Apoptosis In Experimental Models Research Articles

Relative Molecular Similarity in Compound Structures Modulating Neurodegenerative Apoptosis

Neurodegeneration is attributable to metabolic disturbances in the various cell types responsible for this condition, in respect of glucose utilisation and dysfunctional mitochondrial oxidative mechanisms. The properties of neurotoxins and antagonists that limit their action are well documented in disease models, whereas effective therapy is very limited. Cell apoptosis, a general marker of neurodegeneration, is also of therapeutic interest in the treatment of cancer. cGMP nucleotide influences apoptosis and has a role in maintaining equilibrium within cell redox parameters. The chemical structure of cGMP provides a comparative template for demonstrating relative molecular similarity within the structures of natural and synthetic compounds influencing tumour cell apoptosis. The present study uses computational software to investigate molecular similarity within the structures of cGMP and compounds that modulate cell apoptosis in experimental models of diabetic peripheral neuropathy (DPN), Parkinson’s and multiple sclerosis. Differential molecular similarity demonstrated in neurotoxin and antagonist structures implicate metabolite impairment of cGMP signaling function as a common mechanism in the initial phases of these neurodegenerative conditions.

AKT, ERK and NFKB Signaling Pathways in Neurons of Hypothalamic Supraoptic and Paraventricular Nuclei in Aging Transgenic Mice with HER2/neu Overexpression

Epidermal growth factor receptors are widely expressed in various tissues, including the brain, and play an important role in the regulation of such biological processes as proliferation, cell migration and death, etc. This study aimed to study the involvement of the cell survival-related signaling pathways (AKT, ERK, NF-kB cascades) in the regulation of hypothalamic neuronal apoptosis in experimental models of physiological and pathological (HER2/neu overexpression) aging, as well as the effect of FASL and TNF-alpha cytokines on these signaling cascades. Experiments were carried out on HER2/neu transgenic mice with accelerated senescence compared to the wild type mice (FVB/N strain). In the hypothalamic supraoptic and paraventricular nuclei, expression of the anti-apoptotic protein survivin was determined by immunohistochemistry, while expression of proteins of the cytokine-dependent cascades (FASL, TNF-alpha) and protein members of the cell survival-related signaling pathways (phosphorylated AKT, ERK, NF-kB) was assayed by Western blotting. It was found that in FVB/N mice the AKT and ERK cascades do not contribute significantly to neuronal senescence, whereas activation of FAS signaling and a decrease in the NF-kB pathway activity can lead to an increased proportion of dying neurons. In HER2/neu mice, neuronal resistance to apoptosis during aging is ensured by a combination of different cell survival-related signaling pathways: the ERK cascade in the supraoptic nucleus and the NF-kB and AKT cascades in the paraventricular nucleus. In addition, a suppression of FAS signaling was revealed, also leading to a low level of apoptosis.

АKT-, ERK-, NF-KB-СИГНАЛЬНЫЕ КАСКАДЫ В НЕЙРОНАХ СУПРАОПТИЧЕСКОГО И ПАРАВЕНТРИКУЛЯРНОГО ЯДЕР ГИПОТАЛАМУСА ПРИ СТАРЕНИИ У МЫШЕЙ СО СВЕРХЭКСПРЕССИЕЙ HER2/NEU

Epidermal growth factor receptors are widely expressed in the various tissues, including the brain, and play an important role in the regulation of such biological processes as proliferation, cell migration, cell death, etc. This study aimed to study the involvement of signaling pathways related with cell survival (AKT, ERK, NF-kB cascades) in the regulation of hypothalamic neuronal apoptosis in experimental models of physiological and pathological (HER2/neu overexpression) aging, as well as the effect of FASL and TNF-alpha cytokines on these signaling cascades. Experiments were carried out on HER2/neu transgenic mice with accelerated cellular senescence compared to the wild type mice (FVB/N strain). In the hypothalamic supraoptic and paraventricular nuclei, expression of the antiapoptotic protein survivin was determined by immunohistochemistry, while expression of proteins of the cytokine-dependent cascades (FASL, TNF-alpha) and protein members of the survival signaling pathways (phosphorylated AKT, ERK, NF-kB) was assayed by Western blotting. It was found that in FVB/N mice the AKT, ERK cascades do not play a significant role in aging, whereas activation of FAS signaling and a decrease in the NF-kB pathway activity can lead to an increased proportion of dying neurons. In HER2/neu mice, neuronal tolerance to apoptosis during aging is provided by a combination of different survival signaling pathways: the ERK cascade is activated in the supraoptic nucleus and the NF-kB and AKT cascades in the paraventricular nucleus. In addition, a suppression of FAS signaling was revealed, also leading to a low level of apoptosis.

