VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons.

PubMed

Tenn, Catherine C; Wang, Yushan

2007-05-01

Exposure of cell cultures to organophosphorous compounds such as VX can result in cell death. However, it is not clear whether VX-induced cell death is necrotic or involves programmed cell death mechanisms. Activation of caspases, a family of cysteine proteases, is often involved in cell death, and in particular, caspase-3 activation appears to be a key event in programmed cell death processes including apoptosis. In this study, we investigated VX-induced neuronal cell death, as well as the underlying mechanism in terms of its effect on caspase-3 activity. Primary cortical neuronal cultures were prepared from gestational days 17 to 19 Sprague Dawley rat fetuses. At maturation, the cells were treated with varying concentrations of VX and cell death was evaluated by lactate dehydrogenase (LDH) release. VX induced an increase in LDH release in a concentration-dependent manner. Morphological VX-induced cell death was also characterized by using nuclear staining with propidium iodide and Hoechst 33342. VX induced a concentration- and time-dependent increase in caspase-3 activation. Caspase-3 activation was also confirmed by the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), an endogenous caspase-3 substrate. These data suggested that in rat cortical neurons, VX-induced cell death via a programmed cell death pathway that involves changes in caspase-3 protease.

Critical involvement of extracellular ATP acting on P2RX7 purinergic receptors in photoreceptor cell death.

PubMed

Notomi, Shoji; Hisatomi, Toshio; Kanemaru, Takaaki; Takeda, Atsunobu; Ikeda, Yasuhiro; Enaida, Hiroshi; Kroemer, Guido; Ishibashi, Tatsuro

2011-12-01

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7(-/-) mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Blockade of TLR3 protects mice from lethal radiation-induced gastrointestinal syndrome

PubMed Central

Takemura, Naoki; Kawasaki, Takumi; Kunisawa, Jun; Sato, Shintaro; Lamichhane, Aayam; Kobiyama, Kouji; Aoshi, Taiki; Ito, Junichi; Mizuguchi, Kenji; Karuppuchamy, Thangaraj; Matsunaga, Kouta; Miyatake, Shoichiro; Mori, Nobuko; Tsujimura, Tohru; Satoh, Takashi; Kumagai, Yutaro; Kawai, Taro; Standley, Daron M.; Ishii, Ken J.; Kiyono, Hiroshi; Akira, Shizuo; Uematsu, Satoshi

2014-01-01

High-dose ionizing radiation induces severe DNA damage in the epithelial stem cells in small intestinal crypts and causes gastrointestinal syndrome (GIS). Although the tumour suppressor p53 is a primary factor inducing death of crypt cells with DNA damage, its essential role in maintaining genome stability means inhibiting p53 to prevent GIS is not a viable strategy. Here we show that the innate immune receptor Toll-like receptor 3 (TLR3) is critical for the pathogenesis of GIS. Tlr3−/− mice show substantial resistance to GIS owing to significantly reduced radiation-induced crypt cell death. Despite showing reduced crypt cell death, p53-dependent crypt cell death is not impaired in Tlr3−/− mice. p53-dependent crypt cell death causes leakage of cellular RNA, which induces extensive cell death via TLR3. An inhibitor of TLR3–RNA binding ameliorates GIS by reducing crypt cell death. Thus, we propose blocking TLR3 activation as a novel approach to treat GIS. PMID:24637670

Ammonium affects cell viability to inhibit root growth in Arabidopsis * #

PubMed Central

Qin, Cheng; Yi, Ke-ke; Wu, Ping

2011-01-01

Ammonium (NH4 +) is an important form of nitrogen nutrient for most plants, yet is also a stressor for many of them. However, the primary events of NH4 + toxicity at the cellular level are still unclear. Here, we showed that NH4 + toxicity can induce the root cell death in a temporal pattern which primarily occurs in the cells of root maturation and elongation zones, and then spreads to the cells in the meristem and root cap. The results from the NH4 +-hypersensitive mutant hsn1 further confirmed our findings. Taken together, NH4 + toxicity inhibits primary root growth by inhibiting cell elongation and division and inducing root cell death. PMID:21634041

DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

PubMed Central

Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

2013-01-01

Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

Augmented trophoblast cell death in preeclampsia can proceed via ceramide-mediated necroptosis

PubMed Central

Bailey, Liane Jennifer; Alahari, Sruthi; Tagliaferro, Andrea; Post, Martin; Caniggia, Isabella

2017-01-01

Preeclampsia, a serious hypertensive disorder of pregnancy, is characterized by elevated ceramide (CER) content that is responsible for heightened trophoblast cell death rates via apoptosis and autophagy. Whether trophoblast cells undergo necroptosis, a newly characterized form of regulated necrosis, and the potential role of CER in this process remain to be established. Herein, we report that exposure of both JEG3 cells and primary isolated cytotrophoblasts to C16:0 CER in conjunction with a caspase-8 inhibitor (Q-VD-OPh) promoted necroptotic cell death, as evidenced by increased expression and association of receptor-interacting protein kinases RIP1 and RIP3, as well as phosphorylation of mixed lineage kinase domain-like (MLKL) protein. MLKL activation and oligomerization could be abrogated by pretreatment with the necroptosis inhibitor necrostatin-1 (Nec-1). CER+Q-VD-OPH-treated primary trophoblasts displayed striking necrotic morphology along with disrupted fusion processes as evidenced by maintenance of E-cadherin-stained membrane boundaries and reduced glial cell missing-1 expression, but these events were effectively reversed using Nec-1. Of clinical relevance, we established an increased susceptibility to necroptotic cell death in preeclamptic placentae relative to normotensive controls. In preeclampsia, increased necrosome (RIP1/RIP3) protein levels, as well as MLKL activation and oligomerization associated with necrotic cytotrophoblast morphology. In addition, caspase-8 activity was reduced in severe early-onset preeclampsia cases. This study is the first to report that trophoblast cells undergo CER-induced necroptotic cell death, thereby contributing to the increased placental dysfunction and cell death found in preeclampsia. PMID:28151467

Comprehensive Evaluation of Programmed Death-Ligand 1 Expression in Primary and Metastatic Prostate Cancer.

PubMed

Haffner, Michael C; Guner, Gunes; Taheri, Diana; Netto, George J; Palsgrove, Doreen N; Zheng, Qizhi; Guedes, Liana Benevides; Kim, Kunhwa; Tsai, Harrison; Esopi, David M; Lotan, Tamara L; Sharma, Rajni; Meeker, Alan K; Chinnaiyan, Arul M; Nelson, William G; Yegnasubramanian, Srinivasan; Luo, Jun; Mehra, Rohit; Antonarakis, Emmanuel S; Drake, Charles G; De Marzo, Angelo M

2018-06-01

Antibodies targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-L1) interaction have shown clinical activity in multiple cancer types. PD-L1 protein expression is a clinically validated predictive biomarker of response for such therapies. Prior studies evaluating the expression of PD-L1 in primary prostate cancers have reported highly variable rates of PD-L1 positivity. In addition, limited data exist on PD-L1 expression in metastatic castrate-resistant prostate cancer (mCRPC). Here, we determined PD-L1 protein expression by immunohistochemistry using a validated PD-L1-specific antibody (SP263) in a large and representative cohort of primary prostate cancers and prostate cancer metastases. The study included 539 primary prostate cancers comprising 508 acinar adenocarcinomas, 24 prostatic duct adenocarcinomas, 7 small-cell carcinomas, and a total of 57 cases of mCRPC. PD-L1 positivity was low in primary acinar adenocarcinoma, with only 7.7% of cases showing detectable PD-L1 staining. Increased levels of PD-L1 expression were noted in 42.9% of small-cell carcinomas. In mCRPC, 31.6% of cases showed PD-L1-specific immunoreactivity. In conclusion, in this comprehensive evaluation of PD-L1 expression in prostate cancer, PD-L1 expression is rare in primary prostate cancers, but increased rates of PD-L1 positivity were observed in mCRPC. These results will be important for the future clinical development of programmed cell death protein 1/PD-L1-targeting therapies in prostate cancer. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

JNK3-Mediated Apoptotic Cell Death in Primary Dopaminergic Neurons

PubMed Central

Choi, Won-Seok; Klintworth, Heather M.; Xia, Zhengui

2012-01-01

Investigation of mechanisms responsible for dopaminergic neuron death is critical for understanding the pathogenesis of Parkinson’s disease, yet this is often quite challenging technically. Here, we describe detailed methods for culturing primary mesencephalic dopaminergic neurons and examining the activation of c-Jun N-terminal protein Kinase (JNK) in these cultures. We utilized immunocytochemistry and computerized analysis to quantify the number of surviving dopaminergic neurons and JNK activation in dopaminergic neurons. TUNEL staining was used to quantify apoptotic cell death. siRNA was used to specifically inhibit JNK3, the neural specific isoform of JNK. Our data implicate the activation of JNK3 in rotenone-induced dopaminergic neuron apoptosis. PMID:21815073

Programmed death ligand 1 expression and CD8+ tumor-infiltrating lymphocyte density differences between paired primary and brain metastatic lesions in non-small cell lung cancer.

PubMed

Zhou, Jie; Gong, Zhihua; Jia, Qingzhu; Wu, Yan; Yang, Zhen-Zhou; Zhu, Bo

2018-04-15

Immunotherapy targeting the programmed cell death-1/programmed death ligand 1(PD-L1) pathway has shown promising antitumor activity in brain metastases (BMs) of non-small cell lung cancer (NSCLC) patients with an acceptable safety profile; however, the response rates often differ between primary lesions and intracranial lesions. Studies are necessary to identify detailed characterizations of the response biomarkers. In this study, we aimed to compare the differences of PD-L1 expression and CD8 + tumor-infiltrating lymphocyte (TIL) density, two major response biomarkers of PD-1/PD-L1 blockade, between paired primary and brain metastatic lesions in advanced NSCLC. We observed that among primary lesions or BMs, only a small number of patients harbored common PD-L1 expression on both tumor cells and tumor-infiltrating immune cells. Additionally, we found that the numbers of CD8 + TILs were significantly fewer in BMs than in primary lung cancers. Low stromal CD8 + TIL numbers in BMs were associated with significantly shorter overall survival compared to high stromal CD8 + TIL counts. Notably, we demonstrated a discrepancy in PD-L1 expression and CD8 + TIL density between primary lung cancers and their corresponding BMs. Such heterogeneities are significantly associated with the time at which BMs occurred. Our study emphasizes the spatial and temporal heterogeneity of biomarkers for anti-PD-1/PD-L1 therapy, which should be concerned in clinical practice. Copyright © 2018 Elsevier Inc. All rights reserved.

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis.

PubMed

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans; Nilsson, Gunnar; Ungerstedt, Johanna

2017-02-07

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation.

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis

PubMed Central

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans

2017-01-01

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation. PMID:28038453

Toxicity of nutritionally available selenium compounds in primary and transformed hepatocytes.

PubMed

Weiller, Markus; Latta, Markus; Kresse, Matthias; Lucas, Rudolf; Wendel, Albrecht

2004-09-01

The essential trace element selenium is also toxic at low doses. Since supplementation of selenium is discussed as cancer prophylaxis, we investigated whether or not bioavailable selenium compounds are selectively toxic on malignant cells by comparing primary and transformed liver cells as to the extent and mode of cell death. Sodium selenite and selenate exclusively induced necrosis in a concentration-dependent manner in all cell types investigated. In primary murine hepatocytes, the EC50 was 20 microM for selenite, 270 microM for selenate, and 30 microM for Se-methionine. In the human carcinoma cell line HepG2, the EC50 for selenite was 40 microM, and for selenate 1.1 mM, whereas Se-methionine was essentially non-toxic up to 10 mM. Similar results were found in murine Hepa1-6 cells. Exposure of primary murine cells to selenate or selenite resulted in increased lipid peroxidation. Toxicity was inhibited by superoxide dismutase plus catalase, indicating an important role for reactive oxygen intermediates. In primary hepatocytes, metabolical depletion of intracellular ATP by the ketohexose tagatose, significantly decreased the cytotoxicity of Se-methionine, while the one of selenite was increased. These data do not provide any in vitro evidence that bioavailable selenium compounds induce preferentially apoptotic cell death or selectively kill transformed hepatocytes.

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, inflammatory and cell death signaling pathways in diabetic cardiomyopathy

PubMed Central

Rajesh, Mohanraj; Mukhopadhyay, Partha; Bátkai, Sándor; Patel, Vivek; Saito, Keita; Matsumoto, Shingo; Kashiwaya, Yoshihiro; Horváth, Béla; Mukhopadhyay, Bani; Becker, Lauren; Haskó, György; Liaudet, Lucas; Wink, David A; Veves, Aristidis; Mechoulam, Raphael; Pacher, Pál

2010-01-01

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrosative stress, cell death and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background CBD, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts antiinflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by pressure-volume system. Oxidative stress, cell death and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrosative stress, NF-κB and MAPK (JNK and p-38, p38α) activation, enhanced expression of adhesion molecules (ICAM-1, VCAM-1), TNF-α, markers of fibrosis (TGF-β, CTGF, fibronectin, collagen-1, MMP-2 and MMP-9), enhanced cell death (caspase 3/7 and PARP activity, chromatin fragmentation and TUNEL) and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrosative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, NF-κB activation and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of cannabidiol in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrosative stress, inflammation, cell death and fibrosis. PMID:21144973

Pathophysiology of primary open-angle glaucoma from a neuroinflammatory and neurotoxicity perspective: a review of the literature.

PubMed

Evangelho, Karine; Mogilevskaya, Maria; Losada-Barragan, Monica; Vargas-Sanchez, Jeinny Karina

2017-12-30

Glaucoma is the leading cause of blindness in humans, affecting 2% of the population. This disorder can be classified into various types including primary, secondary, glaucoma with angle closure and with open angle. The prevalence of distinct types of glaucoma differs for each particular region of the world. One of the most common types of this disease is primary open-angle glaucoma (POAG), which is a complex inherited disorder characterized by progressive retinal ganglion cell death, optic nerve head excavation and visual field loss. Nowadays, POAG is considered an optic neuropathy, while intraocular pressure is proposed to play a fundamental role in its pathophysiology and especially in optic disk damage. However, the exact mechanism of optic nerve head damage remains a topic of debate. This literature review aims to bring together the information on the pathophysiology of primary open-angle glaucoma, particularly focusing on neuroinflammatory mechanisms leading to the death of the retinal ganglion cell. A literature search was done on PubMed using key words including primary open-angle glaucoma, retinal ganglion cells, Müller cells, glutamate, glial cells, ischemia, hypoxia, exitotoxicity, neuroinflammation, axotomy and neurotrophic factors. The literature was reviewed to collect the information published about the pathophysiologic mechanisms of RGC death in the POAG, from a neuroinflammatory and neurotoxicity perspective. Proposed mechanisms for glaucomatous damage are a result of pressure in RGC followed by ischemia, hypoxia of the ONH, and consequently death due to glutamate-induced excitotoxicity, deprivation of energy and oxygen, increase in levels of inflammatory mediators and alteration of trophic factors flow. These events lead to blockage of anterograde and retrograde axonal transport with ensuing axotomy and eventually blindness. The damage to ganglion cells and eventually glaucomatous injury can occur via various mechanisms including baric trauma, ischemia and impact of metabolic toxins, which triggers an inflammatory process and secondary degeneration in the ONH.

Epigallocatechin-3-Gallate Suppresses Human Herpesvirus 8 Replication and Induces ROS Leading to Apoptosis and Autophagy in Primary Effusion Lymphoma Cells

PubMed Central

Tsai, Ching-Yi; Chen, Chang-Yu; Chiou, Yee-Hsuan; Shyu, Huey-Wen; Lin, Kuan-Hua; Chou, Miao-Chen; Huang, Mei-Han; Wang, Yi-Fen

2017-01-01

Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has been shown to induce cell death in cancer cells. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by human herpesvirus 8 (HHV8). In this study, we examined the role of EGCG on PEL cells in cell death and HHV8 replication. We performed trypan blue exclusion assay to assess the cell viability of PEL cells, flow cytometry analysis to examine the cell cycle distribution and reactive oxygen species (ROS) generation, caspase-3 activity to assay apoptosis, acridine orange staining to determine autophagy, and immunoblotting to detect the protein levels involved in apoptosis and autophagy as well as mitogen activated protein kinases (MAPKs) activation upon EGCG treatment. The expression of the HHV8 lytic gene was determined by luciferase reporter assay and reverse transcription-PCR, and viral progeny production was determined by PCR. Results revealed that EGCG induced cell death and ROS generation in PEL cells in a dose-dependent manner. N-acetylcysteine (NAC) inhibited the EGCG-induced ROS and rescued the cell from EGCG-induced cell death. Even though EGCG induced ROS generation in PEL cells, it reduced the production of progeny virus from PEL cells without causing HHV8 reactivation. These results suggest that EGCG may represent a novel strategy for the treatment of HHV8 infection and HHV8-associated lymphomas. PMID:29267216

Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent

PubMed Central

van Erp, Anke E.M.; Versleijen-Jonkers, Yvonne M.H.; Hillebrandt-Roeffen, Melissa H.S.; van Houdt, Laurens; Gorris, Mark A.J.; van Dam, Laura S.; Mentzel, Thomas; Weidema, Marije E.; Savci-Heijink, C. Dilara; Desar, Ingrid M.E.; Merks, Hans H.M.; van Noesel, Max M.; Shipley, Janet; van der Graaf, Winette T.A.; Flucke, Uta E.; Meyer-Wentrup, Friederike A.G.

2017-01-01

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma (n = 46), Ewing sarcoma (n = 32), alveolar rhabdomyosarcoma (n = 20), embryonal rhabdomyosarcoma (n = 77), synovial sarcoma (n = 22) and desmoplastic small round cell tumors (DSRCT) (n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described. PMID:29050367

Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent.

PubMed

van Erp, Anke E M; Versleijen-Jonkers, Yvonne M H; Hillebrandt-Roeffen, Melissa H S; van Houdt, Laurens; Gorris, Mark A J; van Dam, Laura S; Mentzel, Thomas; Weidema, Marije E; Savci-Heijink, C Dilara; Desar, Ingrid M E; Merks, Hans H M; van Noesel, Max M; Shipley, Janet; van der Graaf, Winette T A; Flucke, Uta E; Meyer-Wentrup, Friederike A G

2017-09-19

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma ( n = 46), Ewing sarcoma ( n = 32), alveolar rhabdomyosarcoma ( n = 20), embryonal rhabdomyosarcoma ( n = 77), synovial sarcoma ( n = 22) and desmoplastic small round cell tumors (DSRCT) ( n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described.

Targeting MET kinase with the small-molecule inhibitor amuvatinib induces cytotoxicity in primary myeloma cells and cell lines

PubMed Central

2013-01-01

Background MET is a receptor tyrosine kinase that is activated by the ligand HGF and this pathway promotes cell survival, migration, and motility. In accordance with its oncogenic role, MET is constitutively active, mutated, or over-expressed in many cancers. Corollary to its impact, inhibition of MET kinase activity causes reduction of the downstream signaling and demise of cells. In myeloma, a B-cell plasma malignancy, MET is neither mutated nor over-expressed, however, HGF is increased in plasma or serum obtained from myeloma patients and this was associated with poor prognosis. The small-molecule, amuvatinib, inhibits MET receptor tyrosine kinase. Based on this background, we hypothesized that targeting the HGF/MET signaling pathway is a rational approach to myeloma therapy and that myeloma cells would be sensitive to amuvatinib. Methods Expression of MET and HGF mRNAs in normal versus malignant plasma cells was compared during disease progression. Cell death and growth as well as MET signaling pathway were assessed in amuvatinib treated primary myeloma cells and cell lines. Results There was a progressive increase in the transcript levels of HGF (but not MET) from normal plasma cells to refractory malignant plasma cells. Amuvatinib readily inhibited MET phosphorylation in primary CD138+ cells from myeloma patients and in concordance, increased cell death. A 48-hr amuvatinib treatment in high HGF-expressing myeloma cell line, U266, resulted in growth inhibition. Levels of cytotoxicity were time-dependent; at 24, 48, and 72 h, amuvatinib (25 μM) resulted in 28%, 40%, and 55% cell death. Consistent with these data, there was an amuvatinib-mediated decrease in MET phosphorylation in the cell line. Amuvatinib at concentrations of 5, 10, or 25 μM readily inhibited HGF-dependent MET, AKT, ERK and GSK-3-beta phosphorylation. MET-mediated effects were not observed in myeloma cell line that has low MET and/or HGF expression. Conclusions These data suggest that at the cellular level MET/HGF pathway inclines with myeloma disease progression. Amuvatinib, a small molecule MET kinase inhibitor, is effective in inducing growth inhibition and cell death in myeloma cell lines as well as primary malignant plasma cells. These cytostatic and cytotoxic effects were associated with an impact on MET/HGF pathway. PMID:24326130

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

PubMed Central

Hakansson, Anders P.; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-01-01

Background Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells. PMID:21423701

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

PubMed

Hakansson, Anders P; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-03-10

Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

PubMed Central

Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

2017-01-01

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

PubMed

Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

2015-07-28

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Artemisinin conferred ERK mediated neuroprotection to PC12 cells and cortical neurons exposed to sodium nitroprusside-induced oxidative insult.

PubMed

Zheng, Wenhua; Chong, Cheong-Meng; Wang, Haitao; Zhou, Xuanhe; Zhang, Lang; Wang, Rikang; Meng, Qian; Lazarovici, Philip; Fang, Jiankang

2016-08-01

The production of nitric oxide (NO) is one of the primary mediators of ischemic damage, glutamate neurotoxicity and neurodegeneration and therefore inhibition of NO-induced neurotoxicity may be considered a therapeutic target for reducing neuronal cell death (neuroprotection). In this study, artemisinin, a well-known anti-malaria drug was found to suppress sodium nitroprusside (SNP, a nitric oxide donor)-induced cell death in the PC12 cells and brain primary cortical neuronal cultures. Pretreatment of PC12 cells with artemisinin significantly suppressed SNP-induced cell death by decreasing the extent of oxidation, preventing the decline of mitochondrial membrane potential, restoring abnormal changes in nuclear morphology and reducing lactate dehydrogenase release and inhibiting caspase 3/7 activities. Western blotting analysis revealed that artemisinin was able to activate extracellular regulated protein kinases (ERK) pathway. Furthermore, the ERK inhibitor PD98059 blocked the neuroprotective effect of artemisinin whereas the PI3K inhibitor LY294002 had no effect. Cumulatively these findings support the notion that artemisinin confers neuroprotection from SNP-induce neuronal cell death insult, a phenomenon coincidentally related to activation of ERK phosphorylation. This SNP-induced oxidative insult in PC12 cell culture model may be useful to investigate molecular mechanisms of NO-induced neurotoxicity and drug-induced neuroprotection, and to generate novel therapeutic concepts for ischemic disease treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Inhibition of autophagy by chloroquine induces apoptosis in primary effusion lymphoma in vitro and in vivo through induction of endoplasmic reticulum stress.

PubMed

Masud Alam, Md; Kariya, Ryusho; Kawaguchi, Azusa; Matsuda, Kouki; Kudo, Eriko; Okada, Seiji

2016-10-01

Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.

3-bromopyruvate: a new targeted antiglycolytic agent and a promise for cancer therapy.

PubMed

Ganapathy-Kanniappan, S; Vali, M; Kunjithapatham, R; Buijs, M; Syed, L H; Rao, P P; Ota, S; Kwak, B K; Loffroy, R; Geschwind, J F

2010-08-01

The pyruvate analog, 3-bromopyruvate, is an alkylating agent and a potent inhibitor of glycolysis. This antiglycolytic property of 3-bromopyruvate has recently been exploited to target cancer cells, as most tumors depend on glycolysis for their energy requirements. The anticancer effect of 3-bromopyruvate is achieved by depleting intracellular energy (ATP) resulting in tumor cell death. In this review, we will discuss the principal mechanism of action and primary targets of 3-bromopyruvate, and report the impressive antitumor effects of 3-bromopyruvate in multiple animal tumor models. We describe that the primary mechanism of 3-bromopyruvate is via preferential alkylation of GAPDH and that 3-bromopyruvate mediated cell death is linked to generation of free radicals. Research in our laboratory also revealed that 3-bromopyruvate induces endoplasmic reticulum stress, inhibits global protein synthesis further contributing to cancer cell death. Therefore, these and other studies reveal the tremendous potential of 3-bromopyruvate as an anticancer agent.

Aprotinin, but not ε-aminocaproic acid and tranexamic acid, exerts neuroprotection against excitotoxic injury in an in vitro neuronal cell culture model.

PubMed

Lu, Zhaohui; Korotcova, Ludmila; Murata, Akira; Ishibashi, Nobuyuki; Jonas, Richard A

2014-06-01

Lack of availability of aprotinin has resulted in increased clinical use of the alternative antifibrinolytic agents, ε-aminocaproic acid (EACA) and tranexamic acid (TXA), which are known to be associated with an increased risk of seizures. In contrast, aprotinin has previously been demonstrated to be neuroprotective through suppression of excitotoxicity-mediated neuronal degeneration via the extracellular plasminogen/plasmin system. This study compares the effect of antifibrinolytic agents on neuronal and mixed glial/neuronal cell cultures. Mixed cortical cultures containing neuronal and glial cells were prepared from fetal mice and plated on a layer of confluent astrocytes from postnatal pups. A primary neuronal culture was obtained from the same gestational stage and plated in multiwall vessels. Slowly triggered excitotoxicity was induced by 24-hour exposure to 12.5 mM N-methyl-D-aspartate (NMDA). Apoptotic neuronal cell death was induced by exposure of primary neural cultures to 24 hours of serum deprivation. Compared with NMDA alone, no significant changes in cell death were observed for any dose of TXA or EACA in mixed cultures. Conversely, a clinical dose of aprotinin significantly reduced cell death by -31% on average. Aprotinin reduced apoptotic neuronal cell death from 75% to 37.3%, and to 34.1% at concentrations of 100 and 200 kIU/mL, respectively, and significantly decreased neuronal nuclear damage. These concentrations of aprotinin significantly inhibited caspase 9 and 3/7 activations; 250 kIU/mL aprotinin exerted maximal protection on primary cortical neurons. In contrast to aprotinin, EACA and TXA exert no protective effect against excitotoxic neuronal injury that can occur during cardiac surgery. Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

Aprotinin, but not epsilon aminocaproic acid and tranexamic acid, exerts neuroprotection against excitotoxic injury in an in vitro neuronal cell culture model

PubMed Central

Lu, Zhaohui; Korotcova, Ludmila; Murata, Akira; Ishibashi, Nobuyuki; Jonas, Richard A.

2013-01-01

Objective Lack of availability of aprotinin has resulted in increased clinical use of the alternative antifibrinolytic agents epsilon aminocaproic acid (EACA) and tranexamic acid (TXA) which are known to be associated with an increased risk of seizures. In contrast aprotinin has previously been demonstrated to be neuroprotective through suppression of excitotoxicity-mediated neuronal degeneration via the extracellular plasminogen/plasmin system. We compared the impact of antifibrinolytic agents on neuronal and mixed glial/neuronal cell cultures. Methods Mixed cortical cultures containing neuronal and glial cells were prepared from fetal mice and plated on a layer of confluent astrocytes from postnatal pups. Primary neuronal culture was obtained from the same gestational stage and plated in multiwall vessels. Slowly triggered excitotoxicity was induced by 24-hour exposure to 12.5 mM N-methyl-D-aspartate (NMDA). Apoptotic neuronal cell death was induced by exposure of primary neural cultures to 24 hours of serum deprivation. Results Compared to NMDA alone, no significant changes in cell death were observed for any dose of TXA or EACA in mixed cultures. Conversely, a clinical dose of aprotinin significantly reduced cell death by -31% on average. Aprotinin reduced apoptotic neuronal cell death from 75% to 37.3%, and 34.1% at concentrations of 100 and 200 KIU/mL, and significantly decreased neuronal nuclear damage. These concentrations of aprotinin significantly inhibited caspase 9 and 3/7 activations. 250 KIU/ml aprotinin exerted maximal protection on primary cortical neurons. Conclusions In contrast to aprotinin, EACA and TXA exert no protective effect against excitotoxic neuronal injury that can occur during cardiac surgery. PMID:24237885

Loss of Atrx Sensitizes Cells to DNA Damaging Agents through p53-Mediated Death Pathways

PubMed Central

Conte, Damiano; Huh, Michael; Goodall, Emma; Delorme, Marilyne; Parks, Robin J.; Picketts, David J.

2012-01-01

Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrx f/f mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres) were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors. PMID:23284920

Loss of Atrx sensitizes cells to DNA damaging agents through p53-mediated death pathways.

PubMed

Conte, Damiano; Huh, Michael; Goodall, Emma; Delorme, Marilyne; Parks, Robin J; Picketts, David J

2012-01-01

Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrx(f/f) mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS) activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU). Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres) were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors.

Combating rituximab resistance by inducing ceramide/lysosome-involved cell death through initiation of CD20-TNFR1 co-localization

PubMed Central

Zhang, Fan; Yang, Junlan; Li, Huafei; Liu, Moyan; Zhang, Jie; Zhao, Lichao; Wang, Lingxiong; LingHu, RuiXia; Feng, Fan; Gao, Xudong; Dong, Biqin; Liu, Xiaohan; Zi, Jian; Zhang, Weijing; Hu, Yi; Pan, Jingkun; Tian, Lei; Hu, Yazuo; Han, Zhitao; Zhang, Honghong; Wang, Xiaoning; Zhao, Lei

2016-01-01

ABSTRACT Despite the success of CD20 antibody rituximab in immunotherapy, acquired resistance is one of the prime obstacles for the successful treatment of B-cell malignancies. There is an urgent need to intensify efforts against resistance in cancer treatment. Growing evidence indicated that lysosomes may form an “Achilles heel” for cancer cells by sensitizing them to death pathways. Here, we uncover an important role of CD20 in initiation of ceramide/lysosomal membrane permeabilization (LMP)-mediated cell death, showing that colocalization of CD20-TNFR1 after type II CD20 antibody ligation can stimulate de novo ceramide synthesis by ceramide synthase and consequently induce remarkable lysosomal permeabilization (LMP) and lysosome-mediated cell death. Further studies show that the potent lysosome-mediated cell death induced by CD20 antibodies exhibits a profound killing effect against both rituximab-sensitive and -resistant (RR) lymphoma. Furthermore, engineering of rituximab by introducing a point mutation endows it with the ability to induce potent ceramide/LMP-mediated cell death in both RR lymphoma and primary B-cell malignancies from patients with rituximab-refractory, suggesting the potential clinical application to combat rituximab resistance. PMID:27467962

Oxidant-Induced Cell Death and Nrf2-Dependent Antioxidative Response Are Controlled by Fra-1/AP-1

PubMed Central

Vaz, Michelle; Machireddy, Narsa; Irving, Ashley; Potteti, Haranatha R.; Chevalier, Karinne; Kalvakolanu, Dhananjaya

2012-01-01

AP-1 (Jun/Fos) transcription factors play key roles in various biological processes, including cell death. Here we report a novel role for Fra-1 in oxidant-induced cell death controlled by modulating antioxidant gene expression. Fra-1-deficient (Fra-1Δ/Δ) mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts (PLFs) were remarkably resistant to H2O2- and diquat-induced cell death, compared to their wild-type (Fra-1+/+) counterparts. Fra-1 deficiency ablated oxidant-induced mitochondrion-dependent apoptosis. Fra-1Δ/Δ cells had elevated basal levels of antioxidant enzymes and intracellular glutathione (GSH), which were further stimulated by oxidants. Loss of Fra-1 led to an increased half-life of transcription factor Nrf2 and increased recruitment of this protein to the promoters of antioxidant genes and increased their expression. Depletion of intracellular GSH or RNA interference (RNAi)-mediated knockdown of Nqo1, Hmox1, and Nrf2 restored oxidant-induced cell death in Fra-1Δ/Δ cells. Thus, Fra-1 appears to increase susceptibility to oxidants and promotes cell death by attenuating Nrf2-driven antioxidant responses. PMID:22393254

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

PubMed Central

Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

2014-01-01

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS).We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. PMID:25034532

Cell death in the pathogenesis of systemic lupus erythematosus and lupus nephritis.

PubMed

Mistry, Pragnesh; Kaplan, Mariana J

2017-12-01

Nephritis is one of the most severe complications of systemic lupus erythematosus (SLE). One key characteristic of lupus nephritis (LN) is the deposition of immune complexes containing nucleic acids and/or proteins binding to nucleic acids and autoantibodies recognizing these molecules. A variety of cell death processes are implicated in the generation and externalization of modified nuclear autoantigens and in the development of LN. Among these processes, apoptosis, primary and secondary necrosis, NETosis, necroptosis, pyroptosis, and autophagy have been proposed to play roles in tissue damage and immune dysregulation. Cell death occurs in healthy individuals during conditions of homeostasis yet autoimmunity does not develop, at least in part, because of rapid clearance of dying cells. In SLE, accelerated cell death combined with a clearance deficiency may lead to the accumulation and externalization of nuclear autoantigens and to autoantibody production. In addition, specific types of cell death may modify autoantigens and alter their immunogenicity. These modified molecules may then become novel targets of the immune system and promote autoimmune responses in predisposed hosts. In this review, we examine various cell death pathways and discuss how enhanced cell death, impaired clearance, and post-translational modifications of proteins could contribute to the development of lupus nephritis. Published by Elsevier Inc.

Switch-Hitting Immune Cells: From Tumor Protection to Metastasis Promotion | Center for Cancer Research

Cancer.gov

The leading cause of death from cancer is not a primary tumor but is the metastases, or invasion of tumor cells into other locations in the body, that result from it. A complex and incompletely understood process, metastatic tumor formation is thought to require several steps in which tumor cells invade the tissue surrounding the primary tumor, enter local blood vessels,

Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells.

PubMed

Kim, Seok-Hyun; Kim, Kunhong; Kwagh, Jae G; Dicker, David T; Herlyn, Meenhard; Rustgi, Anil K; Chen, Youhai; El-Deiry, Wafik S

2004-09-17

The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials.

Dying dangerously: Necrotic cell death and chronic inflammation promote tumor growth.

PubMed

Lotze, Michael T; Demarco, Richard A

2004-12-01

Extract: We all shudder about untimely deaths or those that we were not prepared for. As such we perceive such "unscheduled" deaths as dangerous. Similarly, apoptotic death (literally falling leaves) or the programmed cell death of cells in multicellular organisms ranging from slime mold and simple worms through to mammals, has a level of tidiness and well-orchestrated activities with literally hundreds if not thousands of gene products employed with either the primary or secondary purpose of coordinating the orderly death of cells throughout life. During inflammation of any sort, driven by tissue damage or injury or infection by pathogens (virus, bacteria, and parasites), apoptotic death similarly serves to quickly rid the host of damaged cells, promote removal and digestion of the infected cell, and prepare the way for tissue remodeling and repair. When this goes awry, for example during periods of chronic inflammation, tissues are subjected to the contrasting needs of driving apoptotic death whilst maintaining the barrier function of the epithelia (such as skin cells) as well as the selective permeability of mucosal sites (i.e., areas where mucus is secreted to protect the cells from their surroundings, such as gut cells protecting themselves from the gastric acids). Prudently, they need to limit and husband local resources sufficiently for the maintenance of tissue integrity and renewal. It is our provocative and novel contention that cancer in adults (and not children) most often arises in a setting of chronic inflammation and disordered cell death rather than one associated primarily with disordered cell growth as it is popularly imagined by scientists, clinicians, and the general public.

Influence of Berry-Polyphenols on Receptor Signaling and Cell-Death Pathways: Implications for Breast Cancer Prevention

PubMed Central

Aiyer, Harini S; Warri, Anni M; Woode, Denzel R; Hilakivi-Clarke, Leena; Clarke, Robert

2012-01-01

Breast cancer is the most commonly diagnosed cancer among women worldwide. Many women have become more aware of the benefits of increasing fruit consumption, as part of a healthy lifestyle, for the prevention of cancer. The mechanisms by which fruits, including berries, prevent breast cancer can be partially explained by exploring their interactions with pathways known influence cell-proliferation and evasion of cell-death. Two receptor pathways- estrogen receptor (ER) and tyrosine kinase receptors, especially the epidermal growth factor receptor (EGFR) family- are drivers of cell-proliferation and play a significant role in the development of both primary and recurrent breast cancer. There is strong evidence to show that several phytochemicals present in berries such as cyanidin, delphinidin, quercetin, kaempferol, ellagic acid, resveratrol and pterostilbene, interact with and alter the effects of these pathways. Further, they also induce cell death (apoptosis and autophagy) via their influence on kinase signaling. In this review, we summarize in vitro data regarding the interaction of berry polyphenols with the specific receptors and the mechanisms by which they induce cell death. Further, we also present in vivo data of primary breast cancer prevention by individual compounds and whole berries. Finally, we present a possible role for berries and berry compounds in the prevention of breast cancer and our perspective on the areas that require further research. PMID:22300613

Human GAPDH Is a Target of Aspirin’s Primary Metabolite Salicylic Acid and Its Derivatives

PubMed Central

Manohar, Murli; Harraz, Maged M.; Park, Sang-Wook; Schroeder, Frank C.; Snyder, Solomon H.; Klessig, Daniel F.

2015-01-01

The plant hormone salicylic acid (SA) controls several physiological processes and is a key regulator of multiple levels of plant immunity. To decipher the mechanisms through which SA’s multiple physiological effects are mediated, particularly in immunity, two high-throughput screens were developed to identify SA-binding proteins (SABPs). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) from plants (Arabidopsis thaliana) was identified in these screens. Similar screens and subsequent analyses using SA analogs, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology, established that human GAPDH (HsGAPDH) also binds SA. In addition to its central role in glycolysis, HsGAPDH participates in several pathological processes, including viral replication and neuronal cell death. The anti-Parkinson’s drug deprenyl has been shown to suppress nuclear translocation of HsGAPDH, an early step in cell death and the resulting cell death induced by the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine. Here, we demonstrate that SA, which is the primary metabolite of aspirin (acetyl SA) and is likely responsible for many of its pharmacological effects, also suppresses nuclear translocation of HsGAPDH and cell death. Analysis of two synthetic SA derivatives and two classes of compounds from the Chinese medicinal herb Glycyrrhiza foetida (licorice), glycyrrhizin and the SA-derivatives amorfrutins, revealed that they not only appear to bind HsGAPDH more tightly than SA, but also exhibit a greater ability to suppress translocation of HsGAPDH to the nucleus and cell death. PMID:26606248

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells.

PubMed

Autheman, Delphine; Wyder, Marianne; Popoff, Michel; D'Herde, Katharina; Christen, Stephan; Posthaus, Horst

2013-01-01

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis").

Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia.

PubMed

Chen, Yongqiang; Henson, Elizabeth S; Xiao, Wenyan; Huang, Daniel; McMillan-Ward, Eileen M; Israels, Sara J; Gibson, Spencer B

2016-06-02

Autophagy is an intracellular lysosomal degradation pathway where its primary function is to allow cells to survive under stressful conditions. Autophagy is, however, a double-edge sword that can either promote cell survival or cell death. In cancer, hypoxic regions contribute to poor prognosis due to the ability of cancer cells to adapt to hypoxia in part through autophagy. In contrast, autophagy could contribute to hypoxia induced cell death in cancer cells. In this study, we showed that autophagy increased during hypoxia. At 4 h of hypoxia, autophagy promoted cell survival whereas, after 48 h of hypoxia, autophagy increased cell death. Furthermore, we found that the tyrosine phosphorylation of EGFR (epidermal growth factor receptor) decreased after 16 h in hypoxia. Furthermore, EGFR binding to BECN1 in hypoxia was significantly higher at 4 h compared to 72 h. Knocking down or inhibiting EGFR resulted in an increase in autophagy contributing to increased cell death under hypoxia. In contrast, when EGFR was reactivated by the addition of EGF, the level of autophagy was reduced which led to decreased cell death. Hypoxia led to autophagic degradation of the lipid raft protein CAV1 (caveolin 1) that is known to bind and activate EGFR in a ligand-independent manner during hypoxia. By knocking down CAV1, the amount of EGFR phosphorylation was decreased in hypoxia and amount of autophagy and cell death increased. This indicates that the activation of EGFR plays a critical role in the switch between cell survival and cell death induced by autophagy in hypoxia.

mTOR inhibition sensitizes ONC201-induced anti-colorectal cancer cell activity.

PubMed

Jin, Zhe-Zhu; Wang, Wei; Fang, Di-Long; Jin, Yong-Jun

2016-09-30

We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Is Pyruvylated during 3-Bromopyruvate Mediated Cancer Cell Death

PubMed Central

Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H.; Kunjithapatham, Rani; Buijs, Manon; Vossen, Josephina A.; Tchernyshyov, Irina; Cole, Robert N.; Syed, Labiq H.; Rao, Pramod P.; Ota, Shinichi; Vali, Mustafa

2013-01-01

Background The pyruvic acid analog 3-bromopyruvate (3BrPA) is an alkylating agent known to induce cancer cell death by blocking glycolysis. The anti-glycolytic effect of 3BrPA is considered to be the inactivation of glycolytic enzymes. Yet, there is a lack of experimental documentation on the direct interaction of 3BrPA with any of the suggested targets during its anticancer effect. Methods and Results In the current study, using radiolabeled (14C) 3BrPA in multiple cancer cell lines, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the primary intracellular target of 3BrPA, based on two-dimensional (2D) gel electrophoretic autoradiography, mass spectrometry and immunoprecipitation. Furthermore, in vitro enzyme kinetic studies established that 3BrPA has marked affinity to GAPDH. Finally, Annexin V staining and active caspase-3 immunoblotting demonstrated that apoptosis was induced by 3BrPA. Conclusion GAPDH pyruvylation by 3BrPA affects its enzymatic function and is the primary intracellular target in 3BrPA mediated cancer cell death. PMID:20044597

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis

PubMed Central

Zille, Marietta; Karuppagounder, Saravanan S.; Chen, Yingxin; Gough, Peter J.; Phil, D.; Bertin, John; Finger, Joshua; Milner, Teresa A.; Jonas, Elizabeth A.; Ratan, Rajiv R.

2017-01-01

Background and Purpose Intracerebral hemorrhage (ICH) leads to disability or death with few established treatments. Adverse outcomes following ICH result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. Methods We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were employed. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. Results Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased following ICH in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by greater than 80% and had similar therapeutic windows in vitro. Conclusion Experimental ICH shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either one of these pathways can bring cells below that threshold to survival. PMID:28250197

Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

PubMed

Zhang, Li-Min; Zhao, Xiao-Chun; Sun, Wen-Bo; Li, Rui; Jiang, Xiao-Jing

2015-10-15

Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (P<0.05); increased cell death (P<0.05); decreased mitochondrial membrane potential (P<0.05); and decreased Bid, Bim, Puma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2. Copyright © 2015 Elsevier B.V. All rights reserved.

Pathogenicity of Shigella in chickens.

PubMed

Shi, Run; Yang, Xia; Chen, Lu; Chang, Hong-tao; Liu, Hong-ying; Zhao, Jun; Wang, Xin-wei; Wang, Chuan-qing

2014-01-01

Shigellosis in chickens was first reported in 2004. This study aimed to determine the pathogenicity of Shigella in chickens and the possibility of cross-infection between humans and chickens. The pathogenicity of Shigella in chickens was examined via infection of three-day-old SPF chickens with Shigella strain ZD02 isolated from a human patient. The virulence and invasiveness were examined by infection of the chicken intestines and primary chicken intestinal epithelial cells. The results showed Shigella can cause death via intraperitoneal injection in SPF chickens, but only induce depression via crop injection. Immunohistochemistry and transmission electron microscopy revealed the Shigella can invade the intestinal epithelia. Immunohistochemistry of the primary chicken intestinal epithelial cells infected with Shigella showed the bacteria were internalized into the epithelial cells. Electron microscopy also confirmed that Shigella invaded primary chicken intestinal epithelia and was encapsulated by phagosome-like membranes. Our data demonstrate that Shigella can invade primary chicken intestinal epithelial cells in vitro and chicken intestinal mucosa in vivo, resulting in pathogenicity and even death. The findings suggest Shigella isolated from human or chicken share similar pathogenicity as well as the possibility of human-poultry cross-infection, which is of public health significance.

Pathogenicity of Shigella in Chickens

PubMed Central

Chen, Lu; Chang, Hong-tao; Liu, Hong-ying; Zhao, Jun; Wang, Xin-wei; Wang, Chuan-qing

2014-01-01

Shigellosis in chickens was first reported in 2004. This study aimed to determine the pathogenicity of Shigella in chickens and the possibility of cross-infection between humans and chickens. The pathogenicity of Shigella in chickens was examined via infection of three-day-old SPF chickens with Shigella strain ZD02 isolated from a human patient. The virulence and invasiveness were examined by infection of the chicken intestines and primary chicken intestinal epithelial cells. The results showed Shigella can cause death via intraperitoneal injection in SPF chickens, but only induce depression via crop injection. Immunohistochemistry and transmission electron microscopy revealed the Shigella can invade the intestinal epithelia. Immunohistochemistry of the primary chicken intestinal epithelial cells infected with Shigella showed the bacteria were internalized into the epithelial cells. Electron microscopy also confirmed that Shigella invaded primary chicken intestinal epithelia and was encapsulated by phagosome-like membranes. Our data demonstrate that Shigella can invade primary chicken intestinal epithelial cells in vitro and chicken intestinal mucosa in vivo, resulting in pathogenicity and even death. The findings suggest Shigella isolated from human or chicken share similar pathogenicity as well as the possibility of human-poultry cross-infection, which is of public health significance. PMID:24949637

Drug priming enhances radiosensitivity of adamantinomatous craniopharyngioma via downregulation of survivin.

PubMed

Stache, Christina; Bils, Christiane; Fahlbusch, Rudolf; Flitsch, Jörg; Buchfelder, Michael; Stefanits, Harald; Czech, Thomas; Gaipl, Udo; Frey, Benjamin; Buslei, Rolf; Hölsken, Annett

2016-12-01

OBJECTIVE In this study, the authors investigated the underlying mechanisms responsible for high tumor recurrence rates of adamantinomatous craniopharyngioma (ACP) after radiotherapy and developed new targeted treatment protocols to minimize recurrence. ACPs are characterized by the activation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR), known to mediate radioresistance in various tumor entities. The impact of tyrosine kinase inhibitors (TKIs) gefitinib or CUDC-101 on radiation-induced cell death and associated regulation of survivin gene expression was evaluated. METHODS The hypothesis that activated EGFR promotes radioresistance in ACP was investigated in vitro using human primary cell cultures of ACP (n = 10). The effects of radiation (12 Gy) and combined radiochemotherapy on radiosensitivity were assessed via cell death analysis using flow cytometry. Changes in target gene expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Survivin, identified in qRT-PCR to be involved in radioresistance of ACP, was manipulated by small interfering RNA (siRNA), followed by proliferation and vitality assays to further clarify its role in ACP biology. Immunohistochemically, survivin expression was assessed in patient tumors used for primary cell cultures. RESULTS In primary human ACP cultures, activation of EGFR resulted in significantly reduced cell death levels after radiotherapy. Treatment with TKIs alone and in combination with radiotherapy increased cell death response remarkably, assessed by flow cytometry. CUDC-101 was significantly more effective than gefitinib. The authors identified regulation of survivin expression after therapeutic intervention as the underlying molecular mechanism of radioresistance in ACP. EGFR activation promoting ACP cell survival and proliferation in vitro is consistent with enhanced survivin gene expression shown by qRT-PCR. TKI treatment, as well as the combination with radiotherapy, reduced survivin levels in vitro. Accordingly, ACP showed reduced cell viability and proliferation after survivin downregulation by siRNA. CONCLUSIONS These results indicate an impact of EGFR signaling on radioresistance in ACP. Inhibition of EGFR activity by means of TKI treatment acts as a radiosensitizer on ACP tumor cells, leading to increased cell death. Additionally, the results emphasize the antiapoptotic and pro-proliferative role of survivin in ACP biology and its regulation by EGFR signaling. The suppression of survivin by treatment with TKI and combined radiotherapy represents a new promising treatment strategy that will be further assessed in in vivo models of ACP.

Intracranial AAV-sTRAIL combined with lanatoside C prolongs survival in an orthotopic xenograft mouse model of invasive glioblastoma.

PubMed

Crommentuijn, Matheus H W; Maguire, Casey A; Niers, Johanna M; Vandertop, W Peter; Badr, Christian E; Würdinger, Thomas; Tannous, Bakhos A

2016-04-01

Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of secreted, soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) to GBM tumors in mice and combined it with the TRAIL-sensitizing cardiac glycoside, lanatoside C (lan C). We applied this combined therapy to two different GBM models using human U87 glioma cells and primary patient-derived GBM neural spheres in culture and in orthotopic GBM xenograft models in mice. In U87 cells, conditioned medium from AAV2-sTRAIL expressing cells combined with lan C induced 80% cell death. Similarly, lan C sensitized primary GBM spheres to sTRAIL causing over 90% cell death. In mice bearing intracranial U87 tumors treated with AAVrh.8-sTRAIL, administration of lan C caused a decrease in tumor-associated Fluc signal, while tumor size increased within days of stopping the treatment. Another round of lan C treatment re-sensitized GBM tumor to sTRAIL-induced cell death. AAVrh.8-sTRAIL treatment alone and combined with lanatoside C resulted in a significant decrease in tumor growth and longer survival of mice bearing orthotopic invasive GBM brain tumors. In summary, AAV-sTRAIL combined with lanatoside C induced cell death in U87 glioma cells and patient-derived GBM neural spheres in culture and in vivo leading to an increased in overall mice survival. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rawal, Nina; Corti, Olga; CNRS, UMR 7225, Paris

Parkinson's disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates {beta}-catenin protein levels in vivo. Stabilization of {beta}-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neuronsmore » in parkin null animals, suggesting that both increased stabilization and decreased degradation of {beta}-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and {beta}-catenin-induced cell death.« less

Apoptosis-Inducing-Factor-Dependent Mitochondrial Function Is Required for T Cell but Not B Cell Function.

PubMed

Milasta, Sandra; Dillon, Christopher P; Sturm, Oliver E; Verbist, Katherine C; Brewer, Taylor L; Quarato, Giovanni; Brown, Scott A; Frase, Sharon; Janke, Laura J; Perry, S Scott; Thomas, Paul G; Green, Douglas R

2016-01-19

The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells. Copyright © 2016 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Yuchao; McGill, Mitchell R.; Du, Kuo

3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT)more » release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.« less

Effect of Immunosuppressive Agents on Hepatocyte Apoptosis Post-Liver Transplantation

PubMed Central

Lim, Eu Jin; Chin, Ruth; Nachbur, Ueli; Silke, John; Jia, Zhiyuan; Angus, Peter W.; Torresi, Joseph

2015-01-01

Introduction Immunosuppressants are used ubiquitously post-liver transplantation to prevent allograft rejection. However their effects on hepatocytes are unknown. Experimental data from non-liver cells indicate that immunosuppressants may promote cell death thereby driving an inflammatory response that promotes fibrosis and raises concerns that a similar effect may occur within the liver. We evaluated apoptosis within the liver tissue of post-liver transplant patients and correlated these findings with in vitro experiments investigating the effects of immunosuppressants on apoptosis in primary hepatocytes. Methods Hepatocyte apoptosis was assessed using immunohistochemistry for M30 CytoDEATH and cleaved PARP in human liver tissue. Primary mouse hepatocytes were treated with various combinations of cyclosporine, tacrolimus, sirolimus, or MMF. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved PARP and cleaved caspase 3. Results Post-liver transplant patients had a 4.9-fold and 1.7-fold increase in M30 CytoDEATH and cleaved PARP compared to normal subjects. Cyclosporine and tacrolimus at therapeutic concentrations did not affect hepatocyte apoptosis, however when they were combined with MMF, cell death was significantly enhanced. Cell viability was reduced by 46% and 41%, cleaved PARP was increased 2.6-fold and 2.2-fold, and cleaved caspase 3 increased 2.2-fold and 1.8-fold following treatment with Cyclosporine/MMF and Tacrolimus/MMF respectively. By contrast, the sirolimus/MMF combination did not significantly reduce hepatocyte viability or promote apoptosis. Conclusion Commonly used immunosuppressive drug regimens employed after liver transplantation enhance hepatocyte cell death and may thus contribute to the increased liver fibrosis that occurs in a proportion of liver transplant recipients. PMID:26390404

Long-term treatment of anterior pituitary cells with nitric oxide induces programmed cell death.

PubMed

Velardez, Miguel Omar; Poliandri, Ariel Hernán; Cabilla, Jimena Paula; Bodo, Cristian Carlos Armando; Machiavelli, Leticia Inés; Duvilanski, Beatriz Haydeé

2004-04-01

Nitric oxide (NO) plays a complex role in modulating programmed cell death. It can either protect the cell from apoptotic death or mediate apoptosis, depending on its concentration and the cell type and/or status. In this study, we demonstrate that long-term exposition to NO induces cell death of anterior pituitary cells from Wistar female rats. DETA NONOate (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, 1 mm], a NO donor that releases NO for an extended period of time, decreased cellular viability and prolactin release from primary cultures of anterior pituitary cells. Morphological studies showed an increase in the number of cells with chromatin condensation and nuclear fragmentation at 24 and 48 h after DETA/NO exposure. DNA internucleosomal fragmentation was also observed at the same time. Reversibility of the NO effect on cellular viability and prolactin release was observed only when the cells were incubated with DETA/NO for less than 6 h. Most apoptotic cells were immunopositive for prolactin, suggesting a high susceptibility of lactotrophs to the effect of NO. The cytotoxic effect of NO is dependent of caspase-9 and caspase-3, but seems to be independent of oxidative stress or nitrosative stress. Our results show that the exposition of anterior pituitary cells to NO for long periods induces programmed cell death of anterior pituitary cells.

Reagents that block neuronal death from Huntington's disease also curb oxidative stress.

PubMed

Valencia, Antonio; Sapp, Ellen; Reeves, Patrick B; Alexander, Jonathan; Masso, Nicholas; Li, Xueyi; Kegel, Kimberly B; DiFiglia, Marian

2012-01-04

Patients with Huntington's disease suffer severe neuronal loss and signs of oxidative damage in the brain. Previously we found that primary neurons from embryonic cortex of mice bearing the Huntington's disease mutation (140 glutamines inserted into exon 1 of huntingtin) showed higher levels of reactive oxygen species before cell death. Here, we treated mutant neurons with known neuroprotective agents and determined the effects on neuronal survival and levels of reactive oxygen species. Primary neurons were exposed to the neurotrophin, brain derived neurotrophic factor, the antioxidant N-acetyl-cysteine or a specific inhibitor of glycogen synthase kinase 3-β, SB216763. Each reagent increased the survival of the mutant neurons compared with untreated mutant neurons and also reduced the levels of reactive oxygen species to levels of wild-type neurons. These results suggest that reducing the levels of reactive oxygen species may be necessary to protect neurons with the Huntington's disease mutation from cell death.

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase.

PubMed

Kim, H; You, S; Kong, B W; Foster, L K; Farris, J; Foster, D N

2001-08-22

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.

Failure in generating hemopoietic stem cells is the primary cause of death from cytomegalovirus disease in the immunocompromised host

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mutter, W.; Reddehase, M.J.; Busch, F.W.

1988-05-01

We have shown in a murine model system for cytomegalovirus (CMV) disease in the immunocompromised host that CMV infection interferes with the earliest detectable step in hemopoiesis, the generation of the stem cell CFU-S-I, and thereby prevents the autoreconstitution of bone marrow after sublethal irradiation. The antihemopoietic effect could not be ascribed to a direct infection of stem cells. The failure in hemopoiesis was prevented by adoptive transfer of antiviral CD8+ T lymphocytes and could be overcome by syngeneic bone marrow transplantation. CD8+ T lymphocytes and bone marrow cells both mediated survival, although only CD8+ T lymphocytes were able tomore » limit virus multiplication in host tissues. We concluded that not the cytopathic effect of virus replication in host tissues, but the failure in hemopoiesis, is the primary cause of death in murine CMV disease.« less

Programmed Cell Death Ligand 1 Expression in Primary Central Nervous System Lymphomas: A Clinicopathological Study.

PubMed

Hayano, Azusa; Komohara, Yoshihiro; Takashima, Yasuo; Takeya, Hiroto; Homma, Jumpei; Fukai, Junya; Iwadate, Yasuo; Kajiwara, Koji; Ishizawa, Shin; Hondoh, Hiroaki; Yamanaka, Ryuya

2017-10-01

Programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) have been shown to predict response to PD-L1/PD-1-targeted therapy. We analyzed PD-L1 expression in primary central nervous system lymphomas (PCNSLs). PD-L1 protein and mRNA expression were evaluated in 64 PCNSL tissue samples. IFN-γ, IL-10, CD4, and CD8 mRNA expression was also evaluated. PD-L1 protein was detected in tumor cells in 2 (4.1%) cases and in tumor microenvironments in 25 (52%) cases. PD-L1 mRNA positively correlated with IFN-γ (p=0.0024) and CD4 (p=0.0005) mRNA expression. IFN-γ mRNA positively correlated with CD8 mRNA expression (p=0.0001). Furthermore, tumor cell PD-L1 expression correlated positively with overall survival (p=0.0177), whereas microenvironmental PD-L1 expression exhibited an insignificant negative trend with overall survival (p=0.188). PD-L1 was expressed on both tumor and/or tumor-infiltrating immune cells in PCNSL. The biological roles of this marker warrant further investigation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Inhibition of HOX/PBX dimer formation leads to necroptosis in acute myeloid leukemia cells.

PubMed

Alharbi, Raed A; Pandha, Hardev S; Simpson, Guy R; Pettengell, Ruth; Poterlowicz, Krzysztof; Thompson, Alexander; Harrington, Kevin; El-Tanani, Mohamed; Morgan, Richard

2017-10-27

The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9 ), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5 , HOXB2 , HOXB4 , HOXB9 , and HOXC9 , but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone.

Inhibition of HOX/PBX dimer formation leads to necroptosis in acute myeloid leukemia cells

PubMed Central

Alharbi, Raed A.; Pandha, Hardev S.; Simpson, Guy R.; Pettengell, Ruth; Poterlowicz, Krzysztof; Thompson, Alexander; Harrington, Kevin; El-Tanani, Mohamed; Morgan, Richard

2017-01-01

The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5, HOXB2, HOXB4, HOXB9, and HOXC9, but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone. PMID:29163771

Cell-based optical assay for amyloid β-induced neuronal cell dysfunction using femtosecond-pulsed laser

NASA Astrophysics Data System (ADS)

Lee, Seunghee; Yoon, Jonghee; Choi, Chulhee

2015-03-01

Amyloid β-protein (Aβ) is known as a key molecule related to the pathogenesis of Alzheimer's disease (AD). Over time, the amyloid cascade disrupts essential function of mitochondria including Ca2+ homeostasis and reactive oxygen species (ROS) regulation, and eventually leads to neuronal cell death. However, there have been no methods that analyze and measure neuronal dysfuction in pathologic conditions quantitatively. Here, we suggest a cell-based optical assay to investigate neuronal function in AD using femtosecond-pulsed laser stimulation. We observed that laser stimulation on primary rat hippocampal neurons for a few microseconds induced intracellular Ca2+ level increases or produced intracellular ROS which was a primary cause of neuronal cell death depending on delivered energy. Although Aβ treatment alone had little effect on the neuronal morphologies and networks in a few hours, Aβ-treated neurons showed delayed Ca2+ increasing pattern and were more vulnerable to laser-induced cell death compared to normal neurons. Our results collectively indicate that femtosecond laser stimulation can be a useful tool to study neuronal dysfuction related to AD pathologies. We anticipate this optical method to enable studies in the early progression of neuronal impairments and the quantitative evaluation of drug effects on neurons in neurodegenerative diseases, including AD and Parkinson's disease in a preclinical study.

Impact of the p53 status of tumor cells on extrinsic and intrinsic apoptosis signaling.

PubMed

Wachter, Franziska; Grunert, Michaela; Blaj, Cristina; Weinstock, David M; Jeremias, Irmela; Ehrhardt, Harald

2013-04-17

The p53 protein is the best studied target in human cancer. For decades, p53 has been believed to act mainly as a tumor suppressor and by transcriptional regulation. Only recently, the complex and diverse function of p53 has attracted more attention. Using several molecular approaches, we studied the impact of different p53 variants on extrinsic and intrinsic apoptosis signaling. We reproduced the previously published results within intrinsic apoptosis induction: while wild-type p53 promoted cell death, different p53 mutations reduced apoptosis sensitivity. The prediction of the impact of the p53 status on the extrinsic cell death induction was much more complex. The presence of p53 in tumor cell lines and primary xenograft tumor cells resulted in either augmented, unchanged or reduced cell death. The substitution of wild-type p53 by mutant p53 did not affect the extrinsic apoptosis inducing capacity. In summary, we have identified a non-expected impact of p53 on extrinsic cell death induction. We suggest that the impact of the p53 status of tumor cells on extrinsic apoptosis signaling should be studied in detail especially in the context of therapeutic approaches that aim to restore p53 function to facilitate cell death via the extrinsic apoptosis pathway.

UPREGULATION OF BNIP3 AND TRANSLOCATION TO MITOCHONDRIA MEDIATES CYANIDE-INDUCED APOPTOSIS IN CORTICAL CELLS

PubMed Central

Prabhakaran, K.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

2008-01-01

BNIP3, a BH3 domain only Bcl-2 protein, has been identified as a mitochrondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either overexpressed with BNIP3 cDNA (BNIP3+) or knocked down with small interfering RNA (RNAi). In BNIP3+ cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (Δψm), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan caspase inhibitor zVAD-fmk suppressed BNIP3+-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knock down by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3ΔTM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3ΔTM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3+ cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of Δψm and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduceΔψm This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death. PMID:17980495

Neuroprotective effects of curcumin on endothelin-1 mediated cell death in hippocampal neurons.

