Apoptosis In Cultured Cells Research Articles

GABA Protects Human Islet Cells Against the Deleterious Effects of Immunosuppressive Drugs and Exerts Immunoinhibitory Effects Alone

We recently found that γ-aminobutyric acid (GABA) protects mouse islet β cells. It prevented autoimmune type 1 diabetes in mice, induced islet β-cell regeneration, and exerted immunoinhibitory effects. However, it is not known whether GABA would be equally active on human islet and immune cells. In vitro culture of human islets and immune cells with or without GABA and immunosuppressive drugs. In vitro analysis of apoptosis, proliferation, nuclear factor (NF)-κB activation, calcium signaling, and insulin secretion. GABA reduced human islet cell apoptosis in culture, such that the yield of live cells was approximately tripled after 1 week, and it stimulated insulin secretion. It protected against the deleterious effects of rapamycin, tacrolimus, and mycophenolate mofetil. In human immune cells, GABA had inhibitory effects similar to mouse cells, such as suppressed anti-CD3-stimulated T-cell proliferation, in a GABA type A receptor-dependent fashion. The immunosuppressive mechanisms have been unclear, but we found that GABA blocked calcium influx, which is a key activation signal. GABA also suppressed NF-κB activation in both human islet cells and immune cells. We found that it could be combined with rapamycin to increase its suppressive effects. GABA improved human islet cell survival and had suppressive effects on human immune cells. It inhibited canonical NF-κB activation in both islet and immune cells. This is important because activation of this pathway is detrimental to islet cells and likely promotes damaging autoimmunity and alloreactivity against transplanted islets. These findings suggest that GABA might find applications in clinical islet transplantation.

Inhibition of c-Jun N-Terminal Kinase Activity Enhances Vestibular Schwannoma Cell Sensitivity to Gamma Irradiation

Radiosurgery is increasingly used to treat vestibular schwannomas (VSs). Increasing the sensitivity of VS cells to irradiation (IR) could allow for lower and/or more effective doses of IR, improving safety and efficacy. Persistent c-Jun N-terminal kinase (JNK) activity in VS cells reduces cell death by suppressing the accumulation of reactive oxygen species (ROS), raising the possibility that JNK activity protects against IR-induced VS cell death, which is mediated by ROS. To determine the extent to which JNK signaling contributes to VS cell radiosensitivity. Primary human VS cultures, derived from acutely resected tumors, received single doses (5-40 Gy) of gamma irradiation. Histone 2AX phosphorylation, a marker of IR-induced DNA damage, was assayed by Western blot and immunostaining. ROS levels were quantified by measuring 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. Cell apoptosis was determined by terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling. The JNK inhibitors SP6000125 and I-JIP reduced histone 2AX phosphorylation after IR. They also increased H2DCFDA fluorescence in nonirradiated cultures and significantly increased IR-induced (5-10 Gy) H2DCFDA fluorescence 72 hours, but not 2 hours, after IR. Finally, I-JIP (50 μmol/L) significantly increased VS cell apoptosis in cultures treated with 20 to 40 Gy. I-JIP (20 μmol/L), SP600125 (20 μmol/L), and JNK1/2 short interfering RNA knockdown each increased VS cell apoptosis in cultures treated with 30 to 40 Gy, but not lower doses, of IR. Inhibition of JNK signaling decreases histone 2AX phosphorylation and increases ROS and apoptosis in VS cells after gamma irradiation. These results raise the possibility of using JNK inhibitors to increase the effectiveness of radiosurgery for treatment of VSs.

Common Beans and Their Non-Digestible Fraction: Cancer Inhibitory Activity-An Overview.

