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PubMed2004

Critical signaling events during the aminoglycoside-induced death of sensory hair cells in vitro.

Sensory hair cells undergo apoptosis following exposure to aminoglycoside antibiotics. In neurons, apoptosis is associated with a transient increase in intracellular Ca2+, phosphorylation of the transcription factor c-Jun, and the release of cytochrome c from mitochondria into the cytosol, which along with other cofactors results in the activation of caspases. To examine the possible role of these events in the survival and death of the sensory receptors of the inner ear, we examined the effects of neomycin treatment on cytoplasmic calcium, activation of c-Jun-N-Terminal kinases (JNKs), cytochrome c release, and caspase-3 activation in cultured vestibular hair cells. Increased numbers of phospho-c-Jun-labeled hair cells (a downstream indicator of JNK activation) were observed at 3-12 h after neomycin treatment, whereas increased numbers of cells with cytoplasmic cytochrome c were observed at 12-18 h following the onset of neomycin treatment. This was followed by an increase in the number of cells that contained activated caspase-3 and displayed pyknotic nuclei. Treatment with the general caspase inhibitor BAF did not affect the release of cytochrome c and the number of p-c-Jun-labeled cells, but reduced the number of cells with activated caspase-3 and pyknotic nuclei. In contrast, treatment with CEP-11004, an indirect inhibitor of the JNK signaling pathway, promoted hair cell survival following neomycin treatment and reduced the number of cells with phosphorylated JNK and c-Jun, cytoplasmic cytochrome c, and activated caspase 3. These results suggest that JNK activation occurs upstream of the release of cytochrome c and that cytochrome c release precedes caspase activation. Cytochrome c release and JNK activation were also preceded by large changes in cytoplasmic calcium. Cytoplasmic calcium increases may be causally related to the release of cytochrome c, and may also be a potential pathway for activation of JNK in hair cells.

Aminoglycosides↗

PubMed2000

Caspase regulation of neuronal progenitor cell apoptosis.

Programmed cell death (apoptosis) of both proliferating neuroblasts and postmitotic neurons is essential for normal nervous system development. To study the molecular regulation of apoptosis in neuronal progenitor cells, we developed a flow cytometric assay capable of distinguishing between viable, apoptotic, and necrotic cell populations. Incubation of freshly dissociated telencephalic cells from gestational day 12-13 mouse embryos with either cytosine arabinoside (AraC) or staurosporine caused a marked increase in the percentage of apoptotic cells. Both drugs induced caspase-3 activation, as determined by in vitro cleavage of a caspase-3 substrate and immunocytochemical detection of activated caspase-3. Treatment of telencephalic cells with the broad caspase inhibitor BAF, blocked caspase-3 activation and protected cells against both AraC and staurosporine-induced apoptotic death. These results indicate that neuronal progenitors possess a caspase-dependent apoptotic pathway, the activation of which may regulate neuronal progenitor cell numbers in vivo.

Animals↗

PubMed1998

The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release.

This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IkappaB superrepressor, tumor necrosis factor alpha (TNFalpha) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFalpha induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFalpha treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFalpha treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFalpha-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing DeltaFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFalpha, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFalpha-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Activation of the caspase cascade underlies the rat trigeminal primary neuronal apoptosis induced by neonatal capsaicin administration.

The systemic administration of capsaicin is known to cause a massive loss of sensory primary neurons in newborn rats. Here we examined the trigeminal ganglion neurons immunohistochemically for the possible induction of activated forms of caspases-9 and -3 following a subcutaneous injection of capsaicin in newborn rats. The DNA fragmentation signal was labeled by a TUNEL method. TUNEL-positive neurons were rare (< 0.5%) at 24 h after injection of the vehicle without capsaicin. After the capsaicin injection, TUNEL-positive neurons began to increase by 12 h, reached a peak at 24 h (11.4%), and returned to the control level by 120 h. Vehicle control levels of caspase- 9-immunoreactive (ir) and caspase-3-ir neurons were low (< 0.5%). Neonatal capsaicin administration induced caspase-9-immunoreactivity (ir) and -3-ir. The temporal distributions of caspase-9-ir and caspase-3-ir neurons were similar to those of TUNEL-positive neurons with peak expressions at 24 h of 13.2 and 11.1%, respectively. A double-stain analysis at 24 h post-injection indicated 72% of TUNEL-positive neurons were caspase-9-ir, and 70% caspase-3-ir. Conversely, 78 and 68% of caspase-9-ir and caspase-3-ir neurons, respectively, were TUNEL-positive. Comparison of two adjacent sections immunostained for the two different antigens revealed the co-expression of the two caspases. These results suggest that neonatal capsaicin triggers the caspase cascade and, thereby, induces trigeminal primary neuronal apoptosis.