PubMed

Stankowska, Dorota L; Krishnamoorthy, Vignesh R; Ellis, Dorette Z; Krishnamoorthy, Raghu R

2017-06-01

Alzheimer's disease is a progressive neurodegenerative disease characterized by loss of hippocampal neurons leading to memory deficits and cognitive decline. Studies suggest that levels of the vasoactive peptide endothelin-1 (ET-1) are increased in the brain tissue of Alzheimer's patients. Curcumin, the main ingredient of the spice turmeric, has been shown to have anti-inflammatory, anti-cancer, and neuroprotective effects. However, the mechanisms underlying some of these beneficial effects are not completely understood. The objective of this study was to determine if curcumin could protect hippocampal neurons from ET-1 mediated cell death and examine the involvement of c-Jun in this pathway. Primary hippocampal neurons from rat pups were isolated using a previously published protocol. Viability of the cells was measured by the live/dead assay. Immunoblot and immunohistochemical analyses were performed to analyze c-Jun levels in hippocampal neurons treated with either ET-1 or a combination of ET-1 and curcumin. Apoptotic changes were evaluated by immunoblot detection of cleaved caspase-3, cleaved fodrin, and a caspase 3/7 activation assay. ET-1 treatment produced a 2-fold increase in the levels of c-Jun as determined by an immunoblot analysis in hippocampal neurons. Co-treatment with curcumin significantly attenuated the ET-1 mediated increase in c-Jun levels. ET-1 caused increased neuronal cell death of hippocampal neurons indicated by elevation of cleaved caspase-3, cleaved fodrin and an increased activity of caspases 3 and 7 which was attenuated by co-treatment with curcumin. Blockade of JNK, an upstream effector of c-Jun by specific inhibitor SP600125 did not fully protect from ET-1 mediated activation of pro-apoptotic enzymes in primary hippocampal cells. Our data suggests that one mechanism by which curcumin protects against ET-1-mediated cell death is through blocking an increase in c-Jun levels. Other possible mechanisms include decreasing pro-apoptotic signaling activated by ET-1 in primary hippocampal neurons.

Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.

PubMed

McCaig, William D; Patel, Payal S; Sosunov, Sergey A; Shakerley, Nicole L; Smiraglia, Tori A; Craft, Miranda M; Walker, Katharine M; Deragon, Matthew A; Ten, Vadim S; LaRocca, Timothy J

2018-01-01

Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis. Cell death under hyperglycemic conditions was classified as necroptosis via measurement of markers and involvement of RIP1, RIP3, and MLKL. The shift to necroptosis was driven by RIP1, as mutation of this gene using CRISPR-Cas9 caused cell death to revert to apoptosis under hyperglycemic conditions. The shift of apoptosis to necroptosis depended on glycolysis and production of mitochondrial ROS. Importantly, the shift in PCD was observed in primary human T cells. Levels of RIP1 and MLKL increased, while executioner caspases and PARP1 cleavage decreased, in cerebral tissue from hyperglycemic neonatal mice that underwent hypoxia-ischemia (HI) brain injury, suggesting that this cell death shift occurs in vivo . This is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death which may explain the exacerbation of neonatal HI-brain injury during hyperglycemia. These results are distinct from our previous findings where hyperglycemia enhanced necroptosis under conditions where apoptosis was inhibited artificially. Here we demonstrate a shift from apoptosis to necroptosis under hyperglycemic conditions while both pathways are fully active. Therefore, while our previous work documented that intensity of necroptosis is responsive to glucose, this work sheds light on the molecular balance between apoptosis and necroptosis and identifies hyperglycemia as a condition that pushes cells to undergo necroptosis despite the initial activation of apoptosis.

[Possibilities and limitations of fibroblast cultures in the study of animal aging].

PubMed

Van Gansen, P; Van Lerberghe, N

1987-01-01

INTRODUCTION. Aging--the effect of time--occurs in every living organism. Senescence is the last period of the lifespan, leading to death. It happens in all animals, with the exception of a few didermic species (Hydras) having a stock of embryonic cells and being immortal. The causes of animal senescence are badly known. They depend both on genetic characters (maximal lifespan of a species) and on medium factors (mean expectation of life of the animals of a species). Animal senescence could depend on cell aging: 1) by senescence and death of the differentiated cells, 2) by modified proliferation and differentiation of the stem cells of differentiated tissues, 3) by alterations in the extracellular matrices, 4) by interactions between factors 1) 2) and 3) in each tissue, 5) by interactions between the several tissues of an organism. This complexity badly impedes the experimental study of animal senescence. Normal mammal cells are aging when they are cultivated (in vitro ageing): their phenotype varies and depends on the cell generation (in vitro differentiation); the last cell-generation doesn't divide anymore and declines until death of the culture (in vitro senescence). Analysis of these artificial but well controlled systems allows an experimental approach of the proliferation, differentiation, senescence and death of the cells and of the extracellular matrix functions. Present literature upon in vitro aging of cultivated human cells is essentially made of papers where proliferation and differentiation characteristics are compared between early ("young") and late ("old") cell-generations of the cultures. FIBROBLASTIC CELLS OF THE MOUSE SKIN. This cell type has been studied in our laboratory, using different systems: 1) Primary cultures isolated from peeled skins of 19 day old mouse embryos, 2) Mouse dermis analyzed in the animals, 3) Cultivated explants of skins, 4) Serial sub-cultures of fibroblasts isolated from these explants, 5) Cells cultivated comparably on plane substrates (glass, plastic, collagen films) and on tridimensional matrices (collagen fibres). Systems 2), 3), 4) and 5) have been obtained either from 19 day old embryos or from 6 groups of animals of different ages (from 1/2 till 25 month). In primary cultures (system 1) all the cell generations have been analyzed, including the last one until death of the culture. We have shown that many characters are varying with cell-generation: cell form and cell mass, rate of DNA replication and cell division, rate of RNA transcription, nature of the accumulated and of the synthetized proteins, organization of the cytoskeletal elements, organization of the extracellular matrix, type of cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

PubMed

Ziauddin, S M; Yoshimura, A; Montenegro Raudales, J L; Ozaki, Y; Higuchi, K; Ukai, T; Kaneko, T; Miyazaki, T; Latz, E; Hara, Y

2018-06-01

Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1β precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental calculus was significantly inhibited by cytochalasin D, z-YVAD-fmk and glyburide, indicating NLRP3 inflammasome involvement. In permeability assays, dental calculus attenuated the barrier function of HSC-2 cell monolayers. Dental calculus induces pyroptotic cell death in human oral epithelial cells and the crystalline structure plays a major role in this process. Oral epithelial cell death induced by dental calculus might be important for the etiology of periodontitis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

PubMed

Ishikawa, F; Ushida, K; Mori, K; Shibanuma, M

2015-01-22

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT ((Tert)HMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached (Tert)HMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Chenchen; Xing Tairan; Tang Mingliang

2008-06-15

Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronalmore » death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.« less

Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-induced Macrophage Cell Death through Enhancing Ceramide Production

PubMed Central

Zhang, Yuwen; Rao, Enyu; Zeng, Jun; Hao, Jiaqing; Sun, Yanwen; Liu, Shujun; Sauter, Edward R.; Bernlohr, David A.; Cleary, Margot P.; Suttles, Jill; Li, Bing

2016-01-01

Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms underlying the response of macrophages to elevated fatty acids (FAs) and their contribution to metabolic inflammation in obesity remain to be fully elucidated. Here, we report a new mechanism by which dietary FAs, in particular saturated FAs, are able to directly trigger macrophage cell death. We demonstrated that excess saturated FAs, but not unsaturated FAs, induced the production of cytotoxic ceramides in macrophage cell lines. Most importantly, expression of adipose fatty acid binding protein (A-FABP) in macrophages facilitated metabolism of excess saturated FAs for ceramide synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased saturated FA-induced ceramide production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting saturated FA-induced macrophage cell death with primary bone-marrow derived macrophages and high-fat diet-induced obese mice. Altogether, our data reveal that excess dietary saturated FAs may serve as direct triggers in induction of ceramide production and macrophage cell death through elevated expression of A-FABP, thus establishing A-FABP as a new molecular sensor in triggering macrophage-associated sterile inflammation in obesity. PMID:27920274

Primary cultures of human colon cancer as a model to study cancer stem cells.

PubMed

Koshkin, Sergey; Danilova, Anna; Raskin, Grigory; Petrov, Nikolai; Bajenova, Olga; O'Brien, Stephen J; Tomilin, Alexey; Tolkunova, Elena

2016-09-01

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

PubMed Central

Sharma, Jaswinder; Nelluru, Geetha; Ann Wilson, Mary; Johnston, Michael V; Ahamed Hossain, Mir

2011-01-01

Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox. PMID:21382016

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxiainducible factor-1alpha expression via Poly(ADP-ribose) polymerase -1

PubMed Central

Pan, Rong; Chen, Chen; Liu, Wenlan; Liu, Ke Jian

2013-01-01

Aim Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study testes the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Methods Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-hour hypoxic treatment. Results Although 3-hr hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc concentration dependent manner. Exposure of astrocytes to hypoxia for 3-hr remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Conclusions Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. PMID:23582235

Improving Accuracy in Arrhenius Models of Cell Death: Adding a Temperature-Dependent Time Delay.

PubMed

Pearce, John A

2015-12-01

The Arrhenius formulation for single-step irreversible unimolecular reactions has been used for many decades to describe the thermal damage and cell death processes. Arrhenius predictions are acceptably accurate for structural proteins, for some cell death assays, and for cell death at higher temperatures in most cell lines, above about 55 °C. However, in many cases--and particularly at hyperthermic temperatures, between about 43 and 55 °C--the particular intrinsic cell death or damage process under study exhibits a significant "shoulder" region that constant-rate Arrhenius models are unable to represent with acceptable accuracy. The primary limitation is that Arrhenius calculations always overestimate the cell death fraction, which leads to severely overoptimistic predictions of heating effectiveness in tumor treatment. Several more sophisticated mathematical model approaches have been suggested and show much-improved performance. But simpler models that have adequate accuracy would provide useful and practical alternatives to intricate biochemical analyses. Typical transient intrinsic cell death processes at hyperthermic temperatures consist of a slowly developing shoulder region followed by an essentially constant-rate region. The shoulder regions have been demonstrated to arise chiefly from complex functional protein signaling cascades that generate delays in the onset of the constant-rate region, but may involve heat shock protein activity as well. This paper shows that acceptably accurate and much-improved predictions in the simpler Arrhenius models can be obtained by adding a temperature-dependent time delay. Kinetic coefficients and the appropriate time delay are obtained from the constant-rate regions of the measured survival curves. The resulting predictions are seen to provide acceptably accurate results while not overestimating cell death. The method can be relatively easily incorporated into numerical models. Additionally, evidence is presented to support the application of compensation law behavior to the cell death processes--that is, the strong correlation between the kinetic coefficients, ln{A} and E(a), is confirmed.

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dae-Hee, E-mail: leedneo@gmail.com; Kim, Dong-Wook; Jung, Chang-Hwa

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We alsomore » found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.« less

GDC-0941 enhances the lysosomal compartment via TFEB and primes glioblastoma cells to lysosomal membrane permeabilization and cell death.

PubMed

Enzenmüller, Stefanie; Gonzalez, Patrick; Karpel-Massler, Georg; Debatin, Klaus-Michael; Fulda, Simone

2013-02-01

Since phosphatidylinositol-3-kinase (PI3K) inhibitors are primarily cytostatic against glioblastoma, we searched for new drug combinations. Here, we discover that the PI3K inhibitor GDC-0941 acts in concert with the natural compound B10, a glycosylated derivative of betulinic acid, to induce cell death in glioblastoma cells. Importantly, parallel experiments in primary glioblastoma cultures similarly show that GDC-0941 and B10 cooperate to trigger cell death, underscoring the clinical relevance of this finding. Molecular studies revealed that treatment with GDC-0941 stimulates the expression and nuclear translocation of Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, the lysosomal membrane marker LAMP-1 and the mature form of cathepsin B. Also, GDC-0941 triggers a time-dependent increase of the lysosomal compartment in a TFEB-dependent manner, since knockdown of TFEB significantly reduces this GDC-0941-stimulated lysosomal enhancement. Importantly, GDC-0941 cooperates with B10 to trigger lysosomal membrane permeabilization, leading to increased activation of Bax, loss of mitochondrial membrane potential (MMP), caspase-3 activation and cell death. Addition of the cathepsin B inhibitor CA-074me reduces Bax activation, loss of MMP, caspase-3 activation and cell death upon treatment with GDC-0941/B10. By comparison, knockdown of caspase-3 or the broad-range caspase inhibitor zVAD.fmk inhibits GDC-0941/B10-induced DNA fragmentation, but does not prevent cell death, thus pointing to both caspase-dependent and -independent pathways. By identifying the combination of GDC-0941 and B10 as a new, potent strategy to trigger cell death in glioblastoma cells, our findings have important implications for the development of novel treatment approaches for glioblastoma. Copyright © 2012. Published by Elsevier Ireland Ltd.

Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation.

PubMed

Funakoshi, Takeshi; Aki, Toshihiko; Tajiri, Masateru; Unuma, Kana; Uemura, Koichi

2016-11-25

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation*

PubMed Central

Funakoshi, Takeshi; Aki, Toshihiko; Tajiri, Masateru; Unuma, Kana; Uemura, Koichi

2016-01-01

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells. PMID:27756839

Trimethyltin-induced apoptosis is associated with upregulation of inducible nitric oxide synthase and Bax in a hippocampal cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, L.; Li, L.; Prabhakaran, K.

2006-10-01

Trimethyltin (TMT) produces selective neuronal degeneration in the central nervous system (CNS), in which the hippocampus is the most sensitive area. Since previous studies have been conducted in either non-neural cells or mixed primary cultures, an immortalized hippocampal neuronal cell line (HT-22 cell) was used to assess the mechanism and mode of death produced by TMT. The compound produced a time- and concentration-dependent apoptotic death that was caspase-mediated. Excessive generation of reactive oxygen species (ROS) and subsequent reduction of mitochondrial membrane potential ({delta}{psi}{sub m}) were involved in the cytotoxicity{sub .} Scavenging of ROS by a free radical trapping agent ormore » inhibition of the mitochondrial permeability transition (MPT) pore significantly reduced cell death. Additionally, TMT increased expression of inducible nitric oxide synthase (iNOS) by activation of the redox-sensitive transcription factor NF{kappa}B. Pharmacologic inhibition studies showed that the iNOS-mediated NO generation increased expression of Bax and then mitochondrial-mediated apoptosis. It was concluded that excessive ROS generation initiated the apoptotic cell death by upregulating iNOS followed by increased Bax expression which then led to loss of {delta}{psi}{sub m} and caspase-executed cell death. This study is the first to report in a neuronal cell model that TMT stimulates induction of iNOS, which then increases cellular levels of reactive nitrogen species (RNS) to initiate apoptotic death.« less

Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm

PubMed Central

Philippe, Laure; Ceroi, Adam; Bôle-Richard, Elodie; Jenvrin, Alizée; Biichle, Sabeha; Perrin, Sophie; Limat, Samuel; Bonnefoy, Francis; Deconinck, Eric; Saas, Philippe; Garnache-Ottou, Francine; Angelot-Delettre, Fanny

2017-01-01

Blastic plasmacytoid dendritic cell neoplasm is an aggressive hematologic malignancy with a poor prognosis. No consensus regarding optimal treatment modalities is currently available. Targeting the nuclear factor-kappa B pathway is considered a promising approach since blastic plasmacytoid dendritic cell neoplasm has been reported to exhibit constitutive activation of this pathway. Moreover, nuclear factor-kappa B inhibition in blastic plasmacytoid dendritic cell neoplasm cell lines, achieved using either an experimental specific inhibitor JSH23 or the clinical drug bortezomib, interferes in vitro with leukemic cell proliferation and survival. Here we extended these data by showing that primary blastic plasmacytoid dendritic cell neoplasm cells from seven patients were sensitive to bortezomib-induced cell death. We confirmed that bortezomib efficiently inhibits the phosphorylation of the RelA nuclear factor-kappa B subunit in blastic plasmacytoid dendritic cell neoplasm cell lines and primary cells from patients in vitro and in vivo in a mouse model. We then demonstrated that bortezomib can be associated with other drugs used in different chemotherapy regimens to improve its impact on leukemic cell death. Indeed, when primary blastic plasmacytoid dendritic cell neoplasm cells from a patient were grafted into mice, bortezomib treatment significantly increased the animals’ survival, and was associated with a significant decrease of circulating leukemic cells and RelA nuclear factor-kappa B subunit expression. Overall, our results provide a rationale for the use of bortezomib in combination with other chemotherapy for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm. Based on our data, a prospective clinical trial combining proteasome inhibitor with classical drugs could be envisaged. PMID:28798071

Impact of microRNA-134 on neural cell survival against ischemic injury in primary cultured neuronal cells and mouse brain with ischemic stroke by targeting HSPA12B.

PubMed

Chi, Wenying; Meng, Fanjun; Li, Yan; Li, Peilong; Wang, Guizhi; Cheng, Hong; Han, Song; Li, Junfa

2014-12-10

As a newly discovered member of the HSP70 family, heat shock protein A12B (HSPA12B) is involved in brain ischemic injury. According to our previous study, microRNA-134 (miR-134) could target HSPA12B by binding to its 3'-untranslated region (UTR). However, the regulation of miR-134 on HSPA12B and their role in protecting neuronal cells from ischemic injury are unclear. In this study, the miR-134 expression level was manipulated, and the HSPA12B protein levels were also determined in oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells in vitro and mouse brain after middle cerebral artery occlusion (MCAO)-induced ischemic stroke in vivo. The results showed that miR-134 expression levels increased in primary cultured neuronal cells and mouse brain from 12h to 7 day reoxygenation/reperfusion after 1h OGD or 1h MCAO treatment. miR-134 overexpression promoted neuronal cell death and apoptosis by decreasing HSPA12B protein levels. Conversely, downregulating miR-134 reduced neuronal cell death and apoptosis by enhancing HSPA12B protein levels. Also, HSPA12B siRNA could block miR-134 inhibitor-mediated neuroprotection against OGD-induced neuronal cell injury in vitro. Taken together, miR-134 might influence neuronal cell survival against ischemic injury in primary cultured neuronal cells and mouse brain with ischemic stroke by negatively modulating HSPA12B protein expression in a posttranscriptional manner. Copyright © 2014 Elsevier B.V. All rights reserved.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Signaling and Cell Death in the Immature Central Nervous System after Hypoxia-Ischemia and Inflammation*

PubMed Central

Kichev, Anton; Rousset, Catherine I.; Baburamani, Ana A.; Levison, Steven W.; Wood, Teresa L.; Gressens, Pierre; Thornton, Claire; Hagberg, Henrik

2014-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI. PMID:24509861

Programmed Cell Death-1/Programmed Death-ligand 1 Pathway: A New Target for Sepsis.

PubMed

Liu, Qiang; Li, Chun-Sheng

2017-04-20

Sepsis remains a leading cause of death in many Intensive Care Units worldwide. Immunosuppression has been a primary focus of sepsis research as a key pathophysiological mechanism. Given the important role of the negative costimulatory molecules programmed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1) in the occurrence of immunosuppression during sepsis, we reviewed literatures related to the PD-1/PD-L1 pathway to examine its potential as a new target for sepsis treatment. Studies of the association between PD-1/PD-L1 and sepsis published up to January 31, 2017, were obtained by searching the PubMed database. English language studies, including those based on animal models, clinical research, and reviews, with data related to PD-1/PD-L1 and sepsis, were evaluated. Immunomodulatory therapeutics could reverse the deactivation of immune cells caused by sepsis and restore immune cell activation and function. Blockade of the PD-1/PD-L1 pathway could reduce the exhaustion of T-cells and enhance the proliferation and activation of T-cells. The anti-PD-1/PD-L1 pathway shows promise as a new target for sepsis treatment. This review provides a basis for clinical trials and future studies aimed at revaluating the efficacy and safety of this targeted approach.

Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.

2010-09-15

Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibitedmore » both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.« less

AKT1 provides an essential survival signal required for differentiation and stratification of primary human keratinocytes.

PubMed

Thrash, Barry R; Menges, Craig W; Pierce, Robert H; McCance, Dennis J

2006-04-28

Keratinocyte differentiation and stratification are complex processes involving multiple signaling pathways, which convert a basal proliferative cell into an inviable rigid squame. Loss of attachment to the basement membrane triggers keratinocyte differentiation, while in other epithelial cells, detachment from the extracellular matrix leads to rapid programmed cell death or anoikis. The potential role of AKT in providing a survival signal necessary for stratification and differentiation of primary human keratinocytes was investigated. AKT activity increased during keratinocyte differentiation and was attributed to the specific activation of AKT1 and AKT2. Targeted reduction of AKT1 expression, but not AKT2, by RNA interference resulted in an abnormal epidermis in organotypic skin cultures with a thin parabasal region and a pronounced but disorganized cornified layer. This abnormal stratification was due to significant cell death in the suprabasal layers and was alleviated by caspase inhibition. Normal expression patterns of both early and late markers of keratinocyte differentiation were also disrupted, producing a poorly developed stratum corneum.

MK-2206, an AKT Inhibitor, Promotes Caspase-Independent Cell Death and Inhibits Leiomyoma Growth

PubMed Central

Sefton, Elizabeth C.; Qiang, Wenan; Serna, Vanida; Kurita, Takeshi; Wei, Jian-Jun; Chakravarti, Debabrata

2013-01-01

Uterine leiomyomas (ULs), benign tumors of the myometrium, are the number one indication for hysterectomies in the United States due to a lack of an effective alternative therapy. ULs show activation of the pro-survival AKT pathway compared with normal myometrium; however, substantial data directly linking AKT to UL cell survival are lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable target for inhibiting UL growth. We used the investigational AKT inhibitor MK-2206, currently in phase II trials, on cultured primary human UL and myometrial cells, immortalized leiomyoma cells, and in leiomyoma grafts grown under the kidney capsule in mice. MK-2206 inhibited AKT and PRAS40 phosphorylation but did not regulate serum- and glucocorticoid-induced kinase and ERK1/2, demonstrating its specificity for AKT. MK-2206 reduced UL cell viability and decreased UL tumor volumes. UL cells exhibited disruption of mitochondrial structures and underwent cell death that was independent of caspases. Additionally, mammalian target of rapamycin and p70S6K phosphorylation were reduced, indicating that mammalian target of rapamycin complex 1 signaling was compromised by AKT inhibition in UL cells. MK-2206 also induced autophagy in UL cells. Pretreatment of primary UL cells with 3-methyladenine enhanced MK-2206-mediated UL cell death, whereas knockdown of ATG5 and/or ATG7 did not significantly influence UL cell viability in the presence of MK-2206. Our data provide molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option to consider for the treatment of ULs. PMID:24002033

Myricetin Induces Pancreatic Cancer Cell Death via the Induction of Apoptosis and Inhibition of the Phosphatidylinositol 3-Kinase (PI3K) Signaling Pathway

PubMed Central

Phillips, P.A.; Sangwan, V.; Borja-Cacho, D.; Dudeja, V.; Vickers, S.M.; Saluja, A.K.

2011-01-01

Pancreatic cancer is a the four leading cause of cancer related deaths and is adisease with poor prognosis. It is refractory to standard chemotherapeutic drugs or to novel treatment modalities, making it imperative to find new treatments. In this study, using both primary and metastatic pancreatic cancer cell lines, we have demonstrated that the flavonoid myricetin induced pancreatic cancer cell death in vitro via apoptosis, and caused a decrease in PI3 kinase activity. In vivo, treatment of orthotopic pancreatic tumors with myricetin resulted in tumor regression and decreased metastatic spread. Importantly, myricetin was non-toxic, both in vitro and in vivo, underscoring its use as a therapeutic agent against pancreatic cancer. PMID:21676539

Bioenergetic adaptation in response to autophagy regulators during rotenone exposure

PubMed Central

Giordano, Samantha; Dodson, Matthew; Ravi, Saranya; Redmann, Matthew; Ouyang, Xiaosen; Usmar, Victor M Darley; Zhang, Jianhua

2015-01-01

Parkinson’s disease (PD) is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing PD. We previously reported that in differentiated SH-SY5Y cells, rotenone-induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 hr, caused cell death at 24 hr with an LD50 of 10 nM. Overall autophagic flux was decreased by 10 nM rotenone at both 2 and 24 hr, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 hr, suggesting that a mitochondrial-specific lysosomal degradation pathway may be activated. Upregulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine (3-MA) exacerbated cell death. Interestingly, while 3-MA exacerbated the rotenone-dependent effects on bioenergetics, rapamycin did not prevent rotenone-induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor. PMID:25081478

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1.

PubMed

Kuchipudi, Suresh V; Dunham, Stephen P; Nelli, Rahul; White, Gavin A; Coward, Vivien J; Slomka, Marek J; Brown, Ian H; Chang, Kin Chow

2012-01-01

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and 'classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host-pathogen relationships for highly pathogenic and zoonotic avian influenza.

Ursodeoxycholic acid effectively kills drug-resistant gastric cancer cells through induction of autophagic death.

PubMed

Lim, Sung-Chul; Han, Song Iy

2015-09-01

Carcinoma cells that have acquired drug resistance often exhibit cross-resistance to various other cytotoxic stimuli. Here, we investigated the effects of ursodeoxycholic acid (UDCA), a gastrointestinal tumor-suppressor, on a cisplatin‑resistant SNU601 gastric cancer subline (SNU601/R). While other anticancer drugs, including L-OHP, etoposide, and death ligand TRAIL, had minimal effects on the viability of these resistant cells, they were sensitive to UDCA. The UDCA‑induced reduction in the viability of the SNU601/R cells was accomplished through autophagy while the primary means of cell death in the parental SNU601 cells (SNU601/WT) was apoptosis. Previously, we demonstrated that the UDCA-triggered apoptosis of gastric cancer cells was regulated by a cell surface death receptor, TRAIL-R2/DR5, which was upregulated and re-distributed on lipid rafts. The UDCA stimulation of TRAIL-R2/DR5 also occurred in the SNU601/R cells despite the lack of apoptosis. In the present study, we found that CD95/Fas, another cell surface death receptor, was also translocated into lipid rafts in response to UDCA although it was not involved in the decrease in cell viability. Specifically, raft relocalization of CD95/Fas was triggered by UDCA in the SNU601/WT cells in which apoptosis occurred, but not in the SNU601/R cells where autophagic death occurred. Notably, UDCA reduced ATG5 levels, an essential component of autophagy, in the SNU601/WT, but not in the SNU601/R cell line. Moreover, in CD95/Fas-silenced SNU601/WT cells, UDCA did not decrease ATG5 levels and induced autophagic cell death rather than apoptosis. These results imply that raft‑distributed CD95/Fas may support UDCA-induced apoptosis via downregulation of ATG5 levels, preventing the autophagic pathway. Taken together, these results suggest that UDCA induces both apoptotic and autophagic cell death depending on the intracellular signaling environment, thereby conferring the advantage to overcome drug resistance through apoptotic defects.

Switch-Hitting Immune Cells: From Tumor Protection to Metastasis Promotion | Center for Cancer Research

Cancer.gov

The leading cause of death from cancer is not a primary tumor but is the metastases, or invasion of tumor cells into other locations in the body, that result from it. A complex and incompletely understood process, metastatic tumor formation is thought to require several steps in which tumor cells invade the tissue surrounding the primary tumor, enter local blood vessels, navigate the circulation, exit the vasculature, and colonize a new site. Tumor cells do not, however, operate independently, and the role that the immune system plays in this metastatic process is beginning to be appreciated.

Involvement of tumour necrosis factor-α-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells

PubMed Central

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-01-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257–264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand–tumour necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses. PMID:12100718

Involvement of tumour necrosis factor-alpha-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells.

PubMed

Yu, Yizhi; Liu, Shuxun; Wang, Wenya; Song, Wengang; Zhang, Minghui; Zhang, Weiping; Qin, Zhihai; Cao, Xuetao

2002-07-01

Dendritic cells (DC) are potent antigen-presenting cells (APC) specialized in T-cell mediated immune responses, and also play critical roles in the homeostasis of T cells for controlling immune responses. In the present study, we demonstrated that during mouse bone-marrow-derived DC activation of ovalbumin (OVA)-specific Ia-kb-restricted T hybridoma cells, MF2.2D9 and OVA257-264-specific H-2kb-restricted RF33.70 T cells, respectively, both hybridomas undergo cell death, partially mediated via apoptotic ligand-tumour necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL). Lipopolysaccharide enhanced the cytotoxic effect on the two activated T hybridoma cells, which was correlated with up-regulation of TRAIL-expression on DC to some extent. The activation of caspase-3 in activated T hybridoma cells cocultured with DC contributed to the programmed cell death pathway T cells underwent. Therefore, our results show that activation-induced cell death of T hybridoma cells can be influenced by DC, suggesting that DC may be involved in elimination of activated T cells at the end of primary immune responses.

Evaluation of programmed cell death protein 1 (PD-1) expression as a prognostic biomarker in patients with clear cell renal cell carcinoma.

PubMed

Kim, Kyu Seo; Sekar, Rishi R; Patil, Dattatraya; Dimarco, Michelle A; Kissick, Haydn T; Bilen, Mehmet A; Osunkoya, Adeboye O; Master, Viraj A

2018-01-01

Programmed cell death protein 1 (PD-1) immune checkpoint inhibitors have shown activity in patients with advanced renal cell carcinoma (RCC). However, the role of PD-1 expression in tumor-infiltrating lymphocytes (TILs) as a biomarker for poor outcome is not clear. In this study, we evaluated the prognostic value of TIL PD-1 expression in patients with clear cell RCC (ccRCC). 82 patients who underwent nephrectomy for localized or metastatic ccRCC and followed up for at least four years were searched from our database and retrospectively enrolled. Their fixed primary tumor specimens were stained with anti-PD-1 (NAT105). The specimens were classified as negative or positive for PD-1 expression, and the positive specimens were further scored in 10% increments. 37 (45.12%) patients were negative (<1% stained), 26 (31.71%) patients were low (<10 and 10%), and 19 (23.17%) patients were high (20-50%) for PD-1 expression. The prognostic value of TIL PD-1 expression was evaluated by univariate Cox proportional hazards regression on overall and recurrence-free survivals. Higher TIL PD-1 expression was not associated with increased risk of death (P = 0.336) or with increased risk of recurrence (P = 0.572). Higher primary tumor stage was associated with increased risk of recurrence (P = 0.003), and higher Fuhrman nuclear grade was associated with increased risk of death (P <0.001) and with increased risk of recurrence (P <0.001). Our study shows that TIL PD-1 expression by immunohistochemistry (IHC) does not correlate with poor clinical outcome in patients with ccRCC and is inferior to established prognosticating tools.

Coenzyme Q{sub 10} and alpha-tocopherol protect against amitriptyline toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cordero, Mario D.; Dpto. Citologia e Histologia Normal y Patologica, Facultad de Medicina. Universidad de Sevilla. 41009 Sevilla; Moreno-Fernandez, Ana Maria

Since amitriptyline is a very frequently prescribed antidepressant drug, it is not surprising that amitriptyline toxicity is relatively common. Amitriptyline toxic systemic effects include cardiovascular, autonomous nervous, and central nervous systems. To understand the mechanisms of amitriptyline toxicity we studied the cytotoxic effects of amitriptyline treatment on cultured primary human fibroblasts and zebrafish embryos, and the protective role of coenzyme Q{sub 10} and alpha-tocopherol, two membrane antioxidants. We found that amitriptyline treatment induced oxidative stress and mitochondrial dysfunction in primary human fibroblasts. Mitochondrial dysfunction in amitriptyline treatment was characterized by reduced expression levels of mitochondrial proteins and coenzyme Q{sub 10},more » decreased NADH:cytochrome c reductase activity, and a drop in mitochondrial membrane potential. Moreover, and as a consequence of these toxic effects, amitriptyline treatment induced a significant increase in apoptotic cell death activating mitochondrial permeability transition. Coenzyme Q{sub 10} and alpha-tocopherol supplementation attenuated ROS production, lipid peroxidation, mitochondrial dysfunction, and cell death, suggesting that oxidative stress affecting cell membrane components is involved in amitriptyline cytotoxicity. Furthermore, amitriptyline-dependent toxicity and antioxidant protection were also evaluated in zebrafish embryos, a well established vertebrate model to study developmental toxicity. Amitriptyline significantly increased embryonic cell death and apoptosis rate, and both antioxidants provided a significant protection against amitriptyline embryotoxicity.« less

Nonsmall Cell Lung Carcinoma with Giant Cell Features Expressing Programmed Death-Ligand 1: A Report of a Patient Successfully Treated with Pembrolizumab

PubMed Central

Nakayama, Shingo; Sasaki, Mamoru; Morinaga, Shojiroh

2018-01-01

Giant cell carcinoma, a rare variant of nonsmall cell lung carcinoma (NSCLC), is characterized by aggressive progression and poor response to conventional chemotherapy. This report is the first to describe a patient with NSCLC and giant cell features who was successfully treated with pembrolizumab, an antibody targeting programmed death-1 (PD-1). A 69-year-old woman was diagnosed with NSCLC with multiple brain metastases. Histological evaluation of lung biopsy specimens revealed proliferation of pleomorphic giant tumor cells with poor cohesiveness, findings consistent with giant cell carcinoma. Immunostaining showed that a high proportion of the tumor cells were positive for expression of programmed death-ligand 1 (PD-L1). The patient received stereotactic radiotherapy for the brain metastases, followed by administration of pembrolizumab. Treatment with pembrolizumab resulted in the rapid regression of the primary lung nodule, with the progression-free period maintained for at least four treatment cycles. Immunotherapy targeting PD-1/PD-L1 may be an option for patients with PD-L1-positive NSCLC with giant cell features. PMID:29736285

Nonsmall Cell Lung Carcinoma with Giant Cell Features Expressing Programmed Death-Ligand 1: A Report of a Patient Successfully Treated with Pembrolizumab.

PubMed

Nakayama, Shingo; Sasaki, Mamoru; Morinaga, Shojiroh; Minematsu, Naoto

2018-01-01

Giant cell carcinoma, a rare variant of nonsmall cell lung carcinoma (NSCLC), is characterized by aggressive progression and poor response to conventional chemotherapy. This report is the first to describe a patient with NSCLC and giant cell features who was successfully treated with pembrolizumab, an antibody targeting programmed death-1 (PD-1). A 69-year-old woman was diagnosed with NSCLC with multiple brain metastases. Histological evaluation of lung biopsy specimens revealed proliferation of pleomorphic giant tumor cells with poor cohesiveness, findings consistent with giant cell carcinoma. Immunostaining showed that a high proportion of the tumor cells were positive for expression of programmed death-ligand 1 (PD-L1). The patient received stereotactic radiotherapy for the brain metastases, followed by administration of pembrolizumab. Treatment with pembrolizumab resulted in the rapid regression of the primary lung nodule, with the progression-free period maintained for at least four treatment cycles. Immunotherapy targeting PD-1/PD-L1 may be an option for patients with PD-L1-positive NSCLC with giant cell features.

Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death

PubMed Central

1996-01-01

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point. PMID:8642331

Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

PubMed

Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

2008-12-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites

PubMed Central

Kim, Jong-Hyun

2008-01-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death. PMID:19127326

Mitochondrial protection impairs BET bromodomain inhibitor-mediated cell death and provides rationale for combination therapeutic strategies.

PubMed

Lasorsa, E; Smonksey, M; Kirk, J S; Rosario, S; Hernandez-Ilizaliturri, F J; Ellis, L

2015-12-10

Inhibitors of the bromodomain and extraterminal domain family (BETI) have recently entered phase I clinical trials. In patients with advanced leukemia's, potent antileukemia activity was displayed with minimum dose-limiting toxicity. In preclinical models of hematological malignancies, including aggressive B-cell lymphomas, BETI induced cell-cycle arrest and apoptosis. However, the underlying cell death mechanisms are still not well understood. Dissecting the mechanisms required by BETI to mediate cell death would provide strong direction on how to best utilize BETI to treat patients with aggressive hematological malignancies. Herein, we provide understanding of the molecular mechanisms underlying BETI-mediated cell death using I-BET762. Induction of cell death occurred in primary murine and human B-cell lymphomas through apoptosis. Genetic dissection using Eμ-myc B-cell lymphoma compound mutants demonstrated that I-BET762-induced apoptosis does not require the p53 pathway. Furthermore, deletion of Apaf1, and thus the absence of a functional apoptosome, is associated with a delayed drug response but do not provide long-term resistance. Prolonged treatment of this model in fact fails to suppress the therapeutic efficacy of the drug and is associated with biochemical features of autophagy. However, lack of mitochondrial permeability completely inhibited I-BET762-mediated tumor cell death, indicating mitochondrial damage as key events for its activity. Combination of I-BET762 with BH3-only mimetics ABT-263 or obatoclax, restored sensitivity to I-BET762 lymphoma killing; however, success was determined by expression of Bcl-2 family antiapoptotic proteins. Our study provides critical insight for clinical decisions regarding the appropriate strategy for using BETI as a single agent or in combination to treat patients with aggressive B-cell lymphomas.

[The fundamental mechanisms of metastatic spread and chemotherapy resistance in lung cancer].

PubMed

Tomuleasa, Ciprian; Kacso, Gabriel; Soritau, Olga; Susman, Sergiu; Petrushev, Bobe; Aldea, Mihaela; Buiga, Rareş; Irimie, Alexandru

2011-01-01

Lung cancer is the leading cause of cancer-related death in the European Union and the United States, accounting for about one third of all cancer deaths. Primary lung cancer may arise from the central (bronchial) or peripheral (bronchiolo-alveolar) compartment of the lung, but the origins of the different histological types of primary lung tumours are not well understood and described in medical literature. Current investigation in the field of cancer research have focused on the "cancer stem cell" hypothesis as stem cells are belived to be crucial players in the homeostasis of all adult tissues. Even if the role of stem cells in lung carcinogenesis is not clear yet, numerous studies indicate that lung cancer is not the result of a sudden transforming event, but of a multistep process of molecular changes of the primordial stem cell niche, leading to the development of noeplasia. In the current review, we present state-of-the-art research in the field of lung stem cell biology, with a special emphasis on lung cancer emergence, development, metastasis and multidrug resistance.

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Neuroprotective effect of schizandrin A on oxygen and glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons.

PubMed

Wang, Cai-Ping; Li, Gui-Cai; Shi, Yun-Wei; Zhang, Xiao-Chuan; Li, Jian-Long; Wang, Zhi-Wei; Ding, Fei; Liang, Xin-Miao

2014-09-01

Brain ischemia appears to be associated with innate immunity. Recent reports showed that C3a and C5a, as potent targets, might protect against ischemia induced cell death. In traditional Chinese medicine, the fruit of Schizandra chinesis Baill (Fructus schizandrae) has been widely used as a tonic. In the present study, we sought to evaluate the neuroprotective effects of schizandrin A, a composition of S. chinesis Baill, against oxygen and glucose deprivation followed by reperfusion (OGD/R)-induced cell death in primary culture of rat cortical neurons, and to test whether C3a and C5a affected cortical neuron recovery from ischemic injury after schizandrin A treatment. The results showed that schizandrin A significantly reduced cell apoptosis and necrosis, increased cell survival, and decreased intracellular calcium concentration ([Ca(2+)]i) and lactate dehydrogenase (LDH) release in primary culture of rat cortical neurons after OGD/R. Mechanism studies suggested that the modulation of extracellular-regulated kinase (ERK), c-Jun NH2-terminal kinases (JNK), and p38, as well as caspase-3 activity played an important role on the progress of neuronal apoptosis. C5aR participated in the neuroprotective effect of schizandrin A in primary culture of rat cortical neurons after OGD/R. Our findings suggested that schizandrin A might act as a candidate therapeutic target drug used for brain ischemia and related diseases.

Induction of specific micro RNA (miRNA) species by ROS-generating metal sulfates in primary human brain cells

PubMed Central

Lukiw, Walter J.; Pogue, Aileen I.

2007-01-01

Iron- and aluminum-sulfate together, at nanomolar concentrations, trigger the production of reactive oxygen species (ROS) in cultures of human brain cells. Previous studies have shown that following ROS induction, a family of pathogenic brain genes that promote inflammatory signalling, cellular apoptosis and brain cell death is significantly over-expressed. Notably, iron- and aluminum-sulfate induce genes in cultured human brain cells that exhibit expression patterns similar to those observed to be up-regulated in moderate- to late-stage Alzheimer's disease (AD). In this study we have extended our investigations to analyze the expression of micro RNA (miRNA) populations in iron- and aluminum-sulfate treated human neural cells in primary culture. The main finding was that these ROS-generating neurotoxic metal sulfates also up-regulate a specific set of miRNAs that includes miR-9, miR-125b and miR-128. Notably, these same miRNAs are up-regulated in AD brain. These findings further support the idea that iron- and aluminum-sulfates induce genotoxicity via a ROS-mediated up-regulation of specific regulatory elements and pathogenic genes that redirect brain cell fate towards progressive dysfunction and apoptotic cell death. PMID:17629564

Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm.

PubMed

Philippe, Laure; Ceroi, Adam; Bôle-Richard, Elodie; Jenvrin, Alizée; Biichle, Sabeha; Perrin, Sophie; Limat, Samuel; Bonnefoy, Francis; Deconinck, Eric; Saas, Philippe; Garnache-Ottou, Francine; Angelot-Delettre, Fanny

2017-11-01

Blastic plasmacytoid dendritic cell neoplasm is an aggressive hematologic malignancy with a poor prognosis. No consensus regarding optimal treatment modalities is currently available. Targeting the nuclear factor-kappa B pathway is considered a promising approach since blastic plasmacytoid dendritic cell neoplasm has been reported to exhibit constitutive activation of this pathway. Moreover, nuclear factor-kappa B inhibition in blastic plasmacytoid dendritic cell neoplasm cell lines, achieved using either an experimental specific inhibitor JSH23 or the clinical drug bortezomib, interferes in vitro with leukemic cell proliferation and survival. Here we extended these data by showing that primary blastic plasmacytoid dendritic cell neoplasm cells from seven patients were sensitive to bortezomib-induced cell death. We confirmed that bortezomib efficiently inhibits the phosphorylation of the RelA nuclear factor-kappa B subunit in blastic plasmacytoid dendritic cell neoplasm cell lines and primary cells from patients in vitro and in vivo in a mouse model. We then demonstrated that bortezomib can be associated with other drugs used in different chemotherapy regimens to improve its impact on leukemic cell death. Indeed, when primary blastic plasmacytoid dendritic cell neoplasm cells from a patient were grafted into mice, bortezomib treatment significantly increased the animals' survival, and was associated with a significant decrease of circulating leukemic cells and RelA nuclear factor-kappa B subunit expression. Overall, our results provide a rationale for the use of bortezomib in combination with other chemotherapy for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm. Based on our data, a prospective clinical trial combining proteasome inhibitor with classical drugs could be envisaged. Copyright© Ferrata Storti Foundation.

Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye.

PubMed

Gao, Jiangyuan; Cui, Jing Z; To, Eleanor; Cao, Sijia; Matsubara, Joanne A

2018-01-12

Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), the primary cell type that is afflicted in AMD, undergoes programmed cell death in the late stages of the disease. However, the exact mechanisms for RPE degeneration in AMD are still unresolved. The prevailing theories consider that each cell death pathway works independently and without regulation of each other. Building upon our previous work in which we induced a short burst of inflammasome activity in vivo, we now investigate the effects of prolonged inflammasome activity on RPE cell death mechanisms in rats. Long-Evans rats received three intravitreal injections of amyloid beta (Aβ), once every 4 days, and were sacrificed at day 14. The vitreous samples were collected to assess the levels of secreted cytokines. The inflammasome activity was evaluated by both immunohistochemistry and western blot. The types of RPE cell death mechanisms were determined using specific cell death markers and morphological characterizations. We found robust inflammasome activation evident by enhanced caspase-1 immunoreactivity, augmented NF-κB nuclear translocalization, increased IL-1β vitreal secretion, and IL-18 protein levels. Moreover, we observed elevated proteolytic cleavage of caspase-3 and gasdermin D, markers for apoptosis and pyroptosis, respectively, in RPE-choroid tissues. There was also a significant reduction in the anti-apoptotic factor, X-linked inhibitor of apoptosis protein, consistent with the overall changes of RPE cells. Morphological analysis showed phenotypic characteristics of pyroptosis including RPE cell swelling. Our data suggest that two cell death pathways, pyroptosis and apoptosis, were activated in RPE cells after exposure to prolonged inflammasome activation, induced by a drusen component, Aβ. The involvement of two distinct cell death pathways in RPE sheds light on the potential interplay between these pathways and provides insights on the future development of therapeutic strategies for AMD.

GLUTAMATE NEUROTOXICITY IN THE DEVELOPING RAT COCHLEA IS ANTAGONIZED BY KUNURENIC ACID AND MK-801

EPA Science Inventory

Glutamate (GLU) is neurotoxic in the neonatal rat cochlea, producing hearing impairment which is largely due to the death of spiral ganglion cells, whereas the receptor hair cells are spared. endritic fibers of the spiral ganglion are post-synaptic to the primary afferent synapse...

Effect of blueberries and insulin on glucose induced neurotoxicity in brain cells in vitro

USDA-ARS?s Scientific Manuscript database

Introduction Literature had shown that disruption in glucose metabolism seen in metabolic syndrome maybe responsible for neuronal cell-death. Oxidative stress (OS) and inflammation (INF) triggered by the impaired metabolic process are considered to be the primary factors for the toxic neuronal atmos...

Nano-Pulse Stimulation induces immunogenic cell death in human papillomavirus-transformed tumors and initiates an adaptive immune response

PubMed Central

Skeate, Joseph G.; Da Silva, Diane M.; Chavez-Juan, Elena; Anand, Snjezana; Nuccitelli, Richard; Kast, W. Martin

2018-01-01

Nano-Pulse Stimulation (NPS) is a non-thermal pulsed electric field modality that has been shown to have cancer therapeutic effects. Here we applied NPS treatment to the human papillomavirus type 16 (HPV 16)-transformed C3.43 mouse tumor cell model and showed that it is effective at eliminating primary tumors through the induction of immunogenic cell death while subsequently increasing the number of tumor-infiltrating lymphocytes within the tumor microenvironment. In vitro NPS treatment of C3.43 cells resulted in a doubling of activated caspase 3/7 along with the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating programmed cell death activity. Tumor-bearing mice receiving standard NPS treatment showed an initial decrease in tumor volume followed by clearing of tumors in most mice, and a significant increase in overall survival. Intra-tumor analysis of mice that were unable to clear tumors showed an inverse correlation between the number of tumor infiltrating lymphocytes and the size of the tumor. Approximately half of the mice that cleared established tumors were protected against tumor re-challenge on the opposite flank. Selective depletion of CD8+ T cells eliminated this protection, suggesting that NPS treatment induces an adaptive immune response generating CD8+ T cells that recognize tumor antigen(s) associated with the C3.43 tumor model. This method may be utilized in the future to not only ablate primary tumors, but also to induce an anti-tumor response driven by effector CD8+ T cells capable of protecting individuals from disease recurrence. PMID:29324830

Nano-Pulse Stimulation induces immunogenic cell death in human papillomavirus-transformed tumors and initiates an adaptive immune response.

PubMed

Skeate, Joseph G; Da Silva, Diane M; Chavez-Juan, Elena; Anand, Snjezana; Nuccitelli, Richard; Kast, W Martin

2018-01-01

Nano-Pulse Stimulation (NPS) is a non-thermal pulsed electric field modality that has been shown to have cancer therapeutic effects. Here we applied NPS treatment to the human papillomavirus type 16 (HPV 16)-transformed C3.43 mouse tumor cell model and showed that it is effective at eliminating primary tumors through the induction of immunogenic cell death while subsequently increasing the number of tumor-infiltrating lymphocytes within the tumor microenvironment. In vitro NPS treatment of C3.43 cells resulted in a doubling of activated caspase 3/7 along with the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating programmed cell death activity. Tumor-bearing mice receiving standard NPS treatment showed an initial decrease in tumor volume followed by clearing of tumors in most mice, and a significant increase in overall survival. Intra-tumor analysis of mice that were unable to clear tumors showed an inverse correlation between the number of tumor infiltrating lymphocytes and the size of the tumor. Approximately half of the mice that cleared established tumors were protected against tumor re-challenge on the opposite flank. Selective depletion of CD8+ T cells eliminated this protection, suggesting that NPS treatment induces an adaptive immune response generating CD8+ T cells that recognize tumor antigen(s) associated with the C3.43 tumor model. This method may be utilized in the future to not only ablate primary tumors, but also to induce an anti-tumor response driven by effector CD8+ T cells capable of protecting individuals from disease recurrence.

17-AAG and Apoptosis, Autophagy, and Mitophagy in Canine Osteosarcoma Cell Lines.

PubMed

Massimini, M; Palmieri, C; De Maria, R; Romanucci, M; Malatesta, D; De Martinis, M; Maniscalco, L; Ciccarelli, A; Ginaldi, L; Buracco, P; Bongiovanni, L; Della Salda, L

2017-05-01

Canine osteosarcoma is highly resistant to current chemotherapy; thus, clarifying the mechanisms of tumor cell resistance to treatments is an urgent need. We tested the geldanamycin derivative 17-AAG (17-allylamino-17-demethoxygeldanamycin) prototype of Hsp90 (heat shock protein 90) inhibitors in 2 canine osteosarcoma cell lines, D22 and D17, derived from primary and metastatic tumors, respectively. With the aim to understand the interplay between cell death, autophagy, and mitophagy, in light of the dual effect of autophagy in regulating cancer cell viability and death, D22 and D17 cells were treated with different concentrations of 17-AAG (0.5 μM, 1 μM) for 24 and 48 hours. 17-AAG-induced apoptosis, necrosis, autophagy, and mitophagy were assessed by transmission electron microscopy, flow cytometry, and immunofluorescence. A simultaneous increase in apoptosis, autophagy, and mitophagy was observed only in the D22 cell line, while D17 cells showed low levels of apoptotic cell death. These results reveal differential cell response to drug-induced stress depending on tumor cell type. Therefore, pharmacological treatments based on proapoptotic chemotherapy in association with autophagy regulators would benefit from a predictive in vitro screening of the target cell type.

Autophagy in lurcher mice: indicted but yet to be acquitted for the death of Purkinje cells.

PubMed

Yue, Zhenyu

2010-05-01

A recent study published in the Journal of Neuroscience by Nishiyama et al., has revisited an autophagy-neurodegeneration model of lurcher (Lc) mice and promoted further discussion regarding the "autophagic cell death" hypothesis. While the study confirmed the previous report by Yue et al., that GluRD2Lc induces autophagy both in vitro and in vivo, it also suggests that GluRD2 (Lc)-mediated autophagy and cell death occur via pathways outside the nPIST-Beclin 1 pathway. For example, the study makes an interesting observation that GluRD2 (Lc)-induced degeneration is associated with energy crisis and an aberrant AMPK activity. The result provides insight into the downstream events induced by GluRD2 (Lc); however, it is not surprising considering that constitutive ion influx caused by the Lc mutation is expected to cause activation of multiple cellular pathways or responses. In conclusion, the authors state that "constitutive ion flux causes cell death with, but not by, autophagy." The conclusion appears consistent with the primary function of autophagy, from an evolutionary point of view, as a survival mechanism.

High-throughput monitoring of major cell functions by means of lensfree video microscopy

PubMed Central

Kesavan, S. Vinjimore; Momey, F.; Cioni, O.; David-Watine, B.; Dubrulle, N.; Shorte, S.; Sulpice, E.; Freida, D.; Chalmond, B.; Dinten, J. M.; Gidrol, X.; Allier, C.

2014-01-01

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 – 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells. PMID:25096726

Clearance of autophagy-associated dying retinal pigment epithelial cells – a possible source for inflammation in age-related macular degeneration

PubMed Central

Szatmári-Tóth, M; Kristóf, E; Veréb, Z; Akhtar, S; Facskó, A; Fésüs, L; Kauppinen, A; Kaarniranta, K; Petrovski, G

2016-01-01

Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis. PMID:27607582

Involvement of the Electrophilic Isothiocyanate Sulforaphane in Arabidopsis Local Defense Responses1

PubMed Central

Andersson, Mats X.; Nilsson, Anders K.; Johansson, Oskar N.; Boztaş, Gülin; Adolfsson, Lisa E.; Pinosa, Francesco; Petit, Christel Garcia; Aronsson, Henrik; Mackey, David; Tör, Mahmut; Hamberg, Mats; Ellerström, Mats

2015-01-01

Plants defend themselves against microbial pathogens through a range of highly sophisticated and integrated molecular systems. Recognition of pathogen-secreted effector proteins often triggers the hypersensitive response (HR), a complex multicellular defense reaction where programmed cell death of cells surrounding the primary site of infection is a prominent feature. Even though the HR was described almost a century ago, cell-to-cell factors acting at the local level generating the full defense reaction have remained obscure. In this study, we sought to identify diffusible molecules produced during the HR that could induce cell death in naive tissue. We found that 4-methylsulfinylbutyl isothiocyanate (sulforaphane) is released by Arabidopsis (Arabidopsis thaliana) leaf tissue undergoing the HR and that this compound induces cell death as well as primes defense in naive tissue. Two different mutants impaired in the pathogen-induced accumulation of sulforaphane displayed attenuated programmed cell death upon bacterial and oomycete effector recognition as well as decreased resistance to several isolates of the plant pathogen Hyaloperonospora arabidopsidis. Treatment with sulforaphane provided protection against a virulent H. arabidopsidis isolate. Glucosinolate breakdown products are recognized as antifeeding compounds toward insects and recently also as intracellular signaling and bacteriostatic molecules in Arabidopsis. The data presented here indicate that these compounds also trigger local defense responses in Arabidopsis tissue. PMID:25371552

A Novel Method for Assessing Sex-Specific and Genotype-Specific Response to Injury in Astrocyte Culture

PubMed Central

Liu, Mingyue; Oyarzabal, Esteban; Yang, Rui; Murphy, Stephanie J; Hurn, Patricia D.

2008-01-01

Female astrocytes sustain less cell death from oxygen-glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1−3 day-old male and female, WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences. PMID:18436308

TPEN, a Specific Zn2+ Chelator, Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis, Glutamate Signaling, and Voltage-Gated K+ and Na+ Channels.

PubMed

Zhang, Feng; Ma, Xue-Ling; Wang, Yu-Xiang; He, Cong-Cong; Tian, Kun; Wang, Hong-Gang; An, Di; Heng, Bin; Xie, Lai-Hua; Liu, Yan-Qiang

2017-03-01

Hypoxia-ischemia-induced neuronal death is an important pathophysiological process that accompanies ischemic stroke and represents a major challenge in preventing ischemic stroke. To elucidate factors related to and a potential preventative mechanism of hypoxia-ischemia-induced neuronal death, primary neurons were exposed to sodium dithionite and glucose deprivation (SDGD) to mimic hypoxic-ischemic conditions. The effects of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn 2+ -chelating agent, on SDGD-induced neuronal death, glutamate signaling (including the free glutamate concentration and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (GluR2) and N-methyl-D-aspartate (NMDA) receptor subunits (NR2B), and voltage-dependent K + and Na + channel currents were also investigated. Our results demonstrated that TPEN significantly suppressed increases in cell death, apoptosis, neuronal glutamate release into the culture medium, NR2B protein expression, and I K as well as decreased GluR2 protein expression and Na + channel activity in primary cultured neurons exposed to SDGD. These results suggest that TPEN could inhibit SDGD-induced neuronal death by modulating apoptosis, glutamate signaling (via ligand-gated channels such as AMPA and NMDA receptors), and voltage-gated K + and Na + channels in neurons. Hence, Zn 2+ chelation might be a promising approach for counteracting the neuronal loss caused by transient global ischemia. Moreover, TPEN could represent a potential cell-targeted therapy.

The primary culture of mirror carp snout and caudal fin tissues and the isolation of Koi herpesvirus.

PubMed

Zhou, Jingxiang; Wang, Hao; Zhu, Xia; Li, Xingwei; Lv, Wenliang; Zhang, Dongming

2013-10-01

The explosive Koi herpesvirus (KHV) epidemic has caused the deaths of a large number of carp and carp variants and has produced serious economic losses. The mirror carp (Cyprinus carpio var. specularis) exhibits strong environmental adaptability and its primary cells can be used to isolate KHV. This study utilized the tissue explant method to systematically investigate primary cell culture conditions for mirror carp snout and caudal fin tissues. We demonstrated that cells from these two tissue types had strong adaptability, and when cultured in Medium 199 (M199) containing 20% serum at 26 to 30°C, the cells from the snout and caudal fin tissues exhibited the fastest egress and proliferation. Inoculation of these two cell types with KHV-infected fish kidney tissues produced typical cytopathic effects; additionally, identification by electron microscopy, and PCR indicated that KHV could be isolated from both cell types.

Comparison of Analysis and Quantification of Cell Death in vivo and in vitro

DTIC Science & Technology

1985-05-01

mammalian somatic cells appear to have a finite life span that is genetically programmed ( Hayflick , 1977). Following the consummation of this program... limited situations it is possible to evaluate the proliferation kinetics of cell populations in tis- sues by autoradiographically detecting radiolabeled...are, therefore, virtually limited to the analysis of toxicity of directly active chemicals. Primary cultures of target cells retain the ability to

Minocycline causes widespread cell death and increases microglial labeling in the neonatal mouse brain.

PubMed

Strahan, J Alex; Walker, William H; Montgomery, Taylor R; Forger, Nancy G

2017-06-01

Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase-3 (AC3) and ionized calcium-binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a > tenfold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex, septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3-P5. Minocycline treatment from P3 to P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose 1 week later (P10-P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial "inhibitor" increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 753-766, 2017. © 2016 Wiley Periodicals, Inc.

Minocycline Causes Widespread Cell Death and Increases Microglial Labeling in the Neonatal Mouse Brain

PubMed Central

Strahan, J. Alex; Walker, William H.; Montgomery, Taylor R.; Forger, Nancy G.

2016-01-01

Minocycline, an antibiotic of the tetracycline family, inhibits microglia in many paradigms, and is among the most commonly used tools for examining the role of microglia in physiological processes. Microglia may play an active role in triggering developmental neuronal cell death, although findings have been contradictory. To determine whether microglia influence developmental cell death, we treated perinatal mice with minocycline (45 mg/kg) and quantified effects on dying cells and microglial labeling using immunohistochemistry for activated caspase-3 (AC3) and ionized calcium-binding adapter molecule 1 (Iba1), respectively. Contrary to our expectations, minocycline treatment from embryonic day 18 to postnatal day (P)1 caused a >10-fold increase in cell death 8 h after the last injection in all brain regions examined, including the primary sensory cortex (S1), septum, hippocampus and hypothalamus. Iba1 labeling was also increased in most regions. Similar effects, although of smaller magnitude, were seen when treatment was delayed to P3-P5. Minocycline treatment from P3-P5 also decreased overall cell number in the septum at weaning, suggesting lasting effects of the neonatal exposure. When administered at lower doses (4.5 or 22.5 mg/kg), or at the same dose one week later (P10-P12), minocycline no longer increased microglial markers or cell death. Taken together, the most commonly used microglial “inhibitor” increases cell death and Iba1 labeling in the neonatal mouse brain. Minocycline is used clinically in infant and pediatric populations; caution is warrented when using minocycline in developing animals, or extrapolating the effects of this drug across ages. PMID:27706925

Recent advances in metastasis research.

PubMed

Molloy, Tim; van 't Veer, Laura J

2008-02-01

Advances in the early prediction, detection, and treatment of metastatic disease has improved the outlook in cancer patients in recent decades, however, metastasis remains the major cause of death in affected individuals. Metastasis occurs in a series of discreet biological steps in which a single, frequently clinically occult micrometastatic cell travels from the primary tumor to a distant location, where it lodges, grows, and ultimately results in the patient's death. Recent work has provided many new insights in the mechanisms and biology behind metastatic spread. This short review surveys some of the most important recent developments that have helped increase our understanding of the three broad phases of metastasis - the genesis of the metastatic cell through the loss of local constraints in the primary tumor microenvironment, dissemination of the cell to a distant organ while avoiding immune surveillance, and finally lodging and growth of the overt metastasis. These studies are providing mounting evidence that the interactions between tumor and normal cells and tissues are critical for disease progression - a paradigm that will provide a fertile area for future research.

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

PubMed Central

Mahul-Mellier, A-L; Vercruysse, F; Maco, B; Ait-Bouziad, N; De Roo, M; Muller, D; Lashuel, H A

2015-01-01

The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson's disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric β-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies. PMID:26138444

THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla

2011-01-07

Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown butmore » our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.« less

Vorinostat, a histone deacetylase (HDAC) inhibitor, promotes cell cycle arrest and re-sensitizes rituximab- and chemo-resistant lymphoma cells to chemotherapy agents.

PubMed

Xue, Kai; Gu, Juan J; Zhang, Qunling; Mavis, Cory; Hernandez-Ilizaliturri, Francisco J; Czuczman, Myron S; Guo, Ye

2016-02-01

Preclinical models of chemotherapy resistance and clinical observations derived from the prospective multicenter phase III collaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated that primary refractory/relapsed B cell diffuse large B cell lymphoma has a poor clinical outcome with current available second-line treatments. Preclinically, we found that rituximab resistance is associated with a deregulation on the mitochondrial potential rendering lymphoma cells resistant to chemotherapy-induced apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Posttranscriptional histone modification plays an important role in regulating gene transcription and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-proliferation activity in cancer cells. To this end, we studied the anti-proliferation activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, vorinostat) in rituximab-chemotherapy-resistant preclinical models. A panel of rituximab-chemotherapy-sensitive (RSCL) and rituximab-chemotherapy-resistant cell lines (RRCL) and primary tumor cells isolated from relapsed/refractory B cell lymphoma patients were exposed to escalating doses of vorinostat. Changes in mitochondrial potential, ATP synthesis, and cell cycle distribution were determined by Alamar blue reduction, Titer-Glo luminescent assays, and flow cytometric, respectively. Protein lysates were isolated from vorinostat-exposed cells, and changes in members of Bcl-2 family, cell cycle regulatory proteins, and the acetylation status of histone H3 were evaluated by Western blotting. Finally, cell lines were pre-exposed to vorinostat for 48 h and subsequently exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine); changes in cell viability were determined by CellTiter-Glo(®) luminescence assay (Promega, Fitchburg, WI), and synergistic activity was evaluated using the CalcuSyn software. Vorinostat induced dose-dependent cell death in RRCL and in primary tumor cells. In addition, in vitro exposure of RRCL to vorinostat resulted in an increase in p21 and acetylation of histone H3 leading to G1 cell cycle arrest. Vorinostat exposure resulted in apoptosis in RSCL cell lines but not in RRCL. This finding suggests that in RRCL, vorinostat induces cell death by alternative pathways (i.e., irreversible cell cycle arrest). Of interest, vorinostat was found to reverse acquired chemotherapy resistance in RRCL. Our data suggest that vorinostat is active in RRCL with a known defective apoptotic machinery, it can active alternative cell death pathways. Given the multiple pathways affected by HDAC inhibition, vorinostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B cell lymphoma.