The US Department of Agriculture’s MyPyramid guidelines introduced a near doubling of the dietary recommendations for vegetables including dry beans—an important food staple in many traditional diets that can improve public health and nutrition. Populations with high legume (peas, beans, lentils) consumption have a low risk of cancer and chronic degenerative diseases. Common beans (Phaseolus vulgaris L.) are known as a rich, reliable source of non-digested compounds like fiber, phenolics, peptides and phytochemicals that are associated with health benefits. Emerging evidence indicates that common bean consumption is associated with reduced cancer risk in human populations, inhibiting carcinogenesis in animal models and inducing cell cycle arrest and apoptosis in cell cultures. Fiber may reduce the risk of premature death from all causes, whereas the whole non-digestible fraction from common beans exhibits anti-proliferative activity and induces apoptosis in vitro and in vivo colon cancer. The mechanisms responsible for this apparently protective role may include gene-nutrient interactions and modulation of proteins’ expression. This review investigates the potential health benefits and bioactivity of beans on tumor inhibition, highlighting studies involving functional compounds, mainly non-digestible fractions that modulate genes and proteins, thereby, unraveling their preventive role against the development of cancer.

Aberrant regulation of DNA methylation in amyotrophic lateral sclerosis: a new target of disease mechanisms.

Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. A diagnosis is fatal owing to degeneration of motor neurons in brain and spinal cord that control swallowing, breathing, and movement. ALS can be inherited, but most cases are not associated with a family history of the disease. The mechanisms causing motor neuron death in ALS are still unknown. Given the suspected complex interplay between multiple genes, the environment, metabolism, and lifestyle in the pathogenesis of ALS, we have hypothesized that the mechanisms of disease in ALS involve epigenetic contributions that can drive motor neuron degeneration. DNA methylation is an epigenetic mechanism for gene regulation engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to carbon-5 in cytosine residues in gene regulatory promoter and nonpromoter regions. Recent genome-wide analyses have found differential gene methylation in human ALS. Neuropathologic assessments have revealed that motor neurons in human ALS show significant abnormalities in Dnmt1, Dnmt3a, and 5-methylcytosine. Similar changes are seen in mice with motor neuron degeneration, and Dnmt3a was found abundantly at synapses and in mitochondria. During apoptosis of cultured motor neuron-like cells, Dnmt1 and Dnmt3a protein levels increase, and 5-methylcytosine accumulates. Enforced expression of Dnmt3a, but not Dnmt1, induces degeneration of cultured neurons. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocks apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with small molecules RG108 and procainamide protects motor neurons from excessive DNA methylation and apoptosis in cell culture and in a mouse model of ALS. Thus, motor neurons can engage epigenetic mechanisms to cause their degeneration, involving Dnmts and increased DNA methylation. Aberrant DNA methylation in vulnerable cells is a new direction for discovering mechanisms of ALS pathogenesis that could be relevant to new disease target identification and therapies for ALS.

Calcium flux and calpain-mediated activation of the apoptosis-inducing factor contribute to enterovirus 71-induced apoptosis

Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca²⁺)-activated calpains are capable of mediating caspase-independent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis-inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This was accompanied by the release of cytochrome c, and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca²⁺-dependent, calpain isoforms 1 and 2, but not cathepsins, were proven crucial for the altered AIF behaviour as studied by the pharmacological inhibitor and the knockdown approaches. We then analysed Ca²⁺ dynamics in the infected cells and found elevated levels of mitochondrial Ca²⁺. Treatment with ruthenium red, a mitochondrial Ca²⁺ influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca²⁺ flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca²⁺ homeostasis on EV71 infection.