Animals↗

PubMed2002

CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP-cleaved products of caspase activation following middle cerebral artery occlusion in the rat.

Citicoline has been demonstrated to be beneficial in several models of cerebral ischaemia. We tested the hypothesis that citicoline may provide apoptotic pathways following focal cerebral ischaemia. Focal cerebral ischaemia was produced by distal, permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. The animals were randomised into four groups: (B+A) Citicoline 500 mg/kg IP 24 and 1 h before MCAO, and 23 h after MCAO; (A) citicoline 500 mg/kg IP, within 30 min after MCAO, and 23 h after MCAO; (C) vehicle IP; and (D) sham-operated. The animals were sacrificed at 12 h (n=8 per group) and 24 h (n=8 per group) after MCAO. Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase-1, -2, -3, -6 and -8, and in paraffin-embedded sections processed for cleaved caspase-3 (17 kDa) immunohistochemistry. Finally, some sections were stained with the method of in situ end-labelling of nuclear DNA fragmentation. For gel electrophoresis and Western blotting, antibodies to poly (ADP-ribose) polymerase (PARP) products of 89 kDa were used to reveal specific cleavage substrates of caspases. MCAO induced the expression of all procaspases and the expression of PARP products of 89 kDa, as well as cells with nuclear DNA fragmentation, at 12 and 24 h, in the infarcted core and penumbra. Citicoline reduced the expression of all procaspases at 12 and 24 h after MCAO, as well as the expression of cleaved caspase-3 in cells in the penumbra area. This was accompanied by a reduction in the number of cells bearing nuclear DNA fragments. The expression of caspase-cleaved products of PARP (PARP 89 kDa) was reduced in citicoline-treated ischaemic rats. These results show that citicoline inhibits the expression of proteins involved in apoptosis following MCAO.

Animals↗

PubMed2005

Inhibition of caspases alleviates gentamicin-induced cochlear damage in guinea pigs.

The efficacy of caspase inhibitors for protecting the cochlea was evaluated in an in vivo study using guinea pigs, as the animal model system. Gentamicin (12 mg/ml) was delivered via an osmotic pump into the cochlear perilymphatic space of guinea pigs at 0.5 microl/h for 14 days. Additional animals were given either z-Val-Ala-Asp (Ome)-fluoromethyl ketone (z-VAD-FMK) or z-Leu-Glu-His-Asp-FMK (z-LEHD-FMK), a general caspase inhibitor and a caspase 9 inhibitor, respectively, in addition to gentamicin. The elevation in auditory brain stem response thresholds, at 4, 7, and 14 days following gentamicin administration, were decreased in animals that received both z-VAD-FMK and z-LEHD-FMK. Cochlear sensory hair cells survived in greater numbers in animals that received caspase inhibitors in addition to gentamicin, whereas sensory hair cells in animals that received gentamicin only were severely damaged. These results suggest that auditory cell death induced by gentamicin is closely related to the activation of caspases in vivo.

Administration, Topical↗

PubMed2005

Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

Anaplasma phagocytophilum↗

PubMed2003

Molecular mechanisms of neuronal death in the dorsal lateral geniculate nucleus following visual cortical lesions.