Involvement of miR17 pathway in glucocorticoid-induced cell death in pediatric acute lymphoblastic leukemia.

PubMed

Harada, Masako; Pokrovskaja-Tamm, Katja; Söderhäll, Stefan; Heyman, Mats; Grander, Dan; Corcoran, Martin

2012-10-01

Analysis of the microRNA transcriptome following dexa- methasone treatment of the acute lymphocytic leukemia (ALL) cell line RS4;11 showed a global down-regulation of microRNA levels. MIR17HG was rapidly down-regulated following treatment, with chromatin immunoprecipitation (ChIP) analysis demonstrating the promoter to be a direct target of glucocorticoid (GC)-transcriptional repression and revealing the miR17-92 cluster as a prime target for dexamethasone-induced repression. The loss of miR17 family expression and concomitant increases in the miR17 target Bim occurred in an additional ALL cell line SUP-B15 but not in the dexamethasone-resistant REH. Alteration of miR17 levels through up-regulation or inhibition resulted in an decrease and increase, respectively, in Bim protein levels and dexamethasone-induced cell death. Primary ex vivo ALL cells that underwent apoptosis induced by dexamethasone also down-regulated miR17 levels. Thus, down-regulation of miR17 plays an important role in glucocorticoid-induced cell death suggesting that targeting miR17 may improve the current ALL combination therapy.

Clinicopathological parameters, recurrence, locoregional and distant metastasis in 115 T1-T2 oral squamous cell carcinoma patients

PubMed Central

2010-01-01

The incidence of oral squamous cell carcinoma remains high. Oral and oro-pharyngeal carcinomas are the sixth most common cancer in the world. Several clinicopathological parameters have been implicated in prognosis, recurrence and survival, following oral squamous cell carcinoma. In this retrospective analysis, clinicopathological parameters of 115 T1/T2 OSCC were studied and compared to recurrence and death from tumour-related causes. The study protocol was approved by the Joint UCL/UCLH committees of the ethics for human research. The patients' data was entered onto proformas, which were validated and checked by interval sampling. The fields included a range of clinical, operative and histopathological variables related to the status of the surgical margins. Data collection also included recurrence, cause of death, date of death and last clinic review. Causes of death were collated in 4 categories (1) death from locoregional spread, (2) death from distant metastasis, (3) death from bronchopulmonary pneumonia, and (4) death from any non-tumour event that lead to cardiorespiratory failure. The patients' population comprised 65 males and 50 females. Their mean age at the 1st diagnosis of OSCC was 61.7 years. Two-thirds of the patients were Caucasians. Primary sites were mainly identified in the tongue, floor of mouth (FOM), buccal mucosa and alveolus. Most of the identified OSCCs were low-risk (T1N0 and T2N0). All patients underwent primary resection ± neck dissection and reconstruction when necessary. Twenty-two patients needed adjuvant radiotherapy. Pathological analysis revealed that half of the patients had moderately differentiated OSCC. pTNM slightly differed from the cTNM and showed that 70.4% of the patients had low-risk OSCC. Tumour clearance was ultimately achieved in 107 patients. Follow-up resulted in a 3-year survival of 74.8% and a 5-year survival of 72.2%. Recurrence was identified in 23 males and 20 females. The mean age of 1st diagnosis of the recurrence group was 59.53 years. Most common oral sites included the lateral border of tongue and floor of mouth. Recurrence was associated with clinical N-stage disease. The surgical margins in this group was evaluated and found that 17 had non-cohesive invasion, 30 had dysplasia at margin, 21 had vascular invasion, 9 had nerve invasion and 3 had bony invasion. Severe dysplasia was present in 37 patients. Tumour clearance was achieved in only 8 patients. The mean depth of tumour invasion in the recurrence group was 7.6 mm. An interesting finding was that 5/11 patients who died of distant metastasis had their primary disease in the tongue. Nodal disease comparison showed that 8/10 patients who died of locoregional metastasis and 8/11 patients who died from distant metastasis had clinical nodal involvement. Comparing this to pathological nodal disease (pTNM) showed that 10/10 patients and 10/11 patients who died from locoregional and distant metastasis, respectively, had nodal disease. All patients who died from locoregional and distant metastasis were shown to have recurrence after the primary tumour resection. Squamous cell carcinoma of the oral cavity has a poor overall prognosis with a high tendency to recur at the primary site and extend to involve the cervical lymph nodes. Several clinicopathological parameters can be employed to assess outcome, recurrence and overall survival. PMID:20406474

Protective effect of T-type calcium channel blocker flunarizine on cisplatin-induced death of auditory cells.

PubMed

So, Hong-Seob; Park, Channy; Kim, Hyung-Jin; Lee, Jung-Han; Park, Sung-Yeol; Lee, Jai-Hyung; Lee, Zee-Won; Kim, Hyung-Min; Kalinec, Federico; Lim, David J; Park, Raekil

2005-06-01

Changes in intracellular Ca2+ level are involved in a number of intracellular events, including triggering of apoptosis. The role of intracellular calcium mobilization in cisplatin-induced hair cell death, however, is still unknown. In this study, the effect of calcium channel blocker flunarizine (Sibelium), which is used to prescribe for vertigo and tinnitus, on cisplatin-induced hair cell death was investigated in a cochlear organ of Corti-derived cell line, HEI-OC1, and the neonatal (P2) rat organ of Corti explant. Cisplatin induced apoptotic cell death showing nuclear fragmentation, DNA ladder, and TUNEL positive in both HEI-OC1 and primary organ of Corti explant. Flunarizine significantly inhibited the cisplatin-induced apoptosis. Unexpectedly, flunarizine increased the intracellular calcium ([Ca2+]i) levels of HEI-OC1. However, the protective effect of flunarizine against cisplatin was not mediated by modulation of intracellular calcium level. Treatment of cisplatin resulted in ROS generation and lipid peroxidation in HEI-OC1. Flunarizine did not attenuate ROS production but inhibited lipid peroxidation and mitochondrial permeability transition in cisplatin-treated cells. This result suggests that the protective mechanism of flunarizine on cisplatin-induced cytotoxicity is associated with direct inhibition of lipid peroxidation and mitochondrial permeability transition.

Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB*

PubMed Central

Manna, Sunil K.; Bose, Julie S.; Gangan, Vijay; Raviprakash, Nune; Navaneetha, Thota; Raghavendra, Pongali B.; Babajan, Banaganapalli; Kumar, Chitta S.; Jain, Swatantra K.

2010-01-01

The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor κB (NF-κB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-κB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-κB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells. PMID:20472557

Induction of apoptosis by Fe(salen)Cl through caspase-dependent pathway specifically in tumor cells.

PubMed

Pradhan, Nitika; Pratheek, B M; Garai, Antara; Kumar, Ashutosh; Meena, Vikram S; Ghosh, Shyamasree; Singh, Sujay; Kumari, Shikha; Chandrashekar, T K; Goswami, Chandan; Chattopadhyay, Subhasis; Kar, Sanjib; Maiti, Prasanta K

2014-10-01

Iron-based compounds possess the capability of inducing cell death due to their reactivity with oxidant molecules, but their specificity towards cancer cells and the mechanism of action are hitherto less investigated. A Fe(salen)Cl derivative has been synthesized that remains active in monomer form. The efficacy of this compound as an anti-tumor agent has been investigated in mouse and human leukemia cell lines. Fe(salen)Cl induces cell death specifically in tumor cells and not in primary cells. Mouse and human T-cell leukemia cell lines, EL4 and Jurkat cells are found to be susceptible to Fe(salen)Cl and undergo apoptosis, but normal mouse spleen cells and human peripheral blood mononuclear cells (PBMC) remain largely unaffected by Fe(salen)Cl. Fe(salen)Cl treated tumor cells show significantly higher expression level of cytochrome c that might have triggered the cascade of reactions leading to apoptosis in cancer cells. A significant loss of mitochondrial membrane potential upon Fe(salen)Cl treatment suggests that Fe(salen)Cl induces apoptosis by disrupting mitochondrial membrane potential and homeostasis, leading to cytotoxity. We also established that apoptosis in the Fe(salen)Cl-treated tumor cells is mediated through caspase-dependent pathway. This is the first report demonstrating that Fe(salen)Cl can specifically target the tumor cells, leaving the primary cells least affected, indicating an excellent potential for this compound to emerge as a next-generation anti-tumor drug. © 2014 International Federation for Cell Biology.

Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Yuchao; McGill, Mitchell R.; Dorko, Kenneth

Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5 mM, 10 mM or 20 mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24 h, which correlated with the clinicalmore » onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3 h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12 h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3 h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24 h and 48 h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 h after APAP and a partial protection when given at 15 h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic. - Highlights: • APAP reproducibly causes cell death in freshly isolated primary human hepatocytes. • APAP induces adduct formation, JNK activation and mitochondrial dysfunction in PHH. • Mitochondrial adducts and JNK translocation are delayed in PHH compared to mice. • JNK inhibitor SP600125 partially protects against APAP-induced cell death in PHH. • N-acetylcysteine provides significant protection even if administered 6 h after APAP.« less

Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression.

PubMed

Lanuza, Pilar M; Vigueras, Alan; Olivan, Sara; Prats, Anne C; Costas, Santiago; Llamazares, Guillermo; Sanchez-Martinez, Diego; Ayuso, José María; Fernandez, Luis; Ochoa, Ignacio; Pardo, Julián

2018-01-01

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.

Glutamate-mediated excitotoxicity in neonatal hippocampal neurons is mediated by mGluR-induced release of Ca++ from intracellular stores and is prevented by estradiol

PubMed Central

Hilton, Genell D.; Nunez, Joseph L.; Bambrick, Linda; Thompson, Scott M.; McCarthy, Margaret M.

2008-01-01

Hypoxic/ischemic (HI) brain injury in newborn full-term and premature infants is a common and pervasive source of life time disabilities in cognitive and locomotor function. In the adult, HI induces glutamate release and excitotoxic cell death dependent on NMDA receptor activation. In animal models of the premature human infant, glutamate is also released following HI, but neurons are largely insensitive to NMDA or AMPA/kainic acid (KA) receptor-mediated damage. Using primary cultured hippocampal neurons we have determined that glutamate increases intracellular calcium much more than kainic acid. Moreover, glutamate induces cell death by activating Type I metabotropic glutamate receptors (mGluRs). Pretreatment of neurons with the gonadal steroid estradiol reduces the level of the Type I metabotropic glutamate receptors and completely prevents cell death, suggesting a novel therapeutic approach to excitotoxic brain damage in the neonate. PMID:17156362

Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell-like spheroids, and tumor xenografts through multiple cell signaling pathways.

PubMed

Bijangi-Vishehsaraei, Khadijeh; Reza Saadatzadeh, M; Wang, Haiyan; Nguyen, Angie; Kamocka, Malgorzata M; Cai, Wenjing; Cohen-Gadol, Aaron A; Halum, Stacey L; Sarkaria, Jann N; Pollok, Karen E; Safa, Ahmad R

2017-12-01

OBJECTIVE Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)-a constituent of cruciferous vegetables with a multitargeted effect-as a therapeutic agent for GBM. METHODS The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models. RESULTS SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell-induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10. CONCLUSIONS These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.

Imaging Nuclear-Cytoplasmic Dynamics in Primary and Metastatic Colon Cancer in Nude Mice.

PubMed

Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Bouvet, Michael; Shimizu, Masahito; Hoffman, Robert M

2016-05-01

Colon cancer frequently results in metastasis to the liver, where it becomes the main cause of death. However, the cell cycle in primary tumors and metastases is poorly understood. We developed a mouse model of liver metastasis using the human colon cancer cell line HCT-116, which expresses green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP cells were injected into the spleen of nu/nu nude mice. HCT-116-GFP-RFP cells subsequently formed primary tumors in the spleen, as well as metastatic colonies in the liver and retroperitoneum by 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, it was possible to clearly image mitosis of the dual-colored colon cancer cells in the primary tumor as well as liver and other metastases. Multi-nucleate cancer cells, in addition to mono-nucleate cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nucleate HCT-116-GFP-RFP cells were also observed after culture of the primary and metastatic tumors. A similar ratio of mono-nucleate, multi-nucleate, and mitotic cells grew from the primary and metastatic tumors in culture, suggesting similarity of the nuclear-cytoplasmic dynamics of primary and metastatic cancer cells, further emphasizing the stochastic nature of metastasis. Our results demonstrate a similar heterogeneity of nuclear-cytoplasmic dynamics within primary tumors and metastases, which may be an important factor in the stochastic nature of metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Matrix regulation of skeletal cell apoptosis II: role of Arg-Gly-Asp-containing peptides.

PubMed

Perlot, Robert L; Shapiro, Irving M; Mansfield, Kyle; Adams, Christopher S

2002-01-01

This investigation was based on the assumption that arg-gly-asp (RGD)-containing peptides are released from the extracellular matrix of bone and cartilage during the remodeling cycle. We asked the question: Can RGD peptides influence skeletal cell viability? Primary human osteoblasts, mouse MC-3T3-E1 cells, and chick chondrocytes were incubated with purified RGD-containing peptides and cell viability was determined. The RGD peptide did not kill osteoblasts, chondrocytes, or MC-3T3-E1 cells. In contrast, RGDS and GRGDSP peptides killed all three cell types. Osteoblast death was quite rapid, occurring within 6 h of treatment. transferase uridyl mediated nick end labeling (TUNEL) and transmission electron microscopy (TEM) analysis indicated that death was mediated by apoptosis. To learn if mitochondria transduced the death signal, cells were treated with RGDS and organelle function was evaluated using a voltage-sensitive fluorescent probe. It was observed that there was no net loss of fluorescence and, hence, it was concluded that mitochondria were not the primary effectors of the apoptotic response. Experiments were performed with enzyme inhibitors to determine the import of the caspase pathway on RGDS-mediated osteoblast apoptosis. Results of these studies, as well as a study conducted using a fluorescent substrate, pointed to caspase 3 mediating the effector stage of the apoptotic process. Finally, using a purified labeled-RGDS peptide, we showed that the molecule was not restricted by the plasma membrane because it was accumulated in the cytosolic compartment. Results of the investigation support the view that resorption of the extracellular matrix generates peptide products that can induce apoptosis of vicinal cells.

JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas

PubMed Central

Weyerbrock, Astrid; Osterberg, Nadja; Psarras, Nikolaos; Baumer, Brunhilde; Kogias, Evangelos; Werres, Anna; Bette, Stefanie; Saavedra, Joseph E.; Keefer, Larry K.; Papazoglou, Anna

2011-01-01

Background Glutathione S-transferases (GSTs) control multidrug-resistance and are upregulated in many cancers including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy. Objective To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo. Methods U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis and other mechanisms. GST-expression was evaluated by immunocytochemistry, PCR and Western blot and NO release from JS-K using a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo. Results Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis which could be blocked by Z-VAD-FMK and Q-VD-OPH. GST-inhibition by sulfasalazine, cGMP inhibition by ODQ and MEK 1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signalling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was significantly reduced, with immunohistochemical evidence for increased necrosis, apoptosis and reduced proliferation. Conclusion Our data for the first time show the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas. PMID:21849924

JS-K, a glutathione S-transferase-activated nitric oxide donor with antineoplastic activity in malignant gliomas.

PubMed

Weyerbrock, Astrid; Osterberg, Nadja; Psarras, Nikolaos; Baumer, Brunhilde; Kogias, Evangelos; Werres, Anna; Bette, Stefanie; Saavedra, Joseph E; Keefer, Larry K; Papazoglou, Anna

2012-02-01

Glutathione S-transferases (GSTs) control multidrug resistance and are upregulated in many cancers, including malignant gliomas. The diazeniumdiolate JS-K generates nitric oxide (NO) on enzymatic activation by glutathione and GST, showing promising NO-based anticancer efficacy. To evaluate the role of NO-based antitumor therapy with JS-K in U87 gliomas in vitro and in vivo. U87 glioma cells and primary glioblastoma cell lines were exposed to JS-K and a variety of inhibitors to study cell death by necrosis, apoptosis, and other mechanisms. GST expression was evaluated by immunocytochemistry, polymerase chain reaction, and Western blot, and NO release from JS-K was studied with a NO assay. The growth-inhibitory effect of JS-K was studied in a U87 xenograft model in vivo. Dose-dependent inhibition of cell proliferation was observed in human U87 glioma cells and primary glioblastoma cells in vitro. Cell death was partially induced by caspase-dependent apoptosis, which could be blocked by Z-VAD-FMK and Q-VD-OPH. Inhibition of GST by sulfasalazine, cGMP inhibition by ODQ, and MEK1/2 inhibition by UO126 attenuated the antiproliferative effect of JS-K, suggesting the involvement of various intracellular death signaling pathways. Response to JS-K correlated with mRNA and protein expression of GST and the amount of NO released by the glioma cells. Growth of U87 xenografts was reduced significantly, with immunohistochemical evidence for increased necrosis and apoptosis and reduced proliferation. Our data show for the first time the potent antiproliferative effect of JS-K in gliomas in vitro and in vivo. These findings warrant further investigation of this novel NO-releasing prodrug in gliomas.

Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma

PubMed Central

Kumar, Sandeep; Ramakrishnan, Hariharasubramanian; Roy, Kaushambi; Viswanathan, Suresh; Bloomfield, Stewart A.

2017-01-01

The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma. PMID:28604388

Michelob_x is the missing inhibitor of apoptosis protein antagonist in mosquito genomes

PubMed Central

Zhou, Lei; Jiang, Guohua; Chan, Gina; Santos, Carl P; Severson, David W; Xiao, Lei

2005-01-01

Apoptosis is implicated in the life cycle of the malaria parasite in mosquitoes. The genome project for the primary malaria vector Anopheles gambiae showed a significant expansion of the inhibitor of apoptosis protein (IAP) and caspase gene families in comparison with Drosophila. However, because of extensive sequence divergence, no orthologue was identified for the reaper/grim-like IAP antagonist genes that have a pivotal role in cell death regulation in Drosophila. Using a customized searching strategy, we identified michelob_x(mx), a gene not predicted by the genome project, as the missing IAP antagonist in the An. gambiae and other mosquito genomes. Mx has a highly conserved amino-terminal IAP-binding motif. Expression of Mx induces rapid cell death in insect cell lines and is a potent tissue ablator in vivo. Its proapoptotic activity is totally dependent on the IAP-binding motif. Like reaper in Drosophila, mx is transcriptionally induced by ultraviolet irradiation to mediate cell death. PMID:16041319

Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms.

PubMed

Carson, Ross A; Rudine, Anthony C; Tally, Serena J; Franks, Alexis L; Frahm, Krystle A; Waldman, Jacob K; Silswal, Neerupma; Burale, Suban; Phan, James V; Chandran, Uma R; Monaghan, A Paula; DeFranco, Donald B

2018-01-01

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate in vitro. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.

Neuroprotective effect of rotigotine against complex I inhibitors, MPP⁺ and rotenone, in primary mesencephalic cell culture.

PubMed

Radad, Khaled; Scheller, Dieter; Rausch, Wolf-Dieter; Reichmann, Heinz; Gille, Gabrielle

2014-01-01

Dopamine agonists are suggested to be more efficacious in treating Parkinson's disease (PD) as they have neuroprotective properties in addition to their receptor-related actions. The present study was designed to investigate the neuroprotective effects of rotigotine, a D3/D2/D1 dopamine receptor agonist, against the two powerful complex I inhibitors, 1-methyl-4-phenylpyridinium (MPP+) and rotenone, in primary mesencephalic cell culture relevant to PD. Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala at gestation day 14. Three sets of cultures were treated with rotigotine alone, rotigotine and MPP⁺, and rotigotine and rotenone to investigate the effect of rotigotine on the survival of dopaminergic neurons against age-, MPP⁺- and rotenone-induced cell death. At the end of each treatment, cultures were fixed and stained immunohistochemically against tyrosine hydroxylase (TH). The effect of rotigotine against rotenone-induced reactive oxygen species (ROS) production was measured using CH-H2DCFDA fluorescence dye. Rotigotine alone did not influence the survival of tyrosine hydroxylase immunoreactive (THir) neurons except at 10 µM, it significantly decreased the number of THir neurons by 40% compared to untreated controls. Treatment of cultures with 0.01 µM rotigotine rescued 10% of THir neurons against MPP⁺-induced cell death. Rotigotine was also found to significantly rescue 20% of THir neurons at 0.01 µM of rotenone-treated cultures. Using of CH-H2DCFDA fluorescence dye, it was found that rotigotine significantly attenuated ROS production compared to rotenone-treated cultures. Rotigotine provides minor protection against MPP⁺ and rescues a significant number of THir neurons against rotenone in primary mesencephalic cell cultures relevant to PD.

α-Secretase-derived Fragment of Cellular Prion, N1, Protects against Monomeric and Oligomeric Amyloid β (Aβ)-associated Cell Death*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Ferreira, Sergio T.; Marzolo, Maria-Paz; Bauer, Charlotte; Thevenet, Aurélie; Checler, Frédéric

2012-01-01

In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrPc) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APPLDN) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APPLDN-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease. PMID:22184125

Genetically engineered oncolytic Newcastle disease virus mediates cytolysis of prostate cancer stem like cells.

PubMed

Raghunath, Shobana; Pudupakam, Raghavendra Sumanth; Allen, Adria; Biswas, Moanaro; Sriranganathan, Nammalwar

2017-10-20

Oncolytic virotherapy is a promising novel approach that overcomes the limitations posed by radiation and chemotherapy. In this study, the oncolytic efficacy of a recombinant Newcastle disease virus (rNDV) BC-KLQL-GFP, against prostate cancer stem-like/tumor initiating cells was evaluated. Xenograft derived prostaspheres (XPS) induced tumor more efficiently than monolayer cell derived prostaspheres (MCPS) in nude mice. Primary and secondary XPS show enhanced self-renewal and clonogenic potential compared to MCPS. XPS also expressed embryonic stem cell markers, such as Nanog, CD44 and Nestin. Further, prostate specific antigen (PSA) activated recombinant Newcastle Disease Virus (rNDV) was selectively cytotoxic to tumor derived DU145 prostaspheres. An effective concentration (EC 50 ) of 0.11-0.14 multiplicity of infection was sufficient to cause prostasphere cell death in serum free culture. DU145 tumor xenograft derived prostaspheres were used as tumor surrogates as they were enriched for a putative tumor initiating cell population. PSA activated rNDV was efficient in inducing cell death of cells and prostaspheres derived from primary xenografts ex-vivo, thus signifying a potential in vivo efficacy. The EC 50 (∼0.1 MOI) for cytolysis of tumor initiating cells was slightly higher than that was required for the parental cell line, but within the therapeutic margin for safety and efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

Induction of Cell Death through Alteration of Oxidants and Antioxidants in Epithelial Cells Exposed to High Energy Protons

NASA Technical Reports Server (NTRS)

Ramesh, Govindarajan; Wu, Honglu

2012-01-01

Radiation affects several cellular and molecular processes including double strand breakage, modifications of sugar moieties and bases. In outer space, protons are the primary radiation source which poses a range of potential health risks to astronauts. On the other hand, the use of proton radiation for tumor radiation therapy is increasing as it largely spares healthy tissues while killing tumor tissues. Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. Therefore, in the present study, we irradiated rat epithelial cells (LE) with different doses of protons and investigated their effects on cell proliferation and cell death. Our data showed an inhibition of cell proliferation in proton irradiated cells with a significant dose dependent activation and repression of reactive oxygen species (ROS) and antioxidants, glutathione and superoxide dismutase respectively as compared to control cells. In addition, apoptotic related genes such as caspase-3 and -8 activities were induced in a dose dependent manner with corresponding increased levels of DNA fragmentation in proton irradiated cells than control cells. Together, our results show that proton radiation alters oxidant and antioxidant levels in the cells to activate apoptotic pathway for cell death.

Causes of death in long-term lung cancer survivors: a SEER database analysis.

PubMed

Abdel-Rahman, Omar

2017-07-01

Long-term (>5 years) lung cancer survivors represent a small but distinct subgroup of lung cancer patients and information about the causes of death of this subgroup is scarce. The Surveillance, Epidemiology and End Results (SEER) database (1988-2008) was utilized to determine the causes of death of long-term survivors of lung cancer. Survival analysis was conducted using Kaplan-Meier analysis and multivariate analysis was conducted using a Cox proportional hazard model. Clinicopathological characteristics and survival outcomes were assessed for the whole cohort. A total of 78,701 lung cancer patients with >5 years survival were identified. This cohort included 54,488 patients surviving 5-10 years and 24,213 patients surviving >10 years. Among patients surviving 5-10 years, 21.8% were dead because of primary lung cancer, 10.2% were dead because of other cancers, 6.8% were dead because of cardiac disease and 5.3% were dead because of non-malignant pulmonary disease. Among patients surviving >10 years, 12% were dead because of primary lung cancer, 6% were dead because of other cancers, 6.9% were dead because of cardiac disease and 5.6% were dead because of non-malignant pulmonary disease. On multivariate analysis, factors associated with longer cardiac-disease-specific survival in multivariate analysis include younger age at diagnosis (p < .0001), white race (vs. African American race) (p = .005), female gender (p < .0001), right-sided disease (p = .003), adenocarcinoma (vs. large cell or small cell carcinoma), histology and receiving local treatment by surgery rather than radiotherapy (p < .0001). The probability of death from primary lung cancer is still significant among other causes of death even 20 years after diagnosis of lung cancer. Moreover, cardiac as well as non-malignant pulmonary causes contribute a considerable proportion of deaths in long-term lung cancer survivors.

E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

PubMed Central

Hatchi, Elodie; Rodier, Genevieve; Lacroix, Matthieu; Caramel, Julie; Kirsh, Olivier; Jacquet, Chantal; Schrepfer, Emilie; Lagarrigue, Sylviane; Linares, Laetitia Karine; Lledo, Gwendaline; Tondeur, Sylvie; Dubus, Pierre

2011-01-01

The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA–mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest. PMID:21708927

Changing ethnic disparity in ischemic stroke mortality in US children after the STOP trial.

PubMed

Lehman, Laura L; Fullerton, Heather J

2013-08-01

A prior report showed higher stroke mortality in US black children compared with white children (1979-1998), a disparity likely due in part to sickle cell disease, which leads to a high risk of childhood ischemic stroke. We hypothesized that this disparity has diminished since the publication of the Stroke Prevention Trial in Sickle Cell Anemia (STOP trial) in 1998 demonstrating the efficacy of long-term blood transfusions for primary stroke prevention. To evaluate the demographics and secular trends in mortality from ischemic and hemorrhagic stroke (as a primary cause of death) in US children (<20 years) and determine if there has been a decrease in the disparity between white and black children since the publication of the STOP trial in 1998. We used death certificate data from the National Center for Health Statistics, 1988 through 2007. United States. Children who died in 1988 through 2007 in the United States. Publication of the STOP trial. Incidence rate ratios were calculated as the measure of relative risk. Among 1.6 billion person-years of US children (1988-2007), there were 4425 deaths attributed to stroke, yielding an average of 221 deaths per year; 20% were ischemic; 67%, hemorrhagic; and 12%, unspecified. The relative risk of ischemic stroke mortality for black vs white children dropped from 1.74 from 1988 through 1997 to 1.27 from 1998 through 2007. The ethnic disparity in hemorrhagic stroke mortality, however, remained relatively stable between these 2 periods: black vs white relative risk, 1.90 (1988-1997) and 1.97 (1998-2007). The excess risk of death from ischemic, but not hemorrhagic, stroke in US black children has decreased over the past decade. This may be related to the implementation of an effective ischemic stroke prevention strategy for children with sickle cell disease.

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans

PubMed Central

Sun, C-L; Kim, E; Crowder, C M

2014-01-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2–3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death. PMID:24317200

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans.

PubMed

Sun, C-L; Kim, E; Crowder, C M

2014-04-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohammad, Mohammad K.; Alcohol Research Center, University of Louisville; Avila, Diana

2012-11-15

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidantmore » capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. -- Highlights: ► Human primary hepatocytes and cultured cell lines are used. ► Multiple cell death signaling pathways are activated by acrolein. ► Novel finding of acrolein-induced ER stress ► Acrolein fails to activate ER stress-induced protective responses. ► Combinatorial therapies may be needed for preventing acrolein hepatotoxicity.« less

Enhancement of endocannabinoid signaling protects against cocaine-induced neurotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vilela, Luciano R.; Gobira, Pedro H.; Viana, Thercia G.

Cocaine is an addictive substance with a potential to cause deleterious effects in the brain. The strategies for treating its neurotoxicity, however, are limited. Evidence suggests that the endocannabinoid system exerts neuroprotective functions against various stimuli. Thus, we hypothesized that inhibition of fatty acid amide hydrolase (FAAH), the main enzyme responsible for terminating the actions of the endocannabinoid anandamide, reduces seizures and cell death in the hippocampus in a model of cocaine intoxication. Male Swiss mice received injections of endocannabinoid-related compounds followed by the lowest dose of cocaine that induces seizures, electroencephalographic activity and cell death in the hippocampus. Themore » molecular mechanisms were studied in primary cell culture of this structure. The FAAH inhibitor, URB597, reduced cocaine-induced seizures and epileptiform electroencephalographic activity. The cannabinoid CB{sub 1} receptor selective agonist, ACEA, mimicked these effects, whereas the antagonist, AM251, prevented them. URB597 also inhibited cocaine-induced activation and death of hippocampal neurons, both in animals and in primary cell culture. Finally, we investigated if the PI3K/Akt/ERK intracellular pathway, a cell surviving mechanism coupled to CB{sub 1} receptor, mediated these neuroprotective effects. Accordingly, URB597 injection increased ERK and Akt phosphorylation in the hippocampus. Moreover, the neuroprotective effect of this compound was reversed by the PI3K inhibitor, LY294002. In conclusion, the pharmacological facilitation of the anandamide/CB1/PI3K signaling protects the brain against cocaine intoxication in experimental models. This strategy may be further explored in the development of treatments for drug-induced neurotoxicity. - Highlights: • Cocaine toxicity is characterized by seizures and hippocampal cell death. • The endocannabinoid anandamide acts as a brain protective mechanism. • Inhibition of anandamide hydrolysis attenuates cocaine neurotoxicity. • This effect is mediated by the cannabinoid CB1 receptor/PI3K pathway.« less

Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Shiguang; Mao, Li; Ji, Feng, E-mail: huaiaifengjidr@163.com

Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the othermore » hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.« less

Agmatine Attenuates Brain Edema and Apoptotic Cell Death after Traumatic Brain Injury.

PubMed

Kim, Jae Young; Lee, Yong Woo; Kim, Jae Hwan; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

2015-07-01

Traumatic brain injury (TBI) is associated with poor neurological outcome, including necrosis and brain edema. In this study, we investigated whether agmatine treatment reduces edema and apoptotic cell death after TBI. TBI was produced by cold injury to the cerebral primary motor cortex of rats. Agmatine was administered 30 min after injury and once daily until the end of the experiment. Animals were sacrificed for analysis at 1, 2, or 7 days after the injury. Various neurological analyses were performed to investigate disruption of the blood-brain barrier (BBB) and neurological dysfunction after TBI. To examine the extent of brain edema after TBI, the expression of aquaporins (AQPs), phosphorylation of mitogen-activated protein kinases (MAPKs), and nuclear translocation of nuclear factor-κB (NF-κB) were investigated. Our findings demonstrated that agmatine treatment significantly reduces brain edema after TBI by suppressing the expression of AQP1, 4, and 9. In addition, agmatine treatment significantly reduced apoptotic cell death by suppressing the phosphorylation of MAPKs and by increasing the nuclear translocation of NF-κB after TBI. These results suggest that agmatine treatment may have therapeutic potential for brain edema and neural cell death in various central nervous system diseases.

Interplay between autophagy and apoptosis in lead(II)-induced cytotoxicity of primary rat proximal tubular cells.

PubMed

Chu, Bing-Xin; Fan, Rui-Feng; Lin, Shu-Qian; Yang, Du-Bao; Wang, Zhen-Yong; Wang, Lin

2018-05-01

Autophagy and apoptosis are two different biological processes that determine cell fates. We previously reported that autophagy inhibition and apoptosis induction are involved in lead(II)-induced cytotoxicity in primary rat proximal tubular (rPT) cells, but the interplay between them remains to be elucidated. Firstly, data showed that lead(II)-induced elevation of LC3-II protein levels can be significantly modulated by 3-methyladenine or rapamycin; moreover, protein levels of Autophagy-related protein 5 (Atg5) and Beclin-1 were markedly up-regulated by lead(II) treatment, demonstrating that lead(II) could promote the autophagosomes formation in rPT cells. Next, we applied three pharmacological agents and genetic method targeting the early stage of autophagy to validate that enhancement of autophagosomes formation can inhibit lead(II)-induced apoptotic cell death in rPT cells. Simultaneously, lead(II) inhibited the autophagic degradation of rPT cells, while the addition of autophagic degradation inhibitor bafilomycin A1 aggravated lead(II)-induced apoptotic death in rPT cells. Collectively, this study provided us a good model to know about the dynamic process of lead(II)-induced autophagy in rPT cells, and the interplay between autophagy and apoptosis highlights a new sight into the mechanism of lead(II)-induced nephrotoxicity. Copyright © 2018. Published by Elsevier Inc.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

PubMed

Welsbie, Derek S; Mitchell, Katherine L; Jaskula-Ranga, Vinod; Sluch, Valentin M; Yang, Zhiyong; Kim, Jessica; Buehler, Eugen; Patel, Amit; Martin, Scott E; Zhang, Ping-Wu; Ge, Yan; Duan, Yukan; Fuller, John; Kim, Byung-Jin; Hamed, Eman; Chamling, Xitiz; Lei, Lei; Fraser, Iain D C; Ronai, Ze'ev A; Berlinicke, Cynthia A; Zack, Donald J

2017-06-21

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Review: circulating tumor cells in the practice of breast cancer oncology.

PubMed

Ramos-Medina, R; Moreno, F; Lopez-Tarruella, S; Del Monte-Millán, M; Márquez-Rodas, I; Durán, E; Jerez, Y; Garcia-Saenz, J A; Ocaña, I; Andrés, S; Massarrah, T; González-Rivera, M; Martin, M

2016-08-01

The primary cause of tumor-related death in breast cancer is still represented by distant metastasization. The dissemination of tumor cells from the primary tumor to distant sites through bloodstream cannot be early detected by standard imaging methods. Circulating tumor cells (CTCs) play a major role in the metastatic spread of breast cancer. Different analytical systems for CTCs isolation and detection have been developed and novel areas of research are directed towards developing assays for CTCs molecular characterization. This review describes the current state of art on CTCs detection techniques and the present and future clinical implications of CTCs enumeration and characterization.

Pretreatment CD4 Cell Slope and Progression to AIDS or Death in HIV-Infected Patients Initiating Antiretroviral Therapy—The CASCADE Collaboration: A Collaboration of 23 Cohort Studies

PubMed Central

Wolbers, Marcel; Babiker, Abdel; Sabin, Caroline; Young, Jim; Dorrucci, Maria; Chêne, Geneviève; Mussini, Cristina; Porter, Kholoud; Bucher, Heiner C.

2010-01-01

Background CD4 cell count is a strong predictor of the subsequent risk of AIDS or death in HIV-infected patients initiating combination antiretroviral therapy (cART). It is not known whether the rate of CD4 cell decline prior to therapy is related to prognosis and should, therefore, influence the decision on when to initiate cART. Methods and Findings We carried out survival analyses of patients from the 23 cohorts of the CASCADE (Concerted Action on SeroConversion to AIDS and Death in Europe) collaboration with a known date of HIV seroconversion and with at least two CD4 measurements prior to initiating cART. For each patient, a pre-cART CD4 slope was estimated using a linear mixed effects model. Our primary outcome was time from initiating cART to a first new AIDS event or death. We included 2,820 treatment-naïve patients initiating cART with a median (interquartile range) pre-cART CD4 cell decline of 61 (46–81) cells/µl per year; 255 patients subsequently experienced a new AIDS event or death and 125 patients died. In an analysis adjusted for established risk factors, the hazard ratio for AIDS or death was 1.01 (95% confidence interval 0.97–1.04) for each 10 cells/µl per year reduction in pre-cART CD4 cell decline. There was also no association between pre-cART CD4 cell slope and survival. Alternative estimates of CD4 cell slope gave similar results. In 1,731 AIDS-free patients with >350 CD4 cells/µl from the pre-cART era, the rate of CD4 cell decline was also not significantly associated with progression to AIDS or death (hazard ratio 0.99, 95% confidence interval 0.94–1.03, for each 10 cells/µl per year reduction in CD4 cell decline). Conclusions The CD4 cell slope does not improve the prediction of clinical outcome in patients with a CD4 cell count above 350 cells/µl. Knowledge of the current CD4 cell count is sufficient when deciding whether to initiate cART in asymptomatic patients. Please see later in the article for the Editors' Summary PMID:20186270

Role of Caspase-8 and Fas in Cell Death After Spinal Cord Injury

PubMed Central

Sobrido-Cameán, Daniel; Barreiro-Iglesias, Antón

2018-01-01

Spinal cord injury (SCI) causes the death of neurons and glial cells due to the initial mechanical forces (i.e., primary injury) and through a cascade of secondary molecular events (e.g., inflammation or excitotoxicity) that exacerbate cell death. The loss of neurons and glial cells that are not replaced after the injury is one of the main causes of disability after SCI. Evidence accumulated in last decades has shown that the activation of apoptotic mechanisms is one of the factors causing the death of intrinsic spinal cord (SC) cells following SCI. Although this is not as clear for brain descending neurons, some studies have also shown that apoptosis can be activated in the brain following SCI. There are two main apoptotic pathways, the extrinsic and the intrinsic pathways. Activation of caspase-8 is an important step in the initiation of the extrinsic pathway. Studies in rodents have shown that caspase-8 is activated in SC glial cells and neurons and that the Fas receptor plays a key role in its activation following a traumatic SCI. Recent work in the lamprey model of SCI has also shown the retrograde activation of caspase-8 in brain descending neurons following SCI. Here, we review our current knowledge on the role of caspase-8 and the Fas pathway in cell death following SCI. We also provide a perspective for future work on this process, like the importance of studying the possible contribution of Fas/caspase-8 signaling in the degeneration of brain neurons after SCI in mammals. PMID:29666570

Prevention of Excitotoxicity in Primary Retinal Ganglion Cells by (+)-Pentazocine, a Sigma Receptor-1-Specific Ligand

PubMed Central

Dun, Ying; Thangaraju, Muthusamy; Prasad, Puttur; Ganapathy, Vadivel; Smith, Sylvia B.

2013-01-01

Purpose σRs are non-opioid, non-phencyclidine binding sites with robust neuroprotective properties. Previously, we induced death in the RGC-5 cell line using very high concentrations (1 mM) of the excitatory amino acids glutamate (Glu) and homocysteine (Hcy) and demonstrated that the σR1 ligand (+)-pentazocine ((+)-PTZ) could protect against cell death. The purpose of the present study was to establish a physiologically relevant paradigm for testing the neuroprotective effect of (+)-PTZ in retinal ganglion cells. Methods Primary ganglion cells (1°GCs) were isolated by immunopanning from retinas of 1-day-old mice, maintained in culture for 3 days and then exposed to 10, 20, 25 or 50 µM Glu or 10, 25, 50 or 100 µM Hcy for 6 or 18 h in the presence or absence of (+)-PTZ (0.5, 1, 3 µM). Cell viability was measured using the Live/Dead and ApopTag Fluorescein In Situ Assays. Expression of σR1 was assessed by immunocytochemistry, RT-PCR and western blotting. Morphological appearance of live ganglion cells and their processes was examined over time (0, 3, 6, 18 h) by differential interference contrast (DIC) microscopy following exposure to excitotoxins in the presence or absence of (+)-PTZ. Results 1°GCs showed robust σR1 expression. The cells are exquisitely sensitive to Glu or Hcy toxicity (6 h treatment with 25 or 50 µM Glu or 50 or 100 µM Hcy induced marked cell death). 1°GCs pre-treated 1 h with (+)-PTZ followed by 18 h co-treatment with 25 µM Glu and (+)-PTZ showed a marked decrease in cell death: (25 µM Glu alone: 50%; 25 µM Glu/0.5 µM (+)-PTZ: 38%; 25 µM Glu/1 µM (+)-PTZ: 20%; 25 µM Glu/3 µM (+)-PTZ: 18%). Similar results were obtained with Hcy. σR1 mRNA and protein levels did not change in the presence of the excitotoxins. DIC examination of cells exposed to excitotoxins revealed substantial disruption of neuronal processes; co-treatment with (+)-PTZ revealed marked preservation of these processes. The stereoselective effect of (+)-PTZ for σR1 was established in experiments in which (−)-PTZ, the levo-isomer form of pentazocine, had no neuroprotective effect on excitotoxin-induced ganglion cell death. Conclusions 1°GCs express σR1; their marked sensitivity to Glu and Hcy toxicity mimics the sensitivity observed in vivo, making them a highly relevant model for testing neuroprotection. Pre-treatment of cells with 1–3 µM (+)-PTZ, but not (−)-PTZ affords significant protection against Glu- and Hcy-induced cell death. σR1 ligands may be very useful therapeutic agents in retinal diseases in which ganglion cells die. PMID:17898305

Lack of T-cell receptor-induced signaling is crucial for CD95 ligand up-regulation and protects cutaneous T-cell lymphoma cells from activation-induced cell death.

PubMed

Klemke, Claus-Detlev; Brenner, Dirk; Weiss, Eva-Maria; Schmidt, Marc; Leverkus, Martin; Gülow, Karsten; Krammer, Peter H

2009-05-15

Restimulation of previously activated T cells via the T-cell receptor (TCR) leads to activation-induced cell death (AICD), which is, at least in part, dependent on the death receptor CD95 (APO-1, FAS) and its natural ligand (CD95L). Here, we characterize cutaneous T-cell lymphoma (CTCL) cells (CTCL tumor cell lines and primary CTCL tumor cells from CTCL patients) as AICD resistant. We show that CTCL cells have elevated levels of the CD95-inhibitory protein cFLIP. However, cFLIP is not responsible for CTCL AICD resistance. Instead, our data suggest that reduced TCR-proximal signaling in CTCL cells is responsible for the observed AICD resistance. CTCL cells exhibit no PLC-gamma1 activity, resulting in an impaired Ca(2+)release and reduced generation of reactive oxygen species upon TCR stimulation. Ca(2+) and ROS production are crucial for up-regulation of CD95L and reconstitution of both signals resulted in AICD sensitivity of CTCL cells. In accordance with these data, CTCL tumor cells from patients with Sézary syndrome do not up-regulate CD95L upon TCR-stimulation and are therefore resistant to AICD. These results show a novel mechanism of AICD resistance in CTCL that could have future therapeutic implications to overcome apoptosis resistance in CTCL patients.

Lamin A/C deficiency reduces circulating tumor cell resistance to fluid shear stress

PubMed Central

Denais, Celine; Chan, Maxine F.; Wang, Zhexiao; Lammerding, Jan

2015-01-01

Metastasis contributes to over 90% of cancer-related deaths and is initiated when cancer cells detach from the primary tumor, invade the basement membrane, and enter the circulation as circulating tumor cells (CTCs). While metastasis is viewed as an inefficient process with most CTCs dying within the bloodstream, it is evident that some CTCs are capable of resisting hemodynamic shear forces to form secondary tumors in distant tissues. We hypothesized that nuclear lamins A and C (A/C) act as key structural components within CTCs necessary to resist destruction from elevated shear forces of the bloodstream. Herein, we show that, compared with nonmalignant epithelial cells, tumor cells are resistant to elevated fluid shear forces in vitro that mimic those within the bloodstream, as evidenced by significant decreases in cellular apoptosis and necrosis. Knockdown of lamin A/C significantly reduced tumor cell resistance to fluid shear stress, with significantly increased cell death compared with parental tumor cell and nontargeting controls. Interestingly, lamin A/C knockdown increased shear stress-induced tumor cell apoptosis, but did not significantly affect cellular necrosis. These data demonstrate that lamin A/C is an important structural component that enables tumor cell resistance to fluid shear stress-mediated death in the bloodstream, and may thus facilitate survival and hematogenous metastasis of CTCs. PMID:26447202

Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

PubMed

Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

2008-03-01

The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

The ability of walnut extract and fatty acids to protect against the deleterious effects of oxidative stress and inflammation in hippocampal cells.

PubMed

Carey, Amanda N; Fisher, Derek R; Joseph, James A; Shukitt-Hale, Barbara

2013-01-01

Previous research from our lab has demonstrated that dietary walnut supplementation protects against age-related cognitive declines in rats; however, the cellular mechanisms by which walnuts and polyunsaturated fatty acids (PUFAs) may affect neuronal health and functioning in aging are undetermined. We assessed if pretreatment of primary hippocampal neurons with walnut extract or PUFAs would protect cells against dopamine- and lipopolysaccharide-mediated cell death and calcium dysregulation. Rat primary hippocampal neurons were pretreated with varying concentrations of walnut extract, linoleic acid, alpha-linolenic acid, eicosapentaenoic acid, or docosahexaenoic acid prior to exposure to either dopamine or lipopolysaccharide. Viability was assessed using the Live/Dead Cellular Viability/Cytotoxicity Kit. Also, the ability of the cells to return to baseline calcium levels after depolarization was measured with fluorescent imaging. Results indicated that walnut extract, alpha-linolenic acid, and docosahexaenoic acid provided significant protection against cell death and calcium dysregulation; the effects were pretreatment concentration dependent and stressor dependent. Linoleic acid and eicosapentaenoic acid were not as effective at protecting hippocampal cells from these insults. Walnut extract and omega-3 fatty acids may protect against age-related cellular dysfunction, but not all PUFAs are equivalent in their beneficial effects.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress

PubMed Central

Sriskanthadevan, Shrivani; Jeyaraju, Danny V.; Chung, Timothy E.; Prabha, Swayam; Xu, Wei; Skrtic, Marko; Jhas, Bozhena; Hurren, Rose; Gronda, Marcela; Wang, Xiaoming; Jitkova, Yulia; Sukhai, Mahadeo A.; Lin, Feng-Hsu; Maclean, Neil; Laister, Rob; Goard, Carolyn A.; Mullen, Peter J.; Xie, Stephanie; Penn, Linda Z.; Rogers, Ian M.; Dick, John E.; Minden, Mark D.

2015-01-01

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy. PMID:25631767

Induction of apoptosis by HBI-8000 in adult T-cell leukemia/lymphoma is associated with activation of Bim and NLRP3.

PubMed

Hasegawa, Hiroo; Bissonnette, Reid P; Gillings, Mireille; Sasaki, Daisuke; Taniguchi, Hiroaki; Kitanosono, Hideaki; Tsuruda, Kazuto; Kosai, Kousuke; Uno, Naoki; Morinaga, Yoshitomo; Imaizumi, Yoshitaka; Miyazaki, Yasushi; Yanagihara, Katsunori

2016-08-01

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti-chemokine (C-C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI-8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T-cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI-8000 on ATL-derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI-8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI-8000-induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI-8000-induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI-8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI-8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

PubMed Central

2011-01-01

Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

Reactive oxygen species trigger motoneuron death in non-cell-autonomous models of ALS through activation of c-Abl signaling.

PubMed

Rojas, Fabiola; Gonzalez, David; Cortes, Nicole; Ampuero, Estibaliz; Hernández, Diego E; Fritz, Elsa; Abarzua, Sebastián; Martinez, Alexis; Elorza, Alvaro A; Alvarez, Alejandra; Court, Felipe; van Zundert, Brigitte

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1(G93A) (ACM-hSOD1(G93A)) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1(G93A), but not ACM-hSOD1(WT), increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1(G93A)-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1(G86R) or TDP43(A315T). We further find that co-application of ACM-SOD1(G93A) with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1(G93A) induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.

Wood combustion particles induce adverse effects to normal and diseased airway epithelia.

PubMed

Krapf, Manuel; Künzi, Lisa; Allenbach, Sandrine; Bruns, Emily A; Gavarini, Ilaria; El-Haddad, Imad; Slowik, Jay G; Prévôt, André S H; Drinovec, Luka; Močnik, Griša; Dümbgen, Lutz; Salathe, Matthias; Baumlin, Nathalie; Sioutas, Constantinos; Baltensperger, Urs; Dommen, Josef; Geiser, Marianne

2017-04-19

Residential wood burning is a major source of poorly characterized, deleterious particulate matter, whose composition and toxicity may vary with wood type, burning condition and photochemical age. The causative link between ambient wood particle constituents and observed adverse health effects is currently lacking. Here we investigate the relationship between chemical properties of primary and atmospherically aged wood combustion particles and acute toxicity in human airway epithelial cells. Emissions from a log wood burner were diluted and injected into a smog chamber for photochemical aging. After concentration-enrichment and removal of oxidizing gases, directly emitted and atmospherically aged particles were deposited on cell cultures at the air-liquid interface for 2 hours in an aerosol deposition chamber mimicking physiological conditions in lungs. Cell models were fully differentiated normal and diseased (cystic fibrosis and asthma) human bronchial epithelia (HBE) and the bronchial epithelial cell line BEAS-2B. Cell responses were assessed at 24 hours after aerosol exposure. Atmospherically relevant doses of wood combustion particles significantly increased cell death in all but the asthma cell model. Expression of oxidative stress markers increased in HBE from all donors. Increased cell death and inflammatory responses could not be assigned to a single chemical fraction of the particles. Exposure to primary and aged wood combustion particles caused adverse effects to airway epithelia, apparently induced by several interacting components.

Activation of AMPK and inactivation of Akt result in suppression of mTOR-mediated S6K1 and 4E-BP1 pathways leading to neuronal cell death in in vitro models of Parkinson’s disease

PubMed Central

Chen, Sujuan; Ye, Yangjing; Guo, Min; Ren, Qian; Liu, Lei; Zhang, Hai; Xu, Chong; Zhou, Qian; Huang, Shile; Chen, Long

2014-01-01

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Dysregulation of mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of PD. However, the underlying mechanism is incompletely elucidated. Here, we show that PD mimetics (6-hydroxydopamine, N-methyl-4-phenylpyridine or rotenone) suppressed phosphorylation of mTOR, S6K1 and 4E-BP1, reduced cell viability, and activated caspase-3 and PARP in PC12 cells and primary neurons. Overexpression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 in PC12 cells partially prevented cell death in response to the PD toxins, revealing that mTOR-mediated S6K1 and 4E-BP1 pathways due to the PD toxins were inhibited, leading to neuronal cell death. Furthermore, we found that the inhibition of mTOR signaling contributing to neuronal cell death was attributed to suppression of Akt and activation of AMPK. This is supported by the findings that ectopic expression of constitutively active Akt or dominant negative AMPKα, or inhibition of AMPKα with compound C partially attenuated inhibition of phosphorylation of mTOR, S6K1 and 4E-BP1, activation of caspase-3, and neuronal cell death triggered by the PD toxins. The results indicate that PD stresses activate AMPK and inactivate Akt, causing neuronal cell death via inhibiting mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that proper co-manipulation of AMPK/Akt/mTOR signaling may be a potential strategy for prevention and treatment of PD. PMID:24726895

Cancer terminator viruses (CTV): A better solution for viral-based therapy of cancer.

PubMed

Emdad, Luni; Das, Swadesh K; Wang, Xiang-Yang; Sarkar, Devanand; Fisher, Paul B

2018-08-01

In principle, viral gene therapy holds significant potential for the therapy of solid cancers. However, this promise has not been fully realized and systemic administration of viruses has not proven as successful as envisioned in the clinical arena. Our research is focused on developing the next generation of efficacious viruses to specifically treat both primary cancers and a major cause of cancer lethality, metastatic tumors (that have spread from a primary site of origin to other areas in the body and are responsible for an estimated 90% of cancer deaths). We have generated a chimeric tropism-modified type 5 and 3 adenovirus that selectively replicates in cancer cells and simultaneously produces a secreted anti-cancer toxic cytokine, melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24), referred to as a Cancer Terminator Virus (CTV) (Ad.5/3-CTV). In preclinical animal models, injection into a primary tumor causes selective cell death and therapeutic activity is also observed in non-injected distant tumors, that is, "bystander anti-tumor activity." To enhance the impact and therapeutic utility of the CTV, we have pioneered an elegant approach in which viruses are encapsulated in microbubbles allowing "stealth delivery" to tumor cells that when treated with focused ultrasound causes viral release killing tumor cells through viral replication, and producing and secreting MDA-7/IL-24, which stimulates the immune system to attack distant cancers, inhibits tumor angiogenesis and directly promotes apoptosis in distant cancer cells. This strategy is called UTMD (ultrasound-targeted microbubble-destruction). This novel CTV and UTMD approach hold significant promise for the effective therapy of primary and disseminated tumors. © 2017 Wiley Periodicals, Inc.

Neuroprotective effect of α-mangostin and curcumin against iodoacetate-induced cell death.

PubMed

Reyes-Fermín, Laura María; González-Reyes, Susana; Tarco-Álvarez, Nadia Gabriela; Hernández-Nava, Marisol; Orozco-Ibarra, Marisol; Pedraza-Chaverri, José

2012-09-01

Curcumin is a phenolic yellow curry pigment with anti-inflammatory and antioxidant activities and α-mangostin is a xanthone isolated from mangosteen fruit with antioxidant properties. Iodoacetate (IAA) is an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase that induces a model of metabolic inhibition in neurons where reactive oxygen species (ROS) production is a significant mechanism. Furthermore, it has been shown that the induction of heme oxygenase-1 (HO-1) protects against IAA-induced neuronal death. To study the effects of α-mangostin and curcumin against the IAA-induced cell death and on HO-1 expression in primary cultures of cerebellar granule neurons (CGNs). CGNs were treated with curcumin or α-mangostin before the addition of IAA. Cell viability and ROS production were measured 24 and 4 hours after IAA addition, respectively. HO-1 expression was measured by western blot. Both α-mangostin and curcumin pretreatment ameliorated the neuronal death induced by IAA in a concentration-dependent way, which was associated with an amelioration of IAA-induced ROS formation. In addition, it was found that α-mangostin and curcumin induced HO-1 expression. Treatment with α-mangostin and curcumin provided a neuroprotective effect against IAA in primary cultures of CGNs, an effect associated with an amelioration of the IAA-induced ROS production. HO-1 induced by these antioxidants may also be involved in the neuroprotective effect. Future work will be required to determine whether α-mangostin may cross the blood-brain barrier and achieve enough bioavailability to elicit a protective response in the brain being an effective nutraceutical compound for preventive therapy of neurodegenerative diseases.

NAD+ maintenance attenuates light induced photoreceptor degeneration Δ

PubMed Central

Bai, Shi; Sheline, Christian T.

2013-01-01

Light-induced retinal damage (LD) occurs after surgery or sun exposure. We previously showed that zinc (Zn2+) accumulated in photoreceptors and RPE cells after LD but prior to cell death, and pyruvate or nicotinamide attenuated the resultant death perhaps by restoring nicotinamide adenine dinucleotide (NAD+) levels. We first examined the levels of NAD+ and the efficacy of pyruvate or nicotinamide in oxidative toxicities using primary retinal cultures. We next manipulated NAD+ levels in vivo and tested the affect on LD to photoreceptors and RPE. NAD+ levels cycle with a 24-h rhythm in mammals, which is affected by the feeding schedule. Therefore, we tested the affect of increasing NAD+ levels on LD by giving nicotinamide, inverting the feeding schedule, or using transgenic mice which overexpress cytoplasmic nicotinamide mononucleotide adenyl-transferase-1 (cytNMNAT1), an NAD+ synthetic enzyme. Zn2+ accumulation was also assessed in culture and in retinal sections. Retinas of light damaged animals were examined by OCT and plastic sectioning, and retinal NAD levels were measured. Day fed, or nicotinamide treated rats showed less NAD+ loss, and LD compared to night fed rats or untreated rats without changing the Zn2+ staining pattern. CytNMNAT1 showed less Zn2+ staining, NAD+ loss, and cell death after LD. In conclusion, intense light, Zn2+ and oxidative toxicities caused an increase in Zn2+, NAD+ loss, and cell death which were attenuated by NAD+ restoration. Therefore, NAD+ levels play a protective role in LD-induced death of photoreceptors and RPE cells. PMID:23274583

Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis.

PubMed

Zhang, Jian; Li, Xiaoling; Kwansa, Herman; Kim, Yun Tai; Yi, Liye; Hong, Gina; Andrabi, Shaida A; Dawson, Valina L; Dawson, Ted M; Koehler, Raymond C; Yang, Zeng-Jin

2017-06-01

Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl- N'-nitro- N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl- N'-nitro- N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl- N'-nitro- N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

Scratch2 prevents cell cycle re-entry by repressing miR-25 in postmitotic primary neurons.

PubMed

Rodríguez-Aznar, Eva; Barrallo-Gimeno, Alejandro; Nieto, M Angela

2013-03-20

During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry.

[The role of RKIP mediated ERK pathway in hippocampus neurons injured by electromagnetic radiation].

PubMed

Zuo, Hong-Yan; Wang, De-Wen; Peng, Rui-Yun; Wang, Shui-Ming; Gao, Ya-Bing; Zhang, Zhi-Yi; Xiao, Feng-Jun

2008-07-01

To study the effects of electromagnetic radiation on RKIP and phosphorylated ERK in primary cultured hippocampus neurons. The inhibitor of MEK U0126 was applied to investigate the role of RKIP mediated ERK pathway in radiation injury. Primary hippocampus neurons were cultured in vitro. X-HPM, S-HPM and EMP were taken as radiation source respectively to establish three cell models exposed to electromagnetic radiation. RKIP and phosphorylated ERK were measured by immunofluorescent labelling and laser scanning confocal microscope. Apoptosis and death fraction of the cells were detected by Annexin V-PI double labelling and flow cytometry. After three kinds of electromagnetic radiation, the expression of RKIP in hippocampus neurons decreased but the expression of phosphorylated ERK increased, and its nuclear translocation occurred. No significant differences were seen between radiation groups. Apoptosis and death fraction of the neurons in U0126 pretreatment groups was significantly lower than that in radiation groups but they were still higher than those in sham-radiation group. The excessive activation of RKIP mediated ERK pathway is one of the important mechanisms for the apoptosis and death of hippocampus neurons induced by electromagnetic radiation. U0126 have some protective effects on radiation injury.

CDK6 protects epithelial ovarian cancer from platinum-induced death via FOXO3 regulation.

PubMed

Dall'Acqua, Alessandra; Sonego, Maura; Pellizzari, Ilenia; Pellarin, Ilenia; Canzonieri, Vincenzo; D'Andrea, Sara; Benevol, Sara; Sorio, Roberto; Giorda, Giorgio; Califano, Daniela; Bagnoli, Marina; Militello, Loredana; Mezzanzanica, Delia; Chiappetta, Gennaro; Armenia, Joshua; Belletti, Barbara; Schiappacassi, Monica; Baldassarre, Gustavo

2017-10-01

Epithelial ovarian cancer (EOC) is an infrequent but highly lethal disease, almost invariably treated with platinum-based therapies. Improving the response to platinum represents a great challenge, since it could significantly impact on patient survival. Here, we report that silencing or pharmacological inhibition of CDK6 increases EOC cell sensitivity to platinum. We observed that, upon platinum treatment, CDK6 phosphorylated and stabilized the transcription factor FOXO3, eventually inducing ATR transcription. Blockage of this pathway resulted in EOC cell death, due to altered DNA damage response accompanied by increased apoptosis. These observations were recapitulated in EOC cell lines in vitro , in xenografts in vivo , and in primary tumor cells derived from platinum-treated patients. Consistently, high CDK6 and FOXO3 expression levels in primary EOC predict poor patient survival. Our data suggest that CDK6 represents an actionable target that can be exploited to improve platinum efficacy in EOC patients. As CDK4/6 inhibitors are successfully used in cancer patients, our findings can be immediately transferred to the clinic to improve the outcome of EOC patients. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

[Sudden death from hypoglycemia].

PubMed

Asmundo, A; Aragona, M; Gualniera, P; Aragona, F

1995-12-01

The sudden death by hypoglycemia is an aspect of the forensic pathology frequently neglected. Authors initially described the pathogenesis of different hypoglycemia forms, distinguishing the primary ones due to hyperinsulinism and the secondary ones due to functional insufficiency of other organs (hypophysis, thyroid, adrenal gland, liver); after that Authors described three cases of sudden death induced hypoglycemia by hyperinsulinism: two were unweaned with nesidioblastosis and one adolescent. In any form of hypoglycemia the central nervous system damage is present with evident neuronal degenerative-necrotic phenomena, widespread edema with microhemorrhage, swollen and dissociation of myelin sheath, glial cells hyperplasia. Death caused by primary hypoglycemia is histopathologically different from the secondary one because of the maintenance of hepatic glycogen content in the former, that increase in striated muscles, including the heart, in spite of the constant secretion of catecholamine from the adrenal medulla. Glycogen is depleted in secondary hypoglycemia. In the primary form, behind the adrenal medulla hyperfunction, the increased functional activity of the adrenal cortex is moderate, contrasting with the seriousness of the syndrome, due prevalently to inhibit the gluconeogenesis response conditioned by the persistence of stored glycogen in the liver, heart and striated muscles. The rare anoxic processes coming with resynthesis of hepatic glycogen have to be considered in the differential diagnosis. The primary hypoglycemic death, especially in unweaned, is frequently promoted by other processes inducing hypoxia (fetal asphyxia outcome, pneumonia, etc.) or worsening the hypoglycemia (hypothyroidism, etc.). The secondary hypoglycemias are characterized by the normality of exocrine pancreas and by organic alterations that cause glycogen depletion from the liver.

Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals.