Brain-derived neurotrophic factor reduces inflammation and hippocampal apoptosis in experimental Streptococcus pneumoniae meningitis

BackgroundStreptococcus pneumoniae meningitis is a serious inflammatory disease of the central nervous system (CNS) and is associated with high morbidity and mortality rates. The inflammatory processes initiated by recognition of bacterial components contribute to apoptosis in the hippocampal dentate gyrus. Brain-derived neurotrophic factor (BDNF) has long been recommended for the treatment of CNS diseases due to its powerful neuro-survival properties, as well as its recently reported anti-inflammatory and anti-apoptotic effects in vitro and in vivo.MethodsIn this study, we investigated the effects of BDNF-related signaling on the inflammatory response and hippocampal apoptosis in experimental models of pneumococcal meningitis. Pretreatment with exogenous BDNF or the tropomyosin-receptor kinase B (TrkB) inhibitor k252a was performed to assess the activation or inhibition of the BDNF/TrkB-signaling axis prior to intracisternal infection with live S. pneumoniae. At 24 h post-infection, rats were assessed for clinical severity and sacrificed to harvest the brains. Paraffin-embedded brain sections underwent hematoxylin and eosin staining to evaluate pathological severity, and cytokine and chemokine levels in the hippocampus and cortex were evaluated by enzyme-linked immunosorbent assay. Additionally, apoptotic neurons were detected in the hippocampal dentate gyrus by terminal deoxynucleotidyl transferase dUTP-nick-end labeling, key molecules associated with the related signaling pathway were analyzed by real-time polymerase chain reaction and western blot, and the DNA-binding activity of nuclear factor kappa B (NF-κB) was measured by electrophoretic mobility shift assay.ResultsRats administered BDNF exhibited reduced clinical impairment, pathological severity, and hippocampal apoptosis. Furthermore, BDNF pretreatment suppressed the expression of inflammatory factors, including tumor necrosis factor α, interleukin (IL)-1β, and IL-6, and increased the expression of the anti-inflammatory factor IL-10. Moreover, BDNF pretreatment increased TrkB expression, activated downstream phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling, and inhibited the myeloid differentiation primary response gene 88 (MyD88)/NF-κB-signaling pathway.ConclusionsThese data suggested that BDNF administration exerted anti-inflammatory and anti-apoptotic effects on an experimental pneumococcal meningitis model via modulation of MyD88/NF-κB- and PI3K/AKT-signaling pathways. Our results indicated that treatment with exogenous BDNF might constitute a potential therapeutic strategy for the treatment of bacterial meningitis.

Oxidative stress associated with neuronal apoptosis in experimental models of epilepsy.

Epilepsy is considered one of the most common neurological disorders worldwide. Oxidative stress produced by free radicals may play a role in the initiation and progression of epilepsy; the changes in the mitochondrial and the oxidative stress state can lead mechanism associated with neuronal death pathway. Bioenergetics state failure and impaired mitochondrial function include excessive free radical production with impaired synthesis of antioxidants. This review summarizes evidence that suggest what is the role of oxidative stress on induction of apoptosis in experimental models of epilepsy.

Exercise and the brain: a slap on the HAND

Exercise and the brain: a slap on the HAND

ROLE OF HEPATOPROTECTORS AND IMMUNOMODULATORS IN REGULATION OF HEPATOCYTE APOPTOSIS INDUCED BY ANTITUBERCULOSIS TREATMENT

It was currently shown that hepatopathy due to drug toxicity is associated with increased apoptosis of hepatocytes. Therefore, development of drugs which regulate cell death is of great importance. To involve some hepatoprotectors (ademethionine, reamberin, remaxol) and immunomodulators (cycloferon) into regulation of apoptosis in experimental models of liver first-line antituberculousis drugs (isoniazid, rifampicin, pyraztinamide). Levels of apoptoasis (TUNEL), expression of CD95 (receptor of tumor necrosis factor - by immunohistochemistry), expression of caspase-8, caspase-3 and pS3 (Western-blotting) were measured. Exposition offirst-line antituberculousis drugs leads to dysthrophia of liver parenchyma cells with increased apoptosis of hepatocytes and activation of CD95, caspase-8 (external way) and overexpression of p53 and caspase-3. It was found that reamberin, cycloferon and remaxol have hepatoprotective effect improving liver histology; ademethionine administered by intraperitoneal injection showed no positive effects. Reamberin demonstrated apoptosis-inhibiting effect in the experiment whereas other drugs were found to be apoptosis inductors for hepatocytes in toxic hepatopathy. Legulation of apoptosis by cycloferon and remaxol mediated by external and p53-dependent pathway is confirmed by increased expression of CD95 and p53 protein. Ademethionine might induce apoptosis by the intrinsic pathway.