We investigated the molecular mechanisms of cell death in the dorsal lateral geniculate nucleus of the rat, following suction lesion of the visual cortex at birth or in the third postnatal week, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique and immunohistochemistry for caspase-3, -7, -8, and cleaved poly(ADP-ribose) polymerase. Following lesion at birth, TUNEL-positive neurons were found in the dorsal lateral geniculate nucleus between 24 h and 3 days after lesion, with a peak on the second day. Shorter survival times (12-18 h) resulted in labeling of very few neurons in dorsal lateral geniculate nucleus and of several neurons in the perilesional cortex. Activated caspase-3 was expressed from the first to the third days after lesion, whereas cleaved poly(ADP-ribose) polymerase and activated caspase-8 were expressed on the second and third day. Activated caspase-7 was expressed mainly in pretectal nuclei. Caspase-3 activation coincided with the appearance of TUNEL-positive profiles, but decreased earlier than TUNEL. In the ipsi- and contralateral cerebral cortex, all parameters were unchanged. In animals lesioned in the third week, rare apoptotic thalamic neurons were detected as TUNEL- and activated caspase-3-positive profiles 2 days after cortical ablation, and were still present 1 week after lesion.Thus, early target ablation has dramatic effects on neonatal thalamic neurons, which die following activation of caspases 3 and 8. In contrast, cortical neurons are relatively unaffected by target deprivation. Compared with early lesions, late lesions induce a limited thalamic cell death, that persists over time.

Animals↗

PubMed2004

A novel isoform of pro-interleukin-18 expressed in ovarian tumors is resistant to caspase-1 and -4 processing.

Interleukin-18 (IL-18) is a proinflammatory cytokine synthesized as a 24 kDa inactive precursor (pro-IL-18) by several cell types, and is processed to a bioactive molecule of 18 kDa by the proteinases caspase-1 or caspase-4. All ovarian carcinoma cell lines express pro-IL-18, only in some instances coexpress caspase-1, and always express caspase-4; in any case, they display a defective processing of IL-18. We analysed whether pro-IL-18, present in two ovarian carcinoma cell lysates, could be processed 'in vitro' by recombinant active caspase-1. While most of pro-IL-18 could be cleaved by caspase-1, a residual of pro-IL-18 appeared to be resistant. Cloning and sequence analysis of the whole pro-IL-18 open reading frame demonstrated the existence of an alternatively spliced mRNA variant, which lacked exon-3 (Delta3pro-IL-18). The 12 bp exon-3 encodes for the AEDD amino-acid sequence, which is N-terminal with respect to the cleavage site of caspase-1. Both pro-IL-18 and Delta3pro-IL-18 mRNA isoforms were detected in all ovarian cancer cell lines analysed, while Delta3pro-IL-18 mRNA was undetectable in normal ovarian epithelial cells. The Delta3pro-IL-18 cDNA induced synthesis of an alternative Delta3pro-IL-18 protein upon transfection into a murine cell line. The Delta3pro-IL-18 protein was resistant to proteolytic activation by caspase-1 and -4, although it was capable to bind caspase-1. Aternative splicing of pro-IL-18 exon-3 may represent a novel mechanism of regulation of bioactive IL-18 production in human ovarian tumors.

Base Sequence↗

PubMed2003

Down-regulation of survivin expression reversed multidrug resistance in adriamycin-resistant HL-60/ADR cell line.

AIM: To investigate the ability of an antisense RNA eukaryotic expression plasmid pcDNA3.1/survivin in down-regulating the expression level of survivin mRNA and survivin protein and reversed multidrug resistance (MDR) in adriamycin-resistant HL-60/ADR cell line. METHODS: The expression of survivin mRNA was measured by RT-PCR and the expression of survivin protein was measured by Western blot. Caspase-3 activity was determined by Phar Mingen colorimetric assay kit. Apoptosis was assessed by flow cytometry. The chemosensitivity of HL-60/ADR cells to adriamycin (ADR) was measured by MTT assay. RESULTS: pcDNA3.1/survivin down-regulated the expression level of survivin mRNA and survivin protein obviously, and induced apoptosis of HL-60/ADR cells in a time-dependent manner during 12-48 h. After transient transfection with pcDNA3.1/survivin for 48 h, survivin mRNA decreased by 67 %, survivin protein decreased by 57 %, caspase-3 activity increased 4.37 times, and the apoptosis rate increased by 4.41 % compared with control. Compared with ADR alone, pcDNA3.1/survivin significantly reversed MDR in HL-60/ADR cells, the chemosensitivity of HL-60/ADR cells to ADR was increased to 5.36 folds. CONCLUSION: pcDNA3.1/survivin down-regulated the expression level of survivin mRNA and survivin protein obviously, the threshold of apoptosis was decreased and MDR was reversed.

Apoptosis↗

PubMed2005

Effects of human cytomegalovirus infection on apoptosis and expression of apoptosis-regulating factors.