PubMed

Chu, Wan-Loy; Lim, Yen-Wei; Radhakrishnan, Ammu Kutty; Lim, Phaik-Eem

2010-09-21

Spirulina is a commercial alga well known to contain various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Most of the previous reports on antioxidant activity of Spirulina were based on chemical rather than cell-based assays. The primary objective of this study was to assess the antioxidant activity of aqueous extract from Spirulina based on its protective effect against cell death induced by free radicals. The antioxidant activity of the cold water extract from food-grade Spirulina platensis was assessed using both chemical and cell-based assays. In the cell-based assay, mouse fibroblast cells (3T3) cells were incubated for 1 h in medium containing aqueous extract of Spirulina or vitamin C (positive control) at 25, 125 and 250 μg/mL before the addition of 50 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The cells were incubated for another 24 h before being assessed for cell death due to apoptosis using the Cell Death Detection ELISA Kit. Spectrophotometric assays based on DPPH and ABTS were also used to assess the antioxidant activity of the extract compared to vitamin C and vitamin E (positive controls). Spirulina extract did not cause cytotoxic effect on 3T3 cells within the range of concentrations tested (0 - 250 μg/mL). The extract reduced significantly (p < 0.05) apoptotic cell death due to DPPH and ABTS by 4 to 5-fold although the activity was less than vitamin C. Based on the DPPH assay, the radical scavenging activity of the extract was higher than phycocyanin and was at least 50% of vitamin C and vitamin E. Based on the ABTS assay, the antioxidant activity of the extract at 50 μmug/mL was as good as vitamin C and vitamin E. The results showed that aqueous extract of Spirulina has a protective effect against apoptotic cell death due to free radicals. The potential application of incorporating Spirulina into food products and beverages to enhance their antioxidant capacity is worth exploring.

Chemical chaperones reduce ionizing radiation-induced endoplasmic reticulum stress and cell death in IEC-6 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Eun Sang; Lee, Hae-June; Lee, Yoon-Jin

Highlights: • UPR activation precedes caspase activation in irradiated IEC-6 cells. • Chemical ER stress inducers radiosensitize IEC-6 cells. • siRNAs that targeted ER stress responses ameliorate IR-induced cell death. • Chemical chaperons prevent cell death in irradiated IEC-6 cells. - Abstract: Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by whichmore » ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.« less

Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction

PubMed Central

Cruz, Anthony C.; Ramaswamy, Madhu; Ouyang, Claudia; Klebanoff, Christopher A.; Sengupta, Prabuddha; Yamamoto, Tori N.; Meylan, Françoise; Thomas, Stacy K.; Richoz, Nathan; Eil, Robert; Price, Susan; Casellas, Rafael; Rao, V. Koneti; Lippincott-Schwartz, Jennifer; Restifo, Nicholas P.; Siegel, Richard M.

2016-01-01

Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis. PMID:28008916

SHOX triggers the lysosomal pathway of apoptosis via oxidative stress.

PubMed

Hristov, Georgi; Marttila, Tiina; Durand, Claudia; Niesler, Beate; Rappold, Gudrun A; Marchini, Antonio

2014-03-15

The SHOX gene encodes for a transcription factor important for normal bone development. Mutations in the gene are associated with idiopathic short stature and are responsible for the growth failure and skeletal defects found in the majority of patients with Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia. SHOX is expressed in growth plate chondrocytes where it is supposed to modulate the proliferation, differentiation and cell death of these cells. Supporting this hypothesis, in vitro studies have shown that SHOX expression induces cell cycle arrest and apoptosis in both transformed and primary cells. In this study, we further characterized the cell death mechanisms triggered by SHOX and compared them with the effects induced by one clinically relevant mutant form of SHOX, detected in LWD patients (SHOX R153L) and a SHOX C-terminally truncated version (L185X). We show that SHOX expression in U2OS osteosarcoma cells leads to oxidative stress that, in turn, induces lysosomal membrane rupture with release of active cathepsin B to the cytosol and subsequent activation of the intrinsic apoptotic pathway characterized by mitochondrial membrane permeabilization and caspase activation. Importantly, cells expressing SHOX R153L or L185X did not display any of these features. Given the fact that many of the events observed in SHOX-expressing cells also characterize the complex cell death process occurring in the growth plate during endochondral ossification, our findings further support the hypothesis that SHOX may play a central role in the regulation of the cell death pathways activated during long bone development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Towata, Tomomi; Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811; Komizu, Yuji

Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by human herpes virus-8 infection, and is generally resistant to chemotherapy. Hybrid liposomes, composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (21) dodecyl ether (C{sub 12}(EO){sub 21}) (HL-21), were rapidly accumulated in the membrane of PEL cells. HL-21 also increased membrane fluidity of PEL cells, and induced caspase-3 activation along with cell death. These results suggest that HL-21 should be an effective and attractive regent for PEL treatment.

SYTO probes: markers of apoptotic cell demise.

PubMed

Wlodkowic, Donald; Skommer, Joanna

2007-10-01

As mechanistic studies on tumor cell death advance towards their ultimate translational goal, there is a need for specific, rapid, and high-throughput analytical tools to detect diverse cell demise modes. Patented DNA-binding SYTO probes, for example, are gaining increasing interest as easy-to-use markers of caspase-dependent apoptotic cell death. They are proving convenient for tracking apoptosis in diverse hematopoietic cell lines and primary tumor samples, and, due to their spectral characteristics, appear to be useful for the development of multiparameter flow cytometry assays. Herein, several protocols for multiparametric assessment of apoptotic events using SYTO probes are provided. There are protocols describing the use of green fluorescent SYTO 16 and red fluorescent SYTO 17 dyes in combination with plasma membrane permeability markers. Another protocol highlights the multiparametric use of SYTO 16 dye in conjunction with the mitochondrial membrane potential sensitive probe, tetramethylrhodamine methyl ester (TMRM), and the plasma membrane permeability marker, 7-aminoactinomycin D (7-AAD).

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; Faley, Shannon; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors. PMID:19298813

Cell death in response to antimetabolites directed at thymidylate synthase.

PubMed

Barbour, Karen W; Berger, Franklin G

2008-02-01

Thymidylate synthase (TS) is an indispensable enzyme in the de novo biosynthesis of TMP during DNA replication and cell growth, and has, therefore, been an important target for several classes of antimetabolites used in cancer chemotherapy. While most investigations of the action of TS-directed agents have focused on apoptosis as the primary means of cell death, little is known regarding the role, if any, of non-apoptotic mechanisms. In the present study, we have examined the mode of cell death induced by several TS inhibitors. Apoptosis and necrosis in response to TS inhibitors was assessed. The roles of caspases and the transcriptional regulator nuclear factor kappa B (NFkappaB) in drug-induced cell death were analyzed. Finally, drug-mediated changes in expression of several proteins involved in regulation of apoptosis were analyzed. Though human colon tumor cells exposed to TS inhibitors undergo classical apoptosis, it is not the predominant mechanism of response; rather, a necrosis-like mechanism prevails. The apoptotic response to TS inhibitors is caspase-dependent, and is promoted by NFkappaB. In contrast, the necrosis-like response is independent of both caspases and NFkappaB. Exposure to TS inhibitors induces PARP cleavage, but does not alter expression of the pro or activated forms of caspases-3 or caspases-8, Fas, or FasL. Treatment with the death-inducing cytokine TNFalpha, like TS inhibitors, results in a limited extent of apoptosis that is both caspase- and NFkappaB-dependent; however, unlike TS inhibitors, the cytokine does not induce necrosis. Classical apoptosis occurs to a limited extent in human colon tumor cells exposed to TS inhibitors, with caspase-independent necrosis being the prinicipal mechanism of cell death. We suggest that the role of necrosis and necrosis-like mechanisms should be considered in future studies of the action of TS-directed antimetabolites, as well as other chemotherapeutic agents.

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes

PubMed Central

Gonçalves, Lígia Antunes; Rodo, Joana; Rodrigues-Duarte, Lurdes; de Moraes, Luciana Vieira; Penha-Gonçalves, Carlos

2017-01-01

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium-infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite. PMID:28220125

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes.

PubMed

Gonçalves, Lígia Antunes; Rodo, Joana; Rodrigues-Duarte, Lurdes; de Moraes, Luciana Vieira; Penha-Gonçalves, Carlos

2017-01-01

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium -infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite.

Fas/CD95 regulatory protein Faim2 is neuroprotective after transient brain ischemia.

PubMed

Reich, Arno; Spering, Christopher; Gertz, Karen; Harms, Christoph; Gerhardt, Ellen; Kronenberg, Golo; Nave, Klaus A; Schwab, Markus; Tauber, Simone C; Drinkut, Anja; Harms, Kristian; Beier, Chrstioph P; Voigt, Aaron; Göbbels, Sandra; Endres, Matthias; Schulz, Jörg B

2011-01-05

Death receptor (DR) signaling has a major impact on the outcome of numerous neurological diseases, including ischemic stroke. DRs mediate not only cell death signals, but also proinflammatory responses and cell proliferation. Identification of regulatory proteins that control the switch between apoptotic and alternative DR signaling opens new therapeutic opportunities. Fas apoptotic inhibitory molecule 2 (Faim2) is an evolutionary conserved, neuron-specific inhibitor of Fas/CD95-mediated apoptosis. To investigate its role during development and in disease models, we generated Faim2-deficient mice. The ubiquitous null mutation displayed a viable and fertile phenotype without overt deficiencies. However, lack of Faim2 caused an increase in susceptibility to combined oxygen-glucose deprivation in primary neurons in vitro as well as in caspase-associated cell death, stroke volume, and neurological impairment after cerebral ischemia in vivo. These processes were rescued by lentiviral Faim2 gene transfer. In summary, we provide evidence that Faim2 is a novel neuroprotective molecule in the context of cerebral ischemia.

Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri.

PubMed

Song, K-J; Jang, Y S; Lee, Y A; Kim, K A; Lee, S K; Shin, M H

2011-07-01

Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cells. © 2011 Blackwell Publishing Ltd.

Inhibition of multidrug resistance protein 1 (MRP1) improves chemotherapy drug response in primary and recurrent glioblastoma multiforme.

PubMed

Tivnan, Amanda; Zakaria, Zaitun; O'Leary, Caitrín; Kögel, Donat; Pokorny, Jenny L; Sarkaria, Jann N; Prehn, Jochen H M

2015-01-01

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer with extremely poor prognostic outcome despite intensive treatment. All chemotherapeutic agents currently used have no greater than 30-40% response rate, many fall into the range of 10-20%, with delivery across the blood brain barrier (BBB) or chemoresistance contributing to the extremely poor outcomes despite treatment. Increased expression of the multidrug resistance protein 1(MRP1) in high grade glioma, and it's role in BBB active transport, highlights this member of the ABC transporter family as a target for improving drug responses in GBM. In this study we show that small molecule inhibitors and gene silencing of MRP1 had a significant effect on GBM cell response to temozolomide (150 μM), vincristine (100 nM), and etoposide (2 μM). Pre-treatment with Reversan (inhibitor of MRP1 and P-glycoprotein) led to a significantly improved response to cell death in the presence of all three chemotherapeutics, in both primary and recurrent GBM cells. The presence of MK571 (inhibitor of MRP1 and multidrug resistance protein 4 (MRP4) led to an enhanced effect of vincristine and etoposide in reducing cell viability over a 72 h period. Specific MRP1 inhibition led to a significant increase in vincristine and etoposide-induced cell death in all three cell lines assessed. Treatment with MK571, or specific MRP1 knockdown, did not have any effect on temozolomide drug response in these cells. These findings have significant implications in providing researchers an opportunity to improve currently used chemotherapeutics for the initial treatment of primary GBM, and improved treatment for recurrent GBM patients.

Putting on the Brakes: Blocking the Growth of Metastases | Center for Cancer Research

Cancer.gov

Most of the suffering and death caused by cancer is due, not to the primary tumor, but to the ability of cancer cells to spread throughout the body and to form metastases in other organs. Breast and prostate cancers often have periods of dormancy, which can extend up to 30 years, between the identification and treatment of a primary tumor and the growth of overt metastases.

In Vivo Detection of Hyperoxia-Induced Pulmonary Endothelial Cell Death Using 99mTc-Duramycin

PubMed Central

Audi, Said H.; Jacobs, Elizabeth R.; Zhao, Ming; Roerig, David L.; Haworth, Steven T.; Clough, Anne V.

2014-01-01

Introduction: 99mTc-duramycin, DU, is a SPECT biomarker of tissue injury identifying cell death. The objective of this study is to investigate the potential of DU imaging to quantify capillary endothelial cell death in rat lung injury resulting from hyperoxia exposure as a model of acute lung injury. Methods: Rats were exposed to room air (normoxic) or >98% O2 for 48 or 60 hours. DU was injected i.v. in anesthetized rats, scintigraphy images were acquired at steady-state, and lung DU uptake was quantified from the images. Post-mortem, the lungs were removed for histological studies. Sequential lung sections were immunostained for caspase activation and endothelial and epithelial cells. Results: Lung DU uptake increased significantly (p < 0.001) by 39% and 146% in 48-hr and 60-hr exposed rats, respectively, compared to normoxic rats. There was strong correlation (r2 = 0.82, p = 0.005) between lung DU uptake and the number of cleaved caspase 3 (CC3) positive cells, and endothelial cells accounted for more than 50% of CC3 positive cells in the hyperoxic lungs. Histology revealed preserved lung morphology through 48 hours. By 60 hours there was evidence of edema, and modest neutrophilic infiltrate. Conclusions: Rat lung DU uptake in vivo increased after just 48 hours of >98% O2 exposure, prior to the onset of any substantial evidence of lung injury. These results suggest that apoptotic endothelial cells are the primary contributors to the enhanced DU lung uptake, and support the utility of DU imaging for detecting early endothelial cell death in vivo. PMID:25218023

Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation.

PubMed

Akbarian, Vahe; Wang, Weijia; Audet, Julie

2012-05-01

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture. Copyright © 2012 International Society for Advancement of Cytometry.

Regulation of human glioblastoma cell death by combined treatment of cannabidiol, γ-radiation and small molecule inhibitors of cell signaling pathways

PubMed Central

Ivanov, Vladimir N.; Wu, Jinhua; Hei, Tom K.

2017-01-01

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. The challenging problem in cancer treatment is to find a way to upregulate radiosensitivity of GBM while protecting neurons and neural stem/progenitor cells in the brain. The goal of the present study was upregulation of the cytotoxic effect of γ-irradiation in GBM by non-psychotropic and non-toxic cannabinoid, cannabidiol (CBD). We emphasized three main aspects of signaling mechanisms induced by CBD treatment (alone or in combination with γ-irradiation) in human GBM that govern cell death: 1) CBD significantly upregulated the active (phosphorylated) JNK1/2 and MAPK p38 levels with the subsequent downregulation of the active phospho-ERK1/2 and phospho-AKT1 levels. MAPK p38 was one of the main drivers of CBD-induced cell death, while death levels after combined treatment of CBD and radiation were dependent on both MAPK p38 and JNK. Both MAPK p38 and JNK regulate the endogenous TRAIL expression. 2) NF-κB p65-P(Ser536) was not the main target of CBD treatment and this transcription factor was found at high levels in CBD-treated GBM cells. Additional suppression of p65-P(Ser536) levels using specific small molecule inhibitors significantly increased CBD-induced apoptosis. 3) CBD treatment substantially upregulated TNF/TNFR1 and TRAIL/TRAIL-R2 signaling by modulation of both ligand and receptor levels followed by apoptosis. Our results demonstrate that radiation-induced death in GBM could be enhanced by CBD-mediated signaling in concert with its marginal effects for neural stem/progenitor cells and astrocytes. It will allow selecting efficient targets for sensitization of GBM and overcoming cancer therapy-induced severe adverse sequelae. PMID:29088769

Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response.

PubMed

Delledonne, M; Zeier, J; Marocco, A; Lamb, C

2001-11-06

Nitric oxide (NO) and reactive oxygen intermediates (ROIs) play key roles in the activation of disease resistance mechanisms both in animals and plants. In animals NO cooperates with ROIs to kill tumor cells and for macrophage killing of bacteria. Such cytotoxic events occur because unregulated NO levels drive a diffusion-limited reaction with O(2)(-) to generate peroxynitrite (ONOO(-)), a mediator of cellular injury in many biological systems. Here we show that in soybean cells unregulated NO production at the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate hypersensitive cell death. The HR is triggered only by balanced production of NO and ROIs. Moreover, hypersensitive cell death is activated after interaction of NO not with O(2)- but with H(2)O(2) generated from O(2)(-) by superoxide dismutase. Increasing the level of O(2)(-) reduces NO-mediated toxicity, and ONOO(-) is not a mediator of hypersensitive cell death. During the HR, superoxide dismutase accelerates O(2)(-) dismutation to H(2)O(2) to minimize the loss of NO by reaction with O(2)(-) and to trigger hypersensitive cell death through NO/H(2)O(2) cooperation. However, O(2)(-) rather than H(2)O(2) is the primary ROI signal for pathogen induction of glutathione S-transferase, and the rates of production and dismutation of O(2)(-) generated during the oxidative burst play a crucial role in the modulation and integration of NO/H(2)O(2) signaling in the HR. Thus although plants and animals use a similar repertoire of signals in disease resistance, ROIs and NO are deployed in strikingly different ways to trigger host cell death.

Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei

2013-10-15

Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore,more » trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.« less

Endoplasmic reticulum chaperone GRP78 mediates cigarette smoke-induced necroptosis and injury in bronchial epithelium.

PubMed

Wang, Yong; Zhou, Jie-Sen; Xu, Xu-Chen; Li, Zhou-Yang; Chen, Hai-Pin; Ying, Song-Min; Li, Wen; Shen, Hua-Hao; Chen, Zhi-Hua

2018-01-01

Bronchial epithelial cell death and airway inflammation induced by cigarette smoke (CS) have been involved in the pathogenesis of COPD. GRP78, belonging to heat shock protein 70 family, has been implicated in cell death and inflammation, while little is known about its roles in COPD. Here, we demonstrate that GRP78 regulates CS-induced necroptosis and injury in bronchial epithelial cells. GRP78 and necroptosis markers were examined in human bronchial epithelial (HBE) cell line, primary mouse tracheal epithelial cells, and mouse lungs. siRNA targeting GRP78 gene and necroptosis inhibitor were used. Expression of inflammatory cytokines, mucin MUC5AC, and related signaling pathways were detected. Exposure to CS significantly increased the expression of GRP78 and necroptosis markers in HBE cell line, primary mouse tracheal epithelial cells, and mouse lungs. Inhibition of GRP78 significantly suppressed CS extract (CSE)-induced necroptosis. Furthermore, GRP78-necroptosis cooperatively regulated CSE-induced inflammatory cytokines such as interleukin 6 (IL6), IL8, and mucin MUC5AC in HBE cells, likely through the activation of nuclear factor (NF-κB) and activator protein 1 (AP-1) pathways, respectively. Taken together, our results demonstrate that GRP78 promotes CSE-induced inflammatory response and mucus hyperproduction in airway epithelial cells, likely through upregulation of necroptosis and subsequent activation of NF-κB and AP-1 pathways. Thus, inhibition of GRP78 and/or inhibition of necroptosis could be the effective therapeutic approaches for the treatment of COPD.

The Netrin-4/ Neogenin-1 axis promotes neuroblastoma cell survival and migration

PubMed Central

Villanueva, Andrea A.; Falcón, Paulina; Espinoza, Natalie; Luis, Solano R.; Milla, Luis A.; Hernandez-SanMiguel, Esther; Torres, Vicente A.; Sanchez-Gomez, Pilar; Palma, Verónica

2017-01-01

Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling. PMID:28038459

Cell death induced by the application of alternating magnetic fields to nanoparticle-loaded dendritic cells.

PubMed

Marcos-Campos, I; Asín, L; Torres, T E; Marquina, C; Tres, A; Ibarra, M R; Goya, G F

2011-05-20

In this work, the capability of primary, monocyte-derived dendritic cells (DCs) to uptake iron oxide magnetic nanoparticles (MNPs) is assessed and a strategy to induce selective cell death in these MNP-loaded DCs using external alternating magnetic fields (AMFs) is reported. No significant decrease in the cell viability of MNP-loaded DCs, compared to the control samples, was observed after five days of culture. The number of MNPs incorporated into the cytoplasm was measured by magnetometry, which confirmed that 1-5 pg of the particles were uploaded per cell. The intracellular distribution of these MNPs, assessed by transmission electron microscopy, was found to be primarily inside the endosomic structures. These cells were then subjected to an AMF for 30 min and the viability of the blank DCs (i.e. without MNPs), which were used as control samples, remained essentially unaffected. However, a remarkable decrease of viability from approximately 90% to 2-5% of DCs previously loaded with MNPs was observed after the same 30 min exposure to an AMF. The same results were obtained using MNPs having either positive (NH(2)(+)) or negative (COOH(-)) surface functional groups. In spite of the massive cell death induced by application of AMF to MNP-loaded DCs, the number of incorporated magnetic particles did not raise the temperature of the cell culture. Clear morphological changes at the cell structure after magnetic field application were observed using scanning electron microscopy. Therefore, local damage produced by the MNPs could be the main mechanism for the selective cell death of MNP-loaded DCs under an AMF. Based on the ability of these cells to evade the reticuloendothelial system, these complexes combined with an AMF should be considered as a potentially powerful tool for tumour therapy.

The ultrastructural surface morphology of oral cancer cells and keratinocytes after exposure to chitosan

NASA Astrophysics Data System (ADS)

Fatimah; Sarsito, A. S.; Wimardhani, Y. S.

2017-08-01

Low-molecular-weight chitosan (LMWC) has the same selective cytotoxic effects on oral cancer cells as cisplatin. The cell deaths caused by the anticancer characteristics of chitosan show that apoptosis is not the death pathway of the primary cells involved. The interactions between LMWC and the cells need to be explored. The objective of this study was to compare the ultrastructural morphology of oral Squamous Cell Carcinoma (SCC Ca)-922 and noncancer keratinocyte HaCaT cell lines after exposure to LMWC and cisplatin. The cells were treated with LMWC and cisplatin, and their ultrastructural morphology was analyzed using scanning electron micrographs. Features of early apoptosis, seen as the loss of microvilli, were detected in the LMWC-exposed Ca9-22 cells, and there was a material surrounding the cells. In contrast, the LMWC-exposed HaCaT cells showed no changes related to apoptosis. The results were the opposite when cisplatin was used. This study confirms that there are differences in the ultrastructural surface morphology of LMWC-exposed and cisplatin-exposed oral cancer cells and keratinocytes that could be correlated with their biological activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Fen; Sun, Hong; Kluz, Thomas

Hexavalent chromium [Cr(VI)] is a human carcinogen that results in the generation of reactive oxygen species (ROS) and a variety of DNA lesions leading to cell death. Epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent antioxidative activity capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. Here we demonstrated that co-treatment with EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death in a dose-dependent manner. Cr(VI) induces apoptosis as the primary mode of cell death. Co-treatment of BEAS-2B cells with EGCG dose-dependently suppressed Cr(VI)-induced apoptosis. Fluorescence microscopic analyses and quantitativemore » measurement revealed that EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. Using a well-established K{sup +}/SDS precipitation assay, we further showed that EGCG was able to dose-dependently reduce DNA–protein cross-links (DPC), lesions that could be partially attributed to Cr(VI)-induced oxidative stress. Finally, analyses of Affymetrix microarray containing 28,869 well-annotated genes revealed that, among the 3412 genes changed more than 1.5-fold by Cr(VI) treatment, changes of 2404 genes (70%) were inhibited by pretreatment of EGCG. Real-time PCR confirmed the induction of 3 genes involved in cell death and apoptosis by Cr(VI), which was eliminated by EGCG. In contrast, Cr(VI) reduced the expression of 3 genes related to cellular defense, and this reduction was inhibited by EGCG. Our results indicate that EGCG protects BEAS-2B cells from Cr(VI)-induced cytotoxicity presumably by scavenging ROS and modulating a subset of genes. EGCG, therefore, might serve as a potential chemopreventive agent against Cr(VI) carcinogenesis. -- Highlights: ► EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death and apoptosis. ► EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. ► EGCG reduced DNA-protein cross-links, lesions that could be partially attributed to Cr(VI)-induced oxidative stress. ► EGCG modulated 70% of the gene expression changes induced by Cr(VI) exposure.« less

Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo, E-mail: parkjw@knu.ac.kr

Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report anmore » autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.« less

The Relationship Between Index Hospitalizations, Sepsis, and Death or Transition to Hospice Care During 30-Day Hospital Readmissions.

PubMed

Dietz, Brett W; Jones, Tiffanie K; Small, Dylan S; Gaieski, David F; Mikkelsen, Mark E

2017-04-01

Hospital readmissions are common, expensive, and increasingly used as a metric for assessing quality of care. The relationship between index hospitalizations and specific outcomes among those readmitted remains largely unknown. Identify risk factors present during the index hospitalization associated with death or transition to hospice care during 30-day readmissions and examine the contribution of infection in readmissions resulting in death. Retrospective cohort study. A total of 17,716 30-day readmissions in an academic health system. We used mixed-effects multivariable logistic regression models to identify risk factors associated with the primary outcome, in-hospital death, or transition to hospice during 30-day readmissions. Of 17,716 30-day readmissions, 1144 readmissions resulted in death or transition to hospice care (6.5%). Risk factors identified included: age, burden, and type of comorbid conditions, recent hospitalizations, nonelective index admission type, outside hospital transfer, low discharge hemoglobin, low discharge sodium, high discharge red blood cell distribution width, and disposition to a setting other than home. Sepsis (OR=1.33; 95% CI, 1.02-1.72; P=0.03) and shock (OR=1.78; 95% CI, 1.22-2.58; P=0.002) during the index admission were associated with the primary outcome, and in-hospital mortality specifically. In patients who died, infection was the primary cause for readmission in 51.6% of readmissions after sepsis and 28.6% of readmissions after a nonsepsis hospitalization (P=0.009). We identified factors, including sepsis and shock during the index hospitalization, associated with death or transition to hospice care during readmission. Infection was frequently implicated as the cause of a readmission that ended in death.

Admission white blood cell count predicts short-term clinical outcomes in patients with uncomplicated Stanford type B acute aortic dissection.

PubMed

Chen, Zhao-Ran; Huang, Bi; Lu, Hai-Song; Zhao, Zhen-Hua; Hui, Ru-Tai; Yang, Yan-Min; Fan, Xiao-Han

2017-01-01

Inflammation has been shown to be related with acute aortic dissection (AAD). The present study aimed to evaluate the association of white blood cell counts (WBCc) on admission with both in-hospital and long-term all-cause mortality in patients with uncomplicated Stanford type B AAD. From 2008 to 2010, a total of 377 consecutive patients with uncomplicated type B AAD were enrolled and then followed up. Clinical data and WBCc on admission were collected. The primary end points were in-hospital death and long-term all-cause death. The in-hospital death rate was 4.2%, and the long-term all-cause mortality rate was 6.9% during a median follow-up of 18.9 months. WBCc on admission was identified as a risk factor for in-hospital death by univariate Cox regression analysis as both a continuous variable and a categorical variable using a cut off of 11.0 × 10 9 cell/L (all P < 0.05). After adjusting for age, sex and other risk factors, elevated admission WBCc was still a significant predictor for in-hospital death as both a continuous variable [hazard ratio (HR): 1.052, 95% CI: 1.024-1.336, P = 0.002] and a categorical variable using a cut off of 11.0 × 10 9 cell/L (HR: 2.056, 95% CI: 1.673-5.253, P = 0.034). No relationship was observed between WBCc on admission and long-term all-cause death. Our results indicate that elevated WBCc upon admission might be used as a predictor for increased risk of in-hospital death in uncomplicated type B AAD. There might be no predictive value of WBCc for the long-term survival of type B AAD.

Regeneration and replacement in the vertebrate inner ear.

PubMed

Matsui, Jonathan I; Parker, Mark A; Ryals, Brenda M; Cotanche, Douglas A

2005-10-01

Deafness affects more than 40 million people in the UK and the USA, and many more world-wide. The primary cause of hearing loss is damage to or death of the sensory receptor cells in the inner ear, the hair cells. Birds can readily regenerate their cochlear hair cells but the mammalian cochlea has shown no ability to regenerate after damage. Current research efforts are focusing on gene manipulation, gene therapy and stem cell transplantation for repairing or replacing damaged mammalian cochlear hair cells, which could lead to therapies for treating deafness in humans.

Patterns of in vitro cell-death, metaloproteinase-9 and pro-inflammatory cytokines in human monocytes induced by the BCG vaccine, Moreau strain.

PubMed

Simas, C J A; Silva, D P H; Ponte, C G G; Castello-Branco, L R R; Antas, P R Z

2011-09-02

Mononuclear cells have been implicated in the primary inflammatory response against mycobacteria. Yet, little is known about the interaction of Mycobacterium bovis bacillus Calmette-Guerin (BCG) with human monocytes. Here, we investigated the potential of BCG Moreau strain to induce in vitro specific cell-death utilizing a flow cytometry approach that revealed an increase in apoptosis events in BCG-stimulated monocytes from healthy adults. We also detected a concomitant release of interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), but not metalloproteinase (MMP)-9. In addition, annexin V-propidium iodide double staining demonstrated an enhancement of monocytes necrosis, but not apoptosis, following BCG Moreau strain stimulation of umbilical vein cells from naïve, neonate. This pattern was paralleled by different pro-inflammatory cytokine levels, as well as MMP-9 induction when compared to the adults. Our findings support the hypothesis that BCG induces distinct cell-death patterns during the maturation of the immune system and that this pattern might set the stage for a subsequent antimycobacterial immune response that might have profound effects during vaccination. Copyright © 2011 Elsevier Ltd. All rights reserved.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases. PMID:21247434

MicroRNA-7 Promotes Glycolysis to Protect against 1-Methyl-4-phenylpyridinium-induced Cell Death.

PubMed

Chaudhuri, Amrita Datta; Kabaria, Savan; Choi, Doo Chul; Mouradian, M Maral; Junn, Eunsung

2015-05-08

Parkinson disease is associated with decreased activity of the mitochondrial electron transport chain. This defect can be recapitulated in vitro by challenging dopaminergic cells with 1-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin that inhibits complex I of electron transport chain. Consequently, oxidative phosphorylation is blocked, and cells become dependent on glycolysis for ATP production. Therefore, increasing the rate of glycolysis might help cells to produce more ATP to meet their energy demands. In the present study, we show that microRNA-7, a non-coding RNA that protects dopaminergic neuronal cells against MPP(+)-induced cell death, promotes glycolysis in dopaminergic SH-SY5Y and differentiated human neural progenitor ReNcell VM cells, as evidenced by increased ATP production, glucose consumption, and lactic acid production. Through a series of experiments, we demonstrate that targeted repression of RelA by microRNA-7, as well as subsequent increase in the neuronal glucose transporter 3 (Glut3), underlies this glycolysis-promoting effect. Consistently, silencing Glut3 expression diminishes the protective effect of microRNA-7 against MPP(+). Further, microRNA-7 fails to prevent MPP(+)-induced cell death when SH-SY5Y cells are cultured in a low glucose medium, as well as when differentiated ReNcell VM cells or primary mouse neurons are treated with the hexokinase inhibitor, 2-deoxy-d-glucose, indicating that a functional glycolytic pathway is required for this protective effect. In conclusion, microRNA-7, by down-regulating RelA, augments Glut3 expression, promotes glycolysis, and subsequently prevents MPP(+)-induced cell death. This protective effect of microRNA-7 could be exploited to correct the defects in oxidative phosphorylation in Parkinson disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

PubMed

LaRocca, Timothy J; Stivison, Elizabeth A; Hod, Eldad A; Spitalnik, Steven L; Cowan, Peter J; Randis, Tara M; Ratner, Adam J

2014-08-26

A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family. Copyright © 2014 LaRocca et al.

Oxidative stress, caspase-3 activation and cleavage of ROCK-1 play an essential role in MeHg-induced cell death in primary astroglial cells.

PubMed

Dos Santos, Alessandra Antunes; López-Granero, Caridad; Farina, Marcelo; Rocha, João B T; Bowman, Aaron B; Aschner, Michael

2018-03-01

Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD + /NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA. Copyright © 2018. Published by Elsevier Ltd.

Mechanisms of Ionizing Radiation-Induced Cell Death in Primary Lung Cells

DTIC Science & Technology

2013-03-05

haemocytometer, and plated on 60 mm dishes coated with 0.5% gelatin (modification to allow attachment of PAEC). The cells were incubated for 14 days...we performed delayed plating where PAEC were first cultured to 70-90% confluence, exposed to varying doses of X-rays, incubated, and seeded then for...calculated as a function of plating efficiency of non-irradiated controls. The plating efficiency (PE) was defined as the percentage of the number

Sudden unexpected death from oligodendroglioma: a case report and review of the literature.

PubMed

Manousaki, Maria; Papadaki, Helen; Papavdi, Asteria; Kranioti, Elena F; Mylonakis, Panagiotis; Varakis, John; Michalodimitrakis, Manolis

2011-12-01

Sudden and unexpected deaths due to asymptomatic 5 primary brain tumors are extremely rare, with an incidence that ranges from 0.16 to 3.2%. Usually, such tumors are glioblastomas or, less commonly, astrocytomas. Asymptomatic oligodendrogliomas causing sudden death are hardly ever reported among medico-legal investigated cases.We report a rare case of sudden and unexpected death from a previously asymptomatic and undiagnosed, well-differentiated, grade II oligodendrogloioma (WHO classification). According to the autopsy and the microscopic findings brain edema as a result of obstruction of the cerebrospinal fluid flow due to hemorrhagic leakage of the oligodendroglioma is incriminated as the most probable physiopathological mechanism for the sudden death. Diagnosis is mainly based on the special microscopic features of the tumor cells (typical "fried-egg" appearance), interrupted by a dense network of branching capillaries. We discuss further the pathophysiological mechanisms of death and present a short review of literature.

Role of microglia in ethanol’s apoptotic action on hypothalamic neuronal cells in primary cultures

PubMed Central

Boyadjieva, Nadka I.; Sarkar, Dipak K.

2010-01-01

Background Microglia are the major inflammatory cells in the central nervous system and play a role in brain injuries as well as brain diseases. In this study, we determined the role of microglia in ethanol’s apoptotic action on neuronal cells obtained from the mediobasal hypothalamus and maintained in primary cultures. We also tested the effect of cAMP, a signaling molecule critically involved in hypothalamic neuronal survival, on microglia-mediated ethanol’s neurotoxic action. Methods Ethanol’s neurotoxic action was determined on enriched fetal mediobasal hypothalamic neuronal cells with or without microglia cells or ethanol-activated microglia conditioned media. Ethanol’s apoptotic action was determined using nucleosome assay. Microglia activation was determined using OX6 histochemistry and by measuring inflammatory cytokines secretion from microglia in cultures using enzyme-linked immunosorbent assay (ELISA). An immunoneutralization study was conducted to identify the role of a cytokine involved in ethanol’s apoptotic action. Results We show here that ethanol at a dose range of 50 and 100 mM induces neuronal death by an apoptotic process. Ethanol’s ability to induce an apoptotic death of neurons is increased by the presence of ethanol-activated microglia conditioned media. In the presence of ethanol, microglia showed elevated secretion of various inflammatory cytokines, of which TNF-α shows significant apoptotic action on mediobasal hypothalamic neuronal cells. Ethanol’s neurotoxic action was completely prevented by cAMP. The cell-signaling molecule also prevented ethanol-activated microglial production of TNF-α. Immunoneutralization of TNF-α prevented microglia-derived media’s ability to induce neuronal death. Conclusions These results suggest that ethanol’s apoptotic action on hypothalamic neuronal cells might be mediated via microglia, possibly via increased production of TNF-α. Furthermore, cAMP reduces TNF-α production from microglia to prevent ethanol’s neurotoxic action. PMID:20662807

Lycium barbarum polysaccharide protects against oxygen glucose deprivation/reoxygenation-induced apoptosis and autophagic cell death via the PI3K/Akt/mTOR signaling pathway in primary cultured hippocampal neurons.

PubMed

Yu, Yang; Wu, Xiuquan; Pu, Jingnan; Luo, Peng; Ma, Wenke; Wang, Jiu; Wei, Jialiang; Wang, Yuanxin; Fei, Zhou

2018-01-01

Lycium barbarum polysaccharide (LBP) is the main active ingredient of Lycium barbarum, which exhibits several beneficial effects, including neuroprotection, anti-aging and anti-oxidation. However, the mechanism by which LBP protects against cerebral ischemia/reperfusion-induced injury remains obscure. In this study, we found that LBP pretreatment greatly attenuated oxygen glucose deprivation/reperfusion (OGD/R) injury in primary cultured hippocampal neurons. LBP also suppressed OGD/R-induced lactate dehydrogenase (LDH) leakage, and ameliorated oxidative stress. In addition, LBP significantly reduced OGD/R-induced apoptosis and autophagic cell death. LBP caused the down-regulation of cleaved Caspase-3/Caspase-3, LC3II/LC3I and Beclin 1, as well as up-regulation of Bcl-2/Bax and p62. Furthermore, mechanistic studies indicated that LBP pretreatment increased p-Akt and p-mTOR levels after OGD/R. In summary, our results indicated that LBP protects against OGD/R-induced neuronal injury in primary hippocampal neurons by activating the PI3K/Akt/mTOR signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Human-Specific Bacterial Pore-Forming Toxins Induce Programmed Necrosis in Erythrocytes

PubMed Central

LaRocca, Timothy J.; Stivison, Elizabeth A.; Hod, Eldad A.; Spitalnik, Steven L.; Cowan, Peter J.; Randis, Tara M.

2014-01-01

ABSTRACT A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. PMID:25161188

Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.

PubMed

Martin, A M; Nirschl, T R; Nirschl, C J; Francica, B J; Kochel, C M; van Bokhoven, A; Meeker, A K; Lucia, M S; Anders, R A; DeMarzo, A M; Drake, C G

2015-12-01

Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation--constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. In vitro, many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

Silencing of cytosolic NADP(+)-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine.

PubMed

Lee, Su-Min; Park, Sin Young; Shin, Seoung Woo; Kil, In Sup; Yang, Eun Sun; Park, Jeen-Woo

2009-02-01

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides:

NASA Astrophysics Data System (ADS)

Chen, Junping; Patil, Swanand; Seal, Sudipta; McGinnis, James F.

2006-11-01

Photoreceptor cells are incessantly bombarded with photons of light, which, along with the cells' high rate of oxygen metabolism, continuously exposes them to elevated levels of toxic reactive oxygen intermediates (ROIs). Vacancy-engineered mixed-valence-state cerium oxide nanoparticles (nanoceria particles) scavenge ROIs. Our data show that nanoceria particles prevent increases in the intracellular concentrations of ROIs in primary cell cultures of rat retina and, in vivo, prevent loss of vision due to light-induced degeneration of photoreceptor cells. These data indicate that the nanoceria particles may be effective in inhibiting the progression of ROI-induced cell death, which is thought to be involved in macular degeneration, retinitis pigmentosa and other blinding diseases, as well as the ROI-induced death of other cell types in diabetes, Alzheimer's disease, atherosclerosis, stroke and so on. The use of nanoceria particles as a direct therapy for multiple diseases represents a novel strategy and suggests that they may represent a unique platform technology.

Prostate Cancer Cell Telomere Length Variability and Stromal Cell Telomere Length as Prognostic Markers for Metastasis and Death

PubMed Central

Heaphy, Christopher M.; Yoon, Ghil Suk; Peskoe, Sarah B.; Joshu, Corinne E.; Lee, Thomas K.; Giovannucci, Edward; Mucci, Lorelei A.; Kenfield, Stacey A.; Stampfer, Meir J.; Hicks, Jessica L.; De Marzo, Angelo M.; Platz, Elizabeth A.; Meeker, Alan K.

2013-01-01

Current prognostic indicators are imperfect predictors of outcome in men with clinicallylocalized prostate cancer. Thus, tissue-based markers are urgently needed to improve treatment and surveillance decision-making. Given that shortened telomeres enhance chromosomal instability and such instability is a hallmark of metastatic lesions, we hypothesized that alterations in telomere length in the primary cancer would predict risk of progression to metastasis and prostate cancer death. To test this hypothesis, we conducted a prospective cohort study of 596 surgically treated men who participated in the ongoing Health Professionals Follow-up Study. Men who had the combination of more variable telomere length among prostate cancer cells (cell-to-cell) and shorter telomere length in prostate cancer-associated stromal cells were substantially more likely to progress to metastasis or die of their prostate cancer. These findings point to the translational potential of this telomere biomarker for prognostication and risk stratification for individualized therapeutic and surveillance strategies. PMID:23779129

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate

PubMed Central

Pele, Laetitia; Haas, Carolin T; Hewitt, Rachel; Faria, Nuno; Brown, Andy; Powell, Jonathan

2015-01-01

Aim To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. Material & methods The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. Results Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. Conclusion In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes. PMID:24991724

Angiotensin II type 1 receptor blocker telmisartan induces apoptosis and autophagy in adult T-cell leukemia cells.

PubMed

Kozako, Tomohiro; Soeda, Shuhei; Yoshimitsu, Makoto; Arima, Naomichi; Kuroki, Ayako; Hirata, Shinya; Tanaka, Hiroaki; Imakyure, Osamu; Tone, Nanako; Honda, Shin-Ichiro; Soeda, Shinji

2016-05-01

Adult T-cell leukemia/lymphoma (ATL), an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1), requires new treatments. Drug repositioning, reuse of a drug previously approved for the treatment of another condition to treat ATL, offers the possibility of reduced time and risk. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate peroxisome proliferator-activated receptor-γ, which plays various roles in lipid metabolism, cellular differentiation, and apoptosis. Here, telmisartan reduced cell viability and enhanced apoptotic cells via caspase activation in ex vivo peripheral blood monocytes from asymptomatic HTLV-1 carriers (ACs) or via caspase-independent cell death in acute-type ATL, which has a poor prognosis. Telmisartan also induced significant growth inhibition and apoptosis in leukemia cell lines via caspase activation, whereas other angiotensin II receptor blockers did not induce cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation and autophagy. Thus, telmisartan simultaneously caused caspase activation and autophagy. A hypertension medication with antiproliferation effects on primary and leukemia cells is intriguing. Patients with an early diagnosis of ATL are generally monitored until the disease progresses; thus, suppression of progression from AC and indolent ATL to acute ATL is important. Our results suggest that telmisartan is highly effective against primary cells and leukemia cell lines in caspase-dependent and -independent manners, and its clinical use may suppress acute transformation and improve prognosis of patients with this mortal disease. This is the first report demonstrating a cell growth-inhibitory effect of telmisartan in fresh peripheral blood mononuclear cells from leukemia patients.

Novel Genome-Wide Screening Method Identifies Genes Important to Breast Cancer Metastasis | Center for Cancer Research

Cancer.gov

For patients with solid tumors, the primary cause of illness and death is metastasis, a complex process involving multiple steps and cooperation between cancerous and normal cells. Many genes must be involved, but few have been found and characterized.

5-Fluorouracil-induced apoptosis in cultured oral cancer cells.

PubMed

Tong, D; Poot, M; Hu, D; Oda, D

2000-03-01

Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC.

Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin

PubMed Central

Kortmann, Jens; Brubaker, Sky W.

2015-01-01

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6. PMID:26109648

Dihydromyricetin promotes hepatocellular carcinoma regression via a p53 activation-dependent mechanism

NASA Astrophysics Data System (ADS)

Zhang, Qingyu; Liu, Jie; Liu, Bin; Xia, Juan; Chen, Nianping; Chen, Xiaofeng; Cao, Yi; Zhang, Chen; Lu, Caijie; Li, Mingyi; Zhu, Runzhi

2014-04-01

The development of antitumor chemotherapy drugs remains a key goal for oncologists, and natural products provide a vast resource for anti-cancer drug discovery. In the current study, we found that the flavonoid dihydromyricetin (DHM) exhibited antitumor activity against liver cancer cells, including primary cells obtained from hepatocellular carcinoma (HCC) patients. In contrast, DHM was not cytotoxic to immortalized normal liver cells. Furthermore, DHM treatment resulted in the growth inhibition and remission of xenotransplanted tumors in nude mice. Our results further demonstrated that this antitumor activity was caused by the activation of the p53-dependent apoptosis pathway via p53 phosphorylation at serine (15Ser). Moreover, our results showed that DHM plays a dual role in the induction of cell death when administered in combination with cisplatin, a common clinical drug that kills primary hepatoma cells but not normal liver cells.

Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation

PubMed Central

Lunt, Sophia Y.; Muralidhar, Vinayak; Hosios, Aaron M.; Israelsen, William J.; Gui, Dan Y.; Newhouse, Lauren; Ogrodzinski, Martin; Hecht, Vivian; Xu, Kali; Acevedo, Paula N. Marín; Hollern, Daniel P.; Bellinger, Gary; Dayton, Talya L.; Christen, Stefan; Elia, Ilaria; Dinh, Anh T.; Stephanopoulos, Gregory; Manalis, Scott R.; Yaffe, Michael B.; Andrechek, Eran R.; Fendt, Sarah-Maria; Heiden, Matthew G. Vander

2014-01-01

SUMMARY Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2-deletion affects proliferation and metabolism in non-transformed, non-immortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis. PMID:25482511

Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis

PubMed Central

Lim, M P; Devi, L A; Rozenfeld, R

2011-01-01

The major cellular event in the development and progression of liver fibrosis is the activation of hepatic stellate cells (HSCs). Activated HSCs proliferate and produce excess collagen, leading to accumulation of scar matrix and fibrotic liver. As such, the induction of activated HSC death has been proposed as a means to achieve resolution of liver fibrosis. Here we demonstrate that cannabidiol (CBD), a major non-psychoactive component of the plant Cannabis sativa, induces apoptosis in activated HSCs through a cannabinoid receptor-independent mechanism. CBD elicits an endoplasmic reticulum (ER) stress response, characterized by changes in ER morphology and the initiation of RNA-dependent protein kinase-like ER kinase-, activating transcription factor-6-, and inositol-requiring ER-to-nucleus signal kinase-1 (IRE1)-mediated signaling cascades. Furthermore, CBD induces downstream activation of the pro-apoptotic IRE1/ASK1/c-Jun N-terminal kinase pathway, leading to HSC death. Importantly, we show that this mechanism of CBD-induced ER stress-mediated apoptosis is specific to activated HSCs, as it occurs in activated human and rat HSC lines, and in primary in vivo-activated mouse HSCs, but not in quiescent HSCs or primary hepatocytes from rat. Finally, we provide evidence that the elevated basal level of ER stress in activated HSCs has a role in their susceptibility to the pro-apoptotic effect of CBD. We propose that CBD, by selectively inducing death of activated HSCs, represents a potential therapeutic agent for the treatment of liver fibrosis. PMID:21654828

Differential Susceptibility of Germ and Leydig Cells to Cadmium-Mediated Toxicity: Impact on Testis Structure, Adiponectin Levels, and Steroidogenesis

PubMed Central

Cupertino, Marli C.; Neves, Ana C.; Oliveira, Juraci A.

2017-01-01

This study investigated the relationship between germ and Leydig cell death, testosterone, and adiponectin levels in cadmium-mediated acute toxicity. Cadmium chloride was administered in a single dose to five groups of rats: G1 (0.9% NaCl) and G2 to G5 (0.67, 0.74, 0.86, and 1.1 mg Cd/kg). After 7 days, the animals were euthanized, and the testosterone and testes were analyzed. Dose-dependent Cd accumulation in the testes was identified. At 0.86 and 1.1 mg/kg, animals exhibited marked inflammatory infiltrate and disorganization of the seminiferous epithelium. While Leydig cells were morphologically resistant to Cd toxicity, massive germ cell death and DNA oxidation and fragmentation were observed. Although numerical density of Leydig cells was unchanged, testosterone levels were significantly impaired in animals exposed to 0.86 and 1.1 mg Cd/kg, occurring in parallel with the reduction in total adiponectins and the increase in high-molecular weight adiponectin levels. Our findings indicated that Leydig and germ cells exhibit differential microstructural resistance to Cd toxicity. While germ cells are a primary target of Cd-induced toxicity, Leydig cells remain resistant to death even when exposed to high doses of Cd. Despite morphological resistance, steroidogenesis was drastically impaired by Cd exposure, an event potentially related to the imbalance in adiponectin production. PMID:29422988

Whole genome expression analysis in primary bronchial epithelial cells after exposure to sulphur mustard.

PubMed

Jowsey, Paul A; Blain, Peter G

2014-11-04

Sulphur mustard (SM) is a highly toxic chemical agent and poses a current threat to both civilians and military personnel in the event of a deliberate malicious release. Acute SM toxicity develops over the course of several hours and mainly affects the skin and mucosal surfaces of the eyes and respiratory system. In cases of acute severe exposure, significant lung injury can result in respiratory failure and death. Systemic levels of SM can also be fatal, frequently due to immunodepletion and the subsequent development of secondary infections. Whilst the physical effects associated with SM exposure are well documented, the molecular mechanisms mediating these changes are poorly understood, hindering the development of an effective therapeutic strategy. To gain a better understanding of the mechanism of SM toxicity, this study investigated whole genome transcriptional changes after SM in primary human bronchial epithelial cells, as a model for inhalation exposure. The analysis revealed >400 transcriptional changes associated with SM exposure. Pathways analysis confirmed the findings of previous studies suggesting that DNA damage, cell cycle arrest, cell death and inflammation were important components of SM toxicity. In addition, several other interesting observations were made, suggesting that protein oxidation as well as effects on the mitotic apparatus may contribute to SM toxicity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Causes of death in long-term survivors of head and neck cancer.

PubMed

Baxi, Shrujal S; Pinheiro, Laura C; Patil, Sujata M; Pfister, David G; Oeffinger, Kevin C; Elkin, Elena B

2014-05-15

Survivors of head and neck squamous cell carcinoma (HNSCC) face excess mortality from multiple causes. We used the population-based Surveillance, Epidemiology, and End Results (SEER) cancer registry data to evaluate the causes of death in patients with nonmetastatic HNSCC diagnosed between 1992 and 2005 who survived at least 3 years from diagnosis (long-term survivors). We used competing-risks proportional hazards regression to estimate probabilities of death from causes: HNSCC, second primary malignancy (SPM) excluding HNSCC, cardiovascular disease, and other causes. We identified 35,958 three-year survivors of HNSCC with a median age at diagnosis of 60 years (range = 18-100 years) and a median follow-up of 7.7 years (range = 3-18 years). There were 13,120 deaths during the study period. Death from any cause at 5 and 10 years was 15.4% (95% confidence interval [CI] = 15.0%-15.8%) and 41.0% (95% CI = 40.4%-41.6%), respectively. There were 3852 HNSCC deaths including both primary and subsequent head and neck tumors. The risk of death from HNSCC was greater in patients with nasopharynx or hypopharynx cancer and in patients with locally advanced disease. SPM was the leading cause of non-HNSCC death, and the most common sites of SPM death were lung (53%), esophagus (10%), and colorectal (5%) cancer. Many long-term HNSCC survivors die from cancers other than HNSCC and from noncancer causes. Routine follow-up care for HNSCC survivors should expand beyond surveillance for recurrent and new head and neck cancers. © 2014 American Cancer Society.

In vivo detection of hyperoxia-induced pulmonary endothelial cell death using (99m)Tc-duramycin.

PubMed

Audi, Said H; Jacobs, Elizabeth R; Zhao, Ming; Roerig, David L; Haworth, Steven T; Clough, Anne V

2015-01-01

(99m)Tc-duramycin, DU, is a SPECT biomarker of tissue injury identifying cell death. The objective of this study is to investigate the potential of DU imaging to quantify capillary endothelial cell death in rat lung injury resulting from hyperoxia exposure as a model of acute lung injury. Rats were exposed to room air (normoxic) or >98% O2 for 48 or 60 hours. DU was injected i.v. in anesthetized rats, scintigraphy images were acquired at steady-state, and lung DU uptake was quantified from the images. Post-mortem, the lungs were removed for histological studies. Sequential lung sections were immunostained for caspase activation and endothelial and epithelial cells. Lung DU uptake increased significantly (p<0.001) by 39% and 146% in 48-hr and 60-hr exposed rats, respectively, compared to normoxic rats. There was strong correlation (r(2)=0.82, p=0.005) between lung DU uptake and the number of cleaved caspase 3 (CC3) positive cells, and endothelial cells accounted for more than 50% of CC3 positive cells in the hyperoxic lungs. Histology revealed preserved lung morphology through 48 hours. By 60 hours there was evidence of edema, and modest neutrophilic infiltrate. Rat lung DU uptake in vivo increased after just 48 hours of >98% O2 exposure, prior to the onset of any substantial evidence of lung injury. These results suggest that apoptotic endothelial cells are the primary contributors to the enhanced DU lung uptake, and support the utility of DU imaging for detecting early endothelial cell death in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

Peptidases released by necrotic cells control CD8+ T cell cross-priming

PubMed Central

Gamrekelashvili, Jaba; Kapanadze, Tamar; Han, Miaojun; Wissing, Josef; Ma, Chi; Jaensch, Lothar; Manns, Michael P.; Armstrong, Todd; Jaffee, Elizabeth; White, Ayla O.; Citrin, Deborah E.; Korangy, Firouzeh; Greten, Tim F.

2013-01-01

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells. PMID:24216478

Peptidases released by necrotic cells control CD8+ T cell cross-priming.

PubMed

Gamrekelashvili, Jaba; Kapanadze, Tamar; Han, Miaojun; Wissing, Josef; Ma, Chi; Jaensch, Lothar; Manns, Michael P; Armstrong, Todd; Jaffee, Elizabeth; White, Ayla O; Citrin, Deborah E; Korangy, Firouzeh; Greten, Tim F

2013-11-01

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.

Deletion of a single allele of the Pex11β gene is sufficient to cause oxidative stress, delayed differentiation and neuronal death in mouse brain

PubMed Central

Ahlemeyer, Barbara; Gottwald, Magdalena; Baumgart-Vogt, Eveline

2012-01-01

SUMMARY Impaired neuronal migration and cell death are commonly observed in patients with peroxisomal biogenesis disorders (PBDs), and in mouse models of this diseases. In Pex11β-deficient mice, we observed that the deletion of a single allele of the Pex11β gene (Pex11β+/− heterozygous mice) caused cell death in primary neuronal cultures prepared from the neocortex and cerebellum, although to a lesser extent as compared with the homozygous-null animals (Pex11β−/− mice). In corresponding brain sections, cell death was rare, but differences between the genotypes were similar to those found in vitro. Because PEX11β has been implicated in peroxisomal proliferation, we searched for alterations in peroxisomal abundance in the brain of heterozygous and homozygous Pex11β-null mice compared with wild-type animals. Deletion of one allele of the Pex11β gene slightly increased the abundance of peroxisomes, whereas the deletion of both alleles caused a 30% reduction in peroxisome number. The size of the peroxisomal compartment did not correlate with neuronal death. Similar to cell death, neuronal development was delayed in Pex11β+/− mice, and to a further extent in Pex11β−/− mice, as measured by a reduced mRNA and protein level of synaptophysin and a reduced protein level of the mature isoform of MAP2. Moreover, a gradual increase in oxidative stress was found in brain sections and primary neuronal cultures from wild-type to heterozygous to homozygous Pex11β-deficient mice. SOD2 was upregulated in neurons from Pex11β+/− mice, but not from Pex11β−/− animals, whereas the level of catalase remained unchanged in neurons from Pex11β+/− mice and was reduced in those from Pex11β−/− mice, suggesting a partial compensation of oxidative stress in the heterozygotes, but a failure thereof in the homozygous Pex11β−/− brain. In conclusion, we report the alterations in the brain caused by the deletion of a single allele of the Pex11β gene. Our data might lead to the reconsideration of the clinical treatment of PBDs and the common way of using knockout mouse models for studying autosomal recessive diseases. PMID:21954064

Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals

PubMed Central

2010-01-01

Background Spirulina is a commercial alga well known to contain various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Most of the previous reports on antioxidant activity of Spirulina were based on chemical rather than cell-based assays. The primary objective of this study was to assess the antioxidant activity of aqueous extract from Spirulina based on its protective effect against cell death induced by free radicals. Methods The antioxidant activity of the cold water extract from food-grade Spirulina platensis was assessed using both chemical and cell-based assays. In the cell-based assay, mouse fibroblast cells (3T3) cells were incubated for 1 h in medium containing aqueous extract of Spirulina or vitamin C (positive control) at 25, 125 and 250 μg/mL before the addition of 50 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The cells were incubated for another 24 h before being assessed for cell death due to apoptosis using the Cell Death Detection ELISA Kit. Spectrophotometric assays based on DPPH and ABTS were also used to assess the antioxidant activity of the extract compared to vitamin C and vitamin E (positive controls). Results Spirulina extract did not cause cytotoxic effect on 3T3 cells within the range of concentrations tested (0 - 250 μg/mL). The extract reduced significantly (p < 0.05) apoptotic cell death due to DPPH and ABTS by 4 to 5-fold although the activity was less than vitamin C. Based on the DPPH assay, the radical scavenging activity of the extract was higher than phycocyanin and was at least 50% of vitamin C and vitamin E. Based on the ABTS assay, the antioxidant activity of the extract at 50 μmug/mL was as good as vitamin C and vitamin E. Conclusions The results showed that aqueous extract of Spirulina has a protective effect against apoptotic cell death due to free radicals. The potential application of incorporating Spirulina into food products and beverages to enhance their antioxidant capacity is worth exploring. PMID:20858231

Does symptomatic primary HIV-1 infection accelerate progression to CDC stage IV disease, CD4 count below 200 x 10(6)/l, AIDS, and death from AIDS?

PubMed Central

Lindbäck, S.; Broström, C.; Karlsson, A.; Gaines, H.

1994-01-01

OBJECTIVE--To investigate the prognostic significance of symptomatic primary HIV-1 infection. DESIGN--Prospective study of homosexual men seroconverting to HIV in 1985 and 1986. Patients were followed up at least three times yearly with clinical examinations and T cell subset determinations for an average of 7.2 years. SETTING--Research project centred on attenders for treatment and screening for HIV at the Karolinska Institute, Stockholm. SUBJECTS--19 patients presenting with a glandular-fever-like illness associated with seroconversion to HIV and 29 asymptomatic seroconverters. MAIN OUTCOME MEASURES--Progression to Centers for Disease Control and Prevention stage IV disease, CD4 cell count below 200 x 10(6)/l, AIDS, and death from AIDS. RESULTS--Symptomatic seroconverters were significantly more likely to develop Centers for Disease Control and Prevention stage IV disease (95% v 66%), CD4 cell counts below 200 x 10(6)/l (84% v 55%), and AIDS (58% v 28%) and die of AIDS (53% v 7%). CONCLUSION--A glandular-fever-like illness associated with seroconversion to HIV-1 predicts accelerated progression to AIDS and other HIV related diseases. PMID:7819891

Reactive oxygen species trigger motoneuron death in non-cell-autonomous models of ALS through activation of c-Abl signaling

PubMed Central

Rojas, Fabiola; Gonzalez, David; Cortes, Nicole; Ampuero, Estibaliz; Hernández, Diego E.; Fritz, Elsa; Abarzua, Sebastián; Martinez, Alexis; Elorza, Alvaro A.; Alvarez, Alejandra; Court, Felipe; van Zundert, Brigitte

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1G93A (ACM-hSOD1G93A) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1G93A, but not ACM-hSOD1WT, increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1G93A-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1G86R or TDP43A315T. We further find that co-application of ACM-SOD1G93A with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1G93A induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death. PMID:26106294

[Cause of death: from primary disease to direct cause of death].

PubMed

Oppewal, F; Smedts, F M M; Meyboom-de Jong, B

2005-07-23

Following the death of a patient, the treating physician in the Netherlands is required to fill out two forms. Form A, which is the certificate of death and Form B, which is used by the Statistics Netherlands to compile data on causes ofdeath. The latter form often poses difficulty for the physician with respect to the primary cause of death. This applies particularly to cases of sudden death, which account for one third of all deaths in the Netherlands. As a result, the statistical analyses appear to lead to an incorrect representation of the distribution of causes of death. A more thorough investigation into the primary cause of death is desirable, if necessary, supported by a request for an autopsy. The primary cause of death is to be regarded as the basic disease from which the cascade of changes ultimately leading to death originated.

Hepatic Stellate Cells Alter Liver Immune Environment to Promote Cancer | Center for Cancer Research

Cancer.gov

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for up to 90 percent of cases, and is the second most common cause of cancer-related deaths worldwide according to the World Health Organization’s 2014 World Cancer Report. Even when caught early, HCC often recurs, either from intra-liver metastases or new primary tumors, and recurrence is the leading cause of death for patients with HCC. The liver microenvironment is an important contributor to HCC initiation and progression and also likely plays a role in tumor recurrence. Xin Wei Wang, Ph.D., of CCR’s Laboratory of Human Carcinogenesis, and his colleagues wondered whether activated hepatic stellate cells (A-HSCs), stromal cells in the liver known to participate in repair following injury and in the development of fibrosis, contribute directly to HCC recurrence.

PD-1 and PD-L1 expression in HNSCC primary cancer and related lymph node metastasis - impact on clinical outcome.

PubMed

Schneider, Sven; Kadletz, Lorenz; Wiebringhaus, Robert; Kenner, Lukas; Selzer, Edgar; Füreder, Thorsten; Rajky, Orsolya; Berghoff, Anna S; Preusser, Matthias; Heiduschka, Gregor

2018-05-09

Expression profiles and clinical impact of programmed cell death ligand 1 (PD-L1) and programmed cell death 1 (PD-1) expressing tumour infiltrating lymphocytes (TILs) in head and neck squamous cell carcinoma (HNSCC) are not fully elucidated. This study evaluates expression patterns in primary HNSCC and related lymph node metastasis and impact on patients' clinical outcome. Immunohistochemical staining patterns of PD-L1 and PD-1 were evaluated in 129 specimens of primary HNSCC and 77 lymph node metastases. Results were correlated to patients' clinical data. PD-L1 expression was observed in 36% of primary carcinoma and 33% of lymph node metastasis and significantly correlates with decreased overall survival (OS) (p=0.01) and disease free survival (DFS) (p=0.001) in oral cavity squamous cell carcinoma patients. PD-L1 expression was associated with presence of lymph node metastasis (p=0.0223). Infiltration of PD-1 expressing lymphocytes significantly correlates with favorable OS (p=0.001) and DFS (p=0.001) in oropharyngeal cancer and hypopharyngeal cancer patients OS (p=0.007) and DFS (p=0.001). Presence of PD-1 TILs significantly correlates with better OS (p=0.005) and DFS (p=0) also in the HPV negative cohort. Cox regression multivariate analysis revealed PD-1 TIL expression as an independent prognostic marker for OS (p=0.004) and DFS (p=0.001) and T stage was validated as negative prognostic marker for OS (p=0.011). PD-1 expressing lymphocytes (p=0.0412) and PD-L1 expression (p=0.0022) patterns correlate significantly in primary cancers and matched lymph node metastases. Our results characterize the expression profiles of PD-1 axis proteins in HNSCC which might serve as possible clinical prognostic markers. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1

PubMed Central

Shah, A; Kumar, S; Simon, S D; Singh, D P; Kumar, A

2013-01-01

HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30–60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton–Weiss–Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS. PMID:24113184

Ca2+ is a key factor in α-synuclein-induced neurotoxicity

PubMed Central

Angelova, Plamena R.; Ludtmann, Marthe H. R.; Horrocks, Mathew H.; Negoda, Alexander; Cremades, Nunilo; Klenerman, David; Dobson, Christopher M.; Wood, Nicholas W.; Pavlov, Evgeny V.; Gandhi, Sonia

2016-01-01

ABSTRACT Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca2+ signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca2+ signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca2+, which prevented the α-synuclein-induced Ca2+ dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca2+ signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca2+ dysregulation and Ca2+-dependent cell death. PMID:26989132

Monocarboxylate transporter-dependent mechanism confers resistance to oxygen- and glucose-deprivation injury in astrocyte-neuron co-cultures.

