Apoptosis-regulating Proteins Research Articles

Antiproliferative evaluation and supramolecular association involving electrostatically enhanced π-π interaction in isostructural coordination solids of Mn(II), Co(II) and Zn(II) chlorobenzoates: Experimental and theoretical studies

Three new isostructural coordination solids viz. [Co(3-CNpy)2(2-ClBz)2(H2O)2] (1), [Mn(3-CNpy)2(2-ClBz)2(H2O)2] (2) and [Zn(3-CNpy)2(2-ClBz)2(H2O)2] (3) (3-CNpy = 3-cyanopyridine, 2-ClBz = 2-chlorobenzoate) have been synthesized from purely aqueous media and characterized by X-ray crystal structure analysis, FT-IR, electronic spectra and TGA. Several non-covalent hydrogen bonding interactions of the types OH⋯O, CH⋯O, CH⋯N, CH⋯Cl and π-π stacking contacts build up the supramolecular networks in the crystal structures. Electrostatically enhanced π-π interactions are observed in 1–3 between the phenyl rings of 2-ClBz and pyridine ring of 3-CNpy of the monomeric units of the complexes. We have used the molecular DFT calculations to evaluate energetically the strength of these contacts and also to analyze the effect of the metal on the interaction energies. The energetic features of the H-bonding and π-stacking interactions for 1–3 reveal that the H-bonded assembly is more favorable than the π-stacked contacts. The electrostatically enhanced energy of the π-stacking interactions is stronger than that usually observed for π-stacking interactions involving arenes. The cytostatic potential of all complexes have been studied in Dalton’s lymphoma (DL) cell line by MTT assay, apoptosis assay and further corroborated with molecular docking simulation. The complexes exhibit cytotoxicity (∼25–30%) through apoptotic cell death with negligible cytotoxicity (∼5–10%) in normal PBMC cells. In silico-docking techniques have been performed with apoptosis regulator protein BCL-2 for the identification of critical amino acids and their possible binding affinity with the synthesized complexes. The pharmacophore features based on structure activity relationship (SAR) of the complexes have been identified and the study reveal that the features viz., hydrophobic, aromatic, positive ionizable, negative ionizable, H-bond donor and acceptor and halogen bond donor properties play important role for the biological activities of the complexes.

Hesperidin ameliorates pancreatic β-cell dysfunction and apoptosis in streptozotocin-induced diabetic rat model

AimsThe current study was conducted to investigate the potential protective effects of hesperidin and its possible mechanisms of action on pancreatic β-cells in diabetes. Main methodsMale Sprague Dawley rats were made diabetic using 65 mg/kg intraperitoneal injection of streptozotocin, and then administered daily with 100 mg/kg of hesperidin over 4 weeks. On conclusion of the experiment, blood and pancreatic tissue were collected to determine the function of β-cells, apoptosis, oxidative stress, ER stress, and inflammation. Key findingsTreatment of diabetic rats with hesperidin, significantly decreased fasting blood glucose and food intake, along with increased body weight, serum and pancreatic insulin levels, and pancreatic-duodenal homeobox-1 (PDX-1) protein expression. The beneficial roles of hesperidin on diabetic pancreatic β-cells exhibited an increment in antioxidant SOD and GPx activities, and a decrement in nitrotyrosine as well as malondialdehyde (MDA) levels. Additionally, the elevated concentration of TNF-α and expressions of ER stress maker GRP78 and CHOP proteins in the pancreas of diabetic rats were significantly diminished by hesperidin treatment. Furthermore, hesperidin effectively modulated expressions of apoptosis-regulatory proteins in diabetic rat pancreas, as revealed by upregulating anti-apoptotic Bcl-xL; with a concomitant downregulating pro-apoptotic Bax, cleaved caspase-3, and inhibiting the activation of DNA repair protein poly (ADP-ribose) polymerase (PARP). SignificanceCollectively, these findings suggest that hesperidin may have the potential to protect pancreatic β-cells and improve their function by suppressing oxidative and ER stress, along with activating its antioxidant, anti-inflammatory, and anti-apoptotic effects.