The present study aimed to find out dynamic changes of apoptosis in human cytomegalovirus (HCMV) infected cells and the influence of HCMV infection on activation of caspase-3 and the expression of apoptosis-regulating genes, bcl-2 and fas mRNA. The sequential changes of apoptotic cell rate in high and low MOI (MOI = 2.5 and 0.25 respectively) of HCMV infected human embryonic lung fibroblasts (HELFs) at 1 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h post-infection were measured by flow cytometry. The expression levels of caspase-3 protein and bcl-2 and fas mRNA in HCMV infected cells (MOI = 0.25) at 72 h post-infection were detected by Western blot and in situ hybridization methods, respectively. It was found that the ratio of apoptotic cells in normal controls was consistently lower, but the rates in low and high MOI infected cells were gradually increased with time prolonged, reached peak at 96 h (8.85%) and 72 h (25.63%), respectively. By Western blot analysis, only a narrow band of 32 kD (1 kD = 0.992 1 ku) procaspase-3 was found in normal cells, but a wider procaspase-3 band and a much wider band of 17 kD proteins (p17) appeared in the infected cells. Meanwhile, the expression of bcl-2 mRNA was higher and that of fas mRNA was lower in the normal HELF cells, whereas there were significantly lower bcl-2 mRNA and higher fas mRNA expression levels in HCMV infected cells. It was concluded that HCMV was a stronger inducer of apoptosis in HELF cells. Caspase-3, as the marker of undergoing apoptosis, was expressed increasingly and activated in the infected cells, indicating its action in HCMV-inducing apoptosis. Down-regulating bcl-2 mRNA expression and up-regulating fas mRNA expression were also involved in the mechanism of HCMV-induced apoptosis.

Apoptosis↗

PubMed2006

Role of suckling in regulating cell turnover and onset and maintenance of lactation in individual mammary glands of sows.

This study addressed the mechanisms by which suckling regulates cell turnover and onset and maintenance of lactation of individual mammary glands of sows. The effects of no, transient (through 12 to 14 h postpartum), or regular suckling of individual glands during d 0 to 6 of lactation were studied in 5 sows. Nonsuckling was obtained by taping the glands to prevent access to the nipples. Visual scores confirmed that regularly suckled glands maintained lactation, whereas transiently suckled and nonsuckled glands regressed during lactation. Mammary gland biopsies were collected on d -5, 1, 2, 4, and 6 relative to farrowing in order to evaluate the cell turnover and to quantify the transcription of genes potentially involved in mammary cell turnover and function. The proportion of proliferating cells was greatest prepartum (13.1%). After farrowing, the proportion of proliferating cells declined in all glands, then remained low (5.6%) in nonsuckled glands and increased (P < 0.01) from an average of 7.5% on d 1 to 9.9% on d 6 in regularly suckled and transiently suckled glands. Transcriptional data were analyzed using a gamma-distributed, generalized linear mixed model. Abundance of alpha-lactalbumin mRNA (P < 0.01) increased in regularly suckled glands within the first day of lactation and remained elevated, whereas the expression in taped glands remained at the prepartum level. Prolactin receptor mRNA abundance decreased (P < 0.001), and IGFBP-5 mRNA abundance increased (P < 0.01) in nonsuckled and transiently suckled glands after parturition compared with regularly suckled glands. Mammary mRNA abundances of IGF-I, IGF-II, type I IGF receptor, and caspase 3 were minimally or inconclusively affected by the suckling regimens. In conclusion, suckling during the first 12 to 14 h postpartum is insufficient to initiate and maintain lactation until 24 to 36 h postpartum but sufficient to induce mammary cell proliferation for at least 6 d postpartum. Furthermore, a high prolactin receptor transcription and a low IGFBP-5 transcription seem important for maintaining a functional mammary gland during lactation.

Animals↗

PubMed2006

Could NF-kappaB and caspase-3 be markers for estimation of post-interval of human traumatic brain injury?