PubMed

Gao, Chen; Zhou, Liya; Zhu, Wenxia; Wang, Hongyun; Wang, Ruijuan; He, Yunfei; Li, Zhiyun

2015-05-06

Hypoxic and low-glucose stressors contribute to neuronal death in many brain diseases. Astrocytes are anatomically well-positioned to shield neurons from hypoxic injury. During hypoxia/ischemia, lactate released from astrocytes is taken up by neurons and stored for energy. This process is mediated by monocarboxylate transporters (MCTs) in the central nervous system. In the present study, we investigated the ability of astrocytes to protect neurons from oxygen- and glucose-deprivation (OGD) injury via an MCT-dependent mechanism in vitro. Primary cultures of neurons, astrocytes, and astrocytes-neurons derived from rat hippocampus were subjected to OGD, MCT inhibition with small interfering (si)RNA. Cell survival and expression of MCT4, MCT2, glial fibrillary acidic protein, and neuronal nuclear antigen were evaluated. OGD significantly increased cell death in neuronal cultures and up-regulated MCT4 expression in astrocyte cultures, but no increased cell death was observed in neuron-astrocyte co-cultures or astrocyte cultures. However, neuronal cell death in co-cultures was increased by exposure to MCT4- or MCT2-specific siRNA, and this effect was attenuated by the addition of lactate into the extracellular medium of neuronal cultures prior to OGD. These findings demonstrate that resistance to OGD injury in astrocyte-neuron co-cultures occurs via an MCT-dependent mechanism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mitochondrial permeability transition pore inhibitors prevent ethanol-induced neuronal death in mice.

PubMed

Lamarche, Frederic; Carcenac, Carole; Gonthier, Brigitte; Cottet-Rousselle, Cecile; Chauvin, Christiane; Barret, Luc; Leverve, Xavier; Savasta, Marc; Fontaine, Eric

2013-01-18

Ethanol induces brain injury by a mechanism that remains partly unknown. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). Here, we tested the effect of ethanol and PTP inhibitors on mitochondrial physiology and cell viability both in vitro and in vivo. Direct addition of ethanol up to 100 mM on isolated mouse brain mitochondria slightly decreased oxygen consumption but did not affect PTP regulation. In comparison, when isolated from ethanol-treated (two doses of 2 g/kg, 2 h apart) 7-day-old mouse pups, brain mitochondria displayed a transient decrease in oxygen consumption but no change in PTP regulation or H2O2 production. Conversely, exposure of primary cultured astrocytes and neurons to 20 mM ethanol for 3 days led to a transient PTP opening in astrocytes without affecting cell viability and to a permanent PTP opening in 10 to 20% neurons with the same percentage of cell death. Ethanol-treated mouse pups displayed a widespread caspase-3 activation in neurons but not in astrocytes and dramatic behavioral alterations. Interestingly, two different PTP inhibitors (namely, cyclosporin A and nortriptyline) prevented both ethanol-induced neuronal death in vivo and ethanol-induced behavioral modifications. We conclude that PTP opening is involved in ethanol-induced neurotoxicity in the mouse.

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro.

PubMed

Peake, Kyle B; Campenot, Robert B; Vance, Dennis E; Vance, Jean E

2011-09-01

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1(-/-) mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1(+/+) and Npc1(-/-) microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1(-/-) microglia is ~60% lower than in Npc1(+/+) microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1(-/-) and Npc1(+/+) neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1(-/-) microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease. Copyright © 2011 Elsevier B.V. All rights reserved.

Higher Vulnerability of Menadione-Exposed Cortical Astrocytes of Glutaryl-CoA Dehydrogenase Deficient Mice to Oxidative Stress, Mitochondrial Dysfunction, and Cell Death: Implications for the Neurodegeneration in Glutaric Aciduria Type I.

PubMed

Rodrigues, Marília Danyelle Nunes; Seminotti, Bianca; Zanatta, Ângela; de Mello Gonçalves, Aline; Bellaver, Bruna; Amaral, Alexandre Umpierrez; Quincozes-Santos, André; Goodman, Stephen Irwin; Woontner, Michael; Souza, Diogo Onofre; Wajner, Moacir

2017-08-01

Patients affected by glutaric aciduria type I (GA-I) show progressive cortical leukoencephalopathy whose pathogenesis is poorly known. In the present work, we exposed cortical astrocytes of wild-type (Gcdh +/+ ) and glutaryl-CoA dehydrogenase knockout (Gcdh -/- ) mice to the oxidative stress inducer menadione and measured mitochondrial bioenergetics, redox homeostasis, and cell viability. Mitochondrial function (MTT and JC1-mitochondrial membrane potential assays), redox homeostasis (DCFH oxidation, nitrate and nitrite production, GSH concentrations and activities of the antioxidant enzymes SOD and GPx), and cell death (propidium iodide incorporation) were evaluated in primary cortical astrocyte cultures of Gcdh +/+ and Gcdh -/- mice unstimulated and stimulated by menadione. We also measured the pro-inflammatory response (TNFα levels, IL1-β and NF-ƙB) in unstimulated astrocytes obtained from these mice. Gcdh -/- mice astrocytes were more vulnerable to menadione-induced oxidative stress (decreased GSH concentrations and altered activities of the antioxidant enzymes), mitochondrial dysfunction (decrease of MTT reduction and JC1 values), and cell death as compared with Gcdh +/+ astrocytes. A higher inflammatory response (TNFα, IL1-β and NF-ƙB) was also observed in Gcdh -/- mice astrocytes. These data indicate a higher susceptibility of Gcdh -/- cortical astrocytes to oxidative stress and mitochondrial dysfunction, probably leading to cell death. It is presumed that these pathomechanisms may contribute to the cortical leukodystrophy observed in GA-I patients.

High-resolution microendoscope for the detection of cervical neoplasia.

PubMed

Grant, Benjamin D; Schwarz, Richard A; Quang, Timothy; Schmeler, Kathleen M; Richards-Kortum, Rebecca

2015-01-01

Cervical cancer causes 275,000 deaths each year with 85 % of these deaths occurring in the developing world. One of the primary reasons for the concentration of deaths in developing countries is a lack of effective screening methods suited for the infrastructure of these countries. In order to address this need, we have developed a high-resolution microendoscope (HRME). The HRME is a fiber-based fluorescence microscope with subcellular resolution. Using the vital stain proflavine, we are able to image cell nuclei in vivo and evaluate metrics such as nuclear-to-cytoplasmic ratio, critical to identifying precancerous epithelial regions. In this chapter, we detail the materials and methods necessary to build this system from commercially available parts.

Transport via SLC5A8 (SMCT1) Is Obligatory for 2-Oxothiazolidine-4-Carboxylate to Enhance Glutathione Production in Retinal Pigment Epithelial Cells

PubMed Central

Babu, Ellappan; Ananth, Sudha; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Smith, Sylvia B.; Boettger, Thomas; Ganapathy, Vadivel

2011-01-01

Purpose. To evaluate the role of SLC5A8 in the transport of 2-oxothiazolidine-4-carboxylate (OTC) and to determine whether OTC augments glutathione production in RPE cells, thereby providing protection against oxidative stress. Methods. SLC5A8-mediated transport of OTC was monitored in Xenopus laevis oocytes by electrophysiological means. Saturation kinetics, Na+-activation kinetics, and inhibition by ibuprofen were analyzed by monitoring OTC-induced currents as a measure of transport activity. Oxidative stress was induced in ARPE-19 cells and primary RPE cells isolated from wild type and Slc5a8-/- mouse retinas using H2O2, and the effects of OTC on cell death and intracellular glutathione concentration were examined. Results. Heterologous expression of human SLC5A8 in X. laevis oocytes induced Na+-dependent inward currents in the presence of OTC under voltage-clamp conditions. The transport of OTC via SLC5A8 was saturable, with a Kt of 104 ± 3 μM. The Na+-activation kinetics was sigmoidal with a Hill coefficient of 1.9 ± 0.1, suggesting involvement of two Na+ in the activation process. Ibuprofen, a blocker of SLC5A8, inhibited SLC5A8-mediated OTC transport; the concentration necessary for half-maximal inhibition was 17 ± 1 μM. OTC increased glutathione levels and protected ARPE-19 and primary RPE cells isolated from wild type mouse retinas from H2O2-induced cell death. These effects were abolished in primary RPE isolated from Slc5a8-/- mouse retinas. Conclusions. OTC is a transportable substrate for SLC5A8. OTC augments glutathione production in RPE cells, thereby protecting them from oxidative damage. Transport via SLC5A8 is obligatory for this process. PMID:21508099

Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia

NASA Technical Reports Server (NTRS)

Fritzsch, Bernd

2003-01-01

The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin 1 (ngn1). An immediate downstream gene is the bHLH gene neuronal differentiation (NeuroD). Targeted null mutations of ngn1 results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn1 null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites growth to sensory epithelia is initially independent of trophic factors released from developing sensory epithelia, but becomes rapidly dependent on those factors. Null mutations of specific neurotrophic factors lose distinct primary neuron populations which undergo rapid embryonic cell death.

Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition.

PubMed

Kosic, Milica; Arsikin-Csordas, Katarina; Paunovic, Verica; Firestone, Raymond A; Ristic, Biljana; Mircic, Aleksandar; Petricevic, Sasa; Bosnjak, Mihajlo; Zogovic, Nevena; Mandic, Milos; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

2016-10-28

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell proliferation is a key determinant of the outcome of FOXO3a activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poulsen, Raewyn C., E-mail: raewyn.poulsen@gmail.com; Carr, Andrew J.; Hulley, Philippa A.

2015-06-19

The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media,more » FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail. - Highlights: • FOXO3a activity can result in either promotion of cell survival or apoptosis. • The outcome of FOXO3a activation differs in proliferating compared to non-proliferating cells. • Proliferating cells are susceptible to FOXO3a-mediated apoptosis. • Inhibition of cell proliferation by FOXO3a promotes cell survival.« less

Annonacin Exerts Antitumor Activity through Induction of Apoptosis and Extracellular Signal-regulated Kinase Inhibition

PubMed Central

Yap, Chee Voon; Subramaniam, Kavita S.; Khor, Sik Wey; Chung, Ivy

2017-01-01

Background: Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Annonacin, a natural pure compound extracted from the seeds of Annona muricata, is a potential alternative therapeutic agent to treat EC. Objective: To study the antitumor activity of annonacin and its mechanism of action in EC cells (ECCs). Materials and Methods: Viability of ECCs treated with annonacin for 72 h was determined using methyl thiazolyl tetrazolium assay. The induction of cell cycle arrest and apoptotic cell death was evaluated using propidium iodide and annexin V-PE/7-AAD assay, respectively. DNA strand breaks were visualized using transferase dUTP nick end labeling assay, and the effects of annonacin on survival signaling were determined using western blotting. Results: Annonacin exhibited antiproliferative effects on EC cell lines (ECC-1 and HEC-1A) and primary cells (EC6-ept and EC14-ept) with EC50values ranging from 4.62 to 4.92 μg/ml. EC cells were shown arrested at G2/M phase after treated with 4 μg/ml of annonacin for 72 h. This led to a significant increase in apoptotic cell death (65.7%) in these cells when compared to vehicle-treated cells (P < 0.005). We further showed that annonacin-mediated apoptotic cell death was associated with an increase in caspase-3 cleavage and DNA fragmentation. Cell apoptosis was accompanied with downregulation of extracellular signal-regulated kinase survival protein expression and induction of G2/M cell cycle arrest. Conclusion: Annonacin may be a potential novel therapeutic agent for EC patients. SUMMARY We aimed to study the antitumor activity of annonacin and its mechanism of action in endometrial cancer cells. Annonacin exerted antiproliferation effects on both endometrial cancer cell lines and primary cells via induction of apoptosis and inhibition of extracellular signal-regulated kinase. Our data represented that annonacin could be an alternative therapeutic treatment to combat endometrial cancer. Abbreviations Used: 7-AAD: 7-Amino-Actinomycin, ATP: Adenosine diphosphate, BSA: Bovine serum albumin, DNA: Deoxyribonucleic acid, EC: Endometrial cancer, ECC-1: Endometrial cancer cell-1, EC50: Half maximal effective concentration, Ept: Epithelial, FBS: Fetal bovine serum, HEC-1A: Human endometrial carcinoma-1A, MTT: Methyl thiazolyl tetrazolium, NaCl: Sodium chloride, NADH: Nicotinamide adenine dinucleotide, RPMI 1640: Roswell Park Memorial Institute Medium, SDS: Sodium dodecyl sulfate PMID:29263632

Rapid determination of antibiotic resistance in E. coli using dielectrophoresis

NASA Astrophysics Data System (ADS)

Hoettges, Kai F.; Dale, Jeremy W.; Hughes, Michael P.

2007-09-01

In recent years, infections due to antibiotic-resistant strains of bacteria such as methillicin-resistant Staphylococcus aureus and ciprofloxacin-resistant Escherichia coli are on the rise, and with them the demand for rapid antibiotic testing is also rising. Conventional tests, such as disc diffusion testing, require a primary sample to be tested in the presence of a number of antibiotics to verify which antibiotics suppress growth, which take approximately 24 h to complete and potentially place the patient at severe risk. In this paper we describe the use of dielectrophoresis as a rapid marker of cell death, by detecting changes in the electrophysiology of the cell caused by the administration of an antibiotic. In contrast to other markers, the electrophysiology of the cell changes rapidly during cell death allowing live cells to be distinguished from dead (or dying) cells without the need for culturing. Using polymyxin B as an example antibiotic, our studies indicate that significant changes in cell characteristics can be observed as soon as 1 h passes after isolating a culture from nutrient broth.

MicroRNA profiling reveals new aspects of HIV neurodegeneration: caspase-6 regulates astrocyte survival.

PubMed

Noorbakhsh, Farshid; Ramachandran, Rithwik; Barsby, Nicola; Ellestad, Kristofor K; LeBlanc, Andrea; Dickie, Peter; Baker, Glen; Hollenberg, Morley D; Cohen, Eric A; Power, Christopher

2010-06-01

MicroRNAs (miRNAs) are small noncoding RNA molecules, which are known to regulate gene expression in physiological and pathological conditions. miRNA profiling was performed using brain tissue from patients with HIV encephalitis (HIVE), a neuroinflammatory/degenerative disorder caused by HIV infection of the brain. Microarray analysis showed differential expression of multiple miRNAs in HIVE compared to control brains. Target prediction and gene ontology enrichment analysis disclosed targeting of several gene families/biological processes by differentially expressed miRNAs (DEMs), with cell death-related genes, including caspase-6, showing a bias toward down-regulated DEMs. Consistent with the miRNA data, HIVE brains exhibited higher levels of caspase-6 transcripts compared with control patients. Immunohistochemical analysis showed localization of the cleaved form of caspase-6 in astrocytes in HIVE brain sections. Exposure of cultured human primary astrocytes to HIV viral protein R (Vpr) induced p53 up-regulation, loss of mitochondrial membrane potential, and caspase-6 activation followed by cell injury. Transgenic mice, expressing Vpr in microglial cells, demonstrated astrocyte apoptosis in brain, which was associated with caspase-6 activation and neurobehavioral abnormalities. Overall, these data point to previously unrecognized alterations in miRNA profile in the brain during HIV infection, which contribute to cell death through dysregulation of cell death machinery.

Upregulation of interleukin 7 receptor alpha and programmed death 1 marks an epitope-specific CD8+ T-cell response that disappears following primary Epstein-Barr virus infection.

PubMed

Sauce, Delphine; Larsen, Martin; Abbott, Rachel J M; Hislop, Andrew D; Leese, Alison M; Khan, Naeem; Papagno, Laura; Freeman, Gordon J; Rickinson, Alan B

2009-09-01

In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8(+) T-cell response. However, we noticed that in HLA-A 0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Ralpha; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Ralpha seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells' disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels.

Metformin, at concentrations corresponding to the treatment of diabetes, potentiates the cytotoxic effects of carboplatin in cultures of ovarian cancer cells.

PubMed

Erices, Rafaela; Bravo, Maria Loreto; Gonzalez, Pamela; Oliva, Bárbara; Racordon, Dusan; Garrido, Marcelo; Ibañez, Carolina; Kato, Sumie; Brañes, Jorge; Pizarro, Javier; Barriga, Maria Isabel; Barra, Alejandro; Bravo, Erasmo; Alonso, Catalina; Bustamente, Eva; Cuello, Mauricio A; Owen, Gareth I

2013-12-01

The use of the type 2 diabetics drug metformin has been correlated with enhanced progression-free survival in ovarian cancer. The literature has speculated that this enhancement is due to the high concentration of metformin directly causing cancer cell death. However, this explanation does not fit with clinical data reporting that the women exposed to constant micromolar concentrations of metformin, as present in the treatment of diabetes, respond better to chemotherapy. Herein, our aim was to examine whether micromolar concentrations of metformin alone could bring about cancer cell death and whether micromolar metformin could increase the cytotoxic effect of commonly used chemotherapies in A2780 and SKOV3 cell lines and primary cultured cancer cells isolated from the peritoneal fluid of patients with advanced ovarian cancer. Our results in cell lines demonstrate that no significant loss of viability or change in cell cycle was observed with micromolar metformin alone; however, we observed cytotoxicity with micromolar metformin in combination with chemotherapy at concentrations where the chemotherapy alone produced no loss in viability. We demonstrate that previous exposure and maintenance of metformin in conjunction with carboplatin produces a synergistic enhancement in cytotoxicity of A2780 and SKOV3 cells (55% and 43%, respectively). Furthermore, in 5 (44%) of the 11 ovarian cancer primary cultures, micromolar metformin improved the cytotoxic response to carboplatin but not paclitaxel or doxorubicin. In conclusion, we present data that support the need for a clinical study to evaluate the adjuvant maintenance or prescription of currently approved doses of metformin during the chemotherapeutic treatment of ovarian cancer.

Mechanisms of Transendothelial Migration of Primary Human Invasive Ductal Carcinoma Cells from ER+, Her2+, and Triple-Negative Disease

DTIC Science & Technology

2016-09-01

cell dissem- ination and, ultimately, patient death (1). The outcome of breast cancer patients with metastatic disease has not improved in the past 30...breast cancer is a heterogeneous disease consisting of several distinct subtypes with substantially different responses to therapy and clinical...and Triple-Negative Disease PRINCIPAL INVESTIGATOR: Jeanine Pignatelli CONTRACTING ORGANIZATION: Albert Einstein College of Medicine Bronx, NY 10461

Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells

PubMed Central

Hsieh, I-Shan; Yang, Rong-Sen; Fu, Wen-Mei

2014-01-01

Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients. PMID:25310823

GDP-mannose-4,6-dehydratase (GMDS) Deficiency Renders Colon Cancer Cells Resistant to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor- and CD95-mediated Apoptosis by Inhibiting Complex II Formation*

PubMed Central

Moriwaki, Kenta; Shinzaki, Shinichiro; Miyoshi, Eiji

2011-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors. PMID:22027835

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and ΔΨm across mitochondrial inner membrane.

PubMed

Lee, Ji Hyung; Amarsanaa, Khulan; Wu, Jinji; Jeon, Sang-Chan; Cui, Yanji; Jung, Sung-Cherl; Park, Deok-Bae; Kim, Se-Jae; Han, Sang-Heon; Kim, Hyun-Wook; Rhyu, Im Joo; Eun, Su-Yong

2018-05-01

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨ m ). Therefore, pharmacological manipulation of ΔΨ m can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨ m against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 µM, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 µM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca 2+ (5 µM). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨ m were completely abolished in K + -free medium on pure isolated mitochondria. Taken together, results demonstrate that K + influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K + influx is probably mediated, at least in part, by activation of mitochondrial K + channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

The Neuroprotective Effect of Klotho is Mediated via Regulation of Members of the Redox System*

PubMed Central

Zeldich, Ella; Chen, Ci-Di; Colvin, Teresa A.; Bove-Fenderson, Erin A.; Liang, Jennifer; Tucker Zhou, Tracey B.; Harris, David A.; Abraham, Carmela R.

2014-01-01

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid β (oAβ)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAβ, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAβ, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect. PMID:25037225

Survival and predictors of death among primary immunodeficient patients: a registry-based study.

PubMed

Al-Herz, Waleed; Moussa, Mohamed A A

2012-06-01

The aims of this study were to investigate survival among patients with primary immunodeficiency disorders (PID) in Kuwait and to determine whether certain variables were associated with increased risk of death. The data of 176 patients (98 males and 78 females) were extracted from the Kuwait National Primary Immunodeficiency Disorders Registry and the observation period was from January 2004 to July 2011. The distribution of the reported patients was combined T- and B-cell immunodeficiencies (30.1%), predominantly antibody immunodeficiency (19.9%), other well-defined immunodeficiencies (25%), diseases of immune dysregulation (14.8%), congenital defects of phagocyte number, function or both (6.25%), and complement deficiencies (4.0%). In a total of 619.1 patient-years at risk, 48 patients died (mortality incidence rate 77.53 per 1,000 person-years). The overall survival in the studied cohort was 72.7% (72.4% for males and 73.1% for females). The most common cause of death was sepsis (46%) followed by pneumonia (29%). The probabilities that a patient survived 2, 4, and 6 years after onset of symptoms were 76%, 73%, and 69%, respectively. The variables that were found to be predictors for death are parental consanguinity, sepsis, adenovirus and CMV infections, failure to thrive, PID category, and onset age <6 months. Patients with PID have decreased probabilities of survival that are variable between PID categories. Early diagnosis and aggressive therapeutic interventions specifically of patients with history of the variables associated with increased risk of death may help increase their chance of survival.

PreSERVE-AMI: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Intracoronary Administration of Autologous CD34+ Cells in Patients With Left Ventricular Dysfunction Post STEMI.

PubMed

Quyyumi, Arshed A; Vasquez, Alejandro; Kereiakes, Dean J; Klapholz, Marc; Schaer, Gary L; Abdel-Latif, Ahmed; Frohwein, Stephen; Henry, Timothy D; Schatz, Richard A; Dib, Nabil; Toma, Catalin; Davidson, Charles J; Barsness, Gregory W; Shavelle, David M; Cohen, Martin; Poole, Joseph; Moss, Thomas; Hyde, Pamela; Kanakaraj, Anna Maria; Druker, Vitaly; Chung, Amy; Junge, Candice; Preti, Robert A; Smith, Robin L; Mazzo, David J; Pecora, Andrew; Losordo, Douglas W

2017-01-20

Despite direct immediate intervention and therapy, ST-segment-elevation myocardial infarction (STEMI) victims remain at risk for infarct expansion, heart failure, reinfarction, repeat revascularization, and death. To evaluate the safety and bioactivity of autologous CD34+ cell (CLBS10) intracoronary infusion in patients with left ventricular dysfunction post STEMI. Patients who underwent successful stenting for STEMI and had left ventricular dysfunction (ejection fraction≤48%) ≥4 days poststent were eligible for enrollment. Subjects (N=161) underwent mini bone marrow harvest and were randomized 1:1 to receive (1) autologous CD34+ cells (minimum 10 mol/L±20% cells; N=78) or (2) diluent alone (N=83), via intracoronary infusion. The primary safety end point was adverse events, serious adverse events, and major adverse cardiac event. The primary efficacy end point was change in resting myocardial perfusion over 6 months. No differences in myocardial perfusion or adverse events were observed between the control and treatment groups, although increased perfusion was observed within each group from baseline to 6 months (P<0.001). In secondary analyses, when adjusted for time of ischemia, a consistently favorable cell dose-dependent effect was observed in the change in left ventricular ejection fraction and infarct size, and the duration of time subjects was alive and out of hospital (P=0.05). At 1 year, 3.6% (N=3) and 0% deaths were observed in the control and treatment group, respectively. This PreSERVE-AMI (Phase 2, randomized, double-blind, placebo-controlled trial) represents the largest study of cell-based therapy for STEMI completed in the United States and provides evidence supporting safety and potential efficacy in patients with left ventricular dysfunction post STEMI who are at risk for death and major morbidity. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01495364. © 2016 American Heart Association, Inc.

Restrictive or Liberal Red-Cell Transfusion for Cardiac Surgery.

PubMed

Mazer, C David; Whitlock, Richard P; Fergusson, Dean A; Hall, Judith; Belley-Cote, Emilie; Connolly, Katherine; Khanykin, Boris; Gregory, Alexander J; de Médicis, Étienne; McGuinness, Shay; Royse, Alistair; Carrier, François M; Young, Paul J; Villar, Juan C; Grocott, Hilary P; Seeberger, Manfred D; Fremes, Stephen; Lellouche, François; Syed, Summer; Byrne, Kelly; Bagshaw, Sean M; Hwang, Nian C; Mehta, Chirag; Painter, Thomas W; Royse, Colin; Verma, Subodh; Hare, Gregory M T; Cohen, Ashley; Thorpe, Kevin E; Jüni, Peter; Shehata, Nadine

2017-11-30

The effect of a restrictive versus liberal red-cell transfusion strategy on clinical outcomes in patients undergoing cardiac surgery remains unclear. In this multicenter, open-label, noninferiority trial, we randomly assigned 5243 adults undergoing cardiac surgery who had a European System for Cardiac Operative Risk Evaluation (EuroSCORE) I of 6 or more (on a scale from 0 to 47, with higher scores indicating a higher risk of death after cardiac surgery) to a restrictive red-cell transfusion threshold (transfuse if hemoglobin level was <7.5 g per deciliter, starting from induction of anesthesia) or a liberal red-cell transfusion threshold (transfuse if hemoglobin level was <9.5 g per deciliter in the operating room or intensive care unit [ICU] or was <8.5 g per deciliter in the non-ICU ward). The primary composite outcome was death from any cause, myocardial infarction, stroke, or new-onset renal failure with dialysis by hospital discharge or by day 28, whichever came first. Secondary outcomes included red-cell transfusion and other clinical outcomes. The primary outcome occurred in 11.4% of the patients in the restrictive-threshold group, as compared with 12.5% of those in the liberal-threshold group (absolute risk difference, -1.11 percentage points; 95% confidence interval [CI], -2.93 to 0.72; odds ratio, 0.90; 95% CI, 0.76 to 1.07; P<0.001 for noninferiority). Mortality was 3.0% in the restrictive-threshold group and 3.6% in the liberal-threshold group (odds ratio, 0.85; 95% CI, 0.62 to 1.16). Red-cell transfusion occurred in 52.3% of the patients in the restrictive-threshold group, as compared with 72.6% of those in the liberal-threshold group (odds ratio, 0.41; 95% CI, 0.37 to 0.47). There were no significant between-group differences with regard to the other secondary outcomes. In patients undergoing cardiac surgery who were at moderate-to-high risk for death, a restrictive strategy regarding red-cell transfusion was noninferior to a liberal strategy with respect to the composite outcome of death from any cause, myocardial infarction, stroke, or new-onset renal failure with dialysis, with less blood transfused. (Funded by the Canadian Institutes of Health Research and others; TRICS III ClinicalTrials.gov number, NCT02042898 .).

Exposure to Cell Phone Radiation Up-Regulates Apoptosis Genes in Primary Cultures of Neurons and Astrocytes

PubMed Central

Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E.

2007-01-01

The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working GSM (Global System for Mobile Communication) cell phone rated at a frequency of 1900 MHz. Primary cultures were exposed to cell phone emissions for 2 hrs. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Upregulation occurred in both “on” and “stand-by” modes in neurons, but only in “on” mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons and astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes. PMID:17187929

Epigenetic therapy potential of suberoylanilide hydroxamic acid on invasive human non-small cell lung cancer cells.

PubMed

Zhang, Shirong; Wu, Kan; Feng, Jianguo; Wu, Zhibing; Deng, Qinghua; Guo, Chao; Xia, Bing; Zhang, Jing; Huang, Haixiu; Zhu, Lucheng; Zhang, Ke; Shen, Binghui; Chen, Xufeng; Ma, Shenglin

2016-10-18

Metastasis is the reason for most cancer death, and a crucial primary step for cancer metastasis is invasion of the surrounding tissue, which may be initiated by some rare tumor cells that escape the heterogeneous primary tumor. In this study, we isolated invasive subpopulations of cancer cells from human non-small cell lung cancer (NSCLC) H460 and H1299 cell lines, and determined the gene expression profiles and the responses of these invasive cancer cells to treatments of ionizing radiation and chemotherapeutic agents. The subpopulation of highly invasive NSCLC cells showed epigenetic signatures of epithelial-mesenchymal transition, cancer cell stemness, increased DNA damage repair and cell survival signaling. We also investigated the epigenetic therapy potential of suberoylanilide hydroxamic acid (SAHA) on invasive cancer cells, and found that SAHA suppresses cancer cell invasiveness and sensitizes cancer cells to treatments of IR and chemotherapeutic agents. Our results provide guidelines for identification of metastatic predictors and for clinical management of NSCLC. This study also suggests a beneficial clinical potential of SAHA as a chemotherapeutic agent for NSCLC patients.

Caspase inhibitors protect neurons by enabling selective necroptosis of inflamed microglia.

PubMed

Fricker, Michael; Vilalta, Anna; Tolkovsky, Aviva M; Brown, Guy C

2013-03-29

Microglia are resident brain macrophages, which can cause neuronal loss when activated in infectious, ischemic, traumatic, and neurodegenerative diseases. Caspase-8 has both prodeath and prosurvival roles, mediating apoptosis and/or preventing RIPK1-mediated necroptosis depending on cell type and stimulus. We found that inflammatory stimuli (LPS, lipoteichoic acid, or TNF-α) caused an increase in caspase-8 IETDase activity in primary rat microglia without inducing apoptosis. Inhibition of caspase-8 with either Z-VAD-fmk or IETD-fmk resulted in necrosis of activated microglia. Inhibition of caspases with Z-VAD-fmk did not kill non-activated microglia, or astrocytes and neurons in any condition. Necrostatin-1, a specific inhibitor of RIPK1, prevented microglial caspase inhibition-induced death, indicating death was by necroptosis. In mixed cerebellar cultures of primary neurons, astrocytes, and microglia, LPS induced neuronal loss that was prevented by inhibition of caspase-8 (resulting in microglial necroptosis), and neuronal death was restored by rescue of microglia with necrostatin-1. We conclude that the activation of caspase-8 in inflamed microglia prevents their death by necroptosis, and thus, caspase-8 inhibitors may protect neurons in the inflamed brain by selectively killing activated microglia.

Primary mucinous carcinoma with rhabdoid cells of the thyroid gland: a case report.

PubMed

Matsuo, Mioko; Tuneyoshi, Masazumi; Mine, Mari

2016-06-10

Primary mucinous carcinoma of the thyroid gland is a rare disease; only 6 cases of primary mucinous carcinoma of the thyroid have been previously reported. Primary mucinous carcinoma of the thyroid gland with incomplete tumor resection tends to be associated with a poor prognosis, resulting in death within a few months. An early and appropriate diagnosis may contribute to improvement in patient prognosis; however, it is extremely difficult to diagnose primary mucinous carcinoma of the thyroid. We present the seventh reported case of primary mucinous carcinoma in the thyroid gland; moreover, rhabdoid cells were detected, which, to our knowledge, is a novel finding. An 81-year-old Japanese woman was initially diagnosed with a poorly differentiated thyroid carcinoma, and she underwent a hemithyroidectomy. Pathological examination revealed the presence of abundant mucus and agglomeration of large atypical cells. Rhabdoid cells were also seen scattered among the tumor cells. Immunostaining was performed for various markers, and on the basis of these results, we diagnosed the lesion as primary mucinous carcinoma with rhabdoid cells in the thyroid gland. Ten months after surgery, recurrence was noted in the paratracheal lymph nodes; therefore, total resection of the residual thyroid gland and paratracheal lymphadenectomy with thyroid-stimulating hormone suppression were performed. The patient is currently alive and disease-free. The current case is of interest not only because of the rare histological findings, but also because the patient achieved long-term survival following diagnosis of a mucinous carcinoma. We believe this report will be helpful for diagnosing future cases of mucinous carcinoma of the thyroid.

Differentiation of a Highly Tumorigenic Basal Cell Compartment in Urothelial Carcinoma

PubMed Central

He, Xiaobing; Marchionni, Luigi; Hansel, Donna E.; Yu, Wayne; Sood, Akshay; Yang, Jie; Parmigiani, Giovanni; Matsui, William; Berman, David M.

2011-01-01

Highly tumorigenic cancer cell (HTC) populations have been identified for a variety of solid tumors and assigned stem cell properties. Strategies for identifying HTCs in solid tumors have been primarily empirical rather than rational, particularly in epithelial tumors, which are responsible for 80% of cancer deaths. We report evidence for a spatially restricted bladder epithelial (urothelial) differentiation program in primary urothelial cancers (UCs) and in UC xenografts. We identified a highly tumorigenic UC cell compartment that resembles benign urothelial stem cells (basal cells), co-expresses the 67-kDa laminin receptor and the basal cell-specific cytokeratin CK17, and lacks the carcinoembryonic antigen family member CEACAM6 (CD66c). This multipotent compartment resides at the tumor-stroma interface, is easily identified on histologic sections, and possesses most, if not all, of the engraftable tumor-forming ability in the parental xenograft. We analyzed differential expression of genes and pathways in basal-like cells versus more differentiated cells. Among these, we found significant enrichment of pathways comprising “hallmarks” of cancer, and pharmacologically targetable signaling pathways, including Janus kinase-signal transducer and activator of transcription, Notch, focal adhesion, mammalian target of rapamycin, epidermal growth factor receptor (erythroblastic leukemia viral oncogene homolog [ErbB]), and wingless-type MMTV integration site family (Wnt). The basal/HTC gene expression signature was essentially invisible within the context of nontumorigenic cell gene expression and overlapped significantly with genes driving progression and death in primary human UC. The spatially restricted epithelial differentiation program described here represents a conceptual advance in understanding cellular heterogeneity of carcinomas and identifies basal-like HTCs as attractive targets for cancer therapy. PMID:19544456

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

PubMed

Crawford, Lisa J; Anderson, Gordon; Johnston, Cliona K; Irvine, Alexandra E

2016-10-25

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

Na(+), K(+)-ATPase: the new face of an old player in pathogenesis and apoptotic/hybrid cell death.

PubMed

Yu, Shan Ping

2003-10-15

The Na(+), K(+)-ATPase is a ubiquitous membrane transport protein in mammalian cells, responsible for establishing and maintaining high K(+) and low Na(+) in the cytoplasm required for normal resting membrane potentials and various cellular activities. The ionic homeostasis maintained by the Na(+), K(+)-ATPase is also critical for cell growth, differentiation, and cell survival. Although the toxic effects of blocking the Na(+), K(+)-ATPase by ouabain and other selective inhibitors have been known for years, the mechanism of action remained unclear. Recent progress in two areas has significantly advanced our understanding of the role and mechanism of Na(+), K(+)-ATPase in cell death. Along with increased recognition of apoptosis in a wide range of disease states, Na(+), K(+)-ATPase deficiency has been identified as a contributor to apoptosis and pathogenesis. More importantly, accumulating evidence now endorses a close relationship between ionic homeostasis and apoptosis, namely the regulation of apoptosis by K(+) homeostasis. Since Na(+), K(+)-ATPase is the primary system for K(+) uptake, dysfunction of the transport enzyme and resultant disruption of ionic homeostasis have been re-evaluated for their critical roles in apoptosis and apoptosis-related diseases. In this review, instead of giving a detailed description of the structure and regulation of Na(+), K(+)-ATPase, the author will focus on the most recent evidence indicating the unique role of Na(+), K(+)-ATPase in cell death, including apoptosis and the newly recognized "hybrid death" of concurrent apoptosis and necrosis in the same cells. It is also hoped that discussion of some seemingly conflicting reports will inspire further debate and benefit future investigation in this important research field.

Astrocytes expressing mutant SOD1 and TDP43 trigger motoneuron death that is mediated via sodium channels and nitroxidative stress

PubMed Central

Rojas, Fabiola; Cortes, Nicole; Abarzua, Sebastian; Dyrda, Agnieszka; van Zundert, Brigitte

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1G93A contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Nav) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1G93A and ACM-SOD1G86R) or TDP43 (ACM-TDP43A315T) mutants; we show that such exposure rapidly (within 30–60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Nav channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Nav channel activity. PMID:24570655

Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level.

PubMed

Ventura, P; Toullec, G; Fricano, C; Chapron, L; Meunier, V; Röttinger, E; Furla, P; Barnay-Verdier, S

2018-04-01

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+ 5 and + 8 °C) during 1 and 7 days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a + 8 °C hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins.

PubMed

Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Häcker, G

2015-11-26

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.

Sonic hedgehog is required for survival of both myogenic and chondrogenic somitic lineages.

PubMed

Teillet, M; Watanabe, Y; Jeffs, P; Duprez, D; Lapointe, F; Le Douarin, N M

1998-06-01

In vertebrates, the medial moieties of the somites give rise to the vertebrae and epaxial muscles, which develop in close relationship with the axial organs, neural tube and notochord. The lateral moieties contribute to the ribs and to limb and body wall muscles (hypaxial muscles) after a phase of lateral and ventral migration. Surgical ablation of the neural tube and notochord in the chick embryo during segmentation and early differentiation of the somites (day 2 of incubation) does not affect primary development of the hypaxial muscles, but leads to a complete absence of epaxial muscles, vertebrae and ribs, due to cell death in the somites. Here we demonstrate that cell death, which occurs within 24 hours of excision of the axial organs, affects both myogenic and chondrogenic cell lineages defined, respectively, by the expression of MyoD and Pax-1 genes. In contrast, Pax-3 transcripts, normally present in cells giving rise to hypaxial muscles, are preserved in the excised embryos. Backgrafting either the ventral neural tube or the notochord allows survival of MyoD- and Pax-1-expressing cells. Similarly, Sonic hedgehog-producing cells grafted in place of axial organs also rescue MyoD- and Pax-1-expressing cells from death and allow epaxial muscles, ribs and vertebrae to undergo organogenesis. These results demonstrate that the ventral neural tube and the notochord promote the survival of both myogenic and chondrogenic cell lineages in the somites and that this action is mediated by Sonic hedgehog.

Targeting Metastasis with Snake Toxins: Molecular Mechanisms

PubMed Central

Urra, Félix A.

2017-01-01

Metastasis involves the migration of cancer cells from a primary tumor to invade and establish secondary tumors in distant organs, and it is the main cause for cancer-related deaths. Currently, the conventional cytostatic drugs target the proliferation of malignant cells, being ineffective in metastatic disease. This highlights the need to find new anti-metastatic drugs. Toxins isolated from snake venoms are a natural source of potentially useful molecular scaffolds to obtain agents with anti-migratory and anti-invasive effects in cancer cells. While there is greater evidence concerning the mechanisms of cell death induction of several snake toxin classes on cancer cells; only a reduced number of toxin classes have been reported (i.e., disintegrins/disintegrin-like proteins, C-type lectin-like proteins, C-type lectins, serinproteases, cardiotoxins, snake venom cystatins) as inhibitors of adhesion, migration, and invasion of cancer cells. Here, we discuss the anti-metastatic mechanisms of snake toxins, distinguishing three targets, which involve (1) inhibition of extracellular matrix components-dependent adhesion and migration, (2) inhibition of epithelial-mesenchymal transition, and (3) inhibition of migration by alterations in the actin/cytoskeleton network. PMID:29189742

Primary hepatocytes and their cultures in liver apoptosis research

PubMed Central

Vinken, Mathieu; Maes, Michaël; Oliveira, André G.; Cogliati, Bruno; Marques, Pedro E.; Menezes, Gustavo B.; Dagli, Maria Lúcia Zaidan; Vanhaecke, Tamara; Rogiers, Vera

2014-01-01

Apoptosis not only plays a key role in physiological demise of defunct hepatocytes, but is also associated with a plethora of acute and chronic liver diseases as well as with hepatotoxicity. The present paper focuses on the modelling of this mode of programmed cell death in primary hepatocyte cultures. Particular attention is paid to the activation of spontaneous apoptosis during the isolation of hepatocytes from the liver, its progressive manifestation upon the subsequent establishment of cell cultures and simultaneously to strategies to counteract this deleterious process. In addition, currently applied approaches to experimentally induce controlled apoptosis in this in vitro setting for mechanistic research purposes and thereby its detection using relevant biomarkers are reviewed. PMID:24013573

Samsoeum, a traditional herbal medicine, elicits apoptotic and autophagic cell death by inhibiting Akt/mTOR and activating the JNK pathway in cancer cells

PubMed Central

2013-01-01

Background Samsoeum (SSE), a traditional herbal formula, has been widely used to treat cough, fever, congestion, and emesis for centuries. Recent studies have demonstrated that SSE retains potent pharmacological efficiency in anti-allergic and anti-inflammatory reactions. However, the anti-cancer activity of SSE and its underlying mechanisms have not been studied. Thus, the present study was designed to determine the effect of SSE on cell death and elucidate its detailed mechanism. Methods Following SSE treatment, cell growth and cell death were measured using an MTT assay and trypan blue exclusion assay, respectively. Cell cycle arrest and YO-PRO-1 uptake were assayed using flow cytometry, and LC3 redistribution was observed using confocal microscope. The mechanisms of anti-cancer effect of SSE were investigated through western blot analysis. Results We initially found that SSE caused dose- and time-dependent cell death in cancer cells but not in normal primary hepatocytes. In addition, during early SSE treatment (6–12 h), cells were arrested in G2/M phase concomitant with up-regulation of p21 and p27 and down-regulation of cyclin D1 and cyclin B1, followed by an increase in apoptotic YO-PRO-1 (+) cells. SSE also induced autophagy via up-regulation of Beclin-1 expression, conversion of microtubule-associated protein light chain 3 (LC3) I to LC3-II, and re-distribution of LC3, indicating autophagosome formation. Moreover, the level of B-cell lymphoma 2 (Bcl-2), which is critical for cross-talk between apoptosis and autophagy, was significantly reduced in SSE-treated cells. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was increased, followed by suppression of the protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathway, and phosphorylation of mitogen-activated protein kinases (MAPKs) in response to SSE treatment. In particular, among MAPKs inhibitors, only the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 nearly blocked SSE-induced increases in Beclin-1, LC3-II, and Bax expression and decreases in Bcl-2 expression, indicating that JNK activation plays critical role in cell death caused by SSE. Conclusions These findings suggest that SSE efficiently induces cancer cell death via apoptosis as well as autophagy through modification of the Akt/mTOR and JNK signaling pathways. SSE may be as a potent traditional herbal medicine for treating malignancies. PMID:24053190

Increasing procaspase 8 expression using repurposed drugs to induce HIV infected cell death in ex vivo patient cells

PubMed Central

Sampath, Rahul; Cummins, Nathan W.; Natesampillai, Sekar; Bren, Gary D.; Chung, Thomas D.; Baker, Jason; Henry, Keith; Pagliuzza, Amélie; Badley, Andrew D.

2017-01-01

HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018) without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches. PMID:28628632

Electromagnetic field therapy delays cellular senescence and death by enhancement of the heat shock response.

PubMed

Perez, Felipe P; Zhou, Ximing; Morisaki, Jorge; Jurivich, Donald

2008-04-01

Hormesis may result when mild repetitive stress increases cellular defense against diverse injuries. This process may also extend in vitro cellular proliferative life span as well as delay and reverse some of the age-dependent changes in both replicative and non-replicative cells. This study evaluated the potential hormetic effect of non-thermal repetitive electromagnetic field shock (REMFS) and its impact on cellular aging and mortality in primary human T lymphocytes and fibroblast cell lines. Unlike previous reports employing electromagnetic radiation, this study used a long wave length, low energy, and non-thermal REMFS (50MHz/0.5W) for various therapeutic regimens. The primary outcomes examined were age-dependent morphological changes in cells over time, cellular death prevention, and stimulation of the heat shock response. REMFS achieved several biological effects that modified the aging process. REMFS extended the total number of population doublings of mouse fibroblasts and contributed to youthful morphology of cells near their replicative lifespan. REMFS also enhanced cellular defenses of human T cells as reflected in lower cell mortality when compared to non-treated T cells. To determine the mechanism of REMFS-induced effects, analysis of the cellular heat shock response revealed Hsp90 release from the heat shock transcription factor (HSF1). Furthermore, REMFS increased HSF1 phosphorylation, enhanced HSF1-DNA binding, and improved Hsp70 expression relative to non-REMFS-treated cells. These results show that non-thermal REMFS activates an anti-aging hormetic effect as well as reduces cell mortality during lethal stress. Because the REMFS configuration employed in this study can potentially be applied to whole body therapy, prospects for translating these data into clinical interventions for Alzheimer's disease and other degenerative conditions with aging are discussed.

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38

PubMed Central

Weilbacher, A; Gutekunst, M; Oren, M; Aulitzky, W E; van der Kuip, H

2014-01-01

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug. PMID:25010984

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38.

PubMed

Weilbacher, A; Gutekunst, M; Oren, M; Aulitzky, W E; van der Kuip, H

2014-07-10

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.

Near infrared radiation protects against oxygen-glucose deprivation-induced neurotoxicity by down-regulating neuronal nitric oxide synthase (nNOS) activity in vitro.

PubMed

Yu, Zhanyang; Li, Zhaoyu; Liu, Ning; Jizhang, Yunneng; McCarthy, Thomas J; Tedford, Clark E; Lo, Eng H; Wang, Xiaoying

2015-06-01

Near infrared radiation (NIR) has been shown to be neuroprotective against neurological diseases including stroke and brain trauma, but the underlying mechanisms remain poorly understood. In the current study we aimed to investigate the hypothesis that NIR may protect neurons by attenuating oxygen-glucose deprivation (OGD)-induced nitric oxide (NO) production and modulating cell survival/death signaling. Primary mouse cortical neurons were subjected to 4 h OGD and NIR was applied at 2 h reoxygenation. OGD significantly increased NO level in primary neurons compared to normal control, which was significantly ameliorated by NIR at 5 and 30 min post-NIR. Neither OGD nor NIR significantly changed neuronal nitric oxide synthase (nNOS) mRNA or total protein levels compared to control groups. However, OGD significantly increased nNOS activity compared to normal control, and this effect was significantly diminished by NIR. Moreover, NIR significantly ameliorated the neuronal death induced by S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor. Finally, NIR significantly rescued OGD-induced suppression of p-Akt and Bcl-2 expression, and attenuated OGD-induced upregulation of Bax, BAD and caspase-3 activation. These results suggest NIR may protect against OGD at least partially through reducing NO production by down-regulating nNOS activity, and modulating cell survival/death signaling.

Differential expression of c-Met between primary and metastatic sites in clear-cell renal cell carcinoma and its association with PD-L1 expression.

PubMed

Lalani, Aly-Khan A; Gray, Kathryn P; Albiges, Laurence; Callea, Marcella; Pignon, Jean-Christophe; Pal, Soumitro; Gupta, Mamta; Bhatt, Rupal S; McDermott, David F; Atkins, Michael B; Woude, G F Vande; Harshman, Lauren C; Choueiri, Toni K; Signoretti, Sabina

2017-11-28

In preclinical models, c-Met promotes survival of renal cancer cells through the regulation of programmed death-ligand 1 (PD-L1). However, this relationship in human clear cell renal cell carcinoma (ccRCC) is not well characterized. We evaluated c-Met expression in ccRCC patients using paired primary and metastatic samples and assessed the association with PD-L1 expression and other clinical features. Areas with predominant and highest Fuhrman nuclear grade (FNG) were selected. c-Met expression was evaluated by IHC using an anti-Met monoclonal antibody (MET4 Ab) and calculated by a combined score (CS, 0-300): intensity of c-Met staining (0-3) x % of positive cells (0-100). PD-L1 expression in tumor cells was previously assessed by IHC and PD-L1+ was defined as PD-L1 > 0% positive cells. Our cohort consisted of 45 pairs of primary and metastatic ccRCC samples. Overall, c-Met expression was higher in metastatic sites compared to primary sites (average c-Met CS: 55 vs. 28, p = 0.0003). Higher c-Met expression was associated with higher FNG (4 vs. 3) in primary tumors (average c-Met CS: 52 vs. 20, p = 0.04). c-Met expression was numerically greater in PD-L1+ vs. PD-L1- tumors. Higher c-Met expression in metastatic sites compared to primary tumors suggests that testing for biomarkers of response to c-Met inhibitors should be conducted in metastases. While higher c-Met expression in PD-L1+ tumors requires further investigation, it supports exploring these targets in combination clinical trials.

Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases.

PubMed

Aldhahrani, Adil; Verdon, Bernard; Ward, Chris; Pearson, Jeffery

2017-01-01

Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B). The immortalised human bronchial epithelial cell line (BEAS-2B) was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL)-8, IL-6 and granulocyte-macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L -1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L -1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B. Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo . This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury.

Discovery of a proteinaceous cellular receptor for a norovirus

PubMed Central

Orchard, Robert C.; Wilen, Craig B.; Doench, John G.; Baldridge, Megan T.; McCune, Broc T.; Lee, Ying-Chiang J.; Lee, Sanghyun; Pruett-Miller, Shondra M.; Nelson, Christopher A.; Fremont, Daved H.; Virgin, Herbert W.

2017-01-01

Human noroviruses (NoV) are a leading cause of gastroenteritis globally, yet host factors required for NoV infection are poorly understood. We identified host molecules essential for murine NoV (MNoV) induced cell death including CD300lf as a proteinaceous receptor. CD300lf is essential for MNoV binding and replication in cell lines and primary cells. Additionally, Cd300lf−/− mice are resistant to MNoV infection. Expression of CD300lf in human cells breaks the species barrier restricting MNoV replication. The crystal structure of the CD300lf ectodomain revealed a potential ligand binding cleft composed of residues critical for MNoV infection. Therefore, the presence of a proteinaceous receptor is the primary determinant of MNoV species tropism while other components of cellular machinery required for NoV replication are conserved between humans and mice. PMID:27540007

What do we know about the mechanisms of elimination of autoreactive T and B cells and what challenges remain.

PubMed

Strasser, Andreas; Puthalakath, Hamsa; O'Reilly, Lorraine A; Bouillet, Philippe

2008-01-01

Tolerance to self-antigens within the adaptive immune system is safeguarded, at least in part, through deletion of autoreactive T and B lymphocytes. This deletion can occur during the development of these cells in primary lymphoid organs, the thymus or bone marrow, respectively, or at the mature stage in peripheral lymphoid tissues. Deletion of autoreactive lymphocytes is achieved to a large extent through apoptotic cell death. This review describes current understanding of the mechanisms that mediate apoptosis of autoreactive lymphocytes during their development in primary lymphoid organs and during their activation in the periphery. In particular, we discuss the roles of the proapoptotic Bcl-2 family member Bim and the small family of Nur77-related transcriptional regulators in lymphocyte negative selection. Finally, we speculate on the processes that may lead to the activation of Bim when antigen receptors are activated on autoreactive T or B cells.

Investigating the biochemical progression of liver disease through fibrosis, cirrhosis, dysplasia, and hepatocellular carcinoma using Fourier transform infrared spectroscopic imaging

NASA Astrophysics Data System (ADS)

Sreedhar, Hari; Pant, Mamta; Ronquillo, Nemencio R.; Davidson, Bennett; Nguyen, Peter; Chennuri, Rohini; Choi, Jacqueline; Herrera, Joaquin A.; Hinojosa, Ana C.; Jin, Ming; Kajdacsy-Balla, Andre; Guzman, Grace; Walsh, Michael J.

2014-03-01

Hepatocellular carcinoma (HCC) is the most common form of primary hepatic carcinoma. HCC ranks the fourth most prevalent malignant tumor and the third leading cause of cancer related death in the world. Hepatocellular carcinoma develops in the context of chronic liver disease and its evolution is characterized by progression through intermediate stages to advanced disease and possibly even death. The primary sequence of hepatocarcinogenesis includes the development of cirrhosis, followed by dysplasia, and hepatocellular carcinoma.1 We addressed the utility of Fourier Transform Infrared (FT-IR) spectroscopic imaging, both as a diagnostic tool of the different stages of the disease and to gain insight into the biochemical process associated with disease progression. Tissue microarrays were obtained from the University of Illinois at Chicago tissue bank consisting of liver explants from 12 transplant patients. Tissue core biopsies were obtained from each explant targeting regions of normal, liver cell dysplasia including large cell change and small cell change, and hepatocellular carcinoma. We obtained FT-IR images of these tissues using a modified FT-IR system with high definition capabilities. Firstly, a supervised spectral classifier was built to discriminate between normal and cancerous hepatocytes. Secondly, an expanded classifier was built to discriminate small cell and large cell changes in liver disease. With the emerging advances in FT-IR instrumentation and computation there is a strong drive to develop this technology as a powerful adjunct to current histopathology approaches to improve disease diagnosis and prognosis.

Aldolase B-Mediated Fructose Metabolism Drives Metabolic Reprogramming of Colon Cancer Liver Metastasis.

PubMed

Bu, Pengcheng; Chen, Kai-Yuan; Xiang, Kun; Johnson, Christelle; Crown, Scott B; Rakhilin, Nikolai; Ai, Yiwei; Wang, Lihua; Xi, Rui; Astapova, Inna; Han, Yan; Li, Jiahe; Barth, Bradley B; Lu, Min; Gao, Ziyang; Mines, Robert; Zhang, Liwen; Herman, Mark; Hsu, David; Zhang, Guo-Fang; Shen, Xiling

2018-06-05

Cancer metastasis accounts for the majority of cancer-related deaths and remains a clinical challenge. Metastatic cancer cells generally resemble cells of the primary cancer, but they may be influenced by the milieu of the organs they colonize. Here, we show that colorectal cancer cells undergo metabolic reprogramming after they metastasize and colonize the liver, a key metabolic organ. In particular, via GATA6, metastatic cells in the liver upregulate the enzyme aldolase B (ALDOB), which enhances fructose metabolism and provides fuel for major pathways of central carbon metabolism during tumor cell proliferation. Targeting ALDOB or reducing dietary fructose significantly reduces liver metastatic growth but has little effect on the primary tumor. Our findings suggest that metastatic cells can take advantage of reprogrammed metabolism in their new microenvironment, especially in a metabolically active organ such as the liver. Manipulation of involved pathways may affect the course of metastatic growth. Copyright © 2018 Elsevier Inc. All rights reserved.

Utilization of the cellular stress response to sensitize cancer cells to TRAIL-mediated apoptosis.

PubMed

Siegelin, Markus David

2012-08-01

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising death ligand who has received significant attention due to its specific anti-cancer activity. Recently, a number of clinical trials involving either recombinant soluble TRAIL or agonistic death receptor (DR) antibodies have even been initiated. One major caveat in TRAIL-based anti-cancer therapies is that a considerable number of cancer cells are notorious resistant to apoptosis induction by TRAIL. Overcoming this primary or secondary evolved resistance is an utmost important goal of present cancer research. The current literature suggests that TRAIL resistance is mediated by a number of endogenous factors. According to recent research, stress-related transcription factors have acquired a pivotal role in the sensitization of highly resistant cancer cells, for example, pancreatic cancer and glioblastoma cells, to TRAIL-mediated cell death. Out of this transcription factor family, C/EBP-homologous protein (CHOP) is linked to the control of DR-mediated apoptosis by modulation of several apoptotic and anti-apoptotic factors. Stress responses in certain organelles, such as endoplasmic reticulum (ER) and mitochondria, are potent inductors of CHOP expression. This report focuses on the influence of stress responses on endogenous or acquired resistance to extrinsic apoptosis in tumor cells and summarizes recent findings and results. The Medline and ClinicalTrials database with key words were used for this review. A potential novel treatment strategy for highly treatment-resistant tumors is the induction of a cellular stress response in cancer cells. The induction of an organelle-related stress response, such as nuclear, ER and mitochondrial stress, leads to a dramatic sensitization of a broad variety of cancer cells of different tumor entities to the apoptotic ligand, TRAIL. Importantly, non-neoplastic cells are not sensitized to TRAIL-mediated cell death through the unfolded protein response in most instances, suggesting that this treatment is not only of high efficacy, but even more less of unwanted toxicity in patients.

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

PubMed Central

Patsialou, Antonia; Bravo-Cordero, Jose Javier; Wang, Yarong; Entenberg, David; Liu, Huiping; Clarke, Michael; Condeelis, John S.

2014-01-01

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: a. single cells and b. multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor. PMID:25013744

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4.

PubMed

Todaro, M; Lombardo, Y; Francipane, M G; Alea, M Perez; Cammareri, P; Iovino, F; Di Stefano, A B; Di Bernardo, C; Agrusa, A; Condorelli, G; Walczak, H; Stassi, G

2008-04-01

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.

Pro-apoptotic BIM is an essential initiator of physiological endothelial cell death independent of regulation by FOXO3.

PubMed

Koenig, M N; Naik, E; Rohrbeck, L; Herold, M J; Trounson, E; Bouillet, P; Thomas, T; Voss, A K; Strasser, A; Coultas, L

2014-11-01

The growth of new blood vessels by angiogenesis is essential for normal development, but can also cause or contribute to the pathology of numerous diseases. Recent studies have shown that BIM, a pro-apoptotic BCL2-family protein, is required for endothelial cell apoptosis in vivo, and can contribute to the anti-angiogenic effect of VEGF-A inhibitors in certain tumor models. Despite its importance, the extent to which BIM is autonomously required for physiological endothelial apoptosis remains unknown and its regulation under such conditions is poorly defined. While the transcription factor FOXO3 has been proposed to induce Bim in response to growth factor withdrawal, evidence for this function is circumstantial. We report that apoptosis was reduced in Bim(-/-) primary endothelial cells, demonstrating a cell-autonomous role for BIM in endothelial death following serum and growth factor withdrawal. In conflict with in vitro studies, BIM-dependent endothelial death in vivo did not require FOXO3. Moreover, endothelial apoptosis proceeded normally in mice lacking FOXO-binding sites in the Bim promoter. Bim mRNA was upregulated in endothelial cells starved of serum and growth factors and this was accompanied by the downregulation of miRNAs of the miR-17∼92 cluster. Bim mRNA levels were also elevated in miR-17∼92(+/-) endothelial cells cultured under steady-state conditions, suggesting that miR-17∼92 cluster miRNAs may contribute to regulating overall Bim mRNA levels in endothelial cells.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

PubMed

Galluzzi, Lorenzo; Vitale, Ilio; Aaronson, Stuart A; Abrams, John M; Adam, Dieter; Agostinis, Patrizia; Alnemri, Emad S; Altucci, Lucia; Amelio, Ivano; Andrews, David W; Annicchiarico-Petruzzelli, Margherita; Antonov, Alexey V; Arama, Eli; Baehrecke, Eric H; Barlev, Nickolai A; Bazan, Nicolas G; Bernassola, Francesca; Bertrand, Mathieu J M; Bianchi, Katiuscia; Blagosklonny, Mikhail V; Blomgren, Klas; Borner, Christoph; Boya, Patricia; Brenner, Catherine; Campanella, Michelangelo; Candi, Eleonora; Carmona-Gutierrez, Didac; Cecconi, Francesco; Chan, Francis K-M; Chandel, Navdeep S; Cheng, Emily H; Chipuk, Jerry E; Cidlowski, John A; Ciechanover, Aaron; Cohen, Gerald M; Conrad, Marcus; Cubillos-Ruiz, Juan R; Czabotar, Peter E; D'Angiolella, Vincenzo; Dawson, Ted M; Dawson, Valina L; De Laurenzi, Vincenzo; De Maria, Ruggero; Debatin, Klaus-Michael; DeBerardinis, Ralph J; Deshmukh, Mohanish; Di Daniele, Nicola; Di Virgilio, Francesco; Dixit, Vishva M; Dixon, Scott J; Duckett, Colin S; Dynlacht, Brian D; El-Deiry, Wafik S; Elrod, John W; Fimia, Gian Maria; Fulda, Simone; García-Sáez, Ana J; Garg, Abhishek D; Garrido, Carmen; Gavathiotis, Evripidis; Golstein, Pierre; Gottlieb, Eyal; Green, Douglas R; Greene, Lloyd A; Gronemeyer, Hinrich; Gross, Atan; Hajnoczky, Gyorgy; Hardwick, J Marie; Harris, Isaac S; Hengartner, Michael O; Hetz, Claudio; Ichijo, Hidenori; Jäättelä, Marja; Joseph, Bertrand; Jost, Philipp J; Juin, Philippe P; Kaiser, William J; Karin, Michael; Kaufmann, Thomas; Kepp, Oliver; Kimchi, Adi; Kitsis, Richard N; Klionsky, Daniel J; Knight, Richard A; Kumar, Sharad; Lee, Sam W; Lemasters, John J; Levine, Beth; Linkermann, Andreas; Lipton, Stuart A; Lockshin, Richard A; López-Otín, Carlos; Lowe, Scott W; Luedde, Tom; Lugli, Enrico; MacFarlane, Marion; Madeo, Frank; Malewicz, Michal; Malorni, Walter; Manic, Gwenola; Marine, Jean-Christophe; Martin, Seamus J; Martinou, Jean-Claude; Medema, Jan Paul; Mehlen, Patrick; Meier, Pascal; Melino, Sonia; Miao, Edward A; Molkentin, Jeffery D; Moll, Ute M; Muñoz-Pinedo, Cristina; Nagata, Shigekazu; Nuñez, Gabriel; Oberst, Andrew; Oren, Moshe; Overholtzer, Michael; Pagano, Michele; Panaretakis, Theocharis; Pasparakis, Manolis; Penninger, Josef M; Pereira, David M; Pervaiz, Shazib; Peter, Marcus E; Piacentini, Mauro; Pinton, Paolo; Prehn, Jochen H M; Puthalakath, Hamsa; Rabinovich, Gabriel A; Rehm, Markus; Rizzuto, Rosario; Rodrigues, Cecilia M P; Rubinsztein, David C; Rudel, Thomas; Ryan, Kevin M; Sayan, Emre; Scorrano, Luca; Shao, Feng; Shi, Yufang; Silke, John; Simon, Hans-Uwe; Sistigu, Antonella; Stockwell, Brent R; Strasser, Andreas; Szabadkai, Gyorgy; Tait, Stephen W G; Tang, Daolin; Tavernarakis, Nektarios; Thorburn, Andrew; Tsujimoto, Yoshihide; Turk, Boris; Vanden Berghe, Tom; Vandenabeele, Peter; Vander Heiden, Matthew G; Villunger, Andreas; Virgin, Herbert W; Vousden, Karen H; Vucic, Domagoj; Wagner, Erwin F; Walczak, Henning; Wallach, David; Wang, Ying; Wells, James A; Wood, Will; Yuan, Junying; Zakeri, Zahra; Zhivotovsky, Boris; Zitvogel, Laurence; Melino, Gerry; Kroemer, Guido

2018-03-01

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Attenuation of excitatory amino acid toxicity by metabotropic glutamate receptor agonists and aniracetam in primary cultures of cerebellar granule cells.