Proapoptotic protein BIM as a novel prognostic marker in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma. Numerous studies have demonstrated many genetic aberrations in MCL in addition to the characteristic t(11:14), including frequent biallelic deletions of Bim, a proapoptotic member of the BCL-2 family. In mice, Bim deletion coupled with cyclin D1 overexpression generates pathologic and molecular features of human MCL. Since the regulation of apoptosis is crucial in MCL pathogenesis, we hypothesize that BIM expression may be associated with tumor cell survival. Clinical data and tissue from 100 nodal MCL cases between 1988 and 2009 were collected from three large academic medical centers. The average patient age of our MCL cohort was 65.5 years old (range, 42-97) with a 2:1 male to female ratio. Immunohistochemistry was performed with a validated anti-BIM antibody. Patients were separated into low and high BIM-expressing categories with a cutoff of 80%. As expected for a proapoptotic tumor suppressor, patients with high BIM expression were less likely to have progressive disease and more likely to have a complete response (P = .022). In addition, high BIM-expressing MCL tumors revealed a trend toward increased overall survival with this trend persisting in sub-analysis of Ann Arbor stages III and IV. No correlation between BIM expression, Ki-67 index, and MIPI score was observed, suggesting a role for BIM as a novel independent prognostic factor. While BIM is only one member of a complex family of apoptosis-regulating proteins, these findings may yield clinically relevant information for the prognosis and therapeutic susceptibility of MCL.

MicroRNA-188 aggravates contrast-induced apoptosis by targeting SRSF7 in novel isotonic contrast-induced acute kidney injury rat models and renal tubular epithelial cells.

Contrast media (CM) is widely used in cardiac catheterization; however, it may cause contrast-induced acute kidney injury (CI-AKI) which severely increases mortality. MicroRNA (miRNA) has been found to participate in the process of acute kidney injury (AKI), and this discovery has great potential for diagnosis and treatment. However, the role of miRNA in CI-AKI is still unclear. This study aimed to investigate the regulatory effect miRNAs exert in CI-AKI. We established a novel, representative, isotonic CI-AKI model by using CM iodixanol, a CM which is commonly used in clinic. Next-generation sequencing and reverse transcription polymerase chain reaction (RT-qPCR) were performed to determine the expression of miRNA-188 in CI-AKI. Western blot analysis of the apoptosis regulator protein and TUNEL assay were ordered to evaluate apoptosis. Bioinformatics and double luciferin reporter gene assay were performed to predict and to confirm the interaction between microRNA-188 and SRSF7. The novel isotonic CI-AKI rat model we established exhibited typical characteristics of CI-AKI in serum creatinine, cystatin C, HE staining, and under electron microscope observation. Sequencing and RT-qPCR demonstrated that miRNA-188 was significantly up-regulated both in CI-AKI rat and HK-2 cell models while overexpression of miRNA-188 significantly aggravated apoptosis in CI-AKI cell models. SRSF7 was identified as a direct target gene of miRNA-188, and dual luciferase reporter assay determined the direct interaction between SRSF7 and miRNA-188. In addition, SRSF7 silencing reduced the cell viability rate of the CI-AKI cell model. The present study's findings indicate that miRNA-188 aggravated contrast-induced apoptosis by regulating SRSF7, which may serve as a potential drug target for CI-AKI intervention.

Anticancer activity of newly synthesized 1,1-disubstituted cyclohexane-1-carboxamides: in vitro caspases mediated apoptosis activators in human cancer cell lines and their molecular modeling.

Novel 1,1-disubstituted cyclohexane-1-carboxamides 6a-h, 7a-e, and 8a-b were designed and synthesized as apoptotic inducers. Cytotoxicity test revealed that some compounds have strong to moderate effect, while others displayed weak action against different cancer cell lines including, MCF-7, HepG2, A549, and HTC-116. A549 carcinoma cell line exhibited higher sensitivity toward all synthesized candidates especially compounds 6a and 8a which offered the lowest IC50 values 3.03 and 5.21 μM, respectively, relative to the positive control doxorubicin with IC50 value of 3.01 μM. Compared to doxorubicin treatment, compounds 6a and 8a induced caspases-3, -8, and -9 activities and G2/M growth arrest in A549 carcinoma cell line. The expression levels of p53 (tumor suppressor protein that in humans is encoded by the TP53 gene), Bax (apoptosis regulator protein in humans that is encoded by bax gene), and the Bax/Bcl-2 ratio were all higher than those in doxorubicin-treated cells (Bcl-2, B-cell lymphoma 2, encoded in humans by the Bcl-2 gene). Additionally, compounds 6a and 8a appeared to exhibit higher selectivity against MCF-10 human breast normal cell line. The synthesized congeners could be considered as potent apoptotic inducers interfering with extrinsic and intrinsic apoptotic pathways. Moreover, compound 6a was able to form complex with zinc ions as indicated by UV spectrophotometry which revealed its ability for being caspase activator. Molecular docking studies expected the interactions and binding modes of the synthesized inhibitors in the caspase-3 active site.