Survivin selective inhibitor YM155 induce apoptosis in SK-NEP-1 Wilms tumor cells

BackgroundSurvivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of mitosis and programmed cell death. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. The aim of this study was to determine the antitumor activity of YM155 in SK-NEP-1 cells.MethodsSK-NEP-1 cell growth in vitro and in vivo was assessed by MTT and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis in cell culture. Then gene expression profile of tumor cells treated with YM155 was analyzed with real-time PCR arrays. We then analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis tool.ResultsYM155 treatment resulted in inhibition of cell proliferation of SK-NEP-1cells in a dose-dependent manner. Annexin V assay, cell cycle, and activation of caspase-3 demonstrates that YM155 induced apoptosis in SK-NEP-1 cells. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ± 0.77 cm3; YM155 10 mg/kg: 0.95 ± 0.55 cm3) compared to DMSO group (DMSO: 3.70 ± 2.4 cm3) or PBS group cells (PBS: 3.78 ± 2.20 cm3, ANOVA P < 0.01). YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ± 0.24 g; YM155 10 mg/kg: 0.72 ± 0.17 g) compared to DMSO group (DMSO: 2.06 ± 0.38 g) or PBS group cells (PBS: 2.36 ± 0.43 g, ANOVA P < 0.01). Real-time PCR array analysis showed between Test group and control group there are 32 genes significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment. Ingenuity pathway analysis (IPA) showed cell death was the highest rated network with 65 focus molecules and the significance score of 44. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to cell death, cellular function maintenance, cell morphology, carbohydrate metabolism and cellular growth and proliferation. Death receptor signaling (3.87E-19), TNFR1 signaling, induction of apoptosis by HIV1, apoptosis signaling and molecular mechanisms of cancer came out to be the top four most significant pathways. IPA analysis also showed top molecules up-regulated were BBC3, BIRC3, BIRC8, BNIP1, CASP7, CASP9, CD5, CDKN1A, CEBPG and COL4A3, top molecules down-regulated were ZNF443, UTP11L, TP73, TNFSF10, TNFRSF1B, TNFRSF25, TIAF1, STK17A, SST and SPP1, upstream regulator were NR3C1, TP53, dexamethasone , TNF and Akt.ConclusionsThe present study demonstrates that YM155 treatment resulted in apoptosis and inhibition of cell proliferation of SK-NEP-1cells. YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors. Real-time PCR array analysis firstly showed expression profile of genes dyes-regulated after YM155 treatment. IPA analysis also represents new molecule mechanism of YM155 treatment, such as NR3C1 and dexamethasone may be new target of YM155. And our results may provide new clues of molecular mechanism of apoptosis induced by YM155.

TNF-α Mediates Macrophage-Induced Bystander Effects through Netrin-1

Macrophage-induced bystander effects have been implicated as an important mediator of chromosomal instability and colon cancer triggered by Enterococcus faecalis, a human intestinal commensal bacteria. There is little understanding about how inflammatory cytokines mediate bystander effects, but questions in this area are important because of the pivotal contributions made by inflammatory processes to cancer initiation and progression. Here, we report that the central proinflammatory cytokine TNF-α acts as a diffusible mediator of the bystander effects induced by macrophages, an effect caused by a proliferation of macrophages that trigger epithelial cell production of Netrin-1, a neuronal guidance molecule. TNF-α-mediated bystander assays used a murine coculture system of primary colonic epithelial cells and E. faecalis-infected macrophages (in vitro), with an interleukin 10 (IL-10)-deficient mouse model of colon cancer that involves long-term colonization with E. faecalis (in vivo). In cell cocultures, we observed increased expression of the TNF-α receptor Tnfrsf1b and Netrin-1. These effects were blocked by anti-TNF-α antibody or by pretreatment with an inhibitor of NF-κB signaling. RNAi-mediated attenuation of Tnfrsf1b decreased TNF-α-induced netrin-1 production and augmented epithelial cell apoptosis in culture. Extending these observations, colon biopsies from E. faecalis-colonized IL-10(-/-) mice exhibited crypt hyperplasia and increased staining for macrophages, TNF-α, netrin-1, NF-κB, Tnfrsf1b, and the proliferation marker proliferating cell nuclear antigen while also displaying a reduction in epithelial cell apoptosis. Together, our results define a pathway for macrophage-induced bystander effects in which TNF-α triggers TNFRSF1b receptor signaling leading to increased production of Netrin-1, crypt hyperplasia, and decreased epithelial cell apoptosis. In elucidating an important commensal-associated proinflammatory mechanism in the intestinal microenvironment, our work highlights the role of Netrin-1 and a specific TNF-α receptor as candidate targets to prevent or treat colorectal cancer.

Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors.

We previously proved that a histone deacetylase inhibitor (valproate, VPA) decreases the number of leukemic cells in bovine leukemia virus (BLV)-infected sheep. Here, we characterize the mechanisms initiated upon interruption of treatment. We observed that VPA treatment is followed by a decrease of the B cell counts and proviral loads (copies per blood volume). However, all sheep eventually relapsed after different periods of time and became refractory to further VPA treatment. Sheep remained persistently infected with BLV. B lymphocytes isolated throughout treatment and relapse were responsive to VPA-induced apoptosis in cell culture. B cell proliferation is only marginally affected by VPA ex vivo. Interestingly, in four out of five sheep, ex vivo viral expression was nearly undetectable at the time of relapse. In two sheep, a new tumoral clone arose, most likely revealing a selection process exerted by VPA in vivo. We conclude that the interruption of VPA treatment leads to the resurgence of the leukemia in BLV-infected sheep and hypothesize that resistance to further treatment might be due to the failure of viral expression induction. The development of more potent HDAC inhibitors and/or the combination with other compounds can overcome chemoresistance. These observations in the BLV model may be important for therapies against the related Human T-lymphotropic virus type 1.

Inhibition of MEK pathway in vestibular schwannoma cell culture

The purpose of this study was to evaluate the Ras GTPase (Ras) to extracellular signal-regulated kinase (ERK) pathway in vestibular schwannoma (VS) cell cultures and patient excised schwannoma tumors. Mitogen-activated protein kinase kinase (MEK) inhibitor CI-1040 (PD184352) was utilized to evaluate the effect of specific MEK inhibition on benign schwannoma cell culture proliferation and apoptosis. Prospective evaluation of human schwannoma cell lines and tumors. Western blotting was completed with phospho-antibodies for proteins in the Ras-ERK pathway. Increasing concentrations of CI-1040 were utilized in schwannoma cell cultures to evaluate cell proliferation and apoptosis. Proliferation was measured with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation assay, and apoptosis was monitored with flow cytometry of annexin V/propidium iodide-stained cells. The most consistent Ras-ERK pathway alterations were found in phospho-MEK and ERK. Phospho-MEK was not overexpressed in the schwannoma cell lines, but six out of 10 VS showed significant increases compared to benign Schwann cell controls. Similarly, nine of 10 VS tumors showed increased phospho-ERK expression. CI-1040 showed significantly reduced schwannoma cell proliferation at the 50 and 100 μM (IC(50) 20 μM and 30 μM) concentrations when compared to carrier only controls in two out three schwannoma cell lines. The remaining schwannoma cell line was relatively refractory to the antiproliferative effects of CI-1040 at doses up to 100 μM (IC(50) 58 μM). Cumulative data of four separate schwannoma cell lines demonstrated that apoptosis was increased in treated schwannoma cells at CI-1040 concentrations of 50 and 100 μM at 72 hours. There is overexpression of phosphorylated (activated) proteins in the Ras-ERK pathway in schwannoma cultures and tumors as compared to benign human Schwann cell culture controls. MEK inhibitor, CI-1040, created significantly decreased schwannoma cell proliferation and increased apoptosis in cell culture. These data justify the use of MEK inhibitors in animal treatment studies of VS.