PubMed

Pizzi, M; Fallacara, C; Arrighi, V; Memo, M; Spano, P F

1993-08-01

Activation of glutamate ionotropic receptors represents the primary event in the neurotoxicity process triggered by excitatory amino acids. We demonstrate here that the concentration-dependent stimulation of metabotropic glutamate receptor (mGluR) by the selective agonist trans-1-aminocyclopentane-1,3-dicarboxylate or by quisqualate counteracts both glutamate- and kainate-induced neurotoxicity in primary cultures of rat cerebellar granule cells. The mGluR-evoked responses are potentiated by aniracetam, which per se also elicits neuroprotection. Aniracetam concentration-dependently counteracted glutamate-, kainate-, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death and greatly facilitated neuroprotective response achieved by different concentrations of both quisqualate and trans-1-aminocyclopentane-1,3-dicarboxylate. In addition, aniracetam potentiated the mGluR-coupled stimulation of phospholipase C, as revealed by the measurement of 3H-inositol phosphate formation. Thus, mGluRs could be a suitable target for novel pharmacological strategies pointing to the treatment of neurodegenerative diseases.

Efficacy of aqueous extract of Hippophae rhamnoides and its bio-active flavonoids against hypoxia-induced cell death.

PubMed

Tulsawani, Rajkumar; Gupta, Rashmi; Misra, Kshipra

2013-01-01

To investigate the protective efficacy of aqueous extract of Hippophae rhamnoides against chronic hypoxic injury using primary rat hepatocytes. The extract was prepared using maceration method and characterized by its phenolic and flavonoid content and chemical antioxidant capacity using ferric reducing antioxidant power assay. Hepatocytes were maintained in hypoxia chamber (3% and 1% oxygen) for 72 h. The cells kept under normoxic condition served as control. The cells were treated with the extract and flavonoids; isorhamentin, kaempferol or qurecetin-3-galactoside. After the end of exposure period; cell survival, reactive oxygen species (ROS), leakage of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were measured. The extract showed presence of high phenolic and flavonoid content with significant antioxidant activity in chemical assay. The cell exposed to hypoxia showed concentration dependent cell death and harbored higher reactive oxygen species. In addition, these cells showed significant leakage of intracellular LDH, ALT, and AST accompanied by the diminished levels/activities of GSH, GPx, and SOD. The treatment of cells with aqueous extract of H. rhamnoides reduced hypoxia-induced cell death and prevented increase in ROS levels and leakage of intracellular LDH, ALT, and AST from cells. Moreover, these cells maintained better levels/activities of GSH, GPx, and SOD in comparison to the respective controls. The major flavonoids present in aqueous extract of H. rhamnoides; quercetin-3-galactoside, kaempferol, and isorhamentin also prevented hypoxia induced cell injury individually or in combination, however, the protection offered by these compounds taken together could not match to that of the extract. Overall the findings reveal significance of aqueous extract of H. rhamnoides in controlling ROS-meditated hypoxic injury in cells and can be useful in many hepatic complications.

Low-Dose Aronia melanocarpa Concentrate Attenuates Paraquat-Induced Neurotoxicity

PubMed Central

Case, A. J.; Agraz, D.; Ahmad, I. M.; Zimmerman, M. C.

2016-01-01

Herbicides containing paraquat may contribute to the pathogenesis of neurodegenerative disorders such as Parkinson's disease. Paraquat induces reactive oxygen species-mediated apoptosis in neurons, which is a primary mechanism behind its toxicity. We sought to test the effectiveness of a commercially available polyphenol-rich Aronia melanocarpa (aronia berry) concentrate in the amelioration of paraquat-induced neurotoxicity. Considering the abundance of antioxidants in aronia berries, we hypothesized that aronia berry concentrate attenuates the paraquat-induced increase in reactive oxygen species and protects against paraquat-mediated neuronal cell death. Using a neuronal cell culture model, we observed that low doses of aronia berry concentrate protected against paraquat-mediated neurotoxicity. Additionally, low doses of the concentrate attenuated the paraquat-induced increase in superoxide, hydrogen peroxide, and oxidized glutathione levels. Interestingly, high doses of aronia berry concentrate increased neuronal superoxide levels independent of paraquat, while at the same time decreasing hydrogen peroxide. Moreover, high-dose aronia berry concentrate potentiated paraquat-induced superoxide production and neuronal cell death. In summary, aronia berry concentrate at low doses restores the homeostatic redox environment of neurons treated with paraquat, while high doses exacerbate the imbalance leading to further cell death. Our findings support that moderate levels of aronia berry concentrate may prevent reactive oxygen species-mediated neurotoxicity. PMID:26770655

Low-Dose Aronia melanocarpa Concentrate Attenuates Paraquat-Induced Neurotoxicity.

PubMed

Case, A J; Agraz, D; Ahmad, I M; Zimmerman, M C

2016-01-01

Herbicides containing paraquat may contribute to the pathogenesis of neurodegenerative disorders such as Parkinson's disease. Paraquat induces reactive oxygen species-mediated apoptosis in neurons, which is a primary mechanism behind its toxicity. We sought to test the effectiveness of a commercially available polyphenol-rich Aronia melanocarpa (aronia berry) concentrate in the amelioration of paraquat-induced neurotoxicity. Considering the abundance of antioxidants in aronia berries, we hypothesized that aronia berry concentrate attenuates the paraquat-induced increase in reactive oxygen species and protects against paraquat-mediated neuronal cell death. Using a neuronal cell culture model, we observed that low doses of aronia berry concentrate protected against paraquat-mediated neurotoxicity. Additionally, low doses of the concentrate attenuated the paraquat-induced increase in superoxide, hydrogen peroxide, and oxidized glutathione levels. Interestingly, high doses of aronia berry concentrate increased neuronal superoxide levels independent of paraquat, while at the same time decreasing hydrogen peroxide. Moreover, high-dose aronia berry concentrate potentiated paraquat-induced superoxide production and neuronal cell death. In summary, aronia berry concentrate at low doses restores the homeostatic redox environment of neurons treated with paraquat, while high doses exacerbate the imbalance leading to further cell death. Our findings support that moderate levels of aronia berry concentrate may prevent reactive oxygen species-mediated neurotoxicity.

Thyroid Hormone Induces Apoptosis in Primary Cell Cultures of Tadpole Intestine: Cell Type Specificity and Effects of Extracellular Matrix

PubMed Central

Su, Yuan; Shi, Yufang; Stolow, Melissa A.; Shi, Yun-Bo

1997-01-01

Thyroid hormone (T3 or 3,5,3′-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T3 induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine. We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we show that T3 can enhance the proliferation of both cell types. However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death. These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog metamorphosis. PMID:9396758

Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques

PubMed Central

Bruel, Timothée; Dupuy, Stéphanie; Démoulins, Thomas; Rogez-Kreuz, Christine; Dutrieux, Jacques; Corneau, Aurélien; Cosma, Antonio; Cheynier, Rémi; Dereuddre-Bosquet, Nathalie; Le Grand, Roger; Vaslin, Bruno

2014-01-01

IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα+ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα+ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα− production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67+-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67+-pDC precursors, none of these being IFNα+ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors. PMID:24497833

Expression of Interferon Lambda 4 Is Associated with Reduced Proliferation and Increased Cell Death in Human Hepatic Cells

PubMed Central

Onabajo, Olusegun O.; Porter-Gill, Patricia; Paquin, Ashley; Rao, Nina; Liu, Luyang; Tang, Wei; Brand, Nathan

2015-01-01

Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells—a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes—activation of ISGs, decreased proliferation, and increased cell death—could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance. PMID:26134097

Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype.

PubMed

Hogan, Alison L; Don, Emily K; Rayner, Stephanie L; Lee, Albert; Laird, Angela S; Watchon, Maxinne; Winnick, Claire; Tarr, Ingrid S; Morsch, Marco; Fifita, Jennifer A; Gwee, Serene S L; Formella, Isabel; Hortle, Elinor; Yuan, Kristy C; Molloy, Mark P; Williams, Kelly L; Nicholson, Garth A; Chung, Roger S; Blair, Ian P; Cole, Nicholas J

2017-07-15

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Exposing Cancer Cells' Secret Aides to Spread Disease to Distant Sites | Center for Cancer Research

Cancer.gov

Researchers are shedding new light on the way cancer spreads from the primary organ to distant sites in the body, a process called metastasis. The assault of metastatic tumors on vital organs too frequently results in death, and until recently, little was known about the molecular mechanisms governing this devastating phenomenon.

Necroptosis Takes Place in Human Immunodeficiency Virus Type-1 (HIV-1)-Infected CD4+ T Lymphocytes

PubMed Central

Pan, Ting; Wu, Shuangxin; He, Xin; Luo, Haihua; Zhang, Yijun; Fan, Miaomiao; Geng, Guannan; Ruiz, Vivian Clarke; Zhang, Jim; Mills, Lisa; Bai, Chuan; Zhang, Hui

2014-01-01

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by progressive depletion of CD4+ T lymphocytes and dysfunction of the immune system. The numbers of CD4+ T lymphocytes in the human body are maintained constantly by homeostatic mechanisms that failed during HIV-1 infection, resulting in progressive loss of CD4+ T cells mainly via apoptosis. Recently, a non-apoptotic form of necrotic programmed cell death, named necroptosis, has been investigated in many biological and pathological processes. We then determine whether HIV-1-infected cells also undergo necroptosis. In this report, we demonstrate that HIV-1 not only induces apoptosis, but also mediates necroptosis in the infected primary CD4+ T lymphocytes and CD4+ T-cell lines. Necroptosis-dependent cytopathic effects are significantly increased in HIV-1-infected Jurkat cells that is lack of Fas-associated protein-containing death domain (FADD), indicating that necroptosis occurs as an alternative cell death mechanism in the absence of apoptosis. Unlike apoptosis, necroptosis mainly occurs in HIV-infected cells and spares bystander damage. Treatment with necrostatin-1(Nec-1), a RIP1 inhibitor that specifically blocks the necroptosis pathway, potently restrains HIV-1-induced cytopathic effect and interestingly, inhibits the formation of HIV-induced syncytia in CD4+ T-cell lines. This suggests that syncytia formation is mediated, at least partially, by necroptosis-related processes. Furthermore, we also found that the HIV-1 infection-augmented tumor necrosis factor-alpha (TNF-α) plays a key role in inducing necroptosis and HIV-1 Envelope and Tat proteins function as its co-factors. Taken together,necroptosis can function as an alternative cell death pathway in lieu of apoptosis during HIV-1 infection, thereby also contributing to HIV-1-induced cytopathic effects. Our results reveal that in addition to apoptosis, necroptosis also plays an important role in HIV-1-induced pathogenesis. PMID:24714696

Protection of acetaminophen induced mitochondrial dysfunctions and hepatic necrosis via Akt-NF-kappaB pathway: role of a novel plant protein.

PubMed

Ghosh, Ayantika; Sil, Parames C

2009-01-27

Oxidative stress is a major cause of drug induced hepatic diseases. The present study aims to investigate the antioxidative signaling mechanism of a protein isolated from the herb, Cajanus indicus against acetaminophen induced necrotic cell death. We found that incubation of hepatocytes with the protein prevented acetaminophen-induced loss in cell viability, reduction in glutathione level and enhancement of reactive oxygen species generation. Treatment of mice with the protein before administration of acetaminophen also reduced serum nitrite and TNF-alpha formation. Moreover, it counteracted acetaminophen-induced loss in mitochondrial membrane potential, loss in adenosine tri phosphate and rise in intracellular calcium. Investigating the cell signaling pathways, we found that the protein exerts its protective action via the activation of NF-kappaB and Akt and deactivation of STAT-1. Surprisingly, no role of ERK1/2 or STAT-3 was found in the protein-mediated protection of hepatocytes during acetaminophen exposure. Finally, we found that acetaminophen introduces necrosis as the primary phenomena of cell death and protein treatment decreased the necrotic process as evident from the DNA fragmentation and flow-cytometry studies. In addition, administration of the protein to mice before acetaminophen application showed fewer number of TUNEL positive cells. Combining, data suggest that the protein possesses cytoprotective activity against acetaminophen-induced oxidative cellular damage and prevents hepatocytes from necrotic death.

Chronological shifts and changing causes of death after radiotherapy for early-stage oral cancer.

PubMed

Fujisawa, Rina; Shibuya, Hitoshi; Harata, Naoki; Yuasa-Nakagawa, Keiko; Toda, Kazuma; Hayashi, Keiji

2014-02-01

Following recent improvements in the curability of oral cancer, chronological shifts and changes in the causes of death after treatment have been observed. We conducted a review of the post-treatment causes of death following radiotherapy for oral cancers. The medical records of 966 patients with early-stage (stage I and II) oral cancer treated at our institute between 1980 and 2001 were reviewed, and the chronological shifts and changes in the causes of death after radiotherapy were assessed. Of the 966 patients enrolled in this study, 365 have died to date. Two hundred and eleven patients died of their primary malignancy; 193 of these deaths occurred within 5 years of treatment for the primary oral cancer. The second most frequent cause of death was second primary cancer (n = 90). Twenty-three patients with head and neck cancers and 18 patients with esophageal cancers died within 10 years of radiotherapy, and six patients with lung cancers died after more than 10 years. Within the first 5 years following treatment, the major cause of death was the primary oral cancer. After 5-10 years, a second primary cancer, such as head and neck cancer or esophageal cancer, became the leading cause of death. Over a 10-year period, the proportion of deaths from a second primary cancer in the lung was significant. We have demonstrated that there are chronological shifts and changes in the causes of death following treatment for early-stage oral cancer.

Hematopoietic stem cell transplantation for non-Hodgkin lymphoma.

PubMed

Bhatt, Vijaya Raj; Vose, Julie M

2014-12-01

Up-front rituximab-based chemotherapy has improved outcomes in non-Hodgkin lymphoma (NHL); refractory or relapsed NHL still accounts for approximately 18,000 deaths in the United States. Autologous hematopoietic stem cell transplantation (SCT) can improve survival in primary refractory or relapsed aggressive NHL and mantle cell lymphoma and in relapsed follicular or peripheral T-cell lymphoma. Autologous SCT as a consolidation therapy after first complete or partial remission in high-risk aggressive NHL, mantle cell lymphoma, and peripheral T-cell lymphoma may improve progression-free survival. Allogeneic SCT offers a lower relapse rate but a higher nonrelapse mortality resulting in overall survival similar to autologous SCT. Copyright © 2014 Elsevier Inc. All rights reserved.

Neuroprotective effects of anthocyanin- and proanthocyanidin-rich extracts in cellular models of Parkinson’s disease

PubMed Central

Strathearn, Katherine E.; Yousef, Gad G.; Grace, Mary H.; Roy, Susan L.; Tambe, Mitali A.; Ferruzzi, Mario G.; Wu, Qing-Li; Simon, James E.; Lila, Mary Ann; Rochet, Jean-Christophe

2014-01-01

Neuropathological evidence indicates that dopaminergic cell death in Parkinson’s disease (PD) involves impairment of mitochondrial complex I, oxidative stress, microglial activation, and the formation of Lewy bodies. Epidemiological findings suggest that the consumption of berries rich in anthocyanins and proanthocyanidins may reduce PD risk. In this study, we investigated whether extracts rich in anthocyanins, proanthocyanidins, or other polyphenols suppress the neurotoxic effects of rotenone in a primary cell culture model of PD. Dopaminergic cell death elicited by rotenone was suppressed by extracts prepared from blueberries, grape seed, hibiscus, blackcurrant, and Chinese mulberry. Extracts rich in anthocyanins and proanthocyanidins exhibited greater neuroprotective activity than extracts rich in other polyphenols, and a number of individual anthocyanins interfered with rotenone neurotoxicity. The blueberry and grape seed extracts rescued rotenone-induced defects in mitochondrial respiration in a dopaminergic cell line, and a purple basal extract attenuated nitrite release from microglial cells stimulated by lipopolysaccharide. These findings suggest that anthocyanin- and proanthocyanidin-rich botanical extracts may alleviate neurodegeneration in PD via enhancement of mitochondrial function. PMID:24502982

Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS.

PubMed

Hall, Claire E; Yao, Zhi; Choi, Minee; Tyzack, Giulia E; Serio, Andrea; Luisier, Raphaelle; Harley, Jasmine; Preza, Elisavet; Arber, Charlie; Crisp, Sarah J; Watson, P Marc D; Kullmann, Dimitri M; Abramov, Andrey Y; Wray, Selina; Burley, Russell; Loh, Samantha H Y; Martins, L Miguel; Stevens, Molly M; Luscombe, Nicholas M; Sibley, Christopher R; Lakatos, Andras; Ule, Jernej; Gandhi, Sonia; Patani, Rickie

2017-05-30

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Role of TRAIL and the pro-apoptotic Bcl-2 homolog Bim in acetaminophen-induced liver damage

PubMed Central

Badmann, A; Keough, A; Kaufmann, T; Bouillet, P; Brunner, T; Corazza, N

2011-01-01

Acetaminophen (N-acetyl-para-aminophenol (APAP), paracetamol) is a commonly used analgesic and antipyretic agent. Although considered safe at therapeutic doses, accidental or intentional overdose causes acute liver failure characterized by centrilobular hepatic necrosis with high morbidity and mortality. Although many molecular aspects of APAP-induced cell death have been described, no conclusive mechanism has been proposed. We recently identified TNF-related apoptosis-inducing ligand (TRAIL) and c-Jun kinase (JNK)-dependent activation of the pro-apoptotic Bcl-2 homolog Bim as an important apoptosis amplification pathway in hepatocytes. In this study, we, thus, investigated the role of TRAIL, c-JNK and Bim in APAP-induced liver damage. Our results demonstrate that TRAIL strongly synergizes with APAP in inducing cell death in hepatocyte-like cells lines and primary hepatocyte. Furthermore, we found that APAP strongly induces the expression of Bim in a c-JNK-dependent manner. Consequently, TRAIL- or Bim-deficient mice were substantially protected from APAP-induced liver damage. This study identifies the TRAIL-JNK-Bim axis as a novel target in the treatment of APAP-induced liver damage and substantiates its general role in hepatocyte death. PMID:21654829

SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Li; Weng, Wei; Sun, Zhi-Xin

Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, andmore » concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent. - Highlights: • SKI-II inhibits proliferation and survival of primary and transformed AML cells. • SKI-II induces apoptotic death of AML cells, but is safe to normal PBMCs. • SKI-II is more efficient than two known SphK1 inhibitors in inhibiting AML cells. • SKI-II inhibits SphK1 activity, while increasing ceramide production in AML cells. • SKI-II dose-dependently inhibits U937 xenograft growth in SCID mice.« less

4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Meili, E-mail: fumeilidrlinyi@tom.com; Wan, Fuqiang; Li, Zhengling

The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation–inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D,more » a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.« less

S-Nitrosylation of parkin as a novel regulator of p53-mediated neuronal cell death in sporadic Parkinson’s disease

PubMed Central

2013-01-01

Background Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson’s disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD. Results We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls. Conclusions Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD. PMID:23985028

Innate myeloid cell TNFR1 mediates first line defence against primary Mycobacterium tuberculosis infection.

PubMed Central

Segueni, Noria; Benmerzoug, Sulayman; Rose, Stéphanie; Gauthier, Amandine; Bourigault, Marie-Laure; Reverchon, Flora; Philippeau, Amandine; Erard, François; Le Bert, Marc; Bouscayrol, Hélène; Wachter, Thierry; Garcia, Irène; Kollias, George; Jacobs, Muazzam; Ryffel, Bernhard; Quesniaux, Valerie F.J.

2016-01-01

TNF is crucial for controlling Mycobacterium tuberculosis infection and understanding how will help immunomodulating the host response. Here we assessed the contribution of TNFR1 pathway from innate myeloid versus T cells. We first established the prominent role of TNFR1 in haematopoietic cells for controlling M. tuberculosis in TNFR1 KO chimera mice. Further, absence of TNFR1 specifically on myeloid cells (M-TNFR1 KO) recapitulated the uncontrolled M. tuberculosis infection seen in fully TNFR1 deficient mice, with increased bacterial burden, exacerbated lung inflammation, and rapid death. Pulmonary IL-12p40 over-expression was attributed to a prominent CD11b+ Gr1high cell population in infected M-TNFR1 KO mice. By contrast, absence of TNFR1 on T-cells did not compromise the control of M. tuberculosis infection over 6-months. Thus, the protective TNF/TNFR1 pathway essential for controlling primary M. tuberculosis infection depends on innate macrophage and neutrophil myeloid cells, while TNFR1 pathway in T cells is dispensable. PMID:26931771

The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma.

PubMed

Evans, Robert P; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Maxwell, Christopher A; Keats, Jonathan J; Belch, Andrew R; Pilarski, Linda M; Lai, Raymond; Reiman, Tony

2008-02-01

Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti-myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152-induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti-myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138(+) BM plasma cells from myeloma patients but also, as expected, was toxic to CD138(-) marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152-inhibited tumour growth at well-tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

Comparison of cause of death between anzdata and the australian national death index.

PubMed

Sypek, Matthew P; Dansie, Kathryn B; Clayton, Phil; Webster, Angela C; McDonald, Stephen

2018-03-01

To understand the differences in how cause of death for patients receiving renal replacement therapy in Australia is recorded in The Australian and New Zealand Dialysis and Transplant Registry (ANZDATA) compared to the National Death Index (NDI). Data linkage was performed between ANZDATA and NDI for all deaths in the period 1980-2013. Cause of death was classified according to ICD-10 chapter. Overall and chapter specific agreement were assessed using the Kappa statistic. Descriptive analysis was used to explore differences where there was disagreement on primary cause of death. The analysis cohort included 28,675 patients. Ninety five percent of ANZDATA reported deaths fell within +/- 3 days of the date recorded by NDI. Circulatory death was the most common cause of death in both databases (ANZDATA 48%, NDI 32%). Overall agreement at ICD chapter level of primary cause was poor (36%, kappa 0.22). Agreement was best for malignancy (kappa 0.71). When there was disagreement on primary cause of death these were most commonly coded as genitourinary (35%) and endocrine (25.0%) in NDI, and circulatory (39%) and withdrawal (24%) in ANZDATA. Sixty-nine percent of patients had a renal related cause documented as either primary or a contributing cause of death in the NDI. There is poor agreement in primary cause of death between ANZDATA and NDI which is in part explained by the absence of diabetes and renal failure as causes of death in ANZDATA and the absence of 'withdrawal' in NDI. These differences should be appreciated when interpreting epidemiological data on cause of death in the Australian end stage kidney disease population. This article is protected by copyright. All rights reserved.

Promotion of Cancer Stem-Like Cell Properties in Hepatitis C Virus-Infected Hepatocytes

PubMed Central

Kwon, Young-Chan; Bose, Sandip K.; Steele, Robert; Meyer, Keith; Di Bisceglie, Adrian M.; Ray, Ratna B.

2015-01-01

ABSTRACT We have previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) induces the epithelial mesenchymal transition (EMT) state and extends hepatocyte life span (S. K. Bose, K. Meyer, A. M. Di Bisceglie, R. B. Ray, and R. Ray, J Virol 86:13621–13628, 2012, http://dx.doi.org/10.1128/JVI.02016-12). These hepatocytes displayed sphere formation on ultralow binding plates and survived for more than 12 weeks. The sphere-forming hepatocytes expressed a number of cancer stem-like cell (CSC) markers, including high levels of the stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in hepatocellular carcinoma (HCC). Analysis of c-Kit mRNA displayed a significant increase in the liver biopsy specimens of chronically HCV-infected patients. We also found c-Kit is highly expressed in transformed human hepatocytes (THH) infected in vitro with cell culture-grown HCV genotype 2a. Further studies suggested that HCV core protein significantly upregulates c-Kit expression at the transcriptional level. HCV infection of THH led to a significant increase in the number of spheres displayed on ultralow binding plates and in enhanced EMT and CSC markers and tumor growth in immunodeficient mice. The use of imatinib or dasatinib as a c-Kit inhibitor reduced the level of sphere-forming cells in culture. The sphere-forming cells were sensitive to treatment with sorafenib, a multikinase inhibitor, that is used for HCC treatment. Further, stattic, an inhibitor of the Stat3 molecule, induced sphere-forming cell death. A combination of sorafenib and stattic had a significantly stronger effect, leading to cell death. These results suggested that HCV infection potentiates CSC generation, and selected drugs can be targeted to efficiently inhibit cell growth. IMPORTANCE HCV infection may develop into HCC as an end-stage liver disease. We focused on understanding the mechanism for the risk of HCC from chronic HCV infection and identified targets for treatment. HCV-infected primary and transformed human hepatocytes (PHH or THH) generated CSC. HCV-induced spheres were highly sensitive to cell death from sorafenib and stattic treatment. Thus, our study is highly significant for HCV-associated HCC, with the potential for developing a target-specific strategy for improved therapies. PMID:26355082

Hydrogen Peroxide Contributes to the Epithelial Cell Death Induced by the Oral Mitis Group of Streptococci

PubMed Central

Okahashi, Nobuo; Sumitomo, Tomoko; Nakata, Masanobu; Sakurai, Atsuo; Kuwata, Hirotaka; Kawabata, Shigetada

2014-01-01

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H2O2) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H2O2 may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H2O2-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited S. oralis cytotoxicity, and H2O2 alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H2O2 production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H2O2 induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H2O2 is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts. PMID:24498253

Putting on the Brakes: Blocking the Growth of Metastases | Center for Cancer Research

Cancer.gov

Most of the suffering and death caused by cancer is due, not to the primary tumor, but to the ability of cancer cells to spread throughout the body and to form metastases in other organs. Breast and prostate cancers often have periods of dormancy, which can extend up to 30 years, between the identification and treatment of a primary tumor and the growth of overt metastases. What induces or inhibits metastatic dormancy is unknown, but prolonging this period may improve the survival of patients with these types of cancer.

Use of primary diagnosis during hospitalization in the Unified Health System (Sistema Único de Saúde) to qualify information regarding the underlying cause of natural deaths among the elderly.

PubMed

Cascão, Angela Maria; Jorge, Maria Helena Prado de Mello; Costa, Antonio José Leal; Kale, Pauline Lorena

2016-01-01

Ill-defined causes of death are common among the elderly owing to the high frequency of comorbidities and, consequently, to the difficulty in defining the underlying cause of death. To analyze the validity and reliability of the "primary diagnosis" in hospitalization to recover the information on the underlying cause of death in natural deaths among the elderly whose deaths were originally assigned to "ill-defined cause" in their Death Certificate. The hospitalizations occurred in the state of Rio de Janeiro, in 2006. The databases obtained in the Information Systems on Mortality and Hospitalization were probabilistically linked. The following data were calculated for hospitalizations of the elderly that evolved into deaths with a natural cause: concordance percentages, Kappa coefficient, sensitivity, specificity, and the positive predictive value of the primary diagnosis. Deaths related to "ill-defined causes" were assigned to a new cause, which was defined based on the primary diagnosis. The reliability of the primary diagnosis was good, according to the total percentage of consistency (50.2%), and fair, according to the Kappa coefficient (k = 0.4; p < 0.0001). Diseases related to the circulatory system and neoplasia occurred with the highest frequency among the deaths and the hospitalizations and presented a higher consistency of positive predictive values per chapter and grouping of the International Classification of Diseases. The recovery of the information on the primary cause occurred in 22.6% of the deaths with ill-defined causes (n = 14). The methodology developed and applied for the recovery of the information on the natural cause of death among the elderly in this study had the advantage of effectiveness and the reduction of costs compared to an investigation of the death that is recommended in situations of non-linked and low positive predictive values. Monitoring the mortality profile by the cause of death is necessary to periodically update the predictive values.

Secondary brain injuries in thalamus and hippocampus after focal ischemia caused by mild, transient extradural compression of the somatosensori cortex in the rat.

PubMed

Holmberg, Per; Liljequist, Sture; Wägner, Anna

2009-02-01

The development and distribution of secondary brain lesions, subsequent to ischemic stroke, are of considerable clinical interest but so far only a limited number of studies have investigated the distribution and development of these secondary lesions in detail. In this study, we used an animal model of focal ischemia caused by extradural compression of the sensorimotor cortex. This paradigm of focal ischemia was shown to produce a consistent pattern of secondary lesions located distally from the primary lesion. Functionally the primary brain lesion produced a transient neurological deficit, which was evaluated by daily beam walking tests. Morphological changes were assessed in parallel after the ischemic event using Fluoro-Jade (FJ) staining as a marker of neuronal cell death. Secondary brain lesions were observed in the thalamus as well as in the hippocampus. The first sign of the slowly developing secondary brain lesions was present on day 3 with subsequent lesions being identified until day 16 after the primary ischemia. In addition to the identification of neuronal cell death by the FJ assays, immunostaining for parvalbumin (PA), a marker of GABAergic interneurons, revealed a loss of PA-staining in the pyramidal layer of CA1 on day 3, thus showing a similar time pattern for loss of PA-staining as for the loss of FJ stained cells. Based upon our present results, we suggest that the current animal model of focal ischemia represents a valuable tool for studies concerning the development of secondary remote brain lesions and their association to impaired motor and cognitive functions.

Treatment of prostate cancer cell lines and primary cells using low temperature plasma

NASA Astrophysics Data System (ADS)

O'Connell, Deborah; Hirst, Adam; Frame, Fiona F.; Maitland, Norman J.

2014-10-01

The mechanisms of cell death after plasma treatment of both benign and cancerous prostate epithelial cells are investigated. Prostate cancer tissue was obtained with patient consent from targeted needle core biopsies following radical prostatectomy. Primary cells were cultured from cancer tissue and plated onto a chamber slide at a density of 10,000 cells per well in 200 microliter of stem cell media (SCM). The treated sample was previously identified as Gleason grade 7 cancer through tissue histo-pathology. A dielectric barrier discharge (DBD) jet configuration, with helium as a carrier gas, and 0.3% O2 admixture was used for treating the cells. Reactive oxygen and nitrogen species (RONS) produced by the plasma are believed to be the main mediators of the plasma-cell interaction and response. We found the concentration of reactive oxygen species (ROS) induced inside the cells increased with plasma exposure. Exposure to the plasma for >3 minutes showed high levels of DNA damage compared to untreated and hydrogen peroxide controls. Cell viability and cellular recovery are also investigated and will be presented. All findings were common to both cell lines, suggesting the potential of LTP therapy for both benign and malignant disease.

Moderate extracellular acidification inhibits capsaicin-induced cell death through regulating calcium mobilization, NF-{kappa}B translocation and ROS production in synoviocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Fen; Yang, Shuang; Zhao, Dan

Highlights: Black-Right-Pointing-Pointer Moderate extracellular acidification regulates intracellular Ca{sup 2+} mobilization. Black-Right-Pointing-Pointer Moderate acidification activates NF-{kappa}B nuclear translocation in synoviocytes. Black-Right-Pointing-Pointer Moderate acidification depresses the ROS production induced by capsaicin. Black-Right-Pointing-Pointer Moderate acidification inhibits capsaicin-caused synoviocyte death. -- Abstract: We previously show the expression of transient receptor potential vanilloid 1 (TRPV1) in primary synoviocytes from collagen-induced arthritis (CIA) rats. Capsaicin and lowered extracellular pH from 7.4 to 5.5 induce cell death through TRPV1-mediated Ca{sup 2+} entry and reactive oxygen species (ROS) production. However, under the pathological condition in rheumatoid arthritis, the synovial fluid is acidified to a moderate level (about pHmore » 6.8). In the present study, we examined the effects of pH 6.8 on the TRPV1-mediated cell death. Our finding is different or even opposite from what was observed at pH 5.5. We found that the moderate extracellular acidification (from pH 7.4 to 6.8) inhibited the capsaicin-induced Ca{sup 2+} entry through attenuating the activity of TRPV1. In the mean time, it triggered a phospholipse C (PLC)-related Ca{sup 2+} release from intracellular stores. The nuclear translocation of NF-{kappa}B was found at pH 6.8, and this also depends on PLC activation. Moreover, the capsaicin-evoked massive ROS production and cell death were depressed at pH 6.8, both of which are dependent on the activation of PLC and NF-{kappa}B. Taken together, these results suggested that the moderate extracellular acidification inhibited the capsaicin-induced synoviocyte death through regulating Ca{sup 2+} mobilization, activating NF-{kappa}B nuclear translocation and depressing ROS production.« less

PD-L1 Is a Therapeutic Target of the Bromodomain Inhibitor JQ1 and, Combined with HLA Class I, a Promising Prognostic Biomarker in Neuroblastoma.

PubMed

Melaiu, Ombretta; Mina, Marco; Chierici, Marco; Boldrini, Renata; Jurman, Giuseppe; Romania, Paolo; D'Alicandro, Valerio; Benedetti, Maria C; Castellano, Aurora; Liu, Tao; Furlanello, Cesare; Locatelli, Franco; Fruci, Doriana

2017-08-01

Purpose: This study sought to evaluate the expression of programmed cell death-ligand-1 (PD-L1) and HLA class I on neuroblastoma cells and programmed cell death-1 (PD-1) and lymphocyte activation gene 3 (LAG3) on tumor-infiltrating lymphocytes to better define patient risk stratification and understand whether this tumor may benefit from therapies targeting immune checkpoint molecules. Experimental Design: In situ IHC staining for PD-L1, HLA class I, PD-1, and LAG3 was assessed in 77 neuroblastoma specimens, previously characterized for tumor-infiltrating T-cell density and correlated with clinical outcome. Surface expression of PD-L1 was evaluated by flow cytometry and IHC in neuroblastoma cell lines and tumors genetically and/or pharmacologically inhibited for MYC and MYCN. A dataset of 477 human primary neuroblastomas from GEO and ArrayExpress databases was explored for PD-L1, MYC, and MYCN correlation. Results: Multivariate Cox regression analysis demonstrated that the combination of PD-L1 and HLA class I tumor cell density is a prognostic biomarker for predicting overall survival in neuroblastoma patients ( P = 0.0448). MYC and MYCN control the expression of PD-L1 in neuroblastoma cells both in vitro and in vivo Consistently, abundance of PD-L1 transcript correlates with MYC expression in primary neuroblastoma. Conclusions: The combination of PD-L1 and HLA class I represents a novel prognostic biomarker for neuroblastoma. Pharmacologic inhibition of MYCN and MYC may be exploited to target PD-L1 and restore an efficient antitumor immunity in high-risk neuroblastoma. Clin Cancer Res; 23(15); 4462-72. ©2017 AACR . ©2017 American Association for Cancer Research.

Dexamethasone Alleviates Tumor-Associated Brain Damage and Angiogenesis

PubMed Central

Fan, Zheng; Sehm, Tina; Rauh, Manfred; Buchfelder, Michael

2014-01-01

Children and adults with the most aggressive form of brain cancer, malignant gliomas or glioblastoma, often develop cerebral edema as a life-threatening complication. This complication is routinely treated with dexamethasone (DEXA), a steroidal anti-inflammatory drug with pleiotropic action profile. Here we show that dexamethasone reduces murine and rodent glioma tumor growth in a concentration-dependent manner. Low concentrations of DEXA are already capable of inhibiting glioma cell proliferation and at higher levels induce cell death. Further, the expression of the glutamate antiporter xCT (system Xc −; SLC7a11) and VEGFA is up-regulated after DEXA treatment indicating early cellular stress responses. However, in human gliomas DEXA exerts differential cytotoxic effects, with some human glioma cells (U251, T98G) resistant to DEXA, a finding corroborated by clinical data of dexamethasone non-responders. Moreover, DEXA-resistant gliomas did not show any xCT alterations, indicating that these gene expressions are associated with DEXA-induced cellular stress. Hence, siRNA-mediated xCT knockdown in glioma cells increased the susceptibility to DEXA. Interestingly, cell viability of primary human astrocytes and primary rodent neurons is not affected by DEXA. We further tested the pharmacological effects of DEXA on brain tissue and showed that DEXA reduces tumor-induced disturbances of the microenvironment such as neuronal cell death and tumor-induced angiogenesis. In conclusion, we demonstrate that DEXA inhibits glioma cell growth in a concentration and species-dependent manner. Further, DEXA executes neuroprotective effects in brains and reduces tumor-induced angiogenesis. Thus, our investigations reveal that DEXA acts pleiotropically and impacts tumor growth, tumor vasculature and tumor-associated brain damage. PMID:24714627

Sigma receptor 1 modulates endoplasmic reticulum stress in retinal neurons.

PubMed

Ha, Yonju; Dun, Ying; Thangaraju, Muthusamy; Duplantier, Jennifer; Dong, Zheng; Liu, Kebin; Ganapathy, Vadivel; Smith, Sylvia B

2011-01-01

To investigate the mechanism of σ receptor 1 (σR1) neuroprotection in retinal neurons. Oxidative stress, which is implicated in diabetic retinopathy, was induced in mouse primary ganglion cells (GCs) and RGC-5 cells, and the effect of the σR1 ligand (+)-pentazocine on pro- and anti-apoptotic and endoplasmic reticulum (ER) stress gene expression was examined. Binding of σR1 to BiP, an ER chaperone protein, and σR1 phosphorylation status were examined by immunoprecipitation. Retinas were harvested from Ins2Akita/+ diabetic mice treated with (+)-pentazocine, and the expression of ER stress genes and of the retinal transcriptome was evaluated. Oxidative stress induced the death of primary GCs and RGC-5 cells. The effect was decreased by the application of (+)-pentazocine. Stress increased σR1 binding to BiP and enhanced σR1 phosphorylation in RGC-5 cells. BiP binding was prevented, and σR1 phosphorylation decreased in the presence of (+)-pentazocine. The ER stress proteins PERK, ATF4, ATF6, IRE1α, and CHOP were upregulated in RGC-5 cells during oxidative stress, but decreased in the presence of (+)-pentazocine. A similar phenomenon was observed in retinas of Ins2Akita/+ diabetic mice. Retinal transcriptome analysis of Ins2Akita/+ mice compared with wild-type revealed differential expression of the genes critically involved in oxidative stress, differentiation, and cell death. The expression profile of those genes was reversed when the Ins2Akita/+ mice were treated with (+)-pentazocine. In retinal neurons, the molecular chaperone σR1 binds BiP under stressful conditions; (+)-pentazocine may exert its effects by dissociating σR1 from BiP. As stress in retinal cells increases, phosphorylation of σR1 is increased, which is attenuated when agonists bind to the receptor.

Methylene Blue Protects Astrocytes against Glucose Oxygen Deprivation by Improving Cellular Respiration

PubMed Central

Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan M.; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yuan, Fang; Yang, Shao-Hua; Liu, Ran

2015-01-01

Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration. PMID:25848957

Synthesis of cytochrome C oxidase 2: a p53-dependent metabolic regulator that promotes respiratory function and protects glioma and colon cancer cells from hypoxia-induced cell death.

PubMed

Wanka, C; Brucker, D P; Bähr, O; Ronellenfitsch, M; Weller, M; Steinbach, J P; Rieger, J

2012-08-16

P53 has an important role in the processing of starvation signals. P53-dependent molecular mediators of the Warburg effect reduce glucose consumption and promote mitochondrial function. We therefore hypothesized that the retention of wild-type p53 characteristic of primary glioblastomas limits metabolic demands induced by deregulated signal transduction in the presence of hypoxia and nutrient depletion. Here we report that short hairpin RNA-mediated gene suppression of wild-type p53 or ectopic expression of mutant temperature-sensitive dominant-negative p53(V135A) increased glucose consumption and lactate production, decreased oxygen consumption and enhanced hypoxia-induced cell death in p53 wild-type human glioblastoma cells. Similarly, genetic knockout of p53 in HCT116 colon carcinoma cells resulted in reduced respiration and hypersensitivity towards hypoxia-induced cell death. Further, wild-type p53 gene silencing reduced the expression of synthesis of cytochrome c oxidase 2 (SCO2), an effector necessary for respiratory chain function. An SCO2 transgene reverted the metabolic phenotype and restored resistance towards hypoxia in p53-depleted and p53 mutant glioma cells in a rotenone-sensitive manner, demonstrating that this effect was dependent on intact oxidative phosphorylation. Supplementation with methyl-pyruvate, a mitochondrial substrate, rescued p53 wild-type but not p53 mutant cells from hypoxic cell death, demonstrating a p53-mediated selective aptitude to metabolize mitochondrial substrates. Further, SCO2 gene silencing in p53 wild-type glioma cells sensitized these cells towards hypoxia. Finally, lentiviral gene suppression of SCO2 significantly enhanced tumor necrosis in a subcutaneous HCT116 xenograft tumor model, compatible with impaired energy metabolism in these cells. These findings demonstrate that glioma and colon cancer cells with p53 wild-type status can skew the Warburg effect and thereby reduce their vulnerability towards tumor hypoxia in an SCO2-dependent manner. Targeting SCO2 may therefore represent a valuable strategy to enhance sensitivity towards hypoxia and may complement strategies targeting glucose metabolism.

Zinc Modulates Nanosilver-Induced Toxicity in Primary Neuronal Cultures.

PubMed

Ziemińska, Elżbieta; Strużyńska, Lidia

2016-02-01

Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl(2), respectively. Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl(2) increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.

PubMed

Lilienthal, Nils; Lohmann, Gregor; Crispatzu, Giuliano; Vasyutina, Elena; Zittrich, Stefan; Mayer, Petra; Herling, Carmen Diana; Tur, Mehmet Kemal; Hallek, Michael; Pfitzer, Gabriele; Barth, Stefan; Herling, Marco

2016-05-01

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR. ©2016 American Association for Cancer Research.

Bufalin Inhibits the Differentiation and Proliferation of Cancer Stem Cells Derived from Primary Osteosarcoma Cells through Mir-148a.

PubMed

Chang, Yuewen; Zhao, Yongfang; Gu, Wei; Cao, Yuelong; Wang, Shuqiang; Pang, Jian; Shi, Yinyu

2015-01-01

Osteosarcoma (OS) is the second leading cause of cancer-related death in children and young adults. Chemoresistance is the most important cause of treatment failure in OS, largely resulting from presence of cancer stem cells (CSCs). However, CSCs isolated from cancer cell lines do not necessarily represent those from primary human tumors due to accumulation of genetic aberrations that increase with passage number. Therefore, studies on CSCs from primary OS may be more important for understanding the mechanisms driving the chemoresistance of CSCs in OS. We established a primary culture of OS cells, known as C1OS, from freshly resected tumor tissue. We further isolated CSCs from C1OS cells (C1OS-CSCs). We analyzed the effects of bufalin, a traditional Chinese medicine, on the stemness of C1OS-CSCs. We also analyzed the microRNA (miR) targets of bufalin on the stemness of C1OS-CSCs. Moreover, we examined these findings in the OS specimen. Bufalin inhibited the stemness of C1OS-CSCs. Moreover, we found that miR-148a appeared to be a target of bufalin, and miR-148a further regulated DNMT1 and p27 to control the stemness of OS cells. This mechanism was further confirmed in OS specimen. Our data suggest that bufalin may be a promising treatment for OS, and its function may be conducted through regulation of miR-148a. © 2015 S. Karger AG, Basel.

A study of the mechanism of in vitro cytotoxicity of metal oxide nanoparticles using catfish primary hepatocytes and human HepG2 cells

PubMed Central

Wang, Yonggang; Aker, Winfred G.; Hwang, Huey-min; Yedjou, Clement G.; Yu, Hongtao; Tchounwou, Paul B.

2011-01-01

Nanoparticles (NPs), including nano metal oxides, are being used in diverse applications such as medicine, clothing, cosmetics and food. In order to promote the safe development of nanotechnology, it is essential to assess the potential adverse health consequences associated with human exposure. The liver is a target site for NP toxicity, due to NP accumulation within it after ingestion, inhalation or absorption. The toxicity of nano-ZnO, TiO2, CuO and Co3O4 was investigated using a primary culture of channel catfish hepatocytes and human HepG2 cells as in vitro model systems for assessing the impact of metal oxide NPs on human and environmental health. Some mechanisms of nanotoxicity were determined by using phase contrast inverted microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, reactive oxygen species (ROS) assays, and flow cytometric assays. Nano-CuO and ZnO showed significant toxicity in both HepG2 cells and catfish primary hepatocytes. The results demonstrate that HepG2 cells are more sensitive than catfish primary hepatocytes to the toxicity of metal oxide NPs. The overall ranking of the toxicity of metal oxides to the test cells is as follows: TiO2 < Co3O4< ZnO < CuO. The toxicity is due not only to ROS-induced cell death, but also damages to cell and mitochondrial membranes. PMID:21851965

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

PubMed Central

Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

2016-01-01

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

PubMed

Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

2016-03-04

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

PubMed

Nakamura, K D M; Tilli, T M; Wanderley, J L; Palumbo, A; Mattos, R M; Ferreira, A C; Klumb, C E; Nasciutti, L E; Gimba, E R

2016-02-01

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes

PubMed Central

Sachdeva, Mohit; Mito, Jeffrey K.; Lee, Chang-Lung; Zhang, Minsi; Li, Zhizhong; Dodd, Rebecca D.; Cason, David; Luo, Lixia; Ma, Yan; Van Mater, David; Gladdy, Rebecca; Lev, Dina C.; Cardona, Diana M.; Kirsch, David G.

2014-01-01

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes. PMID:25180607

Cofilin Inhibition Restores Neuronal Cell Death in Oxygen-Glucose Deprivation Model of Ischemia.

PubMed

Madineni, Anusha; Alhadidi, Qasim; Shah, Zahoor A

2016-03-01

Ischemia is a condition associated with decreased blood supply to the brain, eventually leading to death of neurons. It is associated with a diverse cascade of responses involving both degenerative and regenerative mechanisms. At the cellular level, the changes are initiated prominently in the neuronal cytoskeleton. Cofilin, a cytoskeletal actin severing protein, is known to be involved in the early stages of apoptotic cell death. Evidence supports its intervention in the progression of disease states like Alzheimer's and ischemic kidney disease. In the present study, we have hypothesized the possible involvement of cofilin in ischemia. Using PC12 cells and mouse primary cultures of cortical neurons, we investigated the potential role of cofilin in ischemia in two different in vitro ischemic models: chemical induced oxidative stress and oxygen-glucose deprivation/reperfusion (OGD/R). The expression profile studies demonstrated a decrease in phosphocofilin levels in all models of ischemia, implying stress-induced cofilin activation. Furthermore, calcineurin and slingshot 1L (SSH) phosphatases were found to be the signaling mediators of the cofilin activation. In primary cultures of cortical neurons, cofilin was found to be significantly activated after 1 h of OGD. To delineate the role of activated cofilin in ischemia, we knocked down cofilin by small interfering RNA (siRNA) technique and tested the impact of cofilin silencing on neuronal viability. Cofilin siRNA-treated neurons showed a significant reduction of cofilin levels in all treatment groups (control, OGD, and OGD/R). Additionally, cofilin siRNA-reduced cofilin mitochondrial translocation and caspase 3 cleavage, with a concomitant increase in neuronal viability. These results strongly support the active role of cofilin in ischemia-induced neuronal degeneration and apoptosis. We believe that targeting this protein mediator has a potential for therapeutic intervention in ischemic brain injury and stroke.

Autophagy prevention sensitizes AKTi-1/2-induced anti-hepatocellular carcinoma cell activity in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qi; Yang, Manyi; Qu, Zhan

Molecule-targeted therapy has become the research focus for hepatocellular carcinoma (HCC). Persistent PI3K-AKT activation is often detected in HCC, representing a valuable oncotarget for treatment. Here, we tested the anti-HCC activity by a potent AKT inhibitor: AKT inhibitor 1/2 (AKTi-1/2). In both established (HepG2 and Huh-7) and primary human HCC cells, treatment with AKTi-1/2 inhibited cell survival and proliferation, but induced cell apoptosis. AKTi-1/2 blocked AKT-mTOR activation, yet simultaneously provoked cytoprotective autophagy in HCC cells. The latter was evidenced by ATG-5 and Beclin-1 upregulation, p62 downregulation as well as LC3B-GFP puncta formation. Autophagy inhibition, via pharmacological inhibitors (3-methyladenine, ammonium chloride,more » and bafilomycin A1) or Beclin-1 siRNA knockdown, significantly potentiated AKTi-1/2-induced HepG2 cell death and apoptosis. In nude mice, AKTi-1/2 intraperitoneal injection inhibited HepG2 tumor growth. Significantly, its anti-tumor activity in vivo was further sensitized when combined with Beclin-1 shRNA knockdown in HepG2 tumors. Together, these results demonstrate that autophagy activation serves as a main resistance factor of AKTi-1/2 in HCC cells. Autophagy prevention therefore sensitizes AKTi-1/2-induced anti-HCC activity in vitro and in vivo. - Highlights: • AKTi-1/2 inhibits human HCC cells in vitro. • Autophagy inhibitors sensitize AKTi-1/2-induced HCC cell death and apoptosis. • Beclin-1 siRNA potentiates AKTi-1/2-induced HepG2 cell death and apoptosis. • Beclin-1 knockdown augments AKTi-1/2-induced anti-HepG2 tumor activity in vivo.« less

The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways.

PubMed

Lu, Dah-Yuu; Chang, Chih-Shiang; Yeh, Wei-Lan; Tang, Chih-Hsin; Cheung, Chi-Wai; Leung, Yuk-Man; Liu, Ju-Fang; Wong, Kar-Lok

2012-09-15

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis. Copyright © 2012 Elsevier GmbH. All rights reserved.

Biogenic selenium nanoparticles induce ROS-mediated necroptosis in PC-3 cancer cells through TNF activation.

PubMed

Sonkusre, Praveen; Cameotra, Swaranjit Singh

2017-06-07

Selenium is well documented to inhibit cancer at higher doses; however, the mechanism behind this inhibition varies widely depending on the cell type and selenium species. Previously, we have demonstrated that Bacillus licheniformis JS2 derived biogenic selenium nanoparticles (SeNPs) induce non-apoptotic cell death in prostate adenocarcinoma cell line, PC-3, at a minimal concentration of 2 µg Se/ml, without causing toxicity to the primary cells. However, the mechanism behind its anticancer activity was elusive. Our results have shown that these SeNPs at a concentration of 2 µg Se/ml were able to induce reactive oxygen species (ROS) mediated necroptosis in PC-3 cells by gaining cellular internalization. Real-time qPCR analysis showed increased expression of necroptosis associated tumor necrotic factor (TNF) and interferon regulatory factor 1 (IRF1). An increased expression of RIP1 protein was also observed at the translational level upon SeNP treatment. Moreover, the cell viability was significantly increased in the presence of necroptosis inhibitor, Necrostatin-1. Data suggest that our biogenic SeNPs induce cell death in PC-3 cells by the ROS-mediated activation of necroptosis, independent to RIP3 and MLKL, regulated by a RIP1 kinase.

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation.

PubMed

Allavena, Giulia; Cuomo, Francesca; Baumgartner, Georg; Bele, Tadeja; Sellgren, Alexander Yarar; Oo, Kyaw Soe; Johnson, Kaylee; Gogvadze, Vladimir; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

2018-01-01

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.

An age-structured model of hiv infection that allows for variations in the production rate of viral particles and the death rate of productively infected cells.

PubMed

Nelson, Patrick W; Gilchrist, Michael A; Coombs, Daniel; Hyman, James M; Perelson, Alan S

2004-09-01

Mathematical models of HIV-1 infection can help interpret drug treatment experiments and improve our understanding of the interplay between HIV-1 and the immune system. We develop and analyze an age- structured model of HIV-1 infection that allows for variations in the death rate of productively infected T cells and the production rate of viral particles as a function of the length of time a T cell has been infected. We show that this model is a generalization of the standard differential equation and of delay models previously used to describe HIV-1 infection, and provides a means for exploring fundamental issues of viral production and death. We show that the model has uninfected and infected steady states, linked by a transcritical bifurcation. We perform a local stability analysis of the nontrivial equilibrium solution and provide a general stability condition for models with age structure. We then use numerical methods to study solutions of our model focusing on the analysis of primary HIV infection. We show that the time to reach peak viral levels in the blood depends not only on initial conditions but also on the way in which viral production ramps up. If viral production ramps up slowly, we find that the time to peak viral load is delayed compared to results obtained using the standard (constant viral production) model of HIV infection. We find that data on viral load changing over time is insufficient to identify the functions specifying the dependence of the viral production rate or infected cell death rate on infected cell age. These functions must be determined through new quantitative experiments.

Immunotherapy With Magentorheologic Fluids

DTIC Science & Technology

2011-08-01

anti-tumor effects are weakened by removal of the tumor antigen pool (i.e. surgery) or use of cytoreductive and immunosuppressive therapies (i.e...particles were injected as magneto -rheological fluid (MRF) into an orthotopic primary breast cancer and followed by application of a magnetic field to...SUBJECT TERMS MRF: Magneto -rehological fluid iron particles, IT: immunotherapy, necrotic death, DCs: dendritic cells, cytokines, chemokines

Multiple Modes of Cell Death Discovered in a Prokaryotic (Cyanobacterial) Endosymbiont

PubMed Central

Zheng, Weiwen; Rasmussen, Ulla; Zheng, Siping; Bao, Xiaodong; Chen, Bin; Gao, Yuan; Guan, Xiong; Larsson, John; Bergman, Birgitta

2013-01-01

Programmed cell death (PCD) is a genetically-based cell death mechanism with vital roles in eukaryotes. Although there is limited consensus on similar death mode programs in prokaryotes, emerging evidence suggest that PCD events are operative. Here we present cell death events in a cyanobacterium living endophytically in the fern Azolla microphylla, suggestive of PCD. This symbiosis is characterized by some unique traits such as a synchronized development, a vertical transfer of the cyanobacterium between plant generations, and a highly eroding cyanobacterial genome. A combination of methods was used to identify cell death modes in the cyanobacterium. Light- and electron microscopy analyses showed that the proportion of cells undergoing cell death peaked at 53.6% (average 20%) of the total cell population, depending on the cell type and host developmental stage. Biochemical markers used for early and late programmed cell death events related to apoptosis (Annexin V-EGFP and TUNEL staining assays), together with visualization of cytoskeleton alterations (FITC-phalloidin staining), showed that all cyanobacterial cell categories were affected by cell death. Transmission electron microscopy revealed four modes of cell death: apoptotic-like, autophagic-like, necrotic-like and autolytic-like. Abiotic stresses further enhanced cell death in a dose and time dependent manner. The data also suggest that dynamic changes in the peptidoglycan cell wall layer and in the cytoskeleton distribution patterns may act as markers for the various cell death modes. The presence of a metacaspase homolog (domain p20) further suggests that the death modes are genetically programmed. It is therefore concluded that multiple, likely genetically programmed, cell death modes exist in cyanobacteria, a finding that may be connected with the evolution of cell death in the plant kingdom. PMID:23822984

Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, TongFa; Gao, DaQuan; Fang, Zheng-yu, E-mail: fangzhengyu158@sina.com

In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrinmore » A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. - Highlights: • PF-543 is anti-proliferative and cytotoxic to established and primary CRC cells. • PF-543 induces programmed necrosis, but not apoptosis, in CRC cells. • Modulation of mitochondrial protein cyclophilin-D alters PF-543's sensitivity. • PF-543 inhibits HCT-116 xenograft growth in SCID mice, improving mice survival. • Co-administration of cyclophilin-D inhibitor CsA inhibits PF-543's activity in vivo.« less

New types of metacaspases in phytoplankton reveal diverse origins of cell death proteases

PubMed Central

Choi, C J; Berges, J A

2013-01-01

Metacaspases are evolutionarily distant homologs of caspases that are found outside the metazoan and are known to have key roles in programmed cell death (PCD). Two types of metacaspases (types I and II) have been defined in plants based on their domain structures; these have similarities to metazoan ‘initiator' and ‘executioner' caspases. However, we know little about metacaspases in unicellular organisms and even less about their roles in cell death. We identified a novel group of metacaspases in sequenced phytoplanktonic protists that show domain architectures distinct from either type I or II enzymes; we designate them as type III. Type III metacaspases exhibit a rearrangement of domain structures between N- and C-terminus. In addition, we found a group of metacaspase-like proteases in phytoplankton that show sequence homology with other metacaspases, but defy classification in conventional schemes. These metacaspase-like proteases exist in bacteria alongside a variant of type I metacaspases and we propose these bacterial metacaspases are the origins of eukaryotic metacaspases. Type II and III metacaspases were not detected in bacteria and they might be variants of bacterial type I metacaspases that evolved in plants and phytoplanktonic protists, respectively, during the establishment of plastids through the primary and secondary endosymbiotic events. A complete absence of metacaspases in protists that lost plastids, such as oömycetes and ciliates indicates the gene loss during the plastid-to-nucleus gene transfer. Taken together, our findings suggest endosymbiotic gene transfer (EGT) is a key mechanism resulting in the evolutionary diversity of cell death proteases. PMID:23412383

Upregulation of Interleukin 7 Receptor Alpha and Programmed Death 1 Marks an Epitope-Specific CD8+ T-Cell Response That Disappears following Primary Epstein-Barr Virus Infection▿ †

PubMed Central

Sauce, Delphine; Larsen, Martin; Abbott, Rachel J. M.; Hislop, Andrew D.; Leese, Alison M.; Khan, Naeem; Papagno, Laura; Freeman, Gordon J.; Rickinson, Alan B.

2009-01-01

In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8+ T-cell response. However, we noticed that in HLA-A*0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Rα; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Rα seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells’ disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels. PMID:19605492

Die another way – non-apoptotic mechanisms of cell death

PubMed Central

Tait, Stephen W. G.; Ichim, Gabriel; Green, Douglas R.

2014-01-01

ABSTRACT Regulated, programmed cell death is crucial for all multicellular organisms. Cell death is essential in many processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells. The best-studied form of programmed cell death is apoptosis, a process that requires activation of caspase proteases. Recently it has been appreciated that various non-apoptotic forms of cell death also exist, such as necroptosis and pyroptosis. These non-apoptotic cell death modalities can be either triggered independently of apoptosis or are engaged should apoptosis fail to execute. In this Commentary, we discuss several regulated non-apoptotic forms of cell death including necroptosis, autophagic cell death, pyroptosis and caspase-independent cell death. We outline what we know about their mechanism, potential roles in vivo and define outstanding questions. Finally, we review data arguing that the means by which a cell dies actually matters, focusing our discussion on inflammatory aspects of cell death. PMID:24833670

Cell Death and Cell Death Responses in Liver Disease: Mechanisms and Clinical Relevance

PubMed Central

Luedde, Tom; Kaplowitz, Neil; Schwabe, Robert F.

2015-01-01

Summary Hepatocellular death is present in almost all types of human liver disease and is used as a sensitive parameter for the detection of acute and chronic liver disease of viral, toxic, metabolic, or autoimmune origin. Clinical data and animal models suggest that hepatocyte death is the key trigger of liver disease progression, manifested by the subsequent development of inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Modes of hepatocellular death differ substantially between liver diseases. Different modes of cell death such as apoptosis, necrosis, and necroptosis trigger specific cell death responses and promote progression of liver disease through distinct mechanisms. In this review, we first discuss molecular mechanisms by which different modes of cell death, damage-associated molecular patterns, and specific cell death responses contribute to the development of liver disease. We then review the clinical relevance of cell death, focusing on biomarkers; the contribution of cell death to drug-induced, viral, and fatty liver disease and liver cancer; and evidence for cell death pathways as therapeutic targets. PMID:25046161

Pro-apoptotic BIM is an essential initiator of physiological endothelial cell death independent of regulation by FOXO3

PubMed Central

Koenig, M N; Naik, E; Rohrbeck, L; Herold, M J; Trounson, E; Bouillet, P; Thomas, T; Voss, A K; Strasser, A; Coultas, L

2014-01-01

The growth of new blood vessels by angiogenesis is essential for normal development, but can also cause or contribute to the pathology of numerous diseases. Recent studies have shown that BIM, a pro-apoptotic BCL2-family protein, is required for endothelial cell apoptosis in vivo, and can contribute to the anti-angiogenic effect of VEGF-A inhibitors in certain tumor models. Despite its importance, the extent to which BIM is autonomously required for physiological endothelial apoptosis remains unknown and its regulation under such conditions is poorly defined. While the transcription factor FOXO3 has been proposed to induce Bim in response to growth factor withdrawal, evidence for this function is circumstantial. We report that apoptosis was reduced in Bim−/− primary endothelial cells, demonstrating a cell-autonomous role for BIM in endothelial death following serum and growth factor withdrawal. In conflict with in vitro studies, BIM-dependent endothelial death in vivo did not require FOXO3. Moreover, endothelial apoptosis proceeded normally in mice lacking FOXO-binding sites in the Bim promoter. Bim mRNA was upregulated in endothelial cells starved of serum and growth factors and this was accompanied by the downregulation of miRNAs of the miR-17∼92 cluster. Bim mRNA levels were also elevated in miR-17∼92+/− endothelial cells cultured under steady-state conditions, suggesting that miR-17∼92 cluster miRNAs may contribute to regulating overall Bim mRNA levels in endothelial cells. PMID:24971484

Elucidation of Altered Pathways in Tumor-Initiating Cells of Triple-Negative Breast Cancer: A Useful Cell Model System for Drug Screening.