Effect of Chronic Administration of Morphine on the Gene Expression Level of Sodium-Dependent Vitamin C Transporters in Rat Hippocampus and Lumbar Spinal Cord

Chronic morphine leads to dependence, tolerance, and neural apoptosis. Vitamin C inhibits the withdrawal syndrome in morphine-dependent subjects and prevents apoptosis in experimental models. Sodium-dependent vitamin C transporter (SVCT) type-2 is the main transporter for carrying vitamin C into the brain and neural cells. The mechanism(s) by which vitamin C inhibits morphine dependence in not understood. SVCT activity determines the vitamin C availably within the nervous system. We have examined the alterations in the expression of SVCT1, SVCT2, and its splice variants in morphine-tolerant rats. Morphine (20 mg/kg) was injected twice/day to male rats for either 7 or 14 days. The development of analgesic tolerance was assessed using tail-flick test. Lumbar spinal cord and the hippocampus were isolated for RNA extraction. Semiquantitative reverse transcriptase-polymerase chain reaction method was used to assess the levels of gene expression. Administration of morphine for 7 or 14 days reduced the expression level of SVCT2 in both hippocampus and dorsal lumbar spinal cord of rats. SVCT2 expression was reduced in vitamin C-, and vitamin C combined with morphine-treated animals. Results did not show SVCT2 splice variation. SVCT1 did not express in control or morphine-treated rats. It seems that reduced expression level of SVCT2 might be involved in the development of morphine side effects such as tolerance and dependency.

SB939: A Potent and Orally Active HDAC Inhibitor for the Treatment of Hematological Malignancies.

Histone deacetylases (HDACs) are emerging new molecular targets for cancer therapy. Small-molecule HDAC inhibitors have been developed and shown to induce tumor cell cytostasis, differentiation and apoptosis in experimental models and efficacy in clinical trials in various hematological malignancies following intravenous and/or oral administration. SB939 is a novel HDAC inhibitor with improved metabolic, pharmacokinetic and pharmacological properties compared to other HDAC inhibitors currently in clinical trials1. The objective of this study was to characterize the anti-tumor efficacy of SB939 in preclinical models of hematological malignancies. SB939 selectively inhibits HDAC class I and II isozymes, with Ki values ranging from 16 to 247 nM. It inhibited the proliferation of cell lines from various haematological malignancies, including leukemia, lymphoma and multiple myeloma with IC50 values ranging from 80 nM to 200 nM. It induced cell cycle arrest leading to apoptotic cell death in tumor cell lines as well as primary cells isolated from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). SB939 has excellent pharmacokinetic properties and tolerability after oral administration in mice1. The oral anti-tumor efficacy of SB939 was evaluated in models of AML (MV4-11) and lymphoma (Ramos) with the tumors grown subcutaneously in nude mice. After daily oral treatment at 50mg/kg (21 days for MV4-11; 14 days for Ramos), SB939 significantly reduced tumor growth in both models (%TGI values were 116% and 100% respectively in MV4-11 and Ramos). In the MV4-11 model, SB939 induced complete tumor regression, in 6/10 mice. In conclusion, our data demonstrate that SB939 is a potent, orally active anti-tumor drug with potential for the treatment of various types of hematological malignancies. 1Kanda Sangthongpitag, Haishang Wang, Pauline Yeo, Liu Xin, Evelyn Goh, Lee Sun New, Peizi Zeng, Xiaofeng Wu, Changyong Hu, Tony Ng and Kantharaj Ethirajulu. ADME attributes of SB939, a best-in-class HDAC Inhibitor, and its PK/PD correlation in the Pharmacological Species. EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics Prague Congress Centre, 2006, Nov 7–10; Prague, Czech Republic, Abstract number 166

Abstract 1949: The Protein Kinase MAP4K4 Is Activated in Failing Human Hearts and Mediates Cardiomyocyte Apoptosis in Experimental Models, in vitro and in vivo