An antiapoptotic role of sorting nexin 7 is required for liver development in zebrafish

Sorting nexin (SNX) family proteins are best characterized for their abilities to regulate protein trafficking during processes such as endocytosis of membrane receptors, endosomal sorting, and protein degradation, but their in vivo functions remain largely unknown. We started to investigate the biological functions of SNXs using the zebrafish model. In this study, we demonstrated that SNX7 was essential for embryonic liver development. Hepatoblasts were specified normally, and the proliferation of these cells was not affected when SNX7 was knocked down by gene-specific morpholinos; however, they underwent massive apoptosis during the early budding stage. SNX7 mainly regulated the survival of cells in the embryonic liver and did not affect the viability of cells in other endoderm-derived organs. We further demonstrated that down-regulation of SNX7 by short interfering RNAs induced apoptosis in cell culture. At the molecular level, the cellular FLICE-like inhibitory protein (c-FLIP)/caspase 8 pathway was activated when SNX7 was down-regulated. Furthermore, overexpression of c-FLIP(S) was able to rescue the SNX7 knockdown-induced liver defect. SNX7 is a liver-enriched antiapoptotic protein that is indispensable for the survival of hepatoblasts during zebrafish early embryogenesis.

Nicotinic modulation of therapeutic response in vitro and in vivo

Tobacco use significantly increases the risk of developing cancer. Moreover, there is growing evidence that tobacco use decreases survival in cancer patients. Nicotine, a systemically available component of tobacco, is associated with tumor promotion and decreased apoptosis in cell culture; however, the role of nicotine on response to radiotherapy (RT) or chemoradiotherapy (CRT) in vivo has not been evaluated. Our study evaluated the effects of nicotine administration on cancer cell survival in cell culture and mouse models. Nicotine increased survival in two cell lines following RT in vitro. Nicotine administration in mice during fractionated RT or CRT increased xenograft regrowth as compared to RT or CRT alone. Nicotine increased hypoxia-inducible factor 1-alpha (HIF-1α) expression in tumor xenografts without altering expression of carbonic-anhydrase, a clinical marker of tumor hypoxia. The effects of nicotine on HIF-1α expression were transient, returning to baseline levels within 2-3 days after nicotine removal. Further mechanistic studies indicated that inhibition of phosphoinositide-3-kinase (PI3K) prevented nicotine-mediated increases in HIF-1α expression as well as the prosurvival effects of nicotine on RT. These findings imply that during tobacco use, nicotine may function as a systemic agent through acute and reversible regulation of HIF-1α expression and a decreased therapeutic response.

Abstract 3684: Targeting Aurora kinase in peripheral T-cell non-Hodgkin's lymphomas

Abstract Aurora kinases (A and B) are oncogenic serine/threonine (S/T) kinases that play central roles in the mitotic phase of the eukaryotic cell cycle. Over-expression of Auroras during dysregulated cell cycling and their aberrant activation over-rides mitotic and spindle check points leading to anueploidy in many human cancers make Auroras attractive therapeutic targets. Gene expression profiling in mantle cell lymphoma [MCL] have shown the Auroras are over-expressed and is a poor prognostic marker within the ‘proliferative’ signature. PTCL (NOS) are a heterogeneous group of rare non-Hodgkin's lymphomas (NHL) derived from activated CD4+ or CD8+ T cells. PTCL and anaplastic large cell lymphoma (ALCL) both of T-cell phenotype and ALK negative have an aggressive clinical course with an inferior event free and overall survival relative to B-cell NHL counterparts. The biological explanation for the difference is unknown. Patients with PTCL have a poor prognosis due to a very aggressive disease course coupled to a lack of effective therapies. Therefore, the development of novel and effective treatments based on biologically validated targets is urgently needed for this disease. We hypothesized and investigated that (1). Auroras are over-expressed in human PTCL-NOS, (2). Aurora ATP-site small molecule inhibitor (SMI) is effective in inhibiting cell proliferation and promoting apoptosis in cell culture and (3). Aurora SMI will be safe and effective in treating patients with relapsed aggressive T-cell NHL in early phase clinical trials. In this study, we demonstrate that both aurora A and B are highly over-expressed in PTCL patients and cell lines. MLN8237 (alisertib), an ATP-site small molecule inhibitor of Aurora A had potent activity in inhibiting phosphorylation of Aurora A and histone H3 in PTCL cell lines. Cells treated with MLN8237 exhibited endoreduplication confirming the mechanism of action of an Aurora inhibitor. Also, treatment of PTCL cell lines with MLN8237 induced apoptosis in a dose and time dependent manner (flow cytometry and PARP-cleavage) and inhibited cell proliferation with an IC50 &amp;lt; 0.1µM. Preliminary data from a phase II clinical trial using single-agent MLN8237 in patients with relapsed refractory aggressive NHL has demonstrated notable activity with 4 confirmed responses in 6 evaluable PTCL patients. Cytokine profiling on day 1 (pre-treatment) and day 8 (post-treatment) showed a profile consistent with aurora A activity. Together the data suggest inhibition of Auroras offers a promising treatment strategy for patients with aggressive T-cell NHL [Funded by the Lymphoma SPORE, P50 CAB0805501A1]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3684. doi:1538-7445.AM2012-3684