Apoptosis regulation in the penumbra after ischemic stroke: expression of pro- and antiapoptotic proteins.

Ischemic stroke is the leading cause of human disability and mortality in the world. The main problem in stroke therapy is the search of efficient neuroprotector capable to rescue neurons in the potentially salvageable transition zone (penumbra), which is expanding after brain damage. The data on molecular mechanisms of penumbra formation and expression of diverse signaling proteins in the penumbra during first 24h after ischemic stroke are discussed. Two basic features of cell death regulation in the ischemic penumbra were observed: (1) both apoptotic and anti-apoptotic proteins are simultaneously over-expressed in the penumbra, so that the fate of individual cells is determined by the balance between these opposite tendencies. (2) Similtaneous and concerted up-regulation in the ischemic penumbra of proteins that execute apoptosis (caspases 3, 6, 7; Bcl-10, SMAC/DIABLO, AIF, PSR), signaling proteins that regulate different apoptosis pathways (p38, JNK, DYRK1A, neurotrophin receptor p75); transcription factors that control expression of various apoptosis regulation proteins (E2F1, p53, c-Myc, GADD153); and proteins, which are normally involved in diverse cellular functions, but stimulate apoptosis in specific situations (NMDAR2a, Par4, GAD65/67, caspase 11). Hence, diverse apoptosis initiation and regulation pathways are induced simultaneously in penumbra from very different initial positions. Similarly, various anti-apoptotic proteins (Bcl-x, p21/WAF-1, MDM2, p63, PKBα, ERK1, RAF1, ERK5, MAKAPK2, protein phosphatases 1α and MKP-1, estrogen and EGF receptors, calmodulin, CaMKII, CaMKIV) are upregulated. These data provide an integral view of neurodegeneration and neuroprotection in penumbra. Some discussed proteins may serve as potential targets for anti-stroke therapy.

Effect of Linagliptin on the Ratio of Apoptosis Regulators in the Model of Non-Alcoholic Fatty Liver Disease in db/db Mice

We studied the effects of dipeptidyl peptidase 4 (DPP4) inhibitor linagliptin on the expression of apoptosis regulator proteins Bcl-2 and Bad in the liver of db/db mice with genetically determined obesity and type 2 diabetes mellitus. The mice received daily linagliptin or saline (placebo) by gavage from week 10 to week 18 of life. In the liver of non-treated mice, the area positively stained for Bad was greater than the area of Bcl-2 expression, which created the conditions for apoptosis activation in liver at this age. Administration of linagliptin decreased Bad stained area and increased Bcl-2 stained area in the liver cells. At the same time, Bad stained area remained larger in treated mice than the area of Bcl-2 expression area, which attested to partial normalization of pro- and antiapoptotic protein balance.

The effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells

Objective The aim of current study is to determine the effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells by investigating the apoptotic activity and variation of intracellular phosphorylated protein kinase B (p-Akt), X-linkedinhibitor of apoptosis protein (XIAP), cysteinyl aspartate specific proteinase-3 (caspase-3), with the treatment of thymic stromal lymphopoietin. Methods Mouse lumbar nucleus pulposus cells were cultured and identified under a fluorescence microscope. Second or third passage cells maintained in monolayers were used for the following experiments. The groups were divided randomly into normal group, TNF-α treated group, TSLP treated group, TSLP+LY94002 treated group and TSLP+Embelin treated group. As a control, normal group was treated with PBS. TNF-α treated group was treated with 500 ng/ml TNF-α as a positive control. TSLP treated group was treated with 10 ng/ml rhTSLP. TSLP+LY94002 treated group and TSLP+Embelin treated group were treated with 10 ng/ml TSLP with the pretreatment of different pathway inhibitors for 30 min in different corresponding experiments, for which 10 μmol LY294002 or 50 LY294002 responding experimentsreatment of different pathway inhibitors formouse nucleus pulposus cells was detected by FACS.The expression levels of the intracellular p-Akt, XIAP, caspase-3 were investigated by Western blot analysis. Results As the culture cell type II collagen staining was positive observed by fluorescence microscopy, we confirmed that the cultured cells were nucleus pulposus cells. In comparison with negative control, the levels of p-Akt, XIAP in TSLP treated group were elevated (t=9.510, P=0.001; t=8.851, P=0.001). Thecaspase-3 activity were slightly enhanced and the rate of cells apoptosis was no significance. Compared with TSLP treated group, downregulated level of p-Akt and XIAPand upregulatedcaspase-3 activity in TSLP+LY294002 treated group were observed (t=8.798, P=0.001; t=7.032, P=0.002; t=5.908, P=0.004). Upregulated caspase-3 activity were also observed in TSLP+ Embelin treated group (t=7.990, P=0.001). Furthermore, significant increased apoptotic cell rate was observed in TSLP+LY294002 or TSLP+Embelin treated groups (t=21.268, P=0.001; t=21.279, P=0.001). Conclusion TSLP may have a potential anti-apoptotic effect on mouse NP cells via upregulating XIAP in PI3K/Akt signaling pathway to restrain the activation of caspase-3. Key words: Cell culture techniques; Aopotosis; Apoptosis regulatory proteins

Knockdown of TIGAR Induces Apoptosis and Autophagy with Modulates NF-κB and HO-1 Expression in A549 Lung Cancer Cells

The tp53-induced glycolysis and apoptosis regulator (TIGAR) protein controls fructose-2, 6- bisphosphate (Fru-2, 6-P2) levels during glucose metabolism and helps maintain nicotinamide adenine dinucleotide phosphate (NADPH) levels to recycle glutathione (GSH), a key intracellular antioxidant. The present study was designed to investigate the apoptosis and autophagy mechanisms via reactive oxygen species (ROS) that underlie TIGAR knockdown in the A549 cell line. To detect the influence of siRNA-TIGAR on A549 lung cancer cells, we performed cell viabilty, colony formation, ROS, and NADPH assays. In addition, Western blotting and real-time polymerase chain reaction (PCR) assays were used to measure protein and mRNA expression levels, respectively. After TIGAR knockdown in A549 cell lines, various assay parameters were analyzed and showed that down-regulation of TIGAR inhibited viability and decreased colony formation. We also demonstrated that TIGAR knockdown induced apoptosis and autophagy, followed by an induction of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and heme oxygenase-1 (HO-1) expression. Furthermore, increased ROS levels and decreased NADPH levels were observed. This study supports our understanding of the possibility of employing TIGAR knockdown in lung cancer cells to enhance apoptosis and autophagy with NF-κB and HO-1 expression and then suggest TIGAR as a potential target for the treatment of lung cancer.

A H2AX⁻CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage.

Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and γH2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished γH2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (Δ600–652) mutant. Moreover, cells expressing CARP-1 (Δ600–652) mutant were resistant to apoptosis, and had diminished levels of γH2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1–35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636–650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636–650) peptide bound with H2AX (1–35) peptide with a dissociation constant (Kd) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1–35) peptide or EGFP-tagged CARP-1 (636–650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636–650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds.

Severe Uncontrolled Maternal Hyperglycemia Induces Microsomia and Neurodevelopment Delay Accompanied by Apoptosis, Cellular Survival, and Neuroinflammatory Deregulation in Rat Offspring Hippocampus.

