels (7–79%) in fish liver. Ni also suppressed the activities of superoxide dismutase (by 39–55%) and glutathione peroxidase (16–24%) and decreased ATP levels (13–51%) in livers. Moreover, liver caspase-3, one of the key executioners of apoptosis, was markedly activated by the Ni exposure. Ni exposure also increased expression levels of phosphorylated Jun N-terminal kinases (JNK) in liver, which in turn activated pro-apoptotic signaling events by breaking the balance between pro-apoptotic and anti-apoptotic Bcl-2 proteins. In conclusion, these results suggested that Ni induced oxidative stress and apoptosis, at least, via the JNK signaling pathway.

Novel anti‐tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40–60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

Novel Proteasome Inhibitors Induce Mitochondrial Destabilization and Activate Caspase Mediated Apoptosis In Preclinical Models Of Pediatric B-Cell Cancers

BackgroundIn the U.S. over 14,000 new cancer cases occur annually in people under 21 years of age; approximately 21% of cases are acute lymphoblastic leukemia (ALL) and ∼ 30% classify as Burkitt lymphoma (BL). Most cases of ALL and BL are due to malignant B-cells that have acquired disease-defining oncogenic lesions that facilitate their rapid proliferation. To maintain a state of homeostasis, the malignant cells rely heavily on the ability of the internal degradation and recycling systems to match the high rate of protein turnover. As such, the ubiquitin proteasome system (UPS) is central in this operation where it prevents buildup of misfolded proteins while also regulating the availability of anti-apoptotic proteins such as BCL2 and MCL1. These observations coupled with the observation that UPS disruption leads to cellular demise have resulted in the development of several proteasome inhibitors (PI). These PI have primarily been examined in adult B-cell malignancies. The feasibility of a bortezomib-containing regimen for use in pediatric malignancies has been examined by the TACL consortium and demonstrated a 73% response rate in relapsed/refractory ALL patients. However, a major limitation of bortezomib-based therapy is an increased incidence of peripheral neuropathy (PN), which was also noted in the TACL study. As such, more potent and reportedly less toxic PI have been developed that seem to carry considerably lower risk for development of PN. Such agents that maintain a high response rate and carry reduced long-term adverse effects are highly desirable in pediatric age patients. AimInvestigation of the novel proteasome inhibitors carfilzomib and MLN9708 (Millennium Pharmaceuticals) in preclinical models of pediatric B-lymphocytic cancer. MethodsRepresentative preclinical models of BL (Raji cell line) and B-ALL (ALL-1 cells) were used in this study. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Cell viability was measured by MTS assay. Apoptosis was determined by annexin-v/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was performed via immunoblot. ResultsIn an effort to understand the effect of carfilzomib and MLN9708 in our pediatric cancer models, we first assessed their ability to inhibit proteasomal activity. While chymotrypsin-like activity, which is the target of both PIs, is inhibited by more than 90% in both cell lines, inhibition (∼ 35%) of caspase-like function conferred by the b1 subunit was more significantly pronounced in MLN9708 treated Raji and ALL-1 cells. Next we performed an MTS assay to determine the growth inhibitory effects of these PI in vitro. Both PI inhibited the growth of the Raji and ALL-1 cells in a dose dependent manner. Carfilzomib showed a more potent effect (IC50 ∼1nM in both cell lines) as compared to MLN9708 (IC50 ∼30nM in ALL-1 cells and ∼50nM in Raji cells). To further understand mechanisms of growth inhibition we examined both tumor cell lines for induction of apoptosis along with corresponding markers. As compared to Raji cells, we observed ALL-1 cells to be more sensitive to the effects of both PI, albeit with MLN9708 (50nM) inducing more cell death (65%) as compared to carfilzomib (10nM, 50% cell death). Further, we observed altered MOMP, cleavage of caspases 9, 3 and PARP-1 were found to occur in both cell lines in presence of carfilzomib or MLN9708. These changes indicate the apoptotic effects of novel PI in ALL-1 and Raji cells are via mitochondrial destabilization and caspase activation. As inhibition of the UPS aids NOXA and BIM (pro-apoptotic BCL2 proteins) mediated cell death, we examined the expression of BCL2 family proteins changes in Raji and ALL-1 cells in response to novel PI. Indeed protein levels of both BCL2 and MCL1 were reduced in carfilzomib and MLN9708 treated cells. Reduced BCL2 expression was more pronounced in ALL-1 cells as compared to Raji cells and downregulation was noted to be dose-dependent. SummaryOur data show that both carfilzomib and MLN9708 potently inhibit the viability of malignant ALL and BL cells in vitro. We conclude that PI are promising anti-neoplastic therapeutics that target not only the UPS but also modulate the expression of critical anti-apoptotic proteins. Data from mechanistic studies conducted herein underscore the need for further research in in vivo models of these diseases. Disclosures:Foran:Celgene: Research Funding.