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PubMed2003

Delineation of the molecular mechanisms of Francisella tularensis-induced apoptosis in murine macrophages.

Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. Here we analyzed the pathway leading to apoptosis in the murine macrophage-like cell line J774A.1 after infection with F. tularensis strain LVS (named LVS for live vaccine strain). We obtained evidence that the infection affected the mitochondria of the macrophages, since it induced release of the mitochondrial molecule cytochrome c into the cytosol and changed the potential over the mitochondrial membrane. Moreover, activation of caspase 9 and the executioner caspase 3 was also observed in the LVS-infected J774A.1 macrophages. The activated caspase 3 degraded poly(ADP-ribose) polymerase (PARP). All of these events were observed within 9 to 12 h after the initiation of infection, and maximum degradation of a synthetic caspase 3 substrate occurred at 18 h. The internucleosomal fragmentation and PARP degradation resulting from activation of this apoptotic pathway was prevented by the caspase 3 inhibitor Z-DEVD-fmk. No involvement of caspase 1, caspase 8, Bcl-2, or Bid was observed. Thus, the F. tularensis infection induces macrophage apoptosis through a pathway partly resembling the intrinsic apoptotic pathway.

Animals↗
PubMed2001

Expression of redox factor-1, p53-activated gene 608 and caspase-3 messenger RNAs following repeated unilateral common carotid artery occlusion in gerbils--relationship to delayed cell injury and secondary failure of energy state.

The temporospatial expression pattern of the nuclear DNA repair enzyme redox factor-1 (ref-1), the p53-activated gene (pag) 608 and the effector caspase-3 was examined by in situ hybridization histochemistry in gerbils subjected to two 10-min episodes of unilateral common carotid artery occlusion, separated by 5h. Gene responses were correlated with the metabolic state, as revealed by regional adenosine 5'-triphosphate bioluminescent imaging, and with the degree of histological damage, as assessed by haematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated-dUTP nick end labeling (TUNEL), in order to evaluate the role of these genes in the maturation of injury. Focal infarcts developed in the dorsolateral cerebral cortex at the bregma level and the nucleus caudate-putamen within four days after repeated unilateral ischemia, as indicated by a secondary adenosine 5'-triphosphate loss after initial adenosine 5'-triphosphate recovery and by histomorphological signs of pannecrosis. The more caudal cortex at hippocampal levels and the hippocampus (CA1>CA3 area), however, exhibited selective neuronal injury without adenosine 5'-triphosphate depletion. TUNEL+ cells appeared starting 5h after repeated unilateral ischemia. TUNEL+ cells reached maximum levels in the caudate-putamen at 12-24h, but much later in the cortex and hippocampus at two days after ischemia. Remarkably few TUNEL+ cells were noticed in the thalamus, where adenosine 5'-triphosphate state did not recover after reperfusion. Following repeated unilateral ischemia, a transient elevation of ref-1 mRNA was detected after 5h in the cerebral cortex and hippocampal CA1 area. Ref-1 mRNA levels decreased within 12-24h, before the onset of tissue damage. Subsequently, pag608 and caspase-3 mRNA levels increased, closely in parallel with the appearance of DNA fragmented cells, but slightly prior to the deterioration of adenosine 5'-triphosphate state. In the caudate-putamen, pag608 and caspase-3 mRNAs reached maximum levels already 12-24h after repeated common carotid artery occlusion, when DNA fragmentation was most prominent, and declined thereafter. In the cortex and hippocampal CA1-3 areas, where DNA damage appeared more slowly, pag608 and caspase-3 mRNAs were induced starting 24h after ischemia, and remained elevated even after two to four days. The levels of pag608 and caspase-3 mRNAs were similar at rostral and caudal levels of the cortex, as well as in the hippocampal CA1 and CA3 area, although the degree of injury differed considerably between these structures. Notably, pag608 and caspase-3 mRNAs were not elevated in the thalamus after repeated unilateral ischemia. The present report shows a close temporal association between the induction of ref-1, pag608 and caspase-3 mRNAs, the manifestation of cell injury and the secondary adenosine 5'-triphosphate depletion in infarcting brain areas, suggesting (i) that de novo responses of these genes may be involved in the maturation of cell injury and (ii) that apoptotic programs and the secondary deterioration of cerebral energy state may interfere with each other after ischemia.