Abstract 1215: Orexin and their 7-membrane spanning receptors OX1R: a new colon cancer therapeutic target

Abstract Orexins are hypothalamic peptides involved in sleep/wake control. Unexpectedly, we have shown that orexins promote robust apoptosis in various colorectal cancer cells1. The cell death is mediated by the orexin 1 receptor (OX1R) through an original mechanism. OX1R, a G protein-coupled receptor class I (GPCR), is aberrantly expressed in primary colorectal tumors and liver metastases2. We next investigated the molecular mechanism of OX1Rmediated cell death. OX1R makes use of “tyrosine-based motifs” ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif)3 and ITSM (Immunoreceptor Tyrosine-based Switch Motif)4 to drive apoptosis. These “tyrosine-based motifs” are mandatory for recruitment and activation of the tyrosine phosphatase SHP-2, leading to apoptosis. Orexins promote cell death through Mitogen-/Stress-activated Protein Kinase (MAPK/SAPK) p38 activation in a SHP-2 dependent manner. The present work is original for two reasons: 1) OX1R is the first example of the involvement of both ITSM and ITIM in GPCR signal transduction. ITIM and ITSM sequences are more frequent than initially thought in GPCRs. This emphasizes the need to explore this mechanism of signal transduction in various GPCRs for regulating cellular processes including at least cell apoptosis and proliferation; 2) we provide evidence that OX1R is aberrantly expressed in all primary colorectal tumors and liver metastases and that its activation by exogenous orexins result in robust apoptosis of colon cancer cells in culture and strong decrease of tumor development in mice xenografted with colon cancer cells in vivo. OX1R represent a new promising target in colorectal cancer therapy and/or for metastases imaging. 1 J.Biol.Chem, 279: 45875 (2004) 2 Can. Res. 71: 3341 (2011) 3 FASEB J. 22: 1993 (2008) 4 FASEB J. 23: 4069 (2009) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1215. doi:1538-7445.AM2012-1215

Changes in fractal dimension and lacunarity as early markers of UV-induced apoptosis

The aim of our study was to employ fractal analysis for evaluation of ultrastructural changes during early stages of apoptosis. Apoptosis was induced in U251 human glioma cell line by exposure to UVB light. The cells were visualized by optical phase-contrast microscopy and photographed before the UV treatment, immediately after the treatment, as well as at 30min intervals during 5h observation period. For each of the 32 cells analyzed, cellular and nuclear fractal dimension, as well as nuclear lacunarity, were determined at each time point. Our data demonstrate that cellular ultrastructural complexity determined by fractal dimension and lacunarity significantly decreases after the UV irradiation, with the nuclear lacunarity being a particularly sensitive parameter in detecting early apoptosis. Importantly, fractal analysis was able to detect cellular apoptotic changes earlier than conventional flow cytometric analysis of phosphatidylserine exposure, DNA fragmentation and cell membrane permeabilization. These results indicate that fractal analysis might be a powerful and affordable method for non-invasive early identification of apoptosis in cell cultures.