Maternal diabetes constitutes an unfavorable intrauterine environment for offspring development. Although it is known that diabetes can cause brain alterations and increased risk for neurologic disorders, the relationship between neuroimmune activation, brain changes, and neurodevelopment deficits in the offspring remains unclear. In order to elucidate the short- and long-term biological basis of the developmental outcomes caused by the severe uncontrolled maternal hyperglycemia, we studied apoptosis, neurogenesis, and neuroinflammation pathways in the hippocampus of neonates and young rats born to diabetic dams. Diabetes was induced on gestational day 5 by an injection of streptozotocin. Evaluations of milestones, body growth, and inhibitory avoidance were performed to monitor the offspring development and behavior. Hippocampal modifications were studied through cellular survival by BrdU in the dentate gyrus, expression of apoptosis-regulatory proteins (procaspase 3, caspase 3, and Bcl-2), BDNF, and neuroinflammatory modulation by interleukins, MHC-I, MHC-II, Iba-1, and GFAP proteins. Severe maternal diabetes caused microsomia and neurodevelopmental delay in pups and decrease of Bcl-2, procaspase 3, and caspase 3 in the hippocampus. Moreover, in a later stage of development, it was found an increase of TNF-α and a decrease of procaspase 3, caspase 3, MHC-I, IL-1β, and BDNF in the hippocampus, as well as impairment in cellular survival in the dentate gyrus. This study showed significant short- and long-term commitments on the development, apoptosis, cell survival, and neuroinflammation in the offspring hippocampus induced by severe uncontrolled maternal hyperglycemia. The data reinforce the need for treatment of maternal hyperglycemic states during pregnancy and breast-feeding.

Histone methyltransferase NSD2 regulates apoptosis and chemosensitivity in osteosarcoma

Osteosarcoma (OS) is a primary malignant bone tumour. However, the genetic basis for the pathogenesis of OS remains elusive. In this study, we uncovered the role of the histone methyltransferase NSD2 in regulating tumourigenesis and chemosensitivity in OS. We show that NSD2 knockdown leads to increased apoptosis in OS cells in vitro and in vivo. Additionally, NSD2 knockdown significantly enhances the efficacy of cisplatin against OS cells and accordingly inhibits properties associated with cancer stem cells (CSCs). Furthermore, RNA sequencing (RNAseq) and Gene Ontology (GO) analysis revealed that NSD2 promotes transcription of genes associated with negative regulation of apoptotic signalling pathways and CSC properties. The results of chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) assays indicated that NSD2 knockdown leads to decreased H3K36me2 modification at BCL2 and SOX2 loci, thus inhibiting the transcription of these two genes that are closely correlated with apoptosis, CSC properties and chemosensitivity in OS cells. Pathway analysis demonstrated that the ERK and AKT pathways mediate the regulation of OS progression and chemosensitivity by NSD2. Overall, our study is the first to uncover the function of NSD2 in OS chemosensitivity. NSD2 regulates the expression of the apoptosis regulatory proteins BCL2 and SOX2 through the ERK and AKT pathways. Our results suggest that NSD2 is a new target for combined chemotherapy and is a prognostic factor for OS.

Inhibitory Effects of the Extracts of Juglans sigillata Green Husks on the Proliferation, Migration and Survival of KYSE150 and EC9706 Human Esophageal Cancer Cell Lines

This study evaluated the antitumor activity of the extracts of green husks of Juglans sigillata Dode on esophageal cancer. KYSE150 EC9706 cells were treated with different concentrations of six components of the extracts of J. sigillata green husks. Cell viability was measured by MTT. Cell migration and cell invasion were measured by wound-healing assays and transwell assays, respectively. Cell apoptosis and cycle were measured by flow cytometry. The expression of cell migration, cell cycle and cell apoptosis regulatory proteins was analyzed by Western blotting. Only the three constituents, including EtOH extractives, EtOAc soluble fraction and gallic acid (GA), exhibited inhibitory effects on the cell viability, migration and invasion by decreasing MMP2 and MMP9 expression (all P < 0.05). Flow cytometry revealed that these three constituents also induced cell apoptosis by increasing Bax and cleaved caspase-3 but decreasing Bcl-2 in KYSE150 and EC9706 cells. Furthermore, these constituents arrested the cell cycle at G0/G1 by downregulating the expression of Cyclin D1 but upregulating p53 and phospho-p53 expression in KYSE150 cells. In conclusion, the green husks of J. sigillata may act as a potential inhibitor on esophageal cancer growth. GA was the major single active constituent of the extracts.