Adenosine Triphosphate↗
PubMed2001

Expansion of an ablated site and induction of apoptosis after microwave coagulation therapy in rat liver.

BACKGROUND/PURPOSE: Microwave coagulation therapy (MCT), one of the ablation therapies, has been widely used to treat liver cancers, yielding excellent clinical results. Despite its efficacy, the precise events that take place in the ablated liver after MCT remain unknown. We investigated sequential histologic changes around MCT sites and the relationship between MCT and apoptosis in rat liver. METHODS: One session of MCT at 30 W was applied to rat liver. The rats were killed at 0, 2, 6, 12, 24, 72, and 168 h after MCT. The liver surface area was measured for sequential evaluations of the size of the impaired field (i.e., the liver surface affected by MCT). The size of the impaired field was assessed by measuring the area that showed macroscopic changes in color with a relatively clear border. To assess apoptosis, we examined terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling stained sections, determined the positive cell count for DNA fragmentation, and observed DNA ladder formation by gel electrophoresis. Caspase-3 activity at the ablated margin was measured for the enzymatic evaluation of apoptosis. RESULTS: The impaired field gradually expanded through 12 h after MCT. Caspase-3 activity increased four fold from the baseline, peaking at 2 h after MCT, and DNA fragmentation, confirmed by DNA ladder formation, was significantly increased at 6 h. CONCLUSIONS: Alterations in the ablated liver tissue indicated that the activation of caspase-3 around the MCT site was followed by apoptosis and expansion of the impaired field. The expansion continued until 12 h after MCT, and this may be beneficial for the local control of liver cancer.

Animals↗
PubMed2006

Impact of 17beta-estradiol on cytokine-mediated apoptotic effects in primary hippocampal and neocortical cell cultures.

Estrogens are developmental regulators of mitochondrial apoptotic pathway in the central nervous system, but little is known about their involvement in cytokine-induced apoptosis. In the present study, we evaluated effects of 17beta-estradiol on pro-inflammatory cytokine- and staurosporine-mediated activation of caspase-3 and LDH-release in primary neuronal/glial cell cultures of mouse hippocampal and neocortical cells at different stages of their development in vitro. Enzyme activities were determined with colorimetric methods 6 h, 14 h, 24 h, and 48 h after exposure to the apoptotic agents. Biochemical data were supported at the cellular level by Hoechst 33342 and MAP-2 stainings, which were carried out 48 h after the treatment. Cytokines (co-treatment with Il-1beta and TNFalpha; 1 ng/ml) increased caspase-3 activity in the hippocampal and neocortical cells up to over 200% of control values, and these effects were mostly observed on 2 and 7 days in vitro (DIV). Moderate, but significant cytokine-mediated increase in LDH-release was demonstrated in both tissues, especially on 7 and 12 DIV. Estradiol (100 nM) inhibited the activation of caspase-3 at early stage of development (2 DIV) in the hippocampal, but not in the neocortical cultures. The cytokine-induced activation of caspase-3 and LDH-release was inhibited by estradiol in estrogen receptor-independent way. These data point to a possible role of estrogens as non-estrogen receptor-related inhibitors of cytokine-activated apoptotic pathway in the developing central nervous system.

Animals↗
PubMed2006

Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate.

The relative importance of the different proteolytic sites in mammalian proteasomes in protein degradation has not been studied systematically. Nevertheless, it is widely assumed that inhibition of the chymotrypsin-like site, the primary target of the proteasome inhibitors used in research and cancer therapy, reflects the degree of inhibition of protein breakdown. Here we demonstrate that selective inactivation of the chymotrypsin-like site reduced degradation of model proteins by pure 26 S proteasomes by only 11-50% and decreased only slightly the breakdown of proteins in HeLa cells. Inactivation of the caspase-like site decreased breakdown of model proteins by 12-22% and of the trypsin-like site by 3-35%. The relative contributions of these different sites depended on the protein substrate, and the importance of the trypsin-like sites depended on the substrate's content of basic residues. Simultaneous inhibition of the chymotrypsin-like and the caspase- or trypsin-like sites was needed to reduce degradation by >50%. Thus, 1) all three types of active sites contribute significantly to protein breakdown, 2) their relative importance varies widely with the substrate, 3) assaying the chymotrypsin-like activity overestimates the actual reduction in protein degradation, and 4) inhibition of multiple sites is required to markedly decrease proteolysis.