PubMed

Christensen, Anne G; Ehmsen, Sidse; Terp, Mikkel G; Batra, Richa; Alcaraz, Nicolas; Baumbach, Jan; Noer, Julie B; Moreira, José; Leth-Larsen, Rikke; Larsen, Martin R; Ditzel, Henrik J

2017-08-01

A limited number of cancer cells within a tumor are thought to have self-renewing and tumor-initiating capabilities that produce the remaining cancer cells in a heterogeneous tumor mass. Elucidation of central pathways preferentially used by tumor-initiating cells/cancer stem cells (CSCs) may allow their exploitation as potential cancer therapy targets. We used single cell cloning to isolate and characterize four isogenic cell clones from a triple-negative breast cancer cell line; two exhibited mesenchymal-like and two epithelial-like characteristics. Within these pairs, one, but not the other, resulted in tumors in immunodeficient NOD/Shi-scid/IL-2 Rγ null mice and efficiently formed mammospheres. Quantitative proteomics and phosphoproteomics were used to map signaling pathways associated with the tumor-initiating ability. Signaling associated with apoptosis was suppressed in tumor-initiating versus nontumorigenic counterparts with pro-apoptotic proteins, such as Bcl2-associated agonist of cell death (BAD), FAS-associated death domain protein (FADD), and myeloid differentiation primary response protein (MYD88), downregulated in tumor-initiating epithelial-like cells. Functional studies confirmed significantly lower apoptosis in tumor-initiating versus nontumorigenic cells. Moreover, central pathways, including β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signaling, exhibited increased activation in the tumor-initiating cells. To evaluate the CSC model as a tool for drug screening, we assessed the effect of separately blocking NF-κB and Wnt/β-catenin signaling and found markedly reduced mammosphere formation, particularly for tumor-initiating cells. Similar reduction was also observed using patient-derived primary cancer cells. Furthermore, blocking NF-κB signaling in mice transplanted with tumor-initiating cells significantly reduced tumor outgrowth. Our study demonstrates that suppressed apoptosis, activation of pathways associated with cell viability, and CSCs are the major differences between tumor-initiating and nontumorigenic cells independent of their epithelial-like/mesenchymal-like phenotype. These altered pathways may provide targets for future drug development to eliminate CSCs, and the cell model may be a useful tool in such drug screenings. Stem Cells 2017;35:1898-1912. © 2017 AlphaMed Press.

Vena cava thrombectomy and primary repair after radical nephrectomy for renal cell carcinoma: single-center experience.

PubMed

Helfand, Brian T; Smith, Norm D; Kozlowski, James M; Eskandari, Mark K

2011-01-01

Inferior vena cava (IVC) reconstruction for locally advanced renal cell carcinoma (RCC) includes resection with and without interposition grafting, patch graft, or primary repair. The proposed benefits of lateral venorrhaphy and primary repair are avoidance of foreign material, a more expeditious repair, and preservation of lower extremity venous outflow. A single-center retrospective review of 22 patients with RCC and IVC tumor thrombus treated with radical nephrectomy, lateral venorrhaphy, thrombectomy, and primary vena cava repair between July 2002 and June 2009 was carried out. Demographic data, diagnostic information, radiographic cross-sectional imaging, and procedural outcomes were examined. Among the 13 men and nine women, the mean age was 62.1 years (42-83); mean tumor size was 9.8 cm (3-17 cm), and 90% (n = 18) of the cases with RCC were identified pathologically as clear cell adenocarcinoma; on the basis of the classification system adopted by Neves, level I was for 50% (n = 11), level II for 32% (n = 7), level III for 9% (n = 2), and level IV for 9% (n = 2) of the patients. All patients underwent en bloc radical nephrectomy with tumor thrombus removal and primary IVC repair. Mean total operative time was 547.9 ± 138.5 minutes, whereas mean IVC cross-clamp time was 10.8 minutes (6-29 minutes). There were no intraoperative deaths or pulmonary embolism and all IVC margins were found to be pathologically negative. Postoperative complications included one pulmonary embolism, one exacerbation of chronic lymphedema, and two cases of new onset erectile dysfunction. Mean follow-up was 36.4 ± 23.2 months (6-92 months). There were no radiographic or clinically significant changes in mean IVC diameter during follow-up. Five late deaths (23%) occurred as a result of metastatic RCC over a mean period of 24 months (range, 12-48), but without any local recurrences. For advanced RCC with tumor thrombus extension into the IVC, lateral venorrhaphy and primary IVC repair avoids complicated caval reconstructions and results in high patency rates with a low local tumor recurrence rate. Copyright © 2011 Annals of Vascular Surgery Inc. Published by Elsevier Inc. All rights reserved.

Mitochondrial fission proteins regulate programmed cell death in yeast.

PubMed

Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

2004-11-15

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

TRAF6 and p62 inhibit amyloid β-induced neuronal death through p75 neurotrophin receptor

PubMed Central

Geetha, Thangiah; Zheng, Chen; McGregor, Wade C.; White, B. Douglas; Diaz-Meco, Maria T.; Moscat, Jorge; Babu, Jeganathan Ramesh

2014-01-01

Amyloid β (Aβ) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient’s brain. Aβ is known to bind p75 neurotrophin receptor (p75NTR) and mediates Aβ-induced neuronal death. Recently, we showed that NGF leads to p75NTR polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that Aβ stimulation impaired the p75NTR polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75NTR on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75NTR polyubiquitination upon Aβ/NGF treatment. Aβ significantly reduced NF-κB activity by attenuating the interaction of p75NTR with IKKβ. p75NTR increased NF-κB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the Aβ-mediated inhibition of p75NTR polyubiquitination and restored neuronal cell survival. PMID:23017601

Berberine induces FasL-related apoptosis through p38 activation in KB human oral cancer cells

PubMed Central

KIM, JAE-SUNG; OH, DAHYE; YIM, MIN-JI; PARK, JIN-JU; KANG, KYEONG-ROK; CHO, IN-A; MOON, SUNG-MIN; OH, JI-SU; YOU, JAE-SEEK; KIM, CHUN SUNG; KIM, DO KYUNG; LEE, SOOK-YOUNG; LEE, GYEONG-JE; IM, HEE-JEONG; KIM, SU-GWAN

2015-01-01

In the present study, we examined the anticancer properties of berberine in KB oral cancer cells with a specific focus on its cellular mechanism. Berberine did not affect the cell viability of the primary human normal oral keratinocytes that were used as a control. However, the viability of KB cells was found to decrease significantly in the presence of berberine in a dose-dependent manner. Furthermore, in KB cells, berberine induced the fragmentation of genomic DNA, changes in cell morphology, and nuclear condensation. In addition, caspase-3 and -7 activation, and an increase in apoptosis were observed. Berberine was also found to upregulate significantly the expression of the death receptor ligand, FasL. In turn, this upregulation triggered the activation of pro-apoptotic factors such as caspase-8, -9 and -3 and poly(ADP-ribose) polymerase (PARP). Furthermore, pro-apoptotic factors such as Bax, Bad and Apaf-1 were also significantly upregulated by berberine. Anti-apoptotic factors such as Bcl-2 and Bcl-xL were downregulated. Z-VAD-FMK, a cell-permeable pan-caspase inhibitor, suppressed the activation of caspase-3 and PARP. These results clearly indicate that berberine-induced cell death of KB oral cancer cells was mediated by both extrinsic death receptor-dependent and intrinsic mitochondrial-dependent apoptotic signaling pathways. In addition, berberine-induced upregulation of FasL was shown to be mediated by the p38 MAPK signaling pathway. We also found that berberine-induced migration suppression was mediated by downregulation of MMP-2 and MMP-9 through phosphorylation of p38 MAPK. In summary, berberine has the potential to be used as a chemotherapeutic agent, with limited side-effects, for the management of oral cancer. PMID:25634589

The importance of being dead: cell death mechanisms assessment in anti-sarcoma therapy.

PubMed

Rello-Varona, Santiago; Herrero-Martín, David; Lagares-Tena, Laura; López-Alemany, Roser; Mulet-Margalef, Núria; Huertas-Martínez, Juan; Garcia-Monclús, Silvia; García Del Muro, Xavier; Muñoz-Pinedo, Cristina; Tirado, Oscar Martínez

2015-01-01

Cell death can occur through different mechanisms, defined by their nature and physiological implications. Correct assessment of cell death is crucial for cancer therapy success. Sarcomas are a large and diverse group of neoplasias from mesenchymal origin. Among cell death types, apoptosis is by far the most studied in sarcomas. Albeit very promising in other fields, regulated necrosis and other cell death circumstances (as so-called "autophagic cell death" or "mitotic catastrophe") have not been yet properly addressed in sarcomas. Cell death is usually quantified in sarcomas by unspecific assays and in most cases the precise sequence of events remains poorly characterized. In this review, our main objective is to put into context the most recent sarcoma cell death findings in the more general landscape of different cell death modalities.

Cell Death in C. elegans Development.

PubMed

Malin, Jennifer Zuckerman; Shaham, Shai

2015-01-01

Cell death is a common and important feature of animal development, and cell death defects underlie many human disease states. The nematode Caenorhabditis elegans has proven fertile ground for uncovering molecular and cellular processes controlling programmed cell death. A core pathway consisting of the conserved proteins EGL-1/BH3-only, CED-9/BCL2, CED-4/APAF1, and CED-3/caspase promotes most cell death in the nematode, and a conserved set of proteins ensures the engulfment and degradation of dying cells. Multiple regulatory pathways control cell death onset in C. elegans, and many reveal similarities with tumor formation pathways in mammals, supporting the idea that cell death plays key roles in malignant progression. Nonetheless, a number of observations suggest that our understanding of developmental cell death in C. elegans is incomplete. The interaction between dying and engulfing cells seems to be more complex than originally appreciated, and it appears that key aspects of cell death initiation are not fully understood. It has also become apparent that the conserved apoptotic pathway is dispensable for the demise of the C. elegans linker cell, leading to the discovery of a previously unexplored gene program promoting cell death. Here, we review studies that formed the foundation of cell death research in C. elegans and describe new observations that expand, and in some cases remodel, this edifice. We raise the possibility that, in some cells, more than one death program may be needed to ensure cell death fidelity. © 2015 Elsevier Inc. All rights reserved.

Metastasis: recent discoveries and novel treatment strategies

PubMed Central

Eccles, Suzanne A; Welch, Danny R

2007-01-01

Most cancer deaths are due to the development of metastases, hence the most important improvements in morbidity and mortality will result from prevention (or elimination) of such disseminated disease. Some would argue that treatments directed against metastasis are too late because cells have already escaped from the primary tumour. Such an assertion runs contrary to the significant but (for many common adult cancers) fairly modest improvements in survival following the use of adjuvant radiation and chemotherapy designed to eliminate disseminated cells after surgical removal of the primary tumour. Nonetheless, the debate raises important issues concerning the accurate early identification of clonogenic, metastatic cells, the discovery of novel, tractable targets for therapy, and the monitoring of minimal residual disease. We focus on recent findings regarding intrinsic and extrinsic molecular mechanisms controlling metastasis that determine how, when, and where cancers metastasise, and their implications for patient management in the 21st century. PMID:17512859

Microelectroporation device for genomic screening

DOEpatents

Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

2014-09-09

We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism

PubMed Central

Rotin, Lianne E.; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L.; Minden, Mark D.; Slassi, Malik; Schimmer, Aaron D.

2016-01-01

Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease. PMID:26624983

Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study

PubMed Central

Feng, Yuan; Zhou, Jihong; Li, Zhanhua; Jiang, Ying; Zhou, Ying

2016-01-01

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively targets cancer cells. The present preclinical study investigated the anti-cancer efficiency of ONC201, a first-in-class small molecule TRAIL inducer, in lung cancer cells. We showed that ONC201 was cytotoxic and anti-proliferative in both established (A549 and H460 lines) and primary human lung cancer cells. It was yet non-cytotoxic to normal lung epithelial cells. Further, ONC201 induced exogenous apoptosis activation in lung cancer cells, which was evidenced by TRAIL/death receptor-5 (DR5) induction and caspase-8 activation. The caspase-8 inhibitor or TRAIL/DR5 siRNA knockdown alleviated ONC201’s cytotoxicity against lung cancer cells. Molecularly, ONC201 in-activated Akt-S6K1 and Erk signalings in lung cancer cells, causing Foxo3a nuclear translocation. For the in vivo studies, intraperitoneal injection of ONC201 at well-tolerated doses significantly inhibited xenografted A549 tumor growth in severe combined immunodeficient (SCID) mice. Further, ONC201 administration induced TRAIL/DR5 expression, yet inactivated Akt-S6K1 and Erk in tumor tissues. These results of the study demonstrates the potent anti-lung cancer activity by ONC201. PMID:27626799

Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study.

PubMed

Feng, Yuan; Zhou, Jihong; Li, Zhanhua; Jiang, Ying; Zhou, Ying

2016-01-01

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively targets cancer cells. The present preclinical study investigated the anti-cancer efficiency of ONC201, a first-in-class small molecule TRAIL inducer, in lung cancer cells. We showed that ONC201 was cytotoxic and anti-proliferative in both established (A549 and H460 lines) and primary human lung cancer cells. It was yet non-cytotoxic to normal lung epithelial cells. Further, ONC201 induced exogenous apoptosis activation in lung cancer cells, which was evidenced by TRAIL/death receptor-5 (DR5) induction and caspase-8 activation. The caspase-8 inhibitor or TRAIL/DR5 siRNA knockdown alleviated ONC201's cytotoxicity against lung cancer cells. Molecularly, ONC201 in-activated Akt-S6K1 and Erk signalings in lung cancer cells, causing Foxo3a nuclear translocation. For the in vivo studies, intraperitoneal injection of ONC201 at well-tolerated doses significantly inhibited xenografted A549 tumor growth in severe combined immunodeficient (SCID) mice. Further, ONC201 administration induced TRAIL/DR5 expression, yet inactivated Akt-S6K1 and Erk in tumor tissues. These results of the study demonstrates the potent anti-lung cancer activity by ONC201.

Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism.

PubMed

Rotin, Lianne E; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L; Minden, Mark D; Slassi, Malik; Schimmer, Aaron D

2016-01-19

Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease.

LBH589, a deacetylase inhibitor, induces apoptosis in adult T-cell leukemia/lymphoma cells via activation of a novel RAIDD-caspase-2 pathway

PubMed Central

Hasegawa, H; Yamada, Y; Tsukasaki, K; Mori, N; Tsuruda, K; Sasaki, D; Usui, T; Osaka, A; Atogami, S; Ishikawa, C; Machijima, Y; Sawada, S; Hayashi, T; Miyazaki, Y; Kamihira, S

2011-01-01

Adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm etiologically associated with human T-lymphotropic virus type-1 (HTLV-1), is resistant to treatment. In this study, we examined the effects of a new inhibitor of deacetylase enzymes, LBH589, on ATLL cells. LBH589 effectively induced apoptosis in ATLL-related cell lines and primary ATLL cells and reduced the size of tumors inoculated in SCID mice. Analyses, including with a DNA microarray, revealed that neither death receptors nor p53 pathways contributed to the apoptosis. Instead, LBH589 activated an intrinsic pathway through the activation of caspase-2. Furthermore, small interfering RNA experiments targeting caspase-2, caspase-9, RAIDD, p53-induced protein with a death domain (PIDD) and RIPK1 (RIP) indicated that activation of RAIDD is crucial and an event initiating this pathway. In addition, LBH589 caused a marked decrease in levels of factors involved in ATLL cell proliferation and invasion such as CCR4, IL-2R and HTLV-1 HBZ-SI, a spliced form of the HTLV-1 basic zipper factor HBZ. In conclusion, we showed that LBH589 is a strong inducer of apoptosis in ATLL cells and uncovered a novel apoptotic pathway initiated by activation of RAIDD. PMID:21242994

N-Methyl-d-Aspartate (NMDA) Receptor Blockade Prevents Neuronal Death Induced by Zika Virus Infection

PubMed Central

Costa, Vivian V.; Del Sarto, Juliana L.; Rocha, Rebeca F.; Silva, Flavia R.; Doria, Juliana G.; Olmo, Isabella G.; Marques, Rafael E.; Queiroz-Junior, Celso M.; Foureaux, Giselle; Araújo, Julia Maria S.; Cramer, Allysson; Real, Ana Luíza C. V.; Ribeiro, Lucas S.; Sardi, Silvia I.; Ferreira, Anderson J.; Machado, Fabiana S.; de Oliveira, Antônio C.; Teixeira, Antônio L.; Nakaya, Helder I.; Souza, Danielle G.

2017-01-01

ABSTRACT Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration. PMID:28442607

Meta-Analysis of Cell-based CaRdiac stUdiEs (ACCRUE) in patients with acute myocardial infarction based on individual patient data.

PubMed

Gyöngyösi, Mariann; Wojakowski, Wojciech; Lemarchand, Patricia; Lunde, Ketil; Tendera, Michal; Bartunek, Jozef; Marban, Eduardo; Assmus, Birgit; Henry, Timothy D; Traverse, Jay H; Moyé, Lemuel A; Sürder, Daniel; Corti, Roberto; Huikuri, Heikki; Miettinen, Johanna; Wöhrle, Jochen; Obradovic, Slobodan; Roncalli, Jérome; Malliaras, Konstantinos; Pokushalov, Evgeny; Romanov, Alexander; Kastrup, Jens; Bergmann, Martin W; Atsma, Douwe E; Diederichsen, Axel; Edes, Istvan; Benedek, Imre; Benedek, Theodora; Pejkov, Hristo; Nyolczas, Noemi; Pavo, Noemi; Bergler-Klein, Jutta; Pavo, Imre J; Sylven, Christer; Berti, Sergio; Navarese, Eliano P; Maurer, Gerald

2015-04-10

The meta-Analysis of Cell-based CaRdiac study is the first prospectively declared collaborative multinational database, including individual data of patients with ischemic heart disease treated with cell therapy. We analyzed the safety and efficacy of intracoronary cell therapy after acute myocardial infarction (AMI), including individual patient data from 12 randomized trials (ASTAMI, Aalst, BOOST, BONAMI, CADUCEUS, FINCELL, REGENT, REPAIR-AMI, SCAMI, SWISS-AMI, TIME, LATE-TIME; n=1252). The primary end point was freedom from combined major adverse cardiac and cerebrovascular events (including all-cause death, AMI recurrance, stroke, and target vessel revascularization). The secondary end point was freedom from hard clinical end points (death, AMI recurrence, or stroke), assessed with random-effects meta-analyses and Cox regressions for interactions. Secondary efficacy end points included changes in end-diastolic volume, end-systolic volume, and ejection fraction, analyzed with random-effects meta-analyses and ANCOVA. We reported weighted mean differences between cell therapy and control groups. No effect of cell therapy on major adverse cardiac and cerebrovascular events (14.0% versus 16.3%; hazard ratio, 0.86; 95% confidence interval, 0.63-1.18) or death (1.4% versus 2.1%) or death/AMI recurrence/stroke (2.9% versus 4.7%) was identified in comparison with controls. No changes in ejection fraction (mean difference: 0.96%; 95% confidence interval, -0.2 to 2.1), end-diastolic volume, or systolic volume were observed compared with controls. These results were not influenced by anterior AMI location, reduced baseline ejection fraction, or the use of MRI for assessing left ventricular parameters. This meta-analysis of individual patient data from randomized trials in patients with recent AMI revealed that intracoronary cell therapy provided no benefit, in terms of clinical events or changes in left ventricular function. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01098591. © 2015 American Heart Association, Inc.

Characteristic thickened cell walls of the bracts of the 'eternal flower' Helichrysum bracteatum.

PubMed

Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

2008-07-01

Helichrysum bracteatum is called an 'eternal flower' and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type.

The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

PubMed Central

Rauert-Wunderlich, Hilka; Siegmund, Daniela; Maier, Eduard; Giner, Tina; Bargou, Ralf C.; Wajant, Harald; Stühmer, Thorsten

2013-01-01

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells. PMID:23527154

JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice.

PubMed

Song, Xinxin; Zhu, Shan; Xie, Yangchun; Liu, Jiao; Sun, Lingyi; Zeng, Dexing; Wang, Pengcheng; Ma, Xiaochao; Kroemer, Guido; Bartlett, David L; Billiar, Timothy R; Lotze, Michael T; Zeh, Herbert J; Kang, Rui; Tang, Daolin

2018-04-01

Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRas G12D/+ ;Tp53 R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;Kras G12D/+ mice crossed with Hmgb1 flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax -/- /Bak -/- cells), necroptosis (Ripk1 -/- , Ripk3 -/- , or Mlkl -/- cells), ferroptosis (Gpx4 -/- cells), or autophagy (Atg3 -/- , Atg5 -/- , Atg7 -/- , or Sqstm1 -/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34 + progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability.

PubMed

Beebe, Stephen J; Chen, Yeong-Jer; Sain, Nova M; Schoenbach, Karl H; Xiao, Shu

2012-01-01

It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0-80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.

The Roles of p53 in Mitochondrial Dynamics and Cancer Metabolism: The Pendulum between Survival and Death in Breast Cancer?

PubMed

Moulder, David E; Hatoum, Diana; Tay, Enoch; Lin, Yiguang; McGowan, Eileen M

2018-06-08

Cancer research has been heavily geared towards genomic events in the development and progression of cancer. In contrast, metabolic regulation, such as aberrant metabolism in cancer, is poorly understood. Alteration in cellular metabolism was once regarded simply as a consequence of cancer rather than as playing a primary role in cancer promotion and maintenance. Resurgence of cancer metabolism research has identified critical metabolic reprogramming events within biosynthetic and bioenergetic pathways needed to fulfill the requirements of cancer cell growth and maintenance. The tumor suppressor protein p53 is emerging as a key regulator of metabolic processes and metabolic reprogramming in cancer cells—balancing the pendulum between cell death and survival. This review provides an overview of the classical and emerging non-classical tumor suppressor roles of p53 in regulating mitochondrial dynamics: mitochondrial engagement in cell death processes in the prevention of cancer. On the other hand, we discuss p53 as a key metabolic switch in cellular function and survival. The focus is then on the conceivable roles of p53 in breast cancer metabolism. Understanding the metabolic functions of p53 within breast cancer metabolism will, in due course, reveal critical metabolic hotspots that cancers advantageously re-engineer for sustenance. Illustration of these events will pave the way for finding novel therapeutics that target cancer metabolism and serve to overcome the breast cancer burden.

Nitrosothiol signaling and protein nitrosation in cell death.

PubMed

Iyer, Anand Krishnan V; Rojanasakul, Yon; Azad, Neelam

2014-11-15

Nitric oxide, a reactive free radical, is an important signaling molecule that can lead to a plethora of cellular effects affecting homeostasis. A well-established mechanism by which NO manifests its effect on cellular functions is the post-translational chemical modification of cysteine thiols in substrate proteins by a process known as S-nitrosation. Studies that investigate regulation of cellular functions through NO have increasingly established S-nitrosation as the primary modulatory mechanism in their respective systems. There has been a substantial increase in the number of reports citing various candidate proteins undergoing S-nitrosation, which affects cell-death and -survival pathways in a number of tissues including heart, lung, brain and blood. With an exponentially growing list of proteins being identified as substrates for S-nitrosation, it is important to assimilate this information in different cell/tissue systems in order to gain an overall view of protein regulation of both individual proteins and a class of protein substrates. This will allow for broad mapping of proteins as a function of S-nitrosation, and help delineate their global effects on pathophysiological responses including cell death and survival. This information will not only provide a much better understanding of overall functional relevance of NO in the context of various disease states, it will also facilitate the generation of novel therapeutics to combat specific diseases that are driven by NO-mediated S-nitrosation. Copyright © 2014 Elsevier Inc. All rights reserved.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

PubMed Central

Mukhopadhyay, Partha; Rajesh, Mohanraj; Horváth, Béla; Bátkai, Sándor; Park, Ogyi; Tanashian, Galin; Gao, Rachel Y; Patel, Vivek; Wink, David A.; Liaudet, Lucas; Haskó, György; Mechoulam, Raphael; Pacher, Pál

2011-01-01

Ischemia-reperfusion (I/R) is a pivotal mechanism of liver damage following liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol(CBD), the non-psychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor alpha (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, inter-cellular adhesion molecule 1 mRNA levels, tissue neutrophil infiltration, nuclear factor kappa B (NF-KB) activation), stress signaling (p38MAPK and JNK) and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress and cell death, and also attenuated the bacterial endotoxin-triggered NF-KB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecules expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α, and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent from classical CB1/2 receptors. PMID:21362471

What cell death does in development.

PubMed

Zakeri, Zahra; Penaloza, Carlos G; Smith, Kyle; Ye, Yixia; Lockshin, Richard A

2015-01-01

Cell death is prominent in gametogenesis and shapes and sculpts embryos. In non-mammalian embryos one sees little or no cell death prior to the maternal-zygotic transition, but, in mammalian embryos, characteristic deaths of one or two cells occur at the end of compaction and are apparently necessary for the separation of the trophoblast from the inner cell mass. Considerable sculpting of the embryo occurs by cell deaths during organogenesis, and appropriate cell numbers, especially in the CNS and in the immune system, are generated by massive overproduction of cells and selection of a few, with death of the rest. The timing, identity, and genetic control of specific cells that die have been well documented in Caenorhabditis, but in other embryos the stochastic nature of the deaths limit our ability to do more than identify the regions in which cells will die. Complete disruption of the cell death machinery can be lethal, but many mutations of the regulatory machinery yield only modest or no phenotypes, indicating substantial redundancy and compensation of regulatory mechanisms. Most of the deaths are apoptotic and are identified by techniques used to recognize apoptosis, but techniques identifying lysosomes (whether in dying or involuting cells or in the phagocytes that invade the tissue) also reveal patterns of cell death. Aberrant cell deaths that produce known phenotypes are typically localized, indicating that the mechanism of activating a programmed death in a specific region, rather than the mechanism of death, is aberrant. These results lead us to conclude that we need to know much more about the conversations among cells that lead cells to commit suicide.

Inhibition of caspases prevents ototoxic and ongoing hair cell death

NASA Technical Reports Server (NTRS)

Matsui, Jonathan I.; Ogilvie, Judith M.; Warchol, Mark E.

2002-01-01

Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

Cardiac and Noncardiac Causes of Long-Term Mortality in ST-Segment-Elevation Acute Myocardial Infarction Patients Who Underwent Primary Percutaneous Coronary Intervention.

PubMed

Yamashita, Yugo; Shiomi, Hiroki; Morimoto, Takeshi; Yaku, Hidenori; Furukawa, Yutaka; Nakagawa, Yoshihisa; Ando, Kenji; Kadota, Kazushige; Abe, Mitsuru; Nagao, Kazuya; Shizuta, Satoshi; Ono, Koh; Kimura, Takeshi

2017-01-01

In patients with ST-segment-elevation acute myocardial infarction (STEMI) who underwent primary percutaneous coronary intervention, long-term risks for cardiac and noncardiac death beyond acute phase of STEMI have not been thoroughly evaluated yet. We identified 3942 STEMI patients who had primary percutaneous coronary intervention within 24 hours after onset between January 2005 and December 2007 in the CREDO-Kyoto AMI registry (Coronary Revascularization Demonstrating Outcome study in Kyoto Acute Myocardial Infarction) and evaluated their short-term (within 6-month) and long-term (beyond 6-month) incidences and causes of deaths. The cumulative 5-year incidence of all-cause death in the current study population was 20.4% (cardiac death, 12.2% and noncardiac death, 9.4%, respectively). The vast majority of deaths were cardiac in origin within 6-month (cardiac death, 8.0% and noncardiac death, 0.9%), whereas noncardiac death accounted for nearly two thirds of all-cause death beyond 6-month (cardiac death, 4.6% and noncardiac death, 8.5%). In the stratified analysis according to age, the proportion of noncardiac death was similar regardless of age although the absolute mortality rate was higher with increasing age. By the multivariable Cox regression models, the independent risk factors of all-cause death were advanced age, cardiogenic shock, renal dysfunction, large infarct size, and anterior wall infarction within 6 months after STEMI, and advanced age, previous heart failure, renal dysfunction, and liver cirrhosis beyond 6 months after STEMI, respectively. In STEMI patients who underwent primary percutaneous coronary intervention, the long-term risk for cardiac death was relatively low compared with that for noncardiac death, which accounted for nearly two thirds of all-cause death beyond 6 months. © 2017 American Heart Association, Inc.

The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Weihong; Xu, Bin; Yao, Yiting

In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our in vitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. In vivo, oral administrationmore » of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation in vitro and in vivo, and ceramide production might be the key mechanism responsible for its actions. - Highlights: • AR-42 is anti-proliferative against primary/established colon cancer cells. • AR-42 induces significant apoptotic death in primary/established colon cancer cells. • Ceramide production mediates AR-42-induced cytotoxicity in colon cancer cells. • AR-42 oral administration potently inhibits SW-620 xenograft growth in SCID mice.« less

Detection of the Cyanotoxins L-BMAA Uptake and Accumulation in Primary Neurons and Astrocytes.

PubMed

Tan, Vanessa X; Mazzocco, Claire; Varney, Bianca; Bodet, Dominique; Guillemin, Tristan A; Bessede, Alban; Guillemin, Gilles J

2018-01-01

We show for the first time that a newly developed polyclonal antibody (pAb) can specifically target the cyanotoxin β-methylamino-L-alanine (BMAA) and can be used to enable direct visualization of BMAA entry and accumulation in primary brain cells. We used this pAb to investigate the effect of acute and chronic accumulation, and toxicity of both BMAA and its natural isomer 2,4-diaminobutyric acid (DAB), separately or in combination, on primary cultures of rat neurons. We further present evidence that co-treatment with BMAA and DAB increased neuronal death, as measured by MAP2 fluorescence level, and appeared to reduce BMAA accumulation. DAB is likely to be acting synergistically with BMAA resulting in higher level of cellular toxicity. We also found that glial cells such as microglia and astrocytes are also able to directly uptake BMAA indicating that additional brain cell types are affected by BMAA-induced toxicity. Therefore, BMAA clearly acts at multiple cellular levels to possibly increase the risk of developing neurodegenerative diseases, including neuro- and gliotoxicity and synergetic exacerbation with other cyanotoxins.

Changes of Gene Expression in the Apoptosis Pathway in Lncap and PC3 Cells Exposed to X-Rays or Protons

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Radio-resistant or recurrent prostate cancer represents a serious health risk for approximately 20%-30% of patients treated with primary radiation therapy for clinically localized prostate cancer. In our current studies, we investigated the expressions of apoptosis related gene expression profile (84 genes) in two distinct prostate cell lines Lncap (P53+ and AR+) and PC3 (P53- and AR-) before and after exposure to X-rays or protons, using cDNA PCR arrays. In Lncap cells, 10Gy X-ray radiation significantly induced the expression of 19 out of 84 genes at 4h after irradiation. The changed genes were mostly in death and death receptor domain families, TNF ligand and receptor families, and apoptotic group of the BCL2 family, especially in P53 related genes, such as FAS, BAX, BAK1 and GADD45A. In PC3, X-rays only induced the expression of 3 genes, including an increased expression of BIRC3. There was no difference of the X-ray mediated cell killing in both cell lines using the cell cycle analysis. However, these X-ray-induced gene expression differences between PC3 and Lncap may explain the phenotype of PC3 cells that shows more tolerant not only to radiation, but also to other apoptosis inducing and sensitizing reagents. To compare the effectiveness of cell killing with X-rays, we also exposed PC3 cells to 10Gy protons at the Bragg peak region. Protons did not induce more apoptosis than X-rays for the same dose. In comparison to X-rays, protons significantly altered expressions of 13 genes in PC3, which included decreased expressions of anti-apoptosis genes (BCL2 and BCL2L2), and increased expressions of death and death receptor domain family genes, TNF ligand and receptor family and several kinases (FAS, DAPK1 and RIPK2). These data suggest that proton treatment is more effective in influencing the apoptosis pathways in PC3 cells than X-rays, thus protons may be more effective in the treatment of specific prostate tumor.

Lack of TXNIP protects against mitochondria-mediated apoptosis but not against fatty acid-induced ER stress-mediated beta-cell death.

PubMed

Chen, Junqin; Fontes, Ghislaine; Saxena, Geetu; Poitout, Vincent; Shalev, Anath

2010-02-01

We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced beta-cell apoptosis. Because the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP deficiency also protects beta-cells against lipoapoptosis. RESARCH DESIGN AND METHODS: To determine the effects of fatty acids on beta-cell TXNIP expression, INS-1 cells and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or Intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]), mitochondrial death pathway (cytochrome c release), and endoplasmic reticulum (ER) stress (binding protein [BiP], C/EBP homologous protein [CHOP]). Effects of TXNIP deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER stress). Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP deficiency does not effectively protect against palmitate or thapsigargin-induced beta-cell apoptosis, but specifically prevents staurosporine- or glucose-induced toxicity. Our results demonstrate that unlike glucose, fatty acids do not induce beta-cell expression of proapoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying beta-cell glucotoxicity, whereas it has very few protective effects against ER stress-mediated lipoapoptosis.

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons.

PubMed

de Carvalho, Nathalia Delazeri; Garcia, Raphael CaioTamborelli; Ferreira, Adilson Kleber; Batista, Daniel Rodrigo; Cassola, Antonio Carlos; Maria, Durvanei; Lebrun, Ivo; Carneiro, Sylvia Mendes; Afeche, Solange Castro; Marcourakis, Tania; Sandoval, Maria Regina Lopes

2014-03-13

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca(2+) and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca(2+) and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s. Copyright © 2014 Elsevier B.V. All rights reserved.

JNK Activation Contributes to Oxidative Stress-Induced Parthanatos in Glioma Cells via Increase of Intracellular ROS Production.

PubMed

Zheng, Linjie; Wang, Chen; Luo, Tianfei; Lu, Bin; Ma, Hongxi; Zhou, Zijian; Zhu, Dong; Chi, Guangfan; Ge, Pengfei; Luo, Yinan

2017-07-01

Parthanatos is a form of PARP-1-dependent programmed cell death. The induction of parthanatos is emerging as a new strategy to kill gliomas which are the most common type of primary malignant brain tumor. Oxidative stress is thought to be a critical factor triggering parthanatos, but its underlying mechanism is poorly understood. In this study, we used glioma cell lines and H 2 O 2 to investigate the role of JNK in glioma cell parthanatos induced by oxidative stress. We found that exposure to H 2 O 2 not only induced intracellular accumulation of ROS but also resulted in glioma cell death in a concentration- and incubation time-dependent manner, which was accompanied with cytoplasmic formation of PAR polymer, expressional upregulation of PARP-1, mitochondrial depolarization, and AIF translocation to nucleus. Pharmacological inhibition of PARP-1 with 3AB or genetic knockdown of its level with siRNA rescued glioma cell death, as well as suppressed cytoplasmic accumulation of PAR polymer and nuclear translocation of AIF, which were consistent with the definition of parthanatos. Moreover, the phosphorylated level of JNK increased markedly with the extension of H 2 O 2 exposure time. Either attenuation of intracellular ROS with antioxidant NAC or inhibition of JNK phosphorylation with SP600125 or JNK siRNA could significantly prevent H 2 O 2 -induced parthanatos in glioma cells. Additionally, inhibition of JNK with SP600125 alleviated intracellular accumulation of ROS and attenuated mitochondrial generation of superoxide. Thus, we demonstrated that JNK activation contributes to glioma cell parthanatos caused by oxidative stress via increase of intracellular ROS generation.

Association of Blood Transfusion From Female Donors With and Without a History of Pregnancy With Mortality Among Male and Female Transfusion Recipients

PubMed Central

Caram-Deelder, Camila; Kreuger, Aukje L.; Evers, Dorothea; de Vooght, Karen M. K.; van de Kerkhof, Daan; Visser, Otto; Péquériaux, Nathalie C. V.; Hudig, Francisca; Zwaginga, Jaap Jan; van der Bom, Johanna G.

2017-01-01

Importance Transfusion of red blood cells from female donors has been associated with increased mortality in male recipients. Objective To quantify the association between red blood cell transfusion from female donors with and without a history of pregnancy and mortality of red blood cell recipients. Design, Setting, and Participants Retrospective cohort study of first-time transfusion recipients at 6 major Dutch hospitals enrolled from May 30, 2005, to September 1, 2015; the final follow-up date was September 1, 2015. The primary analysis was the no-donor-mixture cohort (ie, either all red blood cell transfusions exclusively from male donors, or all exclusively from female donors without a history of pregnancy, or all exclusively from female donors with a history of pregnancy). The association between mortality and exposure to transfusions from ever-pregnant or never-pregnant female donors was analyzed using life tables and time-varying Cox proportional hazards models. Exposures Red blood cell transfusions from ever-pregnant or never-pregnant female donors, compared with red blood cell transfusions from male donors. Main Outcomes and Measures All-cause mortality during follow-up. Results The cohort for the primary analyses consisted of 31 118 patients (median age, 65 [interquartile range, 42-77] years; 52% female) who received 59 320 red blood cell transfusions exclusively from 1 of 3 types of donors (88% male; 6% ever-pregnant female; and 6% never-pregnant female). The number of deaths in this cohort was 3969 (13% mortality). For male recipients of red blood cell transfusions, all-cause mortality rates after a red blood cell transfusion from an ever-pregnant female donor vs male donor were 101 vs 80 deaths per 1000 person-years (time-dependent “per transfusion” hazard ratio [HR] for death, 1.13 [95% CI, 1.01-1.26]). For receipt of transfusion from a never-pregnant female donor vs male donor, mortality rates were 78 vs 80 deaths per 1000 person-years (HR, 0.93 [95% CI, 0.81-1.06]). Among female recipients of red blood cell transfusions, mortality rates for an ever-pregnant female donor vs male donor were 74 vs 62 per 1000 person-years (HR, 0.99 [95% CI, 0.87 to 1.13]); for a never-pregnant female donor vs male donor, mortality rates were 74 vs 62 per 1000 person-years (HR, 1.01 [95% CI, 0.88-1.15]). Conclusions and Relevance Among patients who received red blood cell transfusions, receipt of a transfusion from an ever-pregnant female donor, compared with a male donor, was associated with increased all-cause mortality among male recipients but not among female recipients. Transfusions from never-pregnant female donors were not associated with increased mortality among male or female recipients. Further research is needed to replicate these findings, determine their clinical significance, and identify the underlying mechanism. PMID:29049654

Association of Blood Transfusion From Female Donors With and Without a History of Pregnancy With Mortality Among Male and Female Transfusion Recipients.

PubMed

Caram-Deelder, Camila; Kreuger, Aukje L; Evers, Dorothea; de Vooght, Karen M K; van de Kerkhof, Daan; Visser, Otto; Péquériaux, Nathalie C V; Hudig, Francisca; Zwaginga, Jaap Jan; van der Bom, Johanna G; Middelburg, Rutger A

2017-10-17

Transfusion of red blood cells from female donors has been associated with increased mortality in male recipients. To quantify the association between red blood cell transfusion from female donors with and without a history of pregnancy and mortality of red blood cell recipients. Retrospective cohort study of first-time transfusion recipients at 6 major Dutch hospitals enrolled from May 30, 2005, to September 1, 2015; the final follow-up date was September 1, 2015. The primary analysis was the no-donor-mixture cohort (ie, either all red blood cell transfusions exclusively from male donors, or all exclusively from female donors without a history of pregnancy, or all exclusively from female donors with a history of pregnancy). The association between mortality and exposure to transfusions from ever-pregnant or never-pregnant female donors was analyzed using life tables and time-varying Cox proportional hazards models. Red blood cell transfusions from ever-pregnant or never-pregnant female donors, compared with red blood cell transfusions from male donors. All-cause mortality during follow-up. The cohort for the primary analyses consisted of 31 118 patients (median age, 65 [interquartile range, 42-77] years; 52% female) who received 59 320 red blood cell transfusions exclusively from 1 of 3 types of donors (88% male; 6% ever-pregnant female; and 6% never-pregnant female). The number of deaths in this cohort was 3969 (13% mortality). For male recipients of red blood cell transfusions, all-cause mortality rates after a red blood cell transfusion from an ever-pregnant female donor vs male donor were 101 vs 80 deaths per 1000 person-years (time-dependent "per transfusion" hazard ratio [HR] for death, 1.13 [95% CI, 1.01-1.26]). For receipt of transfusion from a never-pregnant female donor vs male donor, mortality rates were 78 vs 80 deaths per 1000 person-years (HR, 0.93 [95% CI, 0.81-1.06]). Among female recipients of red blood cell transfusions, mortality rates for an ever-pregnant female donor vs male donor were 74 vs 62 per 1000 person-years (HR, 0.99 [95% CI, 0.87 to 1.13]); for a never-pregnant female donor vs male donor, mortality rates were 74 vs 62 per 1000 person-years (HR, 1.01 [95% CI, 0.88-1.15]). Among patients who received red blood cell transfusions, receipt of a transfusion from an ever-pregnant female donor, compared with a male donor, was associated with increased all-cause mortality among male recipients but not among female recipients. Transfusions from never-pregnant female donors were not associated with increased mortality among male or female recipients. Further research is needed to replicate these findings, determine their clinical significance, and identify the underlying mechanism.

Stem cell-associated genes are extremely poor prognostic factors for soft-tissue sarcoma patients.

PubMed

Taubert, H; Würl, P; Greither, T; Kappler, M; Bache, M; Bartel, F; Kehlen, A; Lautenschläger, C; Harris, L C; Kaushal, D; Füssel, S; Meye, A; Böhnke, A; Schmidt, H; Holzhausen, H-J; Hauptmann, S

2007-11-01

Cancer stem cells can play an important role in tumorigenesis and tumor progression. However, it is still difficult to detect and isolate cancer stem cells. An alternative approach is to analyse stem cell-associated gene expression. We investigated the coexpression of three stem cell-associated genes, Hiwi, hTERT and survivin, by quantitative real-time-PCR in 104 primary soft-tissue sarcomas (STS). Multivariate Cox's proportional hazards regression analyses allowed correlating gene expression with overall survival for STS patients. Coexpression of all three stem cell-associated genes resulted in a significantly increased risk of tumor-related death. Importantly, tumors of patients with the poorest prognosis were of all four tumor stages, suggesting that their risk is based upon coexpression of stem cell-associated genes rather than on tumor stage.

Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH).

PubMed

Naruto, Hirosuke; Huang, Hongyun; Nishikawa, Masaki; Kojima, Nobuhiko; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

2007-10-01

We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1

PubMed Central

Melloni, Elisabetta; Gilli, Paola; Bertolasi, Valerio; Casciano, Fabio; Rigolin, Gian Matteo; Zauli, Giorgio; Secchiero, Paola

2016-01-01

Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH2++•2DCA− cocrystal exhibited enhanced in vitro anti-leukemic activity, with respect to the treatment with the mix consisting of Metformin plus DCA. In particular, the treatment with the cocrystal MetH2++•2DCA− induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL. PMID:26959881

Synthetic Protection Short Interfering RNA Screen Reveals Glyburide as a Novel Radioprotector

PubMed Central

Jiang, Jianfei; McDonald, Peter R.; Dixon, Tracy M.; Franicola, Darcy; Zhang, Xichen; Nie, Suhua; Epperly, Laura D.; Huang, Zhentai; Kagan, Valerian E.; Lazo, John S.; Epperly, Michael W.; Greenberger, Joel S.

2009-01-01

To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from γ-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P = 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors. PMID:19772462

Matrix detachment and proteasomal inhibitors diminish Sulf-2 expression in breast cancer cell lines and mouse xenografts

PubMed Central

Khurana, Ashwani; Jung-Beom, Deok; He, Xiaoping; Kim, Sung-Hoon; Busby, Robert C.; Lorenzon, Laura; Villa, Massimo; Baldi, Alfonso; Molina, Julian; Goetz, Matthew P.; Shridhar, Viji

2013-01-01

Sulfatase 2 (Sulf-2) has been previously shown to be upregulated in breast cancer. Sulf-2 removes sulfate moieties on heparan sulfate proteoglycans which in turn modulate heparin binding growth factor signaling. Here we report that matrix detachment resulted in decreased Sulf-2 expression in breast cancer cells and increased cleavage of poly ADP-ribose polymerase. Silencing of Sulf-2 promotes matrix detachment induced cell death in MCF10DCIS cells. In an attempt to identify Sulf-2 specific inhibitor, we found that proteasomal inhibitors such as MG132, Lactacystin and Bortezomib treatment abolished Sulf-2 expression in multiple breast cancer cell lines. Additionally, we show that Bortezomib treatment of MCF10DCIS cell xenografts in mouse mammary fat pads significantly reduced tumor size, caused massive apoptosis and more importantly reduced Sulf-2 levels in vivo. Finally, our immunohistochemistry analysis of Sulf-2 expression in cohort of patient derived breast tumors indicates that Sulf-2 is significantly upregulated in autologous metastatic lesions compared to primary tumors (p < 0.037, Pearson correlation, Chi-Square analysis). In all, our data suggest that Sulf-2 might play an important role in breast cancer progression from ductal carcinoma in situ into an invasive ductal carcinoma potentially by resisting cell death. PMID:23412907

Oncolytic Reovirus in Canine Mast Cell Tumor

PubMed Central

Hwang, Chung Chew; Umeki, Saori; Kubo, Masahito; Hayashi, Toshiharu; Shimoda, Hiroshi; Mochizuki, Masami; Maeda, Ken; Baba, Kenji; Hiraoka, Hiroko; Coffey, Matt; Okuda, Masaru; Mizuno, Takuya

2013-01-01

The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility. PMID:24073198

The xipotl Mutant of Arabidopsis Reveals a Critical Role for Phospholipid Metabolism in Root System Development and Epidermal Cell Integrity

PubMed Central

Cruz-Ramírez, Alfredo; López-Bucio, José; Ramírez-Pimentel, Gabriel; Zurita-Silva, Andrés; Sánchez-Calderon, Lenin; Ramírez-Chávez, Enrique; González-Ortega, Emmanuel; Herrera-Estrella, Luis

2004-01-01

Phosphocholine (PCho) is an essential metabolite for plant development because it is the precursor for the biosynthesis of phosphatidylcholine, which is the major lipid component in plant cell membranes. The main step in PCho biosynthesis in Arabidopsis thaliana is the triple, sequential N-methylation of phosphoethanolamine, catalyzed by S-adenosyl-l-methionine:phosphoethanolamine N-methyltransferase (PEAMT). In screenings performed to isolate Arabidopsis mutants with altered root system architecture, a T-DNA mutagenized line showing remarkable alterations in root development was isolated. At the seedling stage, the mutant phenotype is characterized by a short primary root, a high number of lateral roots, and short epidermal cells with aberrant morphology. Genetic and biochemical characterization of this mutant showed that the T-DNA was inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT (XIPOTL1). Further analyses revealed that inhibition of PCho biosynthesis in xpl1 mutants not only alters several root developmental traits but also induces cell death in root epidermal cells. Epidermal cell death could be reversed by phosphatidic acid treatment. Taken together, our results suggest that molecules produced downstream of the PCho biosynthesis pathway play key roles in root development and act as signals for cell integrity. PMID:15295103

A novel protein from edible fungi Cordyceps militaris that induces apoptosis.

PubMed

Bai, Ke-Chun; Sheu, Fuu

2018-01-01

Cordyceps militaris is a dietary therapeutic fungus that is an important model species in Cordyceps research. In this study, we purified a novel protein from the fruit bodies of C. militaris and designated it as Cordyceps militaris protein (CMP). CMP has a molecular mass of 18.0 kDa and is not glycosylated. Interestingly, CMP inhibited cell viability in murine primary cells and other cell lines in a time- and dose-dependent manner. Using trypan blue staining and a lactate dehydrogenase release assay, we showed that CMP caused cell death in the murine hepatoma cell line BNL 1MEA.7R.1. Furthermore, the frequency of BNL 1MEA.7R.1 cells at the sub-G1 stage was increased by CMP. Apoptosis, as determined by Annexin V and propidium iodide analysis, indicated that CMP could mediate BNL 1MEA.7R.1 apoptosis, but not necrosis. After coincubation with CMP, a decrease in mitochondria potential was detected using 3,3'-dihexyloxacarbocyanine iodide. These results suggest that CMP is a harmful protein that induces apoptosis through a mitochondrion-dependent pathway. Stability experiments demonstrated that heat treatment and alkalization degraded CMP and further destroyed its cell-death-inducing ability, implying that cooking is necessary for food containing C. militaris. Copyright © 2017. Published by Elsevier B.V.

PD-1 expression by tumor-associated macrophages inhibits phagocytosis and tumor immunity

PubMed Central

Gordon, Sydney R.; Maute, Roy L.; Dulken, Ben W.; Hutter, Gregor; George, Benson M.; McCracken, Melissa N.; Gupta, Rohit; Tsai, Jonathan M.; Sinha, Rahul; Corey, Daniel; Ring, Aaron M.; Connolly, Andrew J.; Weissman, Irving L.

2017-01-01

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells to induce immune tolerance.1,2 Tumor cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating escape from the immune system.3,4 Monoclonal antibodies blocking PD-1/PD-L1 have shown remarkable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small cell lung cancer, and Hodgkin’s lymphoma.5–9 Although it is well-established that PD-1/PD-L1 blockade activates T cells, little is known about the role that this pathway may have on tumor-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models, and with increasing disease stage in primary human cancers. TAM PD-1 expression negatively correlates with phagocytic potency against tumor cells, and blockade of PD-1/PD-L1 in vivo increases macrophage phagocytosis, reduces tumor growth, and lengthens survival in mouse models of cancer in a macrophage-dependent fashion. Our results suggest that PD-1/PD-L1 therapies may also function through a direct effect on macrophages, with significant implications for treatment with these agents. PMID:28514441

Exogenous α-synuclein hinders synaptic communication in cultured cortical primary rat neurons.

PubMed

Hassink, G C; Raiss, C C; Segers-Nolten, I M J; van Wezel, R J A; Subramaniam, V; le Feber, J; Claessens, M M A E

2018-01-01

Amyloid aggregates of the protein α-synuclein (αS) called Lewy Bodies (LB) and Lewy Neurites (LN) are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. We have previously shown that high extracellular αS concentrations can be toxic to cells and that neurons take up αS. Here we aimed to get more insight into the toxicity mechanism associated with high extracellular αS concentrations (50-100 μM). High extracellular αS concentrations resulted in a reduction of the firing rate of the neuronal network by disrupting synaptic transmission, while the neuronal ability to fire action potentials was still intact. Furthermore, many cells developed αS deposits larger than 500 nm within five days, but otherwise appeared healthy. Synaptic dysfunction clearly occurred before the establishment of large intracellular deposits and neuronal death, suggesting that an excessive extracellular αS concentration caused synaptic failure and which later possibly contributed to neuronal death.

Geary autocorrelation and DCCA coefficient: Application to predict apoptosis protein subcellular localization via PSSM

NASA Astrophysics Data System (ADS)

Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

2017-02-01

Apoptosis is a fundamental process controlling normal tissue homeostasis by regulating a balance between cell proliferation and death. Predicting subcellular location of apoptosis proteins is very helpful for understanding its mechanism of programmed cell death. Prediction of apoptosis protein subcellular location is still a challenging and complicated task, and existing methods mainly based on protein primary sequences. In this paper, we propose a new position-specific scoring matrix (PSSM)-based model by using Geary autocorrelation function and detrended cross-correlation coefficient (DCCA coefficient). Then a 270-dimensional (270D) feature vector is constructed on three widely used datasets: ZD98, ZW225 and CL317, and support vector machine is adopted as classifier. The overall prediction accuracies are significantly improved by rigorous jackknife test. The results show that our model offers a reliable and effective PSSM-based tool for prediction of apoptosis protein subcellular localization.

Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

PubMed Central

Endres, Matthias; Biniszkiewicz, Detlev; Sobol, Robert W.; Harms, Christoph; Ahmadi, Michael; Lipski, Andreas; Katchanov, Juri; Mergenthaler, Philipp; Dirnagl, Ulrich; Wilson, Samuel H.; Meisel, Andreas; Jaenisch, Rudolf

2004-01-01

Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia. PMID:15199406

Endothelial dysfunction in dengue virus pathology.

PubMed

Vervaeke, Peter; Vermeire, Kurt; Liekens, Sandra

2015-01-01

Dengue virus (DENV) is a leading cause of illness and death, mainly in the (sub)tropics, where it causes dengue fever and/or the more serious diseases dengue hemorrhagic fever and dengue shock syndrome that are associated with changes in vascular permeability. Despite extensive research, the pathogenesis of DENV is still poorly understood and, although endothelial cells represent the primary fluid barrier of the blood vessels, the extent to which these cells contribute to DENV pathology is still under debate. The primary target cells for DENV are dendritic cells and monocytes/macrophages that release various chemokines and cytokines upon infection, which can activate the endothelium and are thought to play a major role in DENV-induced vascular permeability. However, recent studies indicate that DENV also replicates in endothelial cells and that DENV-infected endothelial cells may directly contribute to viremia, immune activation, vascular permeability and immune targeting of the endothelium. Also, the viral non-structural protein-1 and antibodies directed against this secreted protein have been reported to be involved in endothelial cell dysfunction. This review provides an extensive overview of the effects of DENV infection on endothelial cell physiology and barrier function. Copyright © 2014 John Wiley & Sons, Ltd.

Nanocage-Therapeutics Prevailing Phagocytosis and Immunogenic Cell Death Awakens Immunity against Cancer.

PubMed

Lee, Eun Jung; Nam, Gi-Hoon; Lee, Na Kyeong; Kih, Minwoo; Koh, Eunee; Kim, Yoon Kyoung; Hong, Yeonsun; Kim, Soyoun; Park, Seung-Yoon; Jeong, Cherlhyun; Yang, Yoosoo; Kim, In-San

2018-03-01

A growing appreciation of the relationship between the immune system and the tumorigenesis has led to the development of strategies aimed at "re-editing" the immune system to kill tumors. Here, a novel tactic is reported for overcoming the activation-energy threshold of the immunosuppressive tumor microenvironment and mediating the delivery and presentation of tumor neoantigens to the host's immune system. This nature-derived nanocage not only efficiently presents ligands that enhance cancer cell phagocytosis, but also delivers drugs that induce immunogenic cancer cell death. The designed nanocage-therapeutics induce the release of neoantigens and danger signals in dying tumor cells, and leads to enhancement of tumor cell phagocytosis and cross-priming of tumor specific T cells by neoantigen peptide-loaded antigen-presenting cells. Potent inhibition of tumor growth and complete eradication of tumors is observed through systemic tumor-specific T cell responses in tumor draining lymph nodes and the spleen and further, infiltration of CD8+ T cells into the tumor site. Remarkably, after removal of the primary tumor, all mice treated with this nanocage-therapeutics are protected against subsequent challenge with the same tumor cells, suggesting development of lasting, tumor-specific responses. This designed nanocage-therapeutics "awakens" the host's immune system and provokes a durable systemic immune response against cancer. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

EGFR trans-activation mediates pleiotrophin-induced activation of Akt and Erk in cultured osteoblasts.

PubMed

Fan, Jian-Bo; Liu, Wei; Yuan, Kun; Zhu, Xin-Hui; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

2014-05-09

Pleiotrophin (Ptn) plays an important role in bone growth through regulating osteoblasts' functions. The underlying signaling mechanisms are not fully understood. In the current study, we found that Ptn induced heparin-binding epidermal growth factor (HB-EGF) release to trans-activate EGF-receptor (EGFR) in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, Ptn activated Akt and Erk signalings in cultured osteoblasts. The EGFR inhibitor AG1478 as well as the monoclonal antibody against HB-EGF (anti-HB-EGF) significantly inhibited Ptn-induced EGFR activation and Akt and Erk phosphorylations in MC3T3-E1 cells and primary osteoblasts. Further, EGFR siRNA depletion or dominant negative mutation suppressed also Akt and Erk activation in MC3T3-E1 cells. Finally, we observed that Ptn increased alkaline phosphatase (ALP) activity and inhibited dexamethasone (Dex)-induced cell death in both MC3T3-E1 cells and primary osteoblasts, such effects were alleviated by AG1478 or anti-HB-EGF. Together, these results suggest that Ptn-induced Akt/Erk activation and some of its pleiotropic functions are mediated by EGFR trans-activation in cultured osteoblasts. Copyright © 2014 Elsevier Inc. All rights reserved.

RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death

PubMed Central

Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

2016-01-01

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL. PMID:27588473

RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death.

PubMed

Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

2016-09-27

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL.

Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

PubMed Central

Villarreal, Alejandro; Rosciszewski, Gerardo; Murta, Veronica; Cadena, Vanesa; Usach, Vanina; Dodes-Traian, Martin M.; Setton-Avruj, Patricia; Barbeito, Luis H.; Ramos, Alberto J.

2016-01-01

Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis. Main Points: Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissue IDA show reduced replicative senescence, increased cell division and spontaneous migration IDA potentiate death of oxygen-glucose deprived cortical neurons IDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivo Inhibition of gamma secretases facilitates IDA differentiation to astrocytes PMID:27313509

Nevirapine- Versus Lopinavir/Ritonavir-Based Initial Therapy for HIV-1 Infection among Women in Africa: A Randomized Trial

PubMed Central

Lockman, Shahin; Hughes, Michael; Sawe, Fred; Zheng, Yu; McIntyre, James; Chipato, Tsungai; Asmelash, Aida; Rassool, Mohammed; Kimaiyo, Sylvester; Shaffer, Douglas; Hosseinipour, Mina; Mohapi, Lerato; Ssali, Francis; Chibowa, Margret; Amod, Farida; Halvas, Elias; Hogg, Evelyn; Alston-Smith, Beverly; Smith, Laura; Schooley, Robert; Mellors, John; Currier, Judith

2012-01-01

Background Nevirapine (NVP) is widely used in antiretroviral treatment (ART) of HIV-1 globally. The primary objective of the AA5208/OCTANE trial was to compare the efficacy of NVP-based versus lopinavir/ritonavir (LPV/r)-based initial ART. Methods and Findings In seven African countries (Botswana, Kenya, Malawi, South Africa, Uganda, Zambia, and Zimbabwe), 500 antiretroviral-naïve HIV-infected women with CD4<200 cells/mm3 were enrolled into a two-arm randomized trial to initiate open-label ART with tenofovir (TDF)/emtricitabine (FTC) once/day plus either NVP (n = 249) or LPV/r (n = 251) twice/day, and followed for ≥48 weeks. The primary endpoint was time from randomization to death or confirmed virologic failure ([VF]) (plasma HIV RNA<1 log10 below baseline 12 weeks after treatment initiation, or ≥400 copies/ml at or after 24 weeks), with comparison between treatments based on hazard ratios (HRs) in intention-to-treat analysis. Equivalence of randomized treatments was defined as finding the 95% CI for HR for virological failure or death in the range 0.5 to 2.0. Baseline characteristics were (median): age = 34 years, CD4 = 121 cells/mm3, HIV RNA = 5.2 log10copies/ml. Median follow-up = 118 weeks; 29 (6%) women were lost to follow-up. 42 women (37 VFs, five deaths; 17%) in the NVP and 50 (43 VFs, seven deaths; 20%) in the LPV/r arm reached the primary endpoint (HR 0.85, 95% CI 0.56–1.29). During initial assigned treatment, 14% and 16% of women receiving NVP and LPV/r experienced grade 3/4 signs/symptoms and 26% and 22% experienced grade 3/4 laboratory abnormalities. However, 35 (14%) women discontinued NVP because of adverse events, most in the first 8 weeks, versus none for LPV/r (p<0.001). VF, death, or permanent treatment discontinuation occurred in 80 (32%) of NVP and 54 (22%) of LPV/r arms (HR = 1.7, 95% CI 1.2–2.4), with the difference primarily due to more treatment discontinuation in the NVP arm. 13 (45%) of 29 women tested in the NVP versus six (15%) of 40 in the LPV/r arm had any drug resistance mutation at time of VF. Conclusions Initial ART with NVP+TDF/FTC demonstrated equivalent virologic efficacy but higher rates of treatment discontinuation and new drug resistance compared with LPV/r+TDF/FTC in antiretroviral-naïve women with CD4<200 cells/mm3. Trial registration ClinicalTrials.gov NCT00089505 Please see later in the article for the Editors' Summary PMID:22719231

Control of non-apoptotic nurse cell death by engulfment genes in Drosophila.

PubMed

Timmons, Allison K; Mondragon, Albert A; Meehan, Tracy L; McCall, Kimberly

2017-04-03

Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.

Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma.

PubMed

Markowitz, Daniel; Powell, Caitlin; Tran, Nhan L; Berens, Michael E; Ryken, Timothy C; Vanan, Magimairajan; Rosen, Lisa; He, Mingzu; Sun, Shan; Symons, Marc; Al-Abed, Yousef; Ruggieri, Rosamaria

2016-08-01

Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiotherapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain. We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell-cycle arrest after ionizing radiation. Here, we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a specific small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radiosensitizes UW228 medulloblastoma cells as well as UI226 patient-derived primary cells, whereas it does not affect the response to radiation of normal brain cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage-induced cell-cycle arrest. Importantly, in an animal model of medulloblastoma that employs orthotopic implantation of primary patient-derived UI226 cells in nude mice, M443 in combination with radiation achieved a synergistic increase in survival. We hypothesize that combining radiotherapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects. Mol Cancer Ther; 15(8); 1799-808. ©2016 AACR. ©2016 American Association for Cancer Research.

Neurotoxicity of Methylmercury in Isolated Astrocytes and Neurons: the Cytoskeleton as a Main Target.