Mitogen-activated protein kinases (MAPKs) have been implicated in cardiac muscle hypertrophy, apoptosis, and heart failure, but relatively little is known of the proximal MAP4Ks that might couple cardiac stress signals to downstream effector kinases. Previously we have shown that epitope-tagged MAP4K4 (hematopoietic progenitor kinase/germinal center kinase-like kinase, HGK) was activated by diverse apoptotic signals in cultured cardiomyocytes and transgenic mouse hearts. Here, we report results for the activation of MAP4K4 in the hearts of patients undergoing therapeutic transplantation, and for the functional consequences of disrupting endogenous MAP4K4 by RNA interference. Compared with 10 control donor hearts obtained at overlapping ages, MAP4K4 activity increased in all subjects with dilated (4.5-fold; N = 13), hypertrophic (4-fold; N = 5), ischemic (2.5-fold; N = 6) and adriamycin-induced cardiomyopathy (3-fold, N = 2; p < 0.001 for all four groups). Activity of the MAP4K4 target, TGFbeta-activated kinase-1 (TAK1/MAP3K7), and the executioner caspase, caspase-3, increased concordantly in all subjects. In mouse myocardium, each of four biological signals for cardiomyocyte apoptosis induced the kinase activity of MAP4K4: biomechanical stress, ischemia/reperfusion injury, and gain-of-function mutations for TNFalpha and Gq. Analogous results were seen in cultured cardiomyocytes, using oxidative stress, ceramide and the cardiotoxic anti-cancer drug doxorubicin. Transgenic expression of MAP4K4 in myocardium produced little or no baseline phenotype, but markedly sensitized the myocardium to apoptosis caused by Gq. In culture, we proved that MAP4K4 induces apoptosis via TAK1, JNK, and the mitochondrial death pathway. As predicted, ceramide-induced and hydrogen peroxide-induced apoptosis was suppressed using RNA interference to silence expression of MAP4K4. Thus, MAP4K4 is activated in response to many specific cardiac death signals, and mediates cardiac apoptosis in vitro and in vivo. The prevalent activation of MAP4K4 in failing human hearts and the pro-death actions of the kinase suggest that MAP4K4 is a possible therapeutic target in the treatment of apoptotic cardiomyopathies.

A novel peptide inhibitor targeted to caspase-3 cleavage site of a proapoptotic kinase protein kinase C delta (PKCδ) protects against dopaminergic neuronal degeneration in Parkinson’s disease models

Oxidative stress and apoptosis are considered common mediators of many neurodegenerative disorders including Parkinson’s disease (PD). Recently, we identified that PKCδ, a member of the novel PKC isoform family, is proteolytically activated by caspase-3 to induce apoptosis in experimental models of PD [ Eur. J. Neurosci. 18 (6):1387–1401, 2003; Antioxid. Redox Signal. 5 (5):609–620, 2003]. Since caspase-3 cleaves PKCδ between proline and aspartate residues at the cleavage site 324DIPD327 to activate the kinase, we developed an irreversible and competitive peptide inhibitor, Z-Asp(OMe)-Ile-Pro-Asp(OMe)-FMK (z-DIPD-fmk), to mimic the caspase-3 cleavage site of PKCδ and tested its efficacy against oxidative stress-induced cell death in PD models. Cotreatment of z-DIPD-fmk with the parkinsonian toxins MPP + and 6-OHDA dose dependently attenuated cytotoxicity, caspase-3 activation, and DNA fragmentation in a mesencephalic dopaminergic neuronal cell model (N27 cells). However, z-DIPD-fmk treatment did not block MPP +-induced increases in caspase-9 enzyme activity. The z-DIPD-fmk peptide was much more potent (IC 50 6 μM) than the most widely used and commercially available caspase-3 inhibitor z-DEVD-fmk (IC 50 18 μM). Additionally, z-DIPD-fmk more effectively blocked PKCδ cleavage and proteolytic activation than the cleavage of another caspase-3 substrate, poly(ADP-ribose) polymerase (PARP). Importantly, the peptide inhibitor z-DIPD-fmk completely rescued TH + neurons from MPP +- and 6-OHDA-induced toxicity in mouse primary mesencephalic cultures. Collectively, these results demonstrate that the PKCδ cleavage site is a novel target for development of a neuroprotective therapeutic strategy for PD.

From castration-induced apoptosis of prostatic epithelium to the use of apoptotic genes in the treatment of prostate cancer.

Current knowledge of the mechanisms regulating androgen-ablation-induced apoptosis is reviewed, and our efforts to develop a system in which genes of the apoptotic pathway are used to induce therapeutic apoptosis in experimental models of prostate cancer are described.

Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease.

Caspase-3 is an effector of apoptosis in experimental models of Parkinson's disease (PD). However, its potential role in the human pathology remains to be demonstrated. Using caspase-3 immunohistochemistry on the postmortem human brain, we observed a positive correlation between the degree of neuronal loss in dopaminergic (DA) cell groups affected in the mesencephalon of PD patients and the percentage of caspase-3-positive neurons in these cell groups in control subjects and a significant decrease of caspase-3-positive pigmented neurons in the substantia nigra pars compacta of PD patients compared with controls that also could be observed in an animal model of PD. This suggests that neurons expressing caspase-3 are more sensitive to the pathological process than those that do not express the protein. In addition, using an antibody raised against activated caspase-3, the percentage of active caspase-3-positive neurons among DA neurons was significantly higher in PD patients than in controls. Finally, electron microscopy analysis in the human brain and in vitro data suggest that caspase-3 activation precedes and is not a consequence of apoptotic cell death in PD.