BACKGROUND: Previous studies in a rat model showed expressions of caspase-3 (p20) and NF-kappaB (p65) in different time after TBI. We test the activation changes of caspase-3 and NF-kappaB of human specimen after traumatic brain injury (TBI) in this study to see whether they could be markers to estimate post-intervals of TBI for forensic practice. MATERIAL AND METHODS: Caspase-3 (p20) and NF-kappaB (p65) immunohistochemical staining were performed on 39 TBI specimens (dead from TBI) and eight controls (dead from none TBI reasons) grouped 0, 12, 24, 48, 72, 168, 264 and 480 h. Positive cells were counted and image analysis techniques were used to determine the morphological changes in each group. RESULTS: Immunohistochemical analysis showed a marked induction of caspase-3 (p20) in every injured group compared with normal controls; each positive group has it's morphological character with variant positive cell count. NF-kappaB (p65) did not showed markedly change compared with normal controls until 168 h after TBI and almost all nerve cells were stained by NF-kappaB (p65) in 264 and 480 h groups. CONCLUSION: NF-kappaB (p65) could contribute to estimate delayed death cases after TBI for forensic applications. Caspase-3 (p20) estimating post-TBI intervals are dubious but could suggest TBI exists.

Adult↗

PubMed2005

Effects of estrone on quisqualate-induced toxicity in primary cultures of rat cortical neurons.

Estrogens exert protective effects against neurotoxic changes induced by over-activation of ionotrophic glutamate receptors, whereas little is known about their interaction with changes mediated by metabotropic glutamate receptors. We evaluated effects of estrone on quisqualate (QA)-induced toxicity in neuronal cell cultures on 7 and 12 day in vitro (DIV). Twenty four hour exposure to QA (150 microM and 300 microM) significantly decreased cell survival in 7 day old cultures, but the 12 day old cultures were more resistant to its toxicity. DNQX (10 microM), an AMPA/kainate receptor antagonist, partly attenuated the toxic effects of QA, whereas LY 367 385 (100 microM), a selective mGluR1a antagonist, completely reversed the above effect. QA did not activate, but suppressed spontaneous caspase-3-like activity. Estrone (100 nM and 500 nM) attenuated QA-mediated neurotoxic effects independently of estrogen receptors, as indicated with ICI 182, 780 and without affecting the caspase-3-like activity. At early stage of development in vitro (7 DIV) toxic effects of QA were more profound and mediated mainly by metabotropic glutamate receptors of group I, whereas later (12 DIV) they were mediated mostly by ionotropic AMPA/kainate receptors. The toxic effects of QA were partly accompanied by anti-apoptotic action against spontaneous caspase-3-like activity, possibly due to modulation of neuronal plasticity.

Animals↗

PubMed2006

Statin-induced proinflammatory response in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes.

Statins, which inhibit 3-hydroxy-3-methylglutaryl CoA reductase, have been shown recently to promote proinflammatory responses. We show in this study that both atorvastatin and simvastatin induced proinflammatory responses in mitogen-activated PBMCs by increasing the number of T cells secreting IFN-gamma. This is abolished by the presence of mevalonate, suggesting that statins act specifically by blocking the mevalonate pathway for cholesterol synthesis to promote the proinflammatory response. Both statins at low concentrations induced a dose-dependent increase in the number of IFN-gamma-secreting T cells in mitogen-activated PBMCs, whereas at higher concentrations the effect was abolished. The proinflammatory effect of statins was not seen in purified T cells per se activated with mitogen. However, conditioned medium derived from statin-treated PBMCs enhanced the number of IFN-gamma-secreting cells in activated purified T cells. This effect was not blocked by mevalonate, but was abolished by neutralizing Abs to IL-18 and IL-12. Similarly, the up-regulation of IFN-gamma-secreting T cells in PBMCs costimulated with statins and mitogens was blocked by the neutralizing anti-IL-18 and anti-IL-12. We showed that simvastatin stimulates the secretion of IL-18 and IL-1beta in monocytes. Active caspase-1, which is required for the processing and secretion of IL-18 and IL-1beta, was activated in simvastatin-treated monocytes. This was blocked by mevalonate and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. Taken together, the proinflammatory response mediated by statins in activated PBMCs is mediated mainly via the activation of caspase-1 and IL-18 secretion in the monocytes and to a lesser extent by IL-12.

Amino Acid Chloromethyl Ketones↗

PubMed2006

Antiproliferative activity of Pteleopsis suberosa leaf extract and its flavonoid components in human prostate carcinoma cells.