PubMed

Pierozan, Paula; Biasibetti, Helena; Schmitz, Felipe; Ávila, Helena; Fernandes, Carolina Gonçalves; Pessoa-Pureur, Regina; Wyse, Angela T S

2017-10-01

In the present work, we focused on mechanisms of methylmercury (MeHg) toxicity in primary astrocytes and neurons of rats. Cortical astrocytes and neurons exposed to 0.5-5 μM MeHg present a link among morphological alterations, glutathione (GSH) depletion, glutamate dyshomeostasis, and cell death. Disrupted neuronal cytoskeleton was assessed by decreased neurite length and neurite/neuron ratio. Astrocytes presented reorganization of actin and glial fibrillary acidic protein (GFAP) networks and reduced cytoplasmic area. Glutamate uptake and Na + K + ATPase activity in MeHg-treated astrocytes were preserved; however, downregulated EAAC1-mediated glutamate uptake was associated with impaired Na + K + ATPase activity in neurons. Oxidative imbalance was found in astrocytes and neurons through increased 2'7'-dichlorofluorescein (DCF) production and misregulated superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GPX) activities. Glutathione (GSH) levels were downregulated in both astrocytes and neurons. MeHg reduced neuronal viability and induced caspase 3-dependent apoptosis together with downregulated PI3K/Akt pathway. In astrocytes, necrotic death was associated with increased TNF-α and JNK/MAPK activities. Cytoskeletal remodeling and cell death were fully prevented in astrocytes and neurons by GSH, but not melatonin or Trolox supplementation. These findings support a role for depleted GSH in the cytotoxicity of MeHg leading to disruption of the cytoskeleton and cell death. Moreover, in neurons, glutamate antagonists also prevented cytoskeletal disruption and neuronal death. We propose that cytoskeleton is an end point in MeHg cytotoxicity. Oxidative imbalance and glutamate mechanisms mediate MeHg cytoskeletal disruption and apoptosis in neurons. Otherwise, redox imbalance and glutamate-independent mechanisms disrupted the cytoskeleton and induced necrosis in MeHg-exposed astrocyte.

ATP7B detoxifies silver in ciliated airway epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ibricevic, Aida, E-mail: aidaibricevic@hotmail.co; Brody, Steven L., E-mail: sbrody@dom.wustl.ed; Youngs, Wiley J., E-mail: youngs@uakron.ed

2010-03-15

Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compoundsmore » but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.« less

Efficacy of aqueous extract of Hippophae rhamnoides and its bio-active flavonoids against hypoxia-induced cell death

PubMed Central

Tulsawani, Rajkumar; Gupta, Rashmi; Misra, Kshipra

2013-01-01

Objectives: To investigate the protective efficacy of aqueous extract of Hippophae rhamnoides against chronic hypoxic injury using primary rat hepatocytes. Materials and Methods: The extract was prepared using maceration method and characterized by its phenolic and flavonoid content and chemical antioxidant capacity using ferric reducing antioxidant power assay. Hepatocytes were maintained in hypoxia chamber (3% and 1% oxygen) for 72 h. The cells kept under normoxic condition served as control. The cells were treated with the extract and flavonoids; isorhamentin, kaempferol or qurecetin-3-galactoside. After the end of exposure period; cell survival, reactive oxygen species (ROS), leakage of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were measured. Results: The extract showed presence of high phenolic and flavonoid content with significant antioxidant activity in chemical assay. The cell exposed to hypoxia showed concentration dependent cell death and harbored higher reactive oxygen species. In addition, these cells showed significant leakage of intracellular LDH, ALT, and AST accompanied by the diminished levels/activities of GSH, GPx, and SOD. The treatment of cells with aqueous extract of H. rhamnoides reduced hypoxia-induced cell death and prevented increase in ROS levels and leakage of intracellular LDH, ALT, and AST from cells. Moreover, these cells maintained better levels/activities of GSH, GPx, and SOD in comparison to the respective controls. The major flavonoids present in aqueous extract of H. rhamnoides; quercetin-3-galactoside, kaempferol, and isorhamentin also prevented hypoxia induced cell injury individually or in combination, however, the protection offered by these compounds taken together could not match to that of the extract. Conclusions: Overall the findings reveal significance of aqueous extract of H. rhamnoides in controlling ROS-meditated hypoxic injury in cells and can be useful in many hepatic complications. PMID:23833369

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathwaysmore » involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.« less

Adult Stem Cell Therapy for Stroke: Challenges and Progress

PubMed Central

Bang, Oh Young; Kim, Eun Hee; Cha, Jae Min; Moon, Gyeong Joon

2016-01-01

Stroke is one of the leading causes of death and physical disability among adults. It has been 15 years since clinical trials of stem cell therapy in patients with stroke have been conducted using adult stem cells like mesenchymal stem cells and bone marrow mononuclear cells. Results of randomized controlled trials showed that adult stem cell therapy was safe but its efficacy was modest, underscoring the need for new stem cell therapy strategies. The primary limitations of current stem cell therapies include (a) the limited source of engraftable stem cells, (b) the presence of optimal time window for stem cell therapies, (c) inherited limitation of stem cells in terms of growth, trophic support, and differentiation potential, and (d) possible transplanted cell-mediated adverse effects, such as tumor formation. Here, we discuss recent advances that overcome these hurdles in adult stem cell therapy for stroke. PMID:27733032

The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

PubMed Central

2013-01-01

Background In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. Results The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. Conclusions The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure. PMID:24090154

A Nuclear Attack on Traumatic Brain Injury: Sequestration of Cell Death in the Nucleus.

PubMed

Tajiri, Naoki; De La Peña, Ike; Acosta, Sandra A; Kaneko, Yuji; Tamir, Sharon; Landesman, Yosef; Carlson, Robert; Shacham, Sharon; Borlongan, Cesar V

2016-04-01

Exportin 1 (XPO1/CRM1) plays prominent roles in the regulation of nuclear protein export. Selective inhibitors of nuclear export (SINE) are small orally bioavailable molecules that serve as drug-like inhibitors of XPO1, with potent anti-cancer properties. Traumatic brain injury (TBI) presents with a secondary cell death characterized by neuroinflammation that is putatively regulated by nuclear receptors. Here, we report that the SINE compounds (KPT-350 or KPT-335) sequestered TBI-induced neuroinflammation-related proteins (NF-(k)B, AKT, FOXP1) within the nucleus of cultured primary rat cortical neurons, which coincided with protection against TNF-α (20 ng/mL)-induced neurotoxicity as shown by at least 50% and 100% increments in preservation of cell viability and cellular enzymatic activity, respectively, compared to non-treated neuronal cells (P's < 0.05). In parallel, using an in vivo controlled cortical impact (CCI) model of TBI, we demonstrate that adult Sprague-Dawley rats treated post-injury with SINE compounds exhibited significant reductions in TBI-induced behavioral and histological deficits. Animals that received KPT-350 orally starting at 2 h post-TBI and once a day thereafter over the next 4 days exhibited significantly better motor coordination, and balance in the rotorod test and motor asymmetry test by 100-200% improvements, as early as 4 h after initial SINE compound injection that was sustained during subsequent KPT-350 dosing, and throughout the 18-day post-TBI study period compared to vehicle treatment (P's < 0.05). Moreover, KPT-350 reduced cortical core impact area and peri-impact cell death compared to vehicle treatment (P's < 0.05). Both in vitro and in vivo experiments revealed that KPT-350 increased XPO1, AKT, and FOXP1 nuclear expression and relegated NF-(k)B expression within the neuronal nuclei. Altogether, these findings advance the utility of SINE compounds to stop trafficking of cell death proteins within the nucleus as an efficacious treatment for TBI. © 2016 John Wiley & Sons Ltd.

Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung

Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis inmore » T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.« less

[Causes of death in patients with HIV infection in two Tunisian medical centers].

PubMed

Chelli, Jihène; Bellazreg, Foued; Aouem, Abir; Hattab, Zouhour; Mesmia, Hèla; Lasfar, Nadia Ben; Hachfi, Wissem; Masmoudi, Tasnim; Chakroun, Mohamed; Letaief, Amel

2016-01-01

Antiretroviral tritherapy has contributed to a considerable reduction in HIV-related mortality. The causes of death are dominated by opportunistic infections in developing countries and by cardiovascular diseases and cancer in developed countries. To determine the causes and risk factors associated with death in HIV-infected patients in two Tunisian medical centers. cross-sectional study of HIV-infected patients over 15 years treated at Sousse and Monastir medical centers between 2000 and 2014. Death was considered related to HIV if its primary cause was AIDS-defining illness or if it was due to an opportunistic infection of unknown etiology with CD4 < 50 cells/mm 3 ; it was considered unrelated to HIV if its primary cause wasn't an AIDS defining illness or if it was due to an unknown cause if no information was available. Two hundred thirteen patients, 130 men (61%) and 83 women (39%), average age 40 ± 11 years were enrolled in the study. Fifty four patients died, the mortality rate was 5.4/100 patients/year. Annual mortality rate decreased from 5.8% in 2000-2003 to 2.3% in 2012-2014. Survival was 72% at 5 years and 67% at 10 years. Death events were associated with HIV in 70.4% of cases. The leading causes of death were pneumocystis carinii pneumonia and cryptococcal meningitis in 6 cases (11%) each. Mortality risk factors were a personal history of opportunistic infections, duration of antiretroviral therapy < 12 months and smoking. Strengthening screening, early initiation of antiretroviral therapy and fight against tobacco are needed to reduce mortality in patients infected with HIV in Tunisia.

Targeting Programmed Cell Death Using Small-Molecule Compounds to Improve Potential Cancer Therapy.

PubMed

Ke, Bowen; Tian, Mao; Li, Jingjing; Liu, Bo; He, Gu

2016-11-01

Evasion of cell death is one of the hallmarks of cancer cells, beginning with long-established apoptosis and extending to other new forms of cell death. An elaboration of cell death pathways thus will contribute to a better understanding of cancer pathogenesis and therapeutics. With the recent substantial biochemical and genetic explorations of cell death subroutines, their classification has switched from primarily morphological to more molecular definitions. According to their measurable biochemical features and intricate mechanisms, cell death subroutines can be divided into apoptosis, autophagic cell death, mitotic catastrophe, necroptosis, parthanatos, ferroptosis, pyroptosis, pyronecrosis, anoikis, cornification, entosis, and NETosis. Supportive evidence has gradually revealed the prime molecular mechanisms of each subroutine and thus providing series of possible targets in cancer therapy, while the intricate relationships between different cell death subroutines still remain to be clarified. Over the past decades, cancer drug discovery has significantly benefited from the use of small-molecule compounds to target classical modalities of cell death such as apoptosis, while newly identified cell death subroutines has also emerging their potential for cancer drug discovery in recent years. In this review, we comprehensively focus on summarizing 12 cell death subroutines and discussing their corresponding small-molecule compounds in potential cancer therapy. Together, these inspiring findings may provide more evidence to fill in the gaps between cell death subroutines and small-molecule compounds to better develop novel cancer therapeutic strategies. © 2016 Wiley Periodicals, Inc.

Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

PubMed

Wiman, K G; Zhivotovsky, B

2017-05-01

Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation. © 2017 The Association for the Publication of the Journal of Internal Medicine.

Primary Enforcement of Mandatory Seat Belt Laws and Motor Vehicle Crash Deaths.

PubMed

Harper, Sam; Strumpf, Erin C

2017-08-01

Policies that allow directly citing motorists for seat belt non-use (primary enforcement) have been shown to reduce motor vehicle crash deaths relative to secondary enforcement, but the evidence base is dated and does not account for recent improvements in vehicle designs and road safety. The purpose of this study was to test whether recent upgrades to primary enforcement still reduce motor vehicle crash deaths. In 2016, researchers used motor vehicle crash death data from the Fatal Analysis Reporting System for 2000-2014 and calculated rates using both person- and exposure-based denominators. Researchers used a difference-in-differences design to estimate the effect of primary enforcement on death rates, and estimated negative binomial regression models, controlling for age, substance use involvement, fixed state characteristics, secular trends, state median household income, and other state-level traffic safety policies. Models adjusted only for crash characteristics and state-level covariates models showed a protective effect of primary enforcement (rate ratio, 0.88, 95% CI=0.77, 0.98; rate difference, -1.47 deaths per 100,000 population, 95% CI= -2.75, -0.19). After adjustment for fixed state characteristics and secular trends, there was no evidence of an effect of upgrading from secondary to primary enforcement in the whole population (rate ratio, 0.98, 95% CI=0.92, 1.04; rate difference, -0.22, 95% CI= -0.90, 0.46) or for any age group. Upgrading to primary enforcement no longer appears protective for motor vehicle crash death rates. Copyright © 2017 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights reserved.

Identification of DNA-PKcs as a primary resistance factor of TIC10 in hepatocellular carcinoma cells.

PubMed

Cheng, Long; Liu, Yuan-Yuan; Lu, Pei-Hua; Peng, Yi; Yuan, Qiang; Gu, Xin-Shi; Jin, Yong; Chen, Min-Bin; Bai, Xu-Ming

2017-04-25

The current study tested the anti-hepatocellular carcinoma (HCC) cell activity of TIC10, a first-in-class small-molecule tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) inducer. TIC10 exerted potent anti-proliferative and pro-apoptotic actions in primary and established human HCC cells. TIC10 blocked Akt-Erk activation, leading to Foxo3a nuclear translocation, as well as TRAIL and death receptor-5 (DR5) transcription in HCC cells. We propose that DNA-PKcs is a major resistance factor of TIC10 possibly via inhibiting Foxo3a nuclear translocation. DNA-PKcs inhibition, knockdown or mutation facilitated TIC10-induced Foxo3a nuclear translocation, TRAIL/DR5 expression and cell apoptosis. Reversely, exogenous DNA-PKcs over-expression inhibited above actions by TIC10. In vivo, oral administration of TIC10 significantly inhibited HepG2 tumor growth in nude mice, which was further potentiated with Nu7026 co-administration. Thus, TIC10 shows promising anti-HCC activity, alone or together with DNA-PKcs inhibitors.

Chlorogenic acid protects against aluminium-induced cytotoxicity through chelation and antioxidant actions in primary hippocampal neuronal cells.

PubMed

Wang, Xiaomei; Fan, Xinguang; Yuan, Shuzhi; Jiao, Wenxiao; Liu, Bangdi; Cao, Jiankang; Jiang, Weibo

2017-08-01

Chlorogenic acid (CGA), a major polyphenolic component of many plants, displays antioxidant and neuroprotective properties in neurodegenerative diseases. To investigate whether CGA may influence aluminium (Al) induced cytotoxicity, aluminium chloride (50 μM Al) was administered in primary hippocampal neuronal cells presupplemented with CGA (10, 50 and 100 μM). Our study shows that the exposure to Al caused cell death, Al 3+ accumulation, reactive oxygen species generation and mitochondrial damage in cells. The administration of CGA (50 μM) increased cell viability by 37.5%, decreased the levels of Al 3+ by 26.0%, together with significantly weakening the oxidative damage compared with Al treatment alone. CGA protected neurons against Al-induced oxidative stress by increasing the expression of nuclear factor-E2-related factor 2 and its target phase 2 enzymes. The administration of CGA remarkably promoted the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, creatine kinase and acetylcholinesterase and attenuated the rate of ATP hydrolysis. Our finding shows that CGA has neuroprotective effects against Al-induced cytotoxicity by chelation and antioxidant activation.

Mutations in PRPF31 Inhibit Pre-mRNA Splicing of Rhodopsin Gene and Cause Apoptosis of Retinal Cells

PubMed Central

Yuan, Liya; Kawada, Mariko; Havlioglu, Necat; Tang, Hao; Wu, Jane Y.

2007-01-01

Mutations in human PRPF31 gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP). To begin to understand mechanisms by which defects in this general splicing factor cause retinal degeneration, we examined the relationship between PRPF31 and pre-mRNA splicing of photoreceptor-specific genes. We used a specific anti-PRPF31 antibody to immunoprecipitate splicing complexes from retinal cells and identified the transcript of rhodopsin gene (RHO) among RNA species associated with PRPF31-containing complexes. Mutant PRPF31 proteins significantly inhibited pre-mRNA splicing of intron 3 in RHO gene. In primary retinal cell cultures, expression of the mutant PRPF31 proteins reduced rhodopsin expression and caused apoptosis of rhodopsin-positive retinal cells. This primary retinal culture assay provides an in vitro model to study photoreceptor cell death caused by PRPF31 mutations. Our results demonstrate that mutations in PRPF31 gene affect RHO pre-mRNA splicing and reveal a link between PRPF31 and RHO, two major adRP genes. PMID:15659613

PLASMA CELL LEUKEMIA

PubMed Central

de Larrea, Carlos Fernandez; Kyle, Robert A.; Durie, Brian GM; Ludwig, Heinz; Usmani, Saad; Vesole, David H.; Hajek, Roman; Miguel, Jésus San; Sezer, Orhan; Sonneveld, Pieter; Kumar, Shaji K.; Mahindra, Anuj; Comenzo, Ray; Palumbo, Antonio; Mazumber, Amitabha; Anderson, Kenneth C.; Richardson, Paul G.; Badros, Ashraf Z.; Caers, Jo; Cavo, Michele; LeLeu, Xavier; Dimopoulos, Meletios A.; Chim, CS; Schots, Rik; Noeul, Amara; Fantl, Dorotea; Mellqvist, Ulf-Henrik; Landgren, Ola; Chanan-Khan, Asher; Moreau, Philippe; Fonseca, Rafael; Merlini, Giampaolo; Lahuerta, JJ; Bladé, Joan; Orlowski, Robert Z.; Shah, Jatin J.

2014-01-01

Plasma cell leukemia (PCL) is a rare and aggressive variant of myeloma characterized by the presence of circulating plasma cells. It is classified as either primary PCL occurring at diagnosis or as secondary PCL in patients with relapsed/refractory myeloma. Primary PCL is a distinct clinic-pathologic entity with different cytogenetic and molecular findings. The clinical course is aggressive with short remissions and survival duration. The diagnosis is based upon the percentage (≥ 20%) and absolute number (≥ 2 × 10 9/L) of plasma cells in the peripheral blood. It is proposed that the thresholds for diagnosis be reexamined and consensus recommendations are made for diagnosis, as well as, response and progression criteria. Induction therapy needs to begin promptly and have high clinical activity leading to rapid disease control in an effort to minimize the risk of early death. Intensive chemotherapy regimens and bortezomib-based regimens are recommended followed by high-dose therapy with autologous stem-cell transplantation (HDT/ASCT) if feasible. Allogeneic transplantation can be considered in younger patients. Prospective multicenter studies are required to provide revised definitions and better understanding of the pathogenesis of PCL. PMID:23288300

Morphodynamics of a growing microbial colony driven by cell death

NASA Astrophysics Data System (ADS)

Ghosh, Pushpita; Levine, Herbert

2017-11-01

Bacterial cells can often self-organize into multicellular structures with complex spatiotemporal morphology. In this work, we study the spatiotemporal dynamics of a growing microbial colony in the presence of cell death. We present an individual-based model of nonmotile bacterial cells which grow and proliferate by consuming diffusing nutrients on a semisolid two-dimensional surface. The colony spreads by growth forces and sliding motility of cells and undergoes cell death followed by subsequent disintegration of the dead cells in the medium. We model cell death by considering two possible situations: In one of the cases, cell death occurs in response to the limitation of local nutrients, while the other case corresponds to an active death process, known as apoptotic or programmed cell death. We demonstrate how the colony morphology is influenced by the presence of cell death. Our results show that cell death facilitates transitions from roughly circular to highly branched structures at the periphery of an expanding colony. Interestingly, our results also reveal that for the colonies which are growing in higher initial nutrient concentrations, cell death occurs much earlier compared to the colonies which are growing in lower initial nutrient concentrations. This work provides new insights into the branched patterning of growing bacterial colonies as a consequence of complex interplay among the biochemical and mechanical effects.

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

PubMed Central

Landreville, Solange; Agapova, Olga A.; Kneass, Zachary T.; Salesse, Christian; Harbour, J. William

2011-01-01

SUMMARY Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1− bulk tumor cells. ABCB1+ cells also exhibited enhanced clonogenicity, anchorage independent growth, tumorigenicity and mitochondrial activity compared to ABCB1− cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1+ cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others, and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma. PMID:21575142

Investigating the role of retinal Müller cells with approaches in genetics and cell biology.

PubMed

Fu, Suhua; Zhu, Meili; Ash, John D; Wang, Yunchang; Le, Yun-Zheng

2014-01-01

Müller cells are major macroglia and play many essential roles as a supporting cell in the retina. As Müller cells only constitute a small portion of retinal cells, investigating the role of Müller glia in retinal biology and diseases is particularly challenging. To overcome this problem, we first generated a Cre/lox-based conditional gene targeting system that permits the genetic manipulation and functional dissection of gene of interests in Müller cells. To investigate diabetes-induced alteration of Müller cells, we recently adopted methods to analyze Müller cells survival/death in vitro and in vivo. We also used normal and genetically altered primary cell cultures to reveal the mechanistic insights for Müller cells in biological and disease processes. In this article, we will discuss the applications and limitations of these methodologies, which may be useful for research in retinal Müller cell biology and pathophysiology.

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

PubMed

Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz

2014-05-01

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

The role of the endoplasmic reticulum stress response following cerebral ischemia.

PubMed

Hadley, Gina; Neuhaus, Ain A; Couch, Yvonne; Beard, Daniel J; Adriaanse, Bryan A; Vekrellis, Kostas; DeLuca, Gabriele C; Papadakis, Michalis; Sutherland, Brad A; Buchan, Alastair M

2018-06-01

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Circulating CD34+ progenitor cells and risk of mortality in a population with coronary artery disease.

PubMed

Patel, Riyaz S; Li, Qunna; Ghasemzadeh, Nima; Eapen, Danny J; Moss, Lauren D; Janjua, A Umair; Manocha, Pankaj; Kassem, Hatem Al; Veledar, Emir; Samady, Habib; Taylor, W Robert; Zafari, A Maziar; Sperling, Laurence; Vaccarino, Viola; Waller, Edmund K; Quyyumi, Arshed A

2015-01-16

Low circulating progenitor cell numbers and activity may reflect impaired intrinsic regenerative/reparative potential, but it remains uncertain whether this translates into a worse prognosis. To investigate whether low numbers of progenitor cells associate with a greater risk of mortality in a population at high cardiovascular risk. Patients undergoing coronary angiography were recruited into 2 cohorts (1, n=502 and 2, n=403) over separate time periods. Progenitor cells were enumerated by flow cytometry as CD45(med+) blood mononuclear cells expressing CD34, with additional quantification of subsets coexpressing CD133, vascular endothelial growth factor receptor 2, and chemokine (C-X-C motif) receptor 4. Coefficient of variation for CD34 cells was 2.9% and 4.8%, 21.6% and 6.5% for the respective subsets. Each cohort was followed for a mean of 2.7 and 1.2 years, respectively, for the primary end point of all-cause death. There was an inverse association between CD34(+) and CD34(+)/CD133(+) cell counts and risk of death in cohort 1 (β=-0.92, P=0.043 and β=-1.64, P=0.019, respectively) that was confirmed in cohort 2 (β=-1.25, P=0.020 and β=-1.81, P=0.015, respectively). Covariate-adjusted hazard ratios in the pooled cohort (n=905) were 3.54 (1.67-7.50) and 2.46 (1.18-5.13), respectively. CD34(+)/CD133(+) cell counts improved risk prediction metrics beyond standard risk factors. Reduced circulating progenitor cell counts, identified primarily as CD34(+) mononuclear cells or its subset expressing CD133, are associated with risk of death in individuals with coronary artery disease, suggesting that impaired endogenous regenerative capacity is associated with increased mortality. These findings have implications for biological understanding, risk prediction, and cell selection for cell-based therapies. © 2014 American Heart Association, Inc.

Two dietary polyphenols, fisetin and luteolin, reduce inflammation but augment DNA damage-induced toxicity in human RPE cells.

PubMed

Hytti, Maria; Szabó, Dora; Piippo, Niina; Korhonen, Eveliina; Honkakoski, Paavo; Kaarniranta, Kai; Petrovski, Goran; Kauppinen, Anu

2017-04-01

Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.

PubMed

Young, Christopher N J; Sinadinos, Anthony; Lefebvre, Alexis; Chan, Philippe; Arkle, Stephen; Vaudry, David; Gorecki, Dariusz C

2015-01-01

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd(mdx) mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca(2+) influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome--autophagy--and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Colourful death: six-parameter classification of cell death by flow cytometry--dead cells tell tales.

PubMed

Munoz, Luis E; Maueröder, Christian; Chaurio, Ricardo; Berens, Christian; Herrmann, Martin; Janko, Christina

2013-08-01

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Inhibition of canonical WNT signaling attenuates human leiomyoma cell growth

PubMed Central

Ono, Masanori; Yin, Ping; Navarro, Antonia; Moravek, Molly B.; Coon, John S.; Druschitz, Stacy A.; Gottardi, Cara J.; Bulun, Serdar E.

2014-01-01

Objective Dysregulation of WNT signaling plays a central role in tumor cell growth and progression. Our goal was to assess the effect of three WNT/β-catenin pathway inhibitors, Inhibitor of β-Catenin And TCF4 (ICAT), niclosamide, and XAV939 on the proliferation of primary cultures of human uterine leiomyoma cells. Design Prospective study of human leiomyoma cells obtained from myomectomy or hysterectomy. Setting University research laboratory. Patient(s) Women (n=38) aged 27–53 years undergoing surgery. Intervention(s) Adenoviral ICAT overexpression or treatment with varying concentrations of niclosamide or XAV939. Main Outcome Measure(s) Cell proliferation, cell death, WNT/β-catenin target gene expression or reporter gene regulation, β-catenin levels and cellular localization. Result(s) ICAT, niclosamide, or XAV939 inhibit WNT/β-catenin pathway activation and exert anti-proliferative effects in primary cultures of human leiomyoma cells. Conclusion(s) Three WNT/β-catenin pathway inhibitors specifically block human leiomyoma growth and proliferation, suggesting that the canonical WNT pathway may be a potential therapeutic target for the treatment of uterine leiomyoma. Our findings provide rationale for further preclinical and clinical evaluation of ICAT, niclosamide, and XAV939 as candidate anti-tumor agents for uterine leiomyoma. PMID:24534281

Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

PubMed Central

Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul

2011-01-01

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response. PMID:22072830

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

PubMed

Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

2011-09-01

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

Transglutaminase 2: Friend or foe? The discordant role in neurons and astrocytes.

PubMed

Quinn, Breandan R; Yunes-Medina, Laura; Johnson, Gail V W

2018-03-23

Members of the transglutaminase family catalyze the formation of isopeptide bonds between a polypeptide-bound glutamine and a low molecular weight amine (e.g., spermidine) or the ɛ-amino group of a polypeptide-bound lysine. Transglutaminase 2 (TG2), a prominent member of this family, is unique because in addition to being a transamidating enzyme, it exhibits numerous other activities. As a result, TG2 plays a role in many physiological processes, and its function is highly cell type specific and relies upon a number of factors, including conformation, cellular compartment location, and local concentrations of Ca 2+ and guanine nucleotides. TG2 is the most abundant transglutaminase in the central nervous system (CNS) and plays a pivotal role in the CNS injury response. How TG2 affects the cell in response to an insult is strikingly different in astrocytes and neurons. In neurons, TG2 supports survival. Overexpression of TG2 in primary neurons protects against oxygen and glucose deprivation (OGD)-induced cell death and in vivo results in a reduction in infarct volume subsequent to a stroke. Knockdown of TG2 in primary neurons results in a loss of viability. In contrast, deletion of TG2 from astrocytes results in increased survival following OGD and improved ability to protect neurons from injury. Here, a brief overview of TG2 is provided, followed by a discussion of the role of TG2 in transcriptional regulation, cellular dynamics, and cell death. The differing roles TG2 plays in neurons and astrocytes are highlighted and compared to how TG2 functions in other cell types. © 2018 Wiley Periodicals, Inc.

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

PubMed

Marlin, Jerry W; Chang, Yu-Wen E; Ober, Margaret; Handy, Amy; Xu, Wenhao; Jakobi, Rolf

2011-06-01

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

Inhibition of the development of metastases by dietary vitamin C:K3 combination.

PubMed

Taper, Henryk S; Jamison, James M; Gilloteaux, Jacques; Summers, Jack L; Calderon, Pedro Buc

2004-07-09

The tumor growth-inhibiting and chemo-potentiating effects of vitamin C and K(3)combinations have been demonstrated both in vitro and in vivo. The purpose of this study was to investigate the influence of orally administered vitamin C and K(3) on the metastasis of mouse liver tumor (T.L.T.) cells implanted in C3H mice. Adult male C3H mice were given water containing vitamin C and K3 (15 g/0.15 g dissolved in 1000 ml) beginning 2 weeks before tumor transplantation until the end of the experiment. T.L.T. cells (106) were implanted intramuscularly in the right thigh of mice. All mice were sacrificed 42 days after tumor transplantation. Primary tumor, lungs, lymph nodes and other organs or tissues suspected of harboring metastases were macroscopically examined. Samples of primary tumors, their local lymph nodes, lungs and main organs such as liver, kidneys, spleen were taken for histological examination. Forty-two percent of control mice exhibited lung metastases and 27% possessed metastases in local lymph nodes whereas 24% of vitamin-treated mice exhibited lung metastases and 10% possessed local lymph nodes metastases. The total number of lung metastases was 19 in control group and 10 in vitamin C and K(3)-treated mice. Histopathological examination of the metastatic tumors from the vitamin-treated mice revealed the presence of many tumor cells undergoing autoschizic cell death. These results demonstrate that oral vitamin C and K(3) significantly inhibited the metastases of T.L.T. tumors in C3H mice. At least a portion of this inhibition was due to tumor cell death by autoschizis.

Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells.

PubMed

Pizato, Nathalia; Luzete, Beatriz Christina; Kiffer, Larissa Fernanda Melo Vasconcelos; Corrêa, Luís Henrique; de Oliveira Santos, Igor; Assumpção, José Antônio Fagundes; Ito, Marina Kiyomi; Magalhães, Kelly Grace

2018-01-31

The implication of inflammation in pathophysiology of several type of cancers has been under intense investigation. Omega-3 fatty acids can modulate inflammation and present anticancer effects, promoting cancer cell death. Pyroptosis is an inflammation related cell death and so far, the function of docosahexaenoic acid (DHA) in pyroptosis cell death has not been described. This study investigated the role of DHA in triggering pyroptosis activation in breast cancer cells. MDA-MB-231 breast cancer cells were supplemented with DHA and inflammation cell death was analyzed. DHA-treated breast cancer cells triggered increased caspase-1and gasdermin D activation, enhanced IL-1β secretion, translocated HMGB1 towards the cytoplasm, and membrane pore formation when compared to untreated cells, suggesting DHA induces pyroptosis programmed cell death in breast cancer cells. Moreover, caspase-1 inhibitor (YVAD) could protect breast cancer cells from DHA-induced pyroptotic cell death. In addition, membrane pore formation showed to be a lysosomal damage and ROS formation-depended event in breast cancer cells. DHA triggered pyroptosis cell death in MDA-MB-231by activating several pyroptosis markers in these cells. This is the first study that shows the effect of DHA triggering pyroptosis programmed cell death in breast cancer cells and it could improve the understanding of the omega-3 supplementation during breast cancer treatment.

A high throughput respirometric assay for mitochondrial biogenesis and toxicity

PubMed Central

Beeson, Craig C.; Beeson, Gyda C.; Schnellmann, Rick G.

2010-01-01

Mitochondria are a common target of toxicity for drugs and other chemicals, and results in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput, real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Biosciences analyzer to measure mitochondrial function in real time in multi-well plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl2 and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTC. The merger of the RPTC model and multi-well respirometry results in a single high throughput assay to measure mitochondrial biogenesis and toxicity, and nephrotoxic potential. PMID:20465991

The Epstein-Barr virus Bcl-2 homolog, BHRF1, blocks apoptosis by binding to a limited amount of Bim.

PubMed

Desbien, Anthony L; Kappler, John W; Marrack, Philippa

2009-04-07

Current knowledge suggests that the balance between life and death within a cell can be controlled by the stable engagement of Bcl-2-related proapoptotic proteins such as Bak, Bax, and Bim by survival proteins such as Bcl-2. BHRF1 is a prosurvival molecule from Epstein-Barr virus that has a high degree of homology to Bcl-2. To understand how BHRF1 blocks apoptosis, BHRF1 and mutants of BHRF1 were expressed in primary cells and an IL-2-dependent T cell line. BHRF1 bound the Executioner Bak and, when cells were cultured without cytokines, BHRF1 associated with Bim. A point mutation that lost the ability to bind Bak retained its ability to bind Bim and to protect cells. This result demonstrated that it was the capacity of BHRF1 to bind Bim, not Bak, that provided protection. Interestingly, the amount of Bim bound by BHRF1 was minimal when compared with the amount of Bim induced by apoptosis. Thus, BHRF1 does not act by simply absorbing the excess Bim produced while cells prepare for death. Rather, BHRF1 may act either by binding preferentially the most lethal form of Bim or by acting catalytically on Bim to block apoptosis.

Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate

PubMed Central

Nilsson, H; Lindgren, D; Mandahl Forsberg, A; Mulder, H; Axelson, H; Johansson, M E

2015-01-01

Changes of cellular metabolism are an integral property of the malignant potential of most cancer cells. Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated in vivo, should be further analyzed for the possible development of selective treatment strategies for patients with ccRCC. PMID:25569102

Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate.

PubMed

Nilsson, H; Lindgren, D; Mandahl Forsberg, A; Mulder, H; Axelson, H; Johansson, M E

2015-01-08

Changes of cellular metabolism are an integral property of the malignant potential of most cancer cells. Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated in vivo, should be further analyzed for the possible development of selective treatment strategies for patients with ccRCC.

Photoreceptor cell death and rescue in retinal detachment and degenerations

PubMed Central

Murakami, Yusuke; Notomi, Shoji; Hisatomi, Toshio; Nakazawa, Toru; Ishibashi, Tatsuro; Miller, Joan W.; Vavvas, Demetrios G.

2013-01-01

Photoreceptor cell death is the ultimate cause of vision loss in various retinal disorders, including retinal detachment (RD). Photoreceptor cell death has been thought to occur mainly through apoptosis, which is the most characterized form of programmed cell death. The caspase family of cysteine proteases plays a central role for inducing apoptosis, and in experimental models of RD, dying photoreceptor cells exhibit caspase activation; however, there is a paradox that caspase inhibition alone does not provide a sufficient protection against photoreceptor cell loss, suggesting that other mechanisms of cell death are involved. Recent accumulating evidence demonstrates that non-apoptotic forms of cell death, such as autophagy and necrosis, are also regulated by specific molecular machinery, such as those mediated by autophagy-related proteins and receptor-interacting protein kinases, respectively. Here we summarize the current knowledge of cell death signaling and its roles in photoreceptor cell death after RD and other retinal degenerative diseases. A body of studies indicate that not only apoptotic but also autophagic and necrotic signaling are involved in photoreceptor cell death, and that combined targeting of these pathways may be an effective neuroprotective strategy for retinal diseases associated with photoreceptor cell loss. PMID:23994436

Characteristic Thickened Cell Walls of the Bracts of the ‘Eternal Flower’ Helichrysum bracteatum

PubMed Central

Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

2008-01-01

Background and Aims Helichrysum bracteatum is called an ‘eternal flower’ and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. Methods DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Key Results Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Conclusions Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type. PMID:18436550

Patterns of cell death in the perinatal mouse forebrain.

PubMed

Mosley, Morgan; Shah, Charisma; Morse, Kiriana A; Miloro, Stephen A; Holmes, Melissa M; Ahern, Todd H; Forger, Nancy G

2017-01-01

The importance of cell death in brain development has long been appreciated, but many basic questions remain, such as what initiates or terminates the cell death period. One obstacle has been the lack of quantitative data defining exactly when cell death occurs. We recently created a "cell death atlas," using the detection of activated caspase-3 (AC3) to quantify apoptosis in the postnatal mouse ventral forebrain and hypothalamus, and found that the highest rates of cell death were seen at the earliest postnatal ages in most regions. Here we have extended these analyses to prenatal ages and additional brain regions. We quantified cell death in 16 forebrain regions across nine perinatal ages from embryonic day (E) 17 to postnatal day (P) 11 and found that cell death peaks just after birth in most regions. We found greater cell death in several regions in offspring delivered vaginally on the day of parturition compared with those of the same postconception age but still in utero at the time of collection. We also found massive cell death in the oriens layer of the hippocampus on P1 and in regions surrounding the anterior crossing of the corpus callosum on E18 as well as the persistence of large numbers of cells in those regions in adult mice lacking the pro-death Bax gene. Together these findings suggest that birth may be an important trigger of neuronal cell death and identify transient cell groups that may undergo wholesale elimination perinatally. J. Comp. Neurol. 525:47-64, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Primary Squamous Cell Carcinoma of the Thyroid Gland

PubMed Central

Ibrahim, Mohd-Irman-Shah; Jusoh, Yusri-Rahimi; Adam, Nurul-Nadhihah; Mohamad, Irfan

2018-01-01

Introduction: Primary squamous cell carcinoma (SCC) of the thyroid gland is one of the rarest types of all reported thyroid malignancies worldwide. It is very aggressive in nature and carries a poor prognosis. The surgical resection with adjuvant radiotherapy and chemotherapy is the most recommended treatment despite its poor reported outcome. Case Report: A 74-year-old woman presented with a rapidly progressive neck swelling, with hoarseness and compressive symptoms. Physical examination revealed a multilobulated firm thyroid mass with unilateral vocal cord palsy. Histopathological findings confirmed the diagnosis of SCC while radiological investigations and panendoscopy findings ruled out the possibility of other primary tumors. A surgical intervention was performed; however, the patient eventually succumbed to death prior to undergoing an oncological treatment. Conclusion: With no standard consensus to guide the management plan, SCC of the thyroid gland presents a great challenge for the managing team to come up with the best treatment option, due to its unfavorable rate of survival. PMID:29387667

Dead Cert: Measuring Cell Death.

PubMed

Crowley, Lisa C; Marfell, Brooke J; Scott, Adrian P; Boughaba, Jeanne A; Chojnowski, Grace; Christensen, Melinda E; Waterhouse, Nigel J

2016-12-01

Many cells in the body die at specific times to facilitate healthy development or because they have become old, damaged, or infected. Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer. The ability to objectively measure cell death in a laboratory setting is therefore essential to understanding and investigating the causes and treatments of many human diseases and disorders. Often, it is sufficient to know the extent of cell death in a sample; however, the mechanism of death may also have implications for disease progression, treatment, and the outcomes of experimental investigations. There are a myriad of assays available for measuring the known forms of cell death, including apoptosis, necrosis, autophagy, necroptosis, anoikis, and pyroptosis. Here, we introduce a range of assays for measuring cell death in cultured cells, and we outline basic techniques for distinguishing healthy cells from apoptotic or necrotic cells-the two most common forms of cell death. We also provide personal insight into where these assays may be useful and how they may or may not be used to distinguish apoptotic cell death from other death modalities. © 2016 Cold Spring Harbor Laboratory Press.

Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis.

PubMed

Kumar, Naveen; Gupta, Sonal; Dabral, Surbhi; Singh, Shailja; Sehrawat, Seema

2017-06-01

Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP-EPAC1-AKAP9 direction to the development of additional biotherapeutics for breast cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Shaojie; Patel, Ananddeep; Chu, Chun

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesismore » that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells. • AhR-deficient lung cells have decreased RelB activation.« less

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

PubMed

Han, Bing; Wang, Tong-Dan; Shen, Shao-Ming; Yu, Yun; Mao, Chan; Yao, Zhu-Jun; Wang, Li-Shun

2015-03-18

Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease.

PubMed

Wang, Tao; Lao, Uyen; Edgar, Bruce A

2009-09-07

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR's ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington's disease and phospholipase C (norpA)-mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.

Primary amebic meningoencephalitis due to Naegleria fowleri in a South American tapir.

PubMed

Lozano-Alarcón, F; Bradley, G A; Houser, B S; Visvesvara, G S

1997-05-01

Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are known to cause fatal central nervous system (CNS) disease in human beings. N. fowleri causes acute, fulminating primary amebic meningoencephalitis (PAM), which generally leads to death within 10 days. Acanthamoeba spp. and B. mandrillaris cause chronic granulomatous amebic encephalitis, which may last for 8 weeks. Acanthamoeba spp. and B. mandrillaris also cause CNS disease in animals. N. fowleri, however, has been described only in human beings. This report is the first of PAM in an animal, a South American tapir. Dry cough, lethargy, and coma developed in the animal, and its condition progressed to death. At necropsy, lesions were seen in the cerebrum, cerebellum, and lungs. The CNS had severe, suppurative meningoencephalitis with many neutrophils, fibrin, plasma cells, and amebas. Amebas were 6.5 microns to 9 microns in diameter and had a nucleus containing a large nucleolus. Amebas in the sections reacted with a monoclonal antibody specific for N. fowleri in the immunofluorescent assay and appeared bright green.

Hepatic Stellate Cells Alter Liver Immune Environment to Promote Cancer | Center for Cancer Research

Cancer.gov

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for up to 90 percent of cases, and is the second most common cause of cancer-related deaths worldwide according to the World Health Organization’s 2014 World Cancer Report. Even when caught early, HCC often recurs, either from intra-liver metastases or new primary tumors, and recurrence is the

Triterpene saponins from Vietnamese ginseng (Panax vietnamensis) and their hepatocytoprotective activity.

PubMed

Tran, Q L; Adnyana, I K; Tezuka, Y; Nagaoka, T; Tran, Q K; Kadota, S

2001-04-01

The methanol extract of Vietnamese ginseng (Panax vietnamensis) was found to possess hepatocytoprotective effects on D-galactosamine (D-GalN)/tumor necrosis factor-alpha (TNF-alpha)-induced cell death in primary cultured mouse hepatocytes. Further chemical investigation of the extract afforded two new dammarane-type triterpene saponins, ginsenoside Rh(5) (1) and vina-ginsenoside R(25) (2), as well as eight known dammarane-type triterpene saponins, majonoside R(2) (3), pseudo-ginsenoside RT(4) (4), vina-ginsenosides R(1) (5), R(2) (6), and R(10) (7), ginsenosides Rg(1) (8), Rh(1) (9), and Rh(4) (10), and a known sapogenin protopanaxatriol oxide II (11). Their structures were elucidated on the basis of spectral analysis. In addition, by the using LC-electrospray ionization (ESI)-MS method, five known saponins, ginsenosides Rb(1), Rb(2), Rc, Rd, and Re (12--16), were also identified in the extract. Among the compounds isolated, majonoside R(2) (3), the main saponin in Vietnamese ginseng, showed strong protective activity against D-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes. This demonstrates that the hepatocytoprotective effect of Vietnamese ginseng is due to dammarane-type triterpene saponins that have an ocotillol-type side chain, a characteristic constituent of Vietnamese ginseng.

Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

PubMed

Okahashi, Nobuo; Okinaga, Toshinori; Sakurai, Atsuo; Terao, Yutaka; Nakata, Masanobu; Nakashima, Keisuke; Shintani, Seikou; Kawabata, Shigetada; Ooshima, Takashi; Nishihara, Tatsuji

2011-10-01

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1β and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Cell Death Pathways and Phthalocyanine as an Efficient Agent for Photodynamic Cancer Therapy

PubMed Central

Mfouo-Tynga, Ivan; Abrahamse, Heidi

2015-01-01

The mechanisms of cell death can be predetermined (programmed) or not and categorized into apoptotic, autophagic and necrotic pathways. The process of Hayflick limits completes the execution of death-related mechanisms. Reactive oxygen species (ROS) are associated with oxidative stress and subsequent cytodamage by oxidizing and degrading cell components. ROS are also involved in immune responses, where they stabilize and activate both hypoxia-inducible factors and phagocytic effectors. ROS production and presence enhance cytodamage and photodynamic-induced cell death. Photodynamic cancer therapy (PDT) uses non-toxic chemotherapeutic agents, photosensitizer (PS), to initiate a light-dependent and ROS-related cell death. Phthalocyanines (PCs) are third generation and stable PSs with improved photochemical abilities. They are effective inducers of cell death in various neoplastic models. The metallated PCs localize in critical cellular organelles and are better inducers of cell death than other previous generation PSs as they favor mainly apoptotic cell death events. PMID:25955645

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells

PubMed Central

Karvela, Maria; Baquero, Pablo; Kuntz, Elodie M.; Mukhopadhyay, Arunima; Mitchell, Rebecca; Allan, Elaine K.; Chan, Edmond; Kranc, Kamil R.; Calabretta, Bruno; Salomoni, Paolo; Gottlieb, Eyal; Holyoake, Tessa L.; Helgason, G. Vignir

2016-01-01

ABSTRACT A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease. PMID:27168493

Biological and metabolic effects of IACS-010759, an OxPhos inhibitor, on chronic lymphocytic leukemia cells

PubMed Central

Vangapandu, Hima V.; Alston, Brandon; Morse, Joshua; Ayres, Mary L.; Wierda, William G.; Keating, Michael J.; Marszalek, Joseph R.; Gandhi, Varsha

2018-01-01

Blood cells from patients with chronic lymphocytic leukemia (CLL) are replicationally quiescent but transcriptionally, translationally, and metabolically active. Recently, we demonstrated that oxidative phosphorylation (OxPhos) is a predominant pathway in CLL for energy production and is further augmented in the presence of the stromal microenvironment. Importantly, CLL cells from patients with poor prognostic markers showed increased OxPhos. From these data, we theorized that OxPhos can be targeted to treat CLL. IACS-010759, currently in clinical development, is a small-molecule, orally bioavailable OxPhos inhibitor that targets mitochondrial complex I. Treatment of primary CLL cells with IACS-010759 greatly inhibited OxPhos but caused only minor cell death at 24 and 48 h. In the presence of stroma, the drug successfully inhibited OxPhos and diminished intracellular ribonucleotide pools. However, glycolysis and glucose uptake were induced as compensatory mechanisms. To mitigate the upregulated glycolytic flux, we used 2-deoxy-D-glucose in combination with IACS-010759. This combination reduced both OxPhos and glycolysis and induced cell death. Consistent with these data, low-glucose culture conditions sensitized CLL cells to IACS-010759. Collectively, these data suggest that CLL cells adapt to use a different metabolic pathway when OxPhos is inhibited and that targeting both OxPhos and glycolysis pathways is necessary for biological effect. PMID:29861847

Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

PubMed

Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

2013-02-01

We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cerebrospinal fluid cytotoxicity does not affect survival in amyotrophic lateral sclerosis.

PubMed

Galán, L; Matías-Guiu, J; Matias-Guiu, J A; Yáñez, M; Pytel, V; Guerrero-Sola, A; Vela-Souto, A; Arranz-Tagarro, J A; Gómez-Pinedo, U; García, A G

2017-09-01

Cerebrospinal fluid (CSF) from some patients with amyotrophic lateral sclerosis (ALS) has been demonstrated to significantly reduce the neuronal viability of primary cell cultures of motor neurons. We aimed to study the potential clinical consequences associated with the cytotoxicity of CSF in a cohort of patients with ALS. We collected CSF from thirty-one patients with ALS. We analysed cytotoxicity by incubating it into the primary cultures of motor cortex neurons. Neural viability was quantified after 24 hours using the colorimetric MTT reduction assay. All patients were followed up from the moment of diagnosis to death, and a complete evaluation during disease progression and survival was performed, including gastrostomy and respiratory assistance. Twenty-one patients (67.7%) presented a cytotoxic CSF. There were no significant differences between patients with and without cytotoxicity regarding mean time from symptom onset to the diagnosis, from the diagnosis to death, from the diagnosis to respiratory assistance with BIPAP, from diagnosis to gastrostomy and from the onset of symptoms to death. In Cox regression analysis, bulbar onset, but not cytotoxicity, gender or age at onset, was associated with a lower risk of survival. Cerebrospinal fluid cytotoxicity was not associated with differential survival rates. This suggests that the presence of cytotoxicity in CSF, measured through neuronal viability in primary cultures of motor cortex neurons, could reflect different mechanisms of the disease, but it does not predict disease outcome. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe.

PubMed

Hara, Yusuke; Sudo, Tatsuya; Togane, Yu; Akagawa, Hiromi; Tsujimura, Hidenobu

2018-04-01

Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. To understand the development of the central nervous system, it is essential to reveal not only molecular mechanisms but also the role of neural cell death (Pinto-Teixeira et al., 2016). To understand the role of cell death in neural development, we investigated the effect of inhibition of cell death on optic lobe development. Our data demonstrate that, in the optic lobe of Drosophila, cell death occurs in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development-e.g., failures in the separation of the lamina and medulla cortices and the medulla rotation. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, we would also expect to observe them in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, we found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers. These results indicate that cell death is required for elimination of the precursor cells composing the proliferation centers. This study substantiates an essential role of early neural cell death for ensuring normal development of the central nervous system. Copyright © 2018 Elsevier Inc. All rights reserved.

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

PubMed

Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

2010-12-15

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

PubMed Central

Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M.; Chandel, Navdeep S.; Vanden Hoek, Terry L.; Schumacker, Paul T.

2010-01-01

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, while other studies implicate activation of the mitochondrial permeability transition poreas the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, while it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetyl cysteine and exogenous glutathione (GSH), or by over-expression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells over-expressing Cu, Zn-SOD or MnSOD. Over-expression of antiapoptotic Bcl-XLprotected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochromec, Bax/Bak, caspase-9 and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. PMID:20937380

Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

PubMed Central

Schwer, Christian I.; Lehane, Cornelius; Guelzow, Timo; Zenker, Simone; Strosing, Karl M.; Spassov, Sashko; Erxleben, Anika; Heimrich, Bernd; Buerkle, Hartmut; Humar, Matjaz

2013-01-01

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited. PMID:24167567

Caspase Inhibition Prevents Tumor Necrosis Factor-α-Induced Apoptosis and Promotes Necrotic Cell Death in Mouse Hepatocytes in Vivo and in Vitro.

PubMed

Ni, Hong-Min; McGill, Mitchell R; Chao, Xiaojuan; Woolbright, Benjamin L; Jaeschke, Hartmut; Ding, Wen-Xing

2016-10-01

How different cell death modes and cell survival pathways cross talk remains elusive. We determined the interrelation of apoptosis, necrosis, and autophagy in tumor necrosis factor (TNF)-α/actinomycin D (ActD) and lipopolysaccharide/D-galactosamine (GalN)-induced hepatotoxicity in vitro and in vivo. We found that TNF-α/ActD-induced apoptosis was completely blocked by a general caspase inhibitor ZVAD-fmk at 24 hours but hepatocytes still died by necrosis at 48 hours. Inhibition of caspases also protected mice against lipopolysaccharide/GalN-induced apoptosis and liver injury at the early time point, but this protection was diminished after prolonged treatment by switching apoptosis to necrosis. Inhibition of receptor-interacting protein kinase (RIP)1 by necrostatin 1 partially inhibited TNF-α/ZVAD-induced necrosis in primary hepatocytes. Pharmacologic inhibition of autophagy or genetic deletion of Atg5 in hepatocytes did not protect against TNF-α/ActD/ZVAD-induced necrosis. Moreover, pharmacologic inhibition of RIP1 or genetic deletion of RIP3 failed to protect and even exacerbated liver injury after mice were treated with lipopolysaccharide/GalN and a pan-caspase inhibitor. In conclusion, our results suggest that different cell death mode and cell survival pathways are closely integrated during TNF-α-induced liver injury when both caspases and NF-κB are blocked. Moreover, results from our study also raised concerns about the safety of currently ongoing clinical trials that use caspase inhibitors. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Oxygen glucose deprivation post-conditioning protects cortical neurons against oxygen-glucose deprivation injury: role of HSP70 and inhibition of apoptosis.

PubMed

Zhao, Jian-hua; Meng, Xian-li; Zhang, Jian; Li, Yong-li; Li, Yue-juan; Fan, Zhe-ming

2014-02-01

In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (P<0.05). These findings suggest that PostC inhibited OGD-induced neuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.

YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

PubMed Central

Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B.

2012-01-01

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity. PMID:22563435

YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense.

PubMed

Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B

2012-01-01

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.

Human islet cells are killed by BID-independent mechanisms in response to FAS ligand.

PubMed

Joglekar, Mugdha V; Trivedi, Prerak M; Kay, Thomas W; Hawthorne, Wayne J; O'Connell, Philip J; Jenkins, Alicia J; Hardikar, Anandwardhan A; Thomas, Helen E

2016-04-01

Cell death via FAS/CD95 can occur either by activation of caspases alone (extrinsic) or by activation of mitochondrial death signalling (intrinsic) depending on the cell type. The BH3-only protein BID is activated in the BCL-2-regulated or mitochondrial apoptosis pathway and acts as a switch between the extrinsic and intrinsic cell death pathways. We have previously demonstrated that islets from BID-deficient mice are protected from FAS ligand-mediated apoptosis in vitro. However, it is not yet known if BID plays a similar role in human beta cell death. We therefore aimed to test the role of BID in human islet cell apoptosis immediately after isolation from human cadaver donors, as well as after de-differentiation in vitro. Freshly isolated human islets or 10-12 day cultured human islet cells exhibited BID transcript knockdown after BID siRNA transfection, however they were not protected from FAS ligand-mediated cell death in vitro as determined by DNA fragmentation analysis using flow cytometry. On the other hand, the same cells transfected with siRNA for FAS-associated via death domain (FADD), a molecule in the extrinsic cell death pathway upstream of BID, showed significant reduction in cell death. De-differentiated islets (human islet-derived progenitor cells) also demonstrated similar results with no difference in cell death after BID knockdown as compared to scramble siRNA transfections. Our results indicate that BID-independent pathways are responsible for FAS-dependent human islet cell death. These results are different from those observed in mouse islets and therefore demonstrate potentially alternate pathways of FAS ligand-induced cell death in human and mouse islet cells.

Regression of vessels in the tunica vasculosa lentis is initiated by coordinated endothelial apoptosis: a role for vascular endothelial growth factor as a survival factor for endothelium.

PubMed

Mitchell, C A; Risau, W; Drexler, H C

1998-11-01

The development of the embryonic lens is dependent on the formation and regression of the tunica vasculosa lentis (TVL), which is a transiently occurring capillary plexus that surrounds the posterior part of the lens. In this study, by using the terminal deoxy-nucleotidyl transferase mediated nick end-labelling technique (TUNEL), electron microscopy, radioactive end-labelling of DNA extracted from TVL, and the Comet assay, we show that widespread apoptosis of the endothelial cells that constitute the TVL is occurring already at embryonic day 17.5 (E17.5) of mouse development, much earlier than was reported previously (Jack [1972a] Am. J. Ophthalmol. 74:261-272; Lang [1997] Cell Death Diff. 4:12-20). In addition to apoptotic cell death, regression of this structure is associated with loss of capillary integrity, leakage of erythrocytes into the vitreal compartment, and phagocytosis of the apoptotic endothelium by tissue macrophages (hyalocytes). In situ hybridization experiments with probes for the flk-1 receptor and its high-affinity ligand, vascular endothelial growth factor (VEGF; Terman et al. [1992] Biochem. Biophys. Res. Commun. 187:1579-1586; Millauer et al. [1993] Cell 72:835-846), revealed strong endothelial cell expression for flk-1 in the eyes of E13.5-E17.5 embryos. VEGF mRNA was detected in lens epithelial cells located at the posterior pole of the developing lens in E13.5 embryos, in close proximity to the TVL capillaries. At later times (E14.5-E17.5), when the lens epithelial cells have differentiated into primary lens fiber cells, and a thick lenticular capsule is formed, the expression of VEGF mRNA becomes restricted to the anterior and equatorial portions of the lens. The physical separation of the VEGF-producing cells from the flk-1-expressing endothelium (due to the differentiation of the lens epithelial cells into lens fiber cells and the formation of the lenticular capsule) may deprive the endothelium of an essential survival factor and, thus, may constitute the primary mechanism that is responsible for the induction of endothelial cell apoptosis in this model.

Utilizing Matrigel Transwell Invasion Assay to Detect and Enumerate Circulating Tumor Cells.

PubMed

Liu, Xingtong; Wu, Xiangwei

2017-01-01

Metastasis is the cause of 90% of human cancer deaths. Circulating tumor cells (CTCs) in the peripheral blood and/or lymphatic vessels are cells shed from primary tumors and considered to be precursors of metastasis. Study of CTCs allows the serial monitoring of tumor progression and may provide predictive and prognostic biomarkers in clinic. Current CTC isolation and detection technologies encounter several challenges, including: heterogeneity of CTCs, low cell viability and/or high rate of contamination post-isolation, and the inability to distinguish viable/invasive from nonviable/nonfunctional CTCs, all of which can limit in vitro and in vivo characterization of CTCs. Here, we describe a new method to detect and enumerate of CTCs based on their invasive property.

Electrochemically Reduced Water Protects Neural Cells from Oxidative Damage

PubMed Central

Hamasaki, Takeki; Kinjo, Tomoya; Nakamichi, Noboru; Teruya, Kiichiro; Kabayama, Shigeru

2014-01-01

Aging-related neurodegenerative disorders are closely associated with mitochondrial dysfunction and oxidative stresses and their incidence tends to increase with aging. Brain is the most vulnerable to reactive species generated by a higher rate of oxygen consumption and glucose utilization compared to other organs. Electrochemically reduced water (ERW) was demonstrated to scavenge reactive oxygen species (ROS) in several cell types. In the present study, the protective effect of ERW against hydrogen peroxide (H2O2) and nitric oxide (NO) was investigated in several rodent neuronal cell lines and primary cells. ERW was found to significantly suppress H2O2 (50–200 μM) induced PC12 and SFME cell deaths. ERW scavenged intracellular ROS and exhibited a protective effect against neuronal network damage caused by 200 μM H2O2 in N1E-115 cells. ERW significantly suppressed NO-induced cytotoxicity in PC12 cells despite the fact that it did not have the ability to scavenge intracellular NO. ERW significantly suppressed both glutamate induced Ca2+ influx and the resulting cytotoxicity in primary cells. These results collectively demonstrated for the first time that ERW protects several types of neuronal cells by scavenging ROS because of the presence of hydrogen and platinum nanoparticles dissolved in ERW. PMID:25383141

Artemisinin protects PC12 cells against β-amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway.

PubMed

Zeng, Zhiwen; Xu, Jinying; Zheng, Wenhua

2017-08-01

Accumulating evidence displays that an abnormal deposition of amyloid beta-peptide (Aβ) is the primary cause of the pathogenesis of Alzheimer's disease (AD). And therefore the elimination of Aβ is regarded as an important strategy for AD treatment. The discovery of drug candidates using culture neuronal cells against Aβ peptide toxicity is believed to be an effective approach to develop drug for the treatment of AD patients. We have previously showed that artemisinin, a FDA-approved anti-malaria drug, has neuroprotective effects recently. In the present study, we aimed to investigate the effects and potential mechanism of artemisinin in protecting neuronal PC12 cells from toxicity of β amyloid peptide. Our studies revealed that artemisinin, in clinical relevant concentration, protected and rescued PC12 cells from Aβ25-35-induced cell death. Further study showed that artemisinin significantly ameliorated cell death due to Aβ25-35 insult by restoring abnormal changes in nuclear morphology, lactate dehydrogenase, intracellular ROS, mitochondrial membrane potential and activity of apoptotic caspase. Western blotting analysis demonstrated that artemisinin activated extracellular regulated kinase ERK1/2 but not Akt survival signaling. Consistent with the role of ERK1/2, preincubation of cells with ERK1/2 pathway inhibitor PD98059 blocked the effect of artemisinin while PI3K inhibitor LY294002 has no effect. Moreover, Aβ1-42 also caused cells death of PC12 cells while artemisinin suppressed Aβ1-42 cytotoxicity in PC12 cells. Taken together, these results, at the first time, suggest that artemisinin is a potential protectant against β amyloid insult through activation of the ERK1/2 pathway. Our finding provides a potential application of artemisinin in prevention and treatment of AD. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Human endothelial progenitor cells rescue cortical neurons from oxygen-glucose deprivation induced death.

PubMed

Bacigaluppi, Susanna; Donzelli, Elisabetta; De Cristofaro, Valentina; Bragazzi, Nicola Luigi; D'Amico, Giovanna; Scuteri, Arianna; Tredici, Giovanni

2016-09-19

Cerebral ischemia is characterized by both acute and delayed neuronal injuries. Neuro-protection is a major issue that should be properly addressed from a pharmacological point of view, and cell-based treatment approaches are of interest due to their potential pleiotropic effects. Endothelial progenitor cells have the advantage of being mobilized from the bone marrow into the circulation, but have been less studied than other stem cells, such as mesenchymal stem cells. Therefore, the comparison between human endothelial progenitor cells (hEPC) and human mesenchymal progenitor cells (hMSC) in terms of efficacy in rescuing neurons from cell death after transitory ischemia is the aim of the current study, in the effort to address further directions. In vitro model of oxygen-glucose deprivation (OGD) on a primary culture of rodent cortical neurons was set up with different durations of exposure: 1, 2 and 3hrs with assessment of neuron survival. The 2hrs OGD was chosen for the subsequent experiments. After 2hrs OGD neurons were either placed in indirect co-culture with hMSC or hEPC or cultured in hMSC or hEPC conditioned medium and cell viability was evaluated by MTT assay. At day 2 after 2hrs OGD exposure, mean neuronal survival was 47.9±24.2%. In contrast, after treatment with hEPC and hMSC indirect co-culture was 74.1±27.3%; and 69.4±18.8%, respectively. In contrast, treatment with conditioned medium did not provide any advantage in terms of survival to OGD neurons The study shows the efficacy of hEPC in indirect co-culture to rescue neurons from cell death after OGD, comparable to that of hMSC. hEPC deserve further studies given their potential interest for ischemia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

PubMed

Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

2018-06-11

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Vascular biology: cellular and molecular profiling.

PubMed

Baird, Alison E; Wright, Violet L

2006-02-01

Our understanding of the mechanisms underlying cerebrovascular atherosclerosis has improved in recent years, but significant gaps remain. New insights into the vascular biological processes that result in ischemic stroke may come from cellular and molecular profiling studies of the peripheral blood. In recent cellular profiling studies, increased levels of a proinflammatory T-cell subset (CD4 (+)CD28 (-)) have been associated with stroke recurrence and death. Expansion of this T-cell subset may occur after ischemic stroke and be a pathogenic mechanism leading to recurrent stroke and death. Increases in certain phenotypes of endothelial cell microparticles have been found in stroke patients relative to controls, possibly indicating a state of increased vascular risk. Molecular profiling approaches include gene expression profiling and proteomic methods that permit large-scale analyses of the transcriptome and the proteome, respectively. Ultimately panels of genes and proteins may be identified that are predictive of stroke risk. Cellular and molecular profiling studies of the peripheral blood and of atherosclerotic plaques may also pave the way for the development of therapeutic agents for primary and secondary stroke prevention.