Wavelength-dependent backscattering measurements for quantitative monitoring of apoptosis, Part 2: early spectral changes during apoptosis are linked to apoptotic volume decrease

Elastic scattering spectroscopy (ESS), in the form of wavelength-dependent backscattering measurements, can be used to monitor apoptosis in cell cultures. Early changes in backscattering upon apoptosis induction are characterized by an overall decrease in spectral slope and begin as early as 10 to 15 min post-treatment, progressing over the next 6 to 8 h. The timescale of early scattering changes is consistent with reports of the onset of apoptotic volume decrease (AVD). Modeling cellular scattering with a fixed distribution of sizes and a decreasing index ratio, as well as an increased contribution of the whole cell to cellular scattering, resulting from increased cytoplasmic density, is also consistent with observed spectral changes. Changes in ESS signal from cells undergoing osmotically-induced volume decrease in the absence of apoptosis were similar, but smaller in magnitude, to those of apoptotic cells. Further, blockage of Cl(-) channels, which blocks AVD and delays apoptosis, blocked the early scattering changes, indicating that the early scattering changes during apoptosis result, at least partially, from AVD. Work continues to identify the additional sources of early spectral scattering changes that result from apoptosis induction.

In vivo adaptation of hepatitis C virus in chimpanzees for efficient virus production and evasion of apoptosis

Hepatitis C virus (HCV) employs various strategies to establish persistent infection that can cause chronic liver disease. Our previous study showed that both the original patient serum from which the HCV JFH-1 strain was isolated and the cell culture-generated JFH-1 virus (JFH-1cc) established infection in chimpanzees, and that infected JFH-1 strains accumulated mutations after passage through chimpanzees. The aim of this study was to compare the in vitro characteristics of JFH-1 strains emerged in each chimpanzee at early and late stages of infection, as it could provide an insight into the phenomenon of viral persistence. We generated full-genome JFH-1 constructs with the mutations detected in patient serum-infected (JFH-1/S1 and S2) and JFH-1cc-infected (JFH-1/C) chimpanzees, and assessed their effect on replication, infectious virus production, and regulation of apoptosis in cell culture. The extracellular HCV core antigen secreted from JFH-1/S1-, S2-, and C-transfected HuH-7 cells was 2.5, 8.9, and 2.1 times higher than that from JFH-1 wild-type (JFH-1/wt) transfected cells, respectively. Single cycle virus production assay with a CD81-negative cell line revealed that the strain JFH-1/S2, isolated from the patient serum-infected chimpanzee at a later time point of infection, showed lower replication and higher capacity to assemble infectious virus particles. This strain also showed productive infection in human hepatocyte-transplanted mice. Furthermore, the cells harboring this strain displayed lower susceptibility to the apoptosis induced by tumor necrosis factor α or Fas ligand compared with the cells replicating JFH-1/wt. The ability of lower replication, higher virus production, and less susceptibility to cytokine-induced apoptosis may be important for prolonged infection in vivo. Such control of viral functions by specific mutations may be a key strategy for establishing persistent infection.

Nonvirion Protein of Novirhabdovirus Suppresses Apoptosis at the Early Stage of Virus Infection

Viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are members of the genus Novirhabdovirus within the Rhabdoviridae family, which can cause severe hemorrhagic disease in fresh- and saltwater fish worldwide. These viruses carry an additional nonvirion (NV) gene, which codes for the nonstructural NV protein that has been implicated to play a role in viral pathogenesis. To determine the precise biological function of this NV gene and its gene product, we generated NV-deficient and NV knockout recombinant VHSVs, using reverse genetics. Comparisons of the replication kinetics and markers for virus-induced apoptosis indicated that the NV-deficient and NV knockout mutant viruses induce apoptosis earlier in cell culture than the wild-type recombinant VHSV. These results suggest that the NV protein has an antiapoptotic function at the early stage of virus infection. Furthermore, we created a chimeric VHSV, in which the NV gene of VHSV was replaced by the IHNV NV gene, which was capable of suppressing apoptosis in cell culture. These results show that the NV protein of other members of Novirhabdovirus can restore the NV protein function. In this study, we also investigated the kinetics of VHSV replication during a single round of viral replication and examined the mechanism of VHSV-induced apoptosis. Our results show that VHSV infection induced caspases 3, 8 and 9 in cell culture.

Endothelin-3/endothelin receptor B(ET-3/ETRB) regulates the NF-κB/Bfl-1 anti-apoptotic pathway in a malignant melanoma cell line A375

Objective To investigate the modulation of ET-3 on the nuclear factor (NF)-κB/Bfl-1 antiapoptotic pathway in a malignant melanoma cell line A375. Methods Flow cytometry was performed to detect the apoptosis in cultured A375 cells after treatment with ET-3 of 100 nmol/L for 24 hours. ET-3 of various concentrations (0, 1, 10, 100 nmol/L) was used to treat some A375 cells with or without the pretreatment with the ETRB antagonist BQ788; after another 24-hour culture, RT-PCR and Western blot were conducted to examine the mRNA expression of Bfl-1 and protein expressions of Bfl-1 and ETRB, respectively. Results The 24-hour treatment with ET-3 of 100 nmol/L significantly reduced the apoptosis rate of A375 cells (F = 10.68, P ＜0.05). The mRNA and protein expressions of Bfl-1 were up-regulated by ET-3 in a concentration dependent manner (both P ＜ 0.01 ), while BQ788 significantly blocked the ET-3-induced up-regulation (F = 420.38,229.49, both P ＜ 0.01 ). The protein expression of pNF-κB in A375 cells was also enhanced by ET-3 of different concentrations (all P ＜ 0.05), but the enhancement was suppressed by BQ788, and there was a significant difference in the protein expression of pNF-κB between cells treated with ET-3 of 100 nmol/L and those treated with the combination of ET-3 of 100 nmol/L and BQ788 (F = 255.46, P ＜ 0.01 ). Conclusion ET-3/ETRB inhibits the apoptosis in A375 cells likely by activating the NF-κB/Bfl-1 anti-apoptotic pathway. Key words: Melanoma, experimental; Endothelin-3; Receptor, endothelin B; NF-kappa B

Sublethal Spinal Ketamine Produces Neuronal Apoptosis in Rat Pups

Sublethal Spinal Ketamine Produces Neuronal Apoptosis in Rat Pups

Metabolic Signatures in Apoptotic Human Cancer Cell Lines

Cancer cells have several specific metabolic features, which have been explored for targeted therapies. Agents that promote apoptosis in tumors are currently considered as a powerful tool for cancer therapeutics. The present study aimed to design a fast, reliable and robust system for metabolite measurements in cells lines to observe impact of apoptosis on the metabolome. For that purpose the NBS (newborn screen) mass spectrometry-based metabolomics assay was adapted for cell culture approach. In HEK 293 and in cancer cell lines HepG2, PC3, and MCF7 we searched for metabolic biomarkers of apoptosis differing from that of necrosis. Already nontreated cell lines revealed distinct concentrations of metabolites. Several metabolites indicative for apoptotic processes in cell culture including aspartate, glutamate, methionine, alanine, glycine, propionyl carnitine (C3-carnitine), and malonyl carnitine (C3DC-carnitine) were observed. In some cell lines metabolite changes were visible as early as 4 h after apoptosis induction and preceeding the detection by caspase 3/7 assay. We demonstrated for the first time that the metabolomic signatures might be used in the tests of efficacy of agents causing apoptosis in cell culture. These signatures could be obtained in fast high-throughput screening.