Blocking of methionine aminopeptidase-2 by TNP-470 induces apoptosis and increases chemosensitivity of cholangiocarcinoma.

Resistance of cancer cells to chemotherapeutic drugs is a major pitfall of the failure of chemotherapy treatment for cholangiocarcinoma (CCA). A new therapeutic strategy that can improve treatment efficacy is mandatory for CCA patients. Our previous findings demonstrated the overexpression of methionine aminopeptidase-2 (MetAP2) in CCA patients. In addition, supplementation of TNP-470, a MetAP2 inhibitor, significantly inhibited the growth and metastatic activities of CCA cell lines. However, the molecular mechanism of antitumor activity of TNP-470 and the synergistic antitumor activity of TNP-470 combined with chemotherapeutic drugs are still unknown. The aim of this study is to evaluate the molecular mechanism of anticancer activity and the potential use of TNP-470 as a chemosensitizing agent in CCA cell lines. Cell cycle and apoptosis of CCA cell lines were evaluated using flow cytometry with propidium iodide staining. Expression of apoptosis regulatory proteins was measured by Western blotting. The chemosensitizing effect of TNP-470 was determined using combination index. TNP-470 inhibited the growth of CCA cells via induction of apoptosis through activation of the p38-phosphorylation and up- and down-regulation of Bax and Bcl-xL, respectively. Furthermore, TNP-470 significantly enhanced the antitumor activity of 5-fluorouracil, cisplatin, doxorubicin, and gemcitabine. The present results show that TNP-470 could be a potential therapeutic or adjuvant agent for CCA.

PLEIOTROPIC ENZYMES OF APOPTOSIS AND SYNAPTIC PLASTICITY IN ALBINO RAT HIPPOCAMPUS AFTER OCCLUSION OF COMMON CAROTID ARTERIES

Aim: the aim of the study was to investigate the pleiotropic properties of the apoptotic enzyme caspase-3 and its associations with the synaptic plasticity of the hippocampus of albino rats in healthy animals and in rats after 20-min occlusion of the common carotid arteries.Material and Methods. Total numerical density of neurons, ultrastructure of synapses, and area of immunohistochemically positive hippocampal synaptic terminals of CA1 stratum radiatum and stratum lucidum CA3 were studied by the methods of optical microscopy (hematoxylin and eosin stain), electron microscopy (uranyl acetate and lead citrate as contrast agents), immunohistochemistry (MAP2, synaptophysin, caspase-3, p53, and bcl-2), and morphometry in the brains of intact rats (n=5) and in animals after acute ischemia at day 1 (n=5), 3 (n=5), 7 (n=5), 14 (n=5), and 30 (n=25).Results and Discussion. The study showed that 33.0% of pyramidal neurons in CA1 region and 17.4% of those in CA3 region underwent irreversible damage within 30 days of the post-ischemic period. Among the irreversibly damaged neurons, the cells with signs of coagulative-ischemic necrosis prevailed. In animals subject to ischemia, the relative area of synaptophysin-positive material initially decreased (at day 1) and then recovered (at days 3, 7). We found that caspase-3 colocalized with synaptophysin, which was especially evident in the giant synapses of the stratum lucidum of the hippocampal CA3 region. In the neurosomes of the hippocampal pyramidal cells, caspase-3 was not detected. However, this enzyme was found in the terminals of the axo-dendritic, axo-spine, and axo-somatic synapses. In the course of th e post-ischemic period, the most pronounced changes in the expression of caspase-3 were observed in the stratum radiatum of the CA1 field. Apoptosis regulatory proteins (p53, bcl-2) were detected in the individual neurons. In this regard, caspase-3 should be viewed in the context of its pleiotropy and involvement in the adaptation and recovery processes due to post-ischemic activation of neuroplasticity at the level of axons and synapses.Conclusion. After acute ischemia caused by 20-min occlusion of the common carotid arteries, the activation of caspase-3 contributes to ischemic preconditioning and neuroprotection.

Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1

Mitochondria-dependent apoptosis plays an important role in the embryonic development of the midbrain dopaminergic system as well as in Parkinson’s disease. Central to mitochondria-dependent apoptosis is the Bcl2 family of apoptosis-regulating proteins. However, it was unclear which Bcl2 proteins are important for the survival of dopaminergic neurons. Here, we identify Mcl1 as a critical Bcl2 pro-survival factor in midbrain dopaminergic neurons. Using a chemical biology approach to inhibit various components of the apoptotic machinery in the dopaminergic MN9D cell line or the control neuroblastoma N2A cell line, we find that functional inhibition of Mcl1 with the high affinity small molecule inhibitor UMI-77 results in a rapid and dose-dependent loss of viability, selectively in dopaminergic cells. In-depth analysis of the apoptotic signaling pathway reveals that chemical inhibition of Mcl1 results in the activation of Bax, activation of cleaved caspase-3 and finally cell death. The dependence of mouse dopaminergic midbrain neurons on Mcl1 was confirmed using ex vivo slice cultures from Pitx3GFP/+ and wildtype mice. In mouse dopaminergic midbrain neurons positive for the midbrain dopaminergic marker Pitx3, or tyrosine hydroxylase, UMI-77 treatment caused a dramatic increase in cleaved caspase 3, indicating that Mcl1 activity is required for basal neuronal survival. Overall, our results suggest that Mcl1 is of critical importance to dopaminergic neurons and is a weak link in the chain controlling cellular survival. Boosting the pro-survival function of Mcl1 should be pursued as a therapeutic approach to augment the resilience of midbrain dopaminergic neurons to apoptotic stress in Parkinson’s disease.

Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression

Ethnopharmacological relevanceXiaochaihutang (XCHT), one of famous Chinese herbal prescription for treating Shaoyang symptom, has been used successfully in depressive disorders for many years. Our laboratory has demonstrated that XCHT remarkably alleviated various depressive behaviors induced by several depressive animal models, but previous studies only focused on one or several protein targets, lacked dynamic change and interrelation of proteins. Therefore, potential protein targets and mechanisms are required further systematic investigation. Aim of the studyTo discover and assess the differentially expressed proteins (DEPs) of hippocampus after oral administration of XCHT in corticosterone (CORT) induced model of depression by using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Materials and methodsThe antidepressant effects of XCHT were assessed by two behavioral despair models (forced swimming test and tail suspension test) in CORT induced model of depression. The DEPs of hippocampus after XCHT treatment were investigated by iTRAQ analysis. Potential protein targets and mechanisms were assessed by gene ontology (GO), Kyoto encyclopedia of gene and genomes (KEGG) and protein-protein interaction (PPI) network. ResultsOur data demonstrated XCHT could significantly improve depressive behaviors. A total of 241 DEPs were identified after XCHT treatment, including 68 up regulation and 173 down regulation proteins. GO enrichment results indicated that XCHT mainly regulated intracellular structural proteins involved in cellular response to stress, transferase activity and steroid hormone. KEGG enrichment analysis showed that endocytosis might be the principal pathway of XCHT on depression. PPI analysis predicted cell division cycle and apoptosis regulator protein 1 (Ccar1) and Calretinin (Calb2) might play the central roles in XCHT's antidepressant network. ConclusionOur results indicate that XCHT plays the important roles in antidepressant action by restoring DEPs, which results in the dysregulation of hippocampal neurogenesis, neurotransmitter and steroid hormone. The current results wish to provide novel perspectives for revealing the potential protein targets of XCHT on depression.

Long non-coding RNA MEG3 suppresses the development of bladder urothelial carcinoma by regulating miR-96 and TPM1

ABSTRACTWe aimed at investigating effects of long non-coding RNA maternally expressed 3 (MEG3) on the proliferation, cell cycle and apoptosis of bladder urothelial carcinoma cells and regulatory relationships among lncRNA MEG3, miR-96 and α-tropomyosin 1 (TPM1). Human clinical data from The Cancer Genome Atlas (TCGA) which contains bladder urothelial carcinoma tissues and adjacent tissues were used for analysis. The expression profiles of MEG3, miR-96, TPM1, cell cycle-related genes and apoptosis-related genes were examined by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Regulating relationship among MEG3, miR-96 and TPM1 was confirmed by dual luciferase reporter assay. MTT assay and flow cytometry were performed to observe cell proliferation, cell cycle and apoptosis. The effects of lncRNA MEG3 on bladder urothelial carcinoma were confirmed both in vivo and in vitro. The mRNA expression and protein expression of MEG3, TPM1 were down-regulated in carcinoma tissues, whereas miR-96 expression was up-regulated. MEG3 overexpression resulted in miR-96 downregulation along with TPM1 upregulation, which inhibited cell proliferation and cell cycle but promoted cell apoptosis of bladder urothelial carcinoma cells in vitro, and at the same time inhibited tumor growth in vivo. In this process, expressions of apoptosis-related protein BCL2 associated X (Bax), cleaved-caspase 3 was up-regulated, whereas apoptosis regulator protein (Bcl-2) expression was suppressed when MEG3 was overexpressed, and cell cycle-related protein Cyclin D1 was down-regulated. LncRNA MEG3 low-expression promotes the proliferation and inhibits apoptosis of bladder urothelial carcinoma cells by regulating miR-96 along with TPM1.