Adenosine Triphosphatases↗
PubMed2006

p44/42 MAP kinase and c-Jun N-terminal kinase contribute to the up-regulation of caspase-3 in manganese-induced apoptosis in PC12 cells.

Caspase-3 (32 kDa) is one of the primary protease executioners of apoptosis and is activated by intra-chain proteolytic cleavage, which generates a large subunit (17 kDa) and a small subunit (12 kDa). Typically, after apoptotic stimuli, the level of cleaved caspase-3 increases while that of caspase-3 decreases. It has been shown that caspase-3 mRNA levels increase in cortex following traumatic brain injury or focal ischemia. In the present study, we demonstrate that both caspase-3 mRNA and protein increase in apoptotic PC12 cells following exposure to manganese which strongly induces caspase-3 cleavage. Up-regulation of caspase-3 protein was evident in manganese-treated PC12 cells and was moderate in cisplatin-, rotenone- and A23187-treated cells but was not observed in serum deprivation-, anisomycin-, camptothecin-, cycloheximide- or staurosporine-treated cells in which all treatments induced extensive DNA fragmentation. Manganese-induced up-regulation of caspase-3 mRNA was partially attenuated by the pretreatment with the MEK inhibitor U0126, but not with the c-Jun N-terminal kinase (JNK) inhibitor SP600125. In contrast, the increase in caspase-3 protein was suppressed by both U0126 and SP600125. These results suggest that p44/42 MAPK contributes to the up-regulation of caspase-3 mRNA and the JNK pathway regulates caspase-3 protein levels posttranslationally in manganese-induced apoptosis in PC12 cells.

Animals↗
PubMed2003

Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells.

Epigallocatechin-3-gallate (EGCG), a major component in green tea polyphenols, has been proven to suppress colonic tumorigenesis in animal models and epidemiological studies. As EGCG is retained in the gastrointestinal tract after oral administration, this pharmacokinetics property gives it the potential to function as a chemopreventive agent against colon cancer. In this study, human colorectal carcinoma HT-29 cells were treated with EGCG to examine the anti-proliferative and pro-apoptotic effects of EGCG, as well as the molecular mechanism underlying these effects. Cell viability assay, nuclear staining, DNA fragmentation, caspase assay, cytochrome c release, DiOC6(3) staining, mitogen-activated protein kinases (MAPK) phosphorylation and trypan blue exclusion assays, were utilized to dissect the signaling pathways induced by EGCG. After 36 h treatment, EGCG inhibited HT-29 cell growth with an IC50 of approximately 100 microM. HT-29 cells treated with doses higher than 100 microM showed apparent nuclear condensation and fragmentation, which was confirmed by DNA laddering. Caspase-3 and -9 activation was detected after 12 h treatment, accompanied by mitochondrial transmembrane potential transition and cytochrome c release. Activation of MAPKs was detected as early signaling event elicited by EGCG. Inhibition of c-Jun N-terminal kinase (JNK) pathway showed the involvement of JNK in EGCG-induced cytochrome c release and cell death. EGCG-induced JNK activation was blocked by the antioxidants glutathione and N-acetyl-l-cysteine, suggesting that the cell death signaling was potentially triggered by oxidative stress. In summary, our results from this study suggest that in HT-29 human colon cancer cells (i) EGCG treatment causes damage to mitochondria, and (ii) JNK mediates EGCG-induced apoptotic cell death.

Adenocarcinoma↗
PubMed2006

Improved islet yields from pancreas preserved in perflurocarbon is via inhibition of apoptosis mediated by mitochondrial pathway.