In this work we describe the chemical composition of Pteleopsis suberosa (Combretaceae) leaf extract and its biological activity against androgen-insensitive human prostate cancer cells (DU-145). The methanol extract of the plant leaves exhibited activity against tumor cell growth. Fractionation of this active extract led to the isolation and identification of sixteen flavonoids, including gallocatechin and flavonols having kaempferol, quercetin, and myricetin as aglycones. Among the myricetin derivatives, myricetin 3-O-(3''-acetyl)-alpha-L-arabinopyranoside (1) and myricetin 3-O-(4''-acetyl)-alpha- L-arabinopyranoside (2) are now reported for the first time. Six compounds, myricetin 3-O-alpha- L-rhamnopyranoside (4), myricetin 3-O-beta-D-galactopyranoside (7), myricetin 3-O-(6''-galloyl)-beta-D-galactopyranoside (9), myricetin 3-O-beta-D-xylopyranoside (10), myricetin 3-O-alpha-L-arabinofuranoside (12), and gallocatechin (14), exhibited significant activity, reducing cell vitality and inducing apoptosis via the caspase-dependent pathway in DU-145 cells that can be, in part, correlated to modulation of redox-sensitive mechanisms.

Carcinoma↗

PubMed2000

Traumatic brain injury alters the molecular fingerprint of TUNEL-positive cortical neurons In vivo: A single-cell analysis.

The cerebral cortex is selectively vulnerable to cell death after traumatic brain injury (TBI). We hypothesized that the ratio of mRNAs encoding proteins important for cell survival and/or cell death is altered in individual damaged neurons after injury that may contribute to the cell's fate. To investigate this possibility, we used amplified antisense mRNA (aRNA) amplification to examine the relative abundance of 31 selected candidate mRNAs in individual cortical neurons with fragmented DNA at 12 or 24 hr after lateral fluid percussion brain injury in anesthetized rats. Only pyramidal neurons characterized by nuclear terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) reactivity with little cytoplasmic staining were analyzed. For controls, non-TUNEL-positive neurons from the cortex of sham-injured animals were obtained and subjected to aRNA amplification. At 12 hr after injury, injured neurons exhibited a decrease in the relative abundance of specific mRNAs including those encoding for endogenous neuroprotective proteins. By 24 hr after injury, many of the mRNAs altered at 12 hr after injury had returned to baseline (sham-injured) levels except for increases in caspase-2 and bax mRNAs. These data suggest that TBI induces a temporal and selective alteration in the gene expression profiles or "molecular fingerprints" of TUNEL-positive neurons in the cerebral cortex. These patterns of gene expression may provide information about the molecular basis of cell death in this region after TBI and may suggest multiple avenues for therapeutic intervention.

Animals↗

PubMed2001

5-Fluorouracil induced Fas upregulation associated with apoptosis in liver metastases of colorectal cancer patients.

BACKGROUND: In vitro, thymidylate synthase (TS) inhibition by 5-fluorouracil (5-FU) induces thymineless apoptosis possibly via Fas receptor Fas ligand interactions and cell-cycle arrest. In colorectal cancer patients we evaluated whether 5-FU administration also resulted in apoptosis and cell-cycle arrest and which proteins might be involved. PATIENTS AND METHODS: Biopsy specimens were taken from 36 patients 2, 22 or 46 hours after administration of 500 mg/m2 5-FU, and from 12 control patients who did not receive 5-FU. In frozen tissue-sections from liver metastases immunohistochemistry was performed with antibodies directed against p53, p21, E2F2, Rb, Ki67 and TS (cell-cycle related) and bax, BCL-2, BCL-x, mcl-1, PARP, caspase-3, Fas receptor and Fas ligand (apoptosis related). Apoptosis was determined by M30 immunostaining, which recognises a cleavage product of cytokeratin 18. RESULTS: Fas receptor expression was 50% higher (P = 0.036) 46 hours after 5-FU administration compared to the control group. This was associated with a 12% increase (P < 0.02) in M30 positive tumour cells and with elevation of caspase-3 and PARP expression. The expression of Ki67 and E2F2 was 30% lower after 46 hours compared to the control group, whereas TS was 56% lower after 2 hours and 32% higher again after 46 hours. No differences in the expression of the other proteins were found. CONCLUSIONS: These results suggest that 5-FU decreases proliferation status and induces apoptosis possibly via the Fas pathway. Since Fas mediated cell killing is important for cytotoxic T cells this indicates that clinical studies combining immunotherapy for activation of T cells and chemotherapy using 5-FU might be very effective.

Adenocarcinoma↗