Phoyunnanin E Induces Apoptosis of Non-small Cell Lung Cancer Cells via p53 Activation and Down-regulation of Survivin

Lung cancer is by far the most common cause of cancer mortality, accounting for nearly 20% of all global cancer deaths. Therefore, potent and effective compounds for treatment of this cancer type are essential. Phoyunnanin E, isolated from Dendrobium venustum (Orchidaceae), has promising pharmacological activities; however, it is unknown if phoyunnanin E affects apoptosis of lung cancer cells. The apoptosis-inducing activity of phoyunnanin E on H460 lung cancer cells was investigated by Hoechst 33342, and annexin V-fluorescein isothiocyanate/propidium iodide staining. The underlying mechanism was determined via monitoring apoptosis-regulatory proteins by western blot analysis. The apoptotic effect of the compound was confirmed in H23 lung cancer cells. Phoyunnanin E significantly induced apoptotic cell death of H460 lung cancer cells, as indicated by condensed and fragmented nuclei with the activation of caspase-3 and -9 and poly (ADP-ribose) polymerase cleavage. Phoyunnanin E mediated apoptosis via a p53-dependent pathway by increasing the accumulation of cellular p53 protein. As a consequence, anti-apoptotic proteins including induced myeloid leukemia cell differentiation protein (MCL1) and B-cell lymphoma 2 (BCL2) were found to be significantly depleted, while pro-apoptotic BCL-2-associated X protein (BAX) protein was up-regulated. Furthermore, it was found that expression of an inhibitor of apoptosis, survivin, markedly reduced in response to phoyunnanin E treatment. The apoptosis-inducting effect was also found in phoyunnanin E-treated H23 lung cancer cells. These results indicate the promising effect of phoyunnanin E in induction of apoptosis, that may be useful for the development of novel anticancer agents.

Discovery of a small-molecule inhibitor of specific serine residue BAD phosphorylation

Human BCL-2-associated death promoter (hBAD) is an apoptosis-regulatory protein mediating survival signals to carcinoma cells upon phosphorylation of Ser99, among other residues. Herein, we screened multiple small-molecule databases queried in a Laplacian-modified naive Bayesian-based cheminformatics platform and identified a Petasis reaction product as a site-specific inhibitor for hBAD phosphorylation. Based on apoptotic efficacy against mammary carcinoma cells, N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB) was identified as a potential lead compound. In vitro biochemical analyses demonstrated that NPB inhibited the phosphorylation of hBAD specifically on Ser99. NPB was observed to exert this effect independently of AKT and other kinase activities despite the demonstration of AKT-mediated BAD-Ser99 phosphorylation. Using a structure-based bioinformatics platform, we observed that NPB exhibited predicted interactions with hBAD in silico and verified the same by direct binding kinetics. NPB reduced phosphorylation of BAD-Ser99 and enhanced caspase 3/7 activity with associated loss of cell viability in various human cancer cell lines derived from mammary, endometrial, ovarian, hepatocellular, colon, prostatic, and pancreatic carcinoma. Furthermore, by use of a xenograft model, it was observed that NPB, as a single agent, markedly diminished BAD phosphorylation in tumor tissue and significantly inhibited tumor growth. Similar doses of NPB utilized in acute toxicity studies in mice did not exhibit significant effects. Hence, we report a site-specific inhibitor of BAD phosphorylation with efficacy in tumor models.