Islet transplantation is a treatment option for type I diabetic patients. Preservation of human pancreata prior to islet isolation using two-layer method with perfluorocarbon (PFC) and University of Wisconsin solution (UW) results in twofold increase in islet yields. The objective of this study was to determine the mechanism by which islets undergo apoptosis and determine PFC's effects on this process. Gene array analysis was used to analyze the expression of pro- and anti-apoptotic genes in islets isolated from pancreata preserved under varying conditions. A 12-fold increase in the expression of inhibitor of apoptosis (IAP) and survivin was observed in islets isolated from pancreata preserved in PFC. This was accompanied by decreased expression of BAD (3.7-fold), BAX (2.7-fold) and caspases (5.2-fold). Levels of activated caspase-9 (77.98%), caspase-2 (61.5%), caspase-3 (68.3%) and caspase-8 (37.2%) were also reduced. 'Rescue' of pancreata after storage (12 h) in UW by preservation using PFC also resulted in a down-regulation of pro-apoptotic genes and inhibition of caspase activation. Apoptosis observed in islets from all groups was mainly mitochondria-dependent, mediated by change in redox potential initiated by hypoxia. We demonstrate that reduction in hypoxia of pancreata preserved using PFC leads to significant up-regulation of anti-apoptotic and inhibition of pro-apoptotic genes.

Apoptosis↗
PubMed2003

Sepsis induces apoptosis and profound depletion of splenic interdigitating and follicular dendritic cells.

Dendritic cells are a phenotypically diverse group of APC that have unique capabilities to regulate the activity and survival of B and T cells. Although proper function of dendritic cells is essential to host control of invading pathogens, few studies have examined the impact of sepsis on dendritic cells. The purpose of this study was to determine the effect of sepsis on splenic interdigitating dendritic cells (IDCs) and follicular dendritic cells (FDCs) using a clinically relevant animal model. Immunohistochemical staining for FDCs showed that sepsis induced an initial marked expansion in FDCs that peaked at 36 h after onset. The FDCs expanded to fill the entire lymphoid zone otherwise occupied by B cells. Between 36 and 48 h after sepsis, there was a profound caspase 3 mediated apoptosis induced depletion of FDCs such that only a small contingent of cells remained. In contrast to the initial increase in FDCs, IDC numbers were decreased to approximately 50% of control by 12 h after onset of sepsis. IDC death occurred by caspase 3-mediated apoptosis. Such profound apoptosis induced loss of FDCs and IDCs may significantly compromise B and T cell function and impair the ability of the host to survive sepsis.

Animals↗
PubMed2005

Porcine circovirus type 2 (PCV2) causes apoptosis in experimentally inoculated BALB/c mice.

BACKGROUND: We have previously described microscopic and electron microscopic alterations in lymphoid organs of PCV2 inoculated mice as apoptosis. In this study we wanted to investigate the molecular pathogenetic mechanism of PCV2-induced apoptosis. Eight-week old BALB/c mice were either sham inoculated (control mice) or inoculated intraperitoneally (ip) and intranasally (in) with a single (sPCV mice) or multiple (mPCV mice) doses of PCV2. Four control mice and 4 sPCV mice were sacrificed 7, 14, 28 and 42 days post inoculation (PI). All 4 mPCV mice were sacrificed 42 days PI. Following necropsy, immunohistochemistry for caspase 3 and in-situ TUNEL assay were performed on sections of spleen, lymph nodes, thymus and ileum from control, sPCV and mPCV mice. In addition, total RNA was extracted from spleens of control, sPCV and mPCV mice for simultaneous detection and semiquantitation of bcl-2 homologues and various caspase mRNAs using a multiprobe RNase protection assay system. RESULTS: PCV2 replicated and was associated with apoptosis in spleens, lymph nodes and Peyer's patches of infected BALB/c mice. Upregulation of caspase 1, 2, 3, 6, 7, 8, 11 and 12 and upregulation for the transcripts of apoptosis inhibitors bcl-2, bcl-w and bcl-X and apoptosis promoters' bax, bak and bad was detected in spleens of sPCV and mPCV mice, but not control mice. Apoptosis was further confirmed by light and electron microscopic morphology as well as by positive TUNEL assay and detection of activated caspase 3. PCV2 nucleic acid was detected by in-situ hybridization in the nuclei and cytoplasm of such apoptotic cells. CONCLUSION: The data presented here support the hypothesis that PCV2 induces apoptosis mediated through the activation of caspases 8 and 3 in the spleens of infected mice.

Journal Article↗
PubMedDate not supplied

Diclofenac induces apoptosis in hepatocytes.

Hepatotoxicity is one of the side effects associated with the administration of diclofenac, a non-steroidal anti-inflammatory drug widely used clinically. The effect of diclofenac on the early events that trigger apoptosis cascade have been evaluated in rat hepatocytes. To do this, early and late apoptotic markers, associated with the pivotal steps of the execution phase, have been evaluated after incubation with the drug. The results show that the apoptotic effect of diclofenac occurs after exposure to sub-cytotoxic concentrations of the drug (maximal non toxic concentration, MNTC, after 24-h treatment was 450 microM), without overlapping with cell necrosis (LDH leakage evaluation). Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 3 activation (3-5-fold control) was maximal after 12 h of exposure to 350 microM of the drug. The involvement of the mitochondrial permeability transition (MPT) in diclofenac-induced apoptosis was investigated. Cyclosporine A and decylubiquinone, MPT specific inhibitor, prevented the activation of caspase 3, thus showing that diclofenac opened the MPT pore. Treatment of hepatocytes with antioxidants (alpha-tocopherol, N,N-dimethylthiourea, superoxide dismutase) were able to prevent caspase cascade activation by diclofenac, revealing that oxidative stress at the mitochondrial level is involved in MPT induction. Finally, the differential cytotoxic and apoptotic effect produced in hepatocytes and non-metabolizing hepatoma cells suggest that CYP-mediated metabolism of diclofenac apoptosis may be related to the apoptotic effect of the drug.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2004

Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosis.

Mcl-1 is an antiapoptotic member of the Bcl-2 family that can promote cell viability. We report here that Mcl-1 is a new substrate for caspases during induction of apoptosis. Mcl-1 cleavage occurs after Asp127 and Asp157 and generates four fragments of 24, 19, 17 and 12 kDa in both intact cells and in vitro, an effect prevented by selective caspase inhibitors. As a consequence, the resulting protein that lacks the first 127 or 157 amino acids contains only the BH1-BH3 domains of Bcl-2 family members. Mutation of Asp127 and Asp157 abolishes the generation of the 24 and 12 kDa fragments and that of the 19 and 17 kDa fragments, respectively. Interestingly, when expressed in HeLa cells Mcl-1 wt and Mcl-1 Delta127 showed a markedly different intracellular distribution. Mcl-1 wt colocalized with alpha-Tubulin near the internal face of the plasma membrane, while Mcl-1 Delta127 coassociated with Bim-EL at the mitochondrial level. Coimmunoprecipitation experiments also demonstrated that Mcl1 Delta127 exhibited increased binding to Bim when compared to Mcl-1 wt. Finally, Mcl-1 wt unlike Mcl-1 Delta127 inhibited Bim-EL-induced caspase activation. Altogether, our findings demonstrate that cleavage of Mcl-1 by caspases modifies its subcellular localization, increases its association with Bim and inhibits its antiapoptotic function.

Apoptosis↗
PubMed2004

The mycotoxin fumonisin B1 transiently activates nuclear factor-kappaB, tumor necrosis factor alpha and caspase 3 via protein kinase Calpha-dependent pathway in porcine renal epithelial cells.

Fumonisin B1 (FB1) is a toxic mycotoxin produced by Fusarium verticillioides, predominantly present in corn. The principal biochemical responses of FB1 involve disruption of sphingolipid metabolism from the inhibition of ceramide synthesis leading to accumulation of free sphingoid bases, particularly sphinganine. The ability of FB1 to modulate signal transduction pathways plays a role in its toxicity. We recently reported that FB1 selectively and transiently activates protein kinase Calpha (PKCalpha) in porcine renal epithelial cells (LLC-PK1). The aim of current study was to investigate the effect of PKCalpha activation by FB1 on NF-kappaB activation and subsequently on TNFalpha gene expression and caspase 3 induction in LLC-PK1 cells. FB1 (1 micromol/L for 5 min) transiently activated PKCalpha and increased nuclear translocation of NF-kappaB, followed by their down-regulation at later time points. Preincubating the cells with the PKC inhibitor, calphostin C, prevented the activation of NF-kappaB by FB1. TNFalpha mRNA expression was increased after 15 min exposure to FB1 or the PKC activator, phorbol 12-myristate 13-acetate. In addition, an increase in caspase 3 activity was observed after addition of FB1 for 1 h. Calphostin C prevented both the FB1-induced increase in TNFalpha expression and caspase 3 activation. In summary, we hereby demonstrate that the FB1 activation of NF-kappaB and sequential induction of TNFalpha expression resulting in the subsequent increase in caspase 3 activity are all dependent on PKCalpha stimulation in LLC-PK1 cells.

Animals↗
PubMed2001

Bovine herpesvirus 1 glycoprotein G is required for prevention of apoptosis and efficient viral growth in rabbit kidney cells.

In rabbit kidney (RK13) cells, gG-negative BHV-1 exhibited significant defects in plaque formation and growth compared to that of gG-positive BHV-1. RK13 cells infected with gG-negative BHV-1 exhibited a distinctive CPE and contained a larger number of cells stained with trypan blue dye compared to those infected with gG-positive strains, suggesting that gG-negative BHV-1 inflicted more damage to the infected cells than gG-positive BHV-1. Apoptotic cell death was induced in RK13 cells infected with gG-negative BHV-1 within 8 h. In contrast, the onset of apoptosis in gG-positive BHV-1-infected RK13 cells was around 12-16 h postinfection. In the presence of caspase inhibitor Z-Asp-CH2-DCB, multiplication of gG-negative minus BHV-1 was significantly increased. These results demonstrate that BHV-1 gG is involved in stabilizing the cell structure, postponing apoptotic process, and efficient BHV-1 replication in infected RK13 cells.

Animals↗
PubMed2006

Acetaldehyde promotes rapamycin-dependent activation of p70(S6K) and glucose uptake despite inhibition of Akt and mTOR in dopaminergic SH-SY5Y human neuroblastoma cells.

Alcohol intake is one of the important lifestyle factors for the risk of insulin resistance and type 2 diabetes. Acetaldehyde, the major ethanol metabolite which is far more reactive than ethanol, has been postulated to participate in alcohol-induced tissue injury although its direct impact on insulin signaling is unclear. This study was designed to examine the effect of acetaldehyde on glucose uptake and insulin signaling in human dopaminergic SH-SY5Y cells. Akt, mammalian target of rapamycin (mTOR), ribosomal-S6 kinase (p70(S6K)), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and insulin receptor substrate (IRS)-2 were evaluated by Western blot analysis. Glucose uptake and apoptosis were measured using [(3)H]-2-deoxyglucose uptake and caspase-3 assay, respectively. Short-term exposure (12 h) of acetaldehyde (150 muM) facilitated glucose uptake in a rapamycin-dependent manner without affecting apoptosis, IRS-2 expression and insulin-stimulated glucose uptake in SH-SY5Y cells. Acetaldehyde suppressed basal and insulin-stimulated Akt phosphorylation without affecting total Akt expression. Acetaldehyde inhibited mTOR phosphorylation without affecting total mTOR and insulin-elicited response on mTOR phosphorylation. Rapamycin, which inhibits mTOR leading to inactivation of p70(S6K), did not affect acetaldehyde-induced inhibition on phosphorylation of Akt and mTOR. Interestingly, acetaldehyde enhanced p70(S6K) activation and depressed 4E-BP1 phosphorylation, the effect of which was blunted and exaggerated, respectively, by rapamycin. Collectively, these data suggested that acetaldehyde did not adversely affect glucose uptake despite inhibition of insulin signaling cascade at the levels of Akt and mTOR, possibly due to presence of certain mechanism(s) responsible for enhanced p70(S6K) phosphorylation.

Acetaldehyde↗
PubMed2003

Recent insights on the mechanisms of liver preconditioning.

Ischemia/reperfusion is the main cause of hepatic damage consequent to temporary clamping of the hepatoduodenal ligament during liver surgery as well as graft failure after liver transplantation. In recent years, a number of animal studies have shown that pre-exposure of the liver to transient ischemia, hyperthermia, or mild oxidative stress increases the tolerance to reperfusion injury, a phenomenon known as hepatic preconditioning. The development of hepatic preconditioning can be differentiated into 2 phases. An immediate phase (early preconditioning) occurs within minutes and involves the direct modulation of energy supplies, pH regulation, Na(+) and Ca(2+) homeostasis, and caspase activation. The subsequent phase (late preconditioning) begins 12-24 hours after the stimulus and requires the synthesis of multiple stress-response proteins, including heat shock proteins HSP70, HSP27, and HSP32/heme oxygenase 1. Hepatic preconditioning is not limited to parenchymal cells but ameliorates sinusoidal perfusion, prevents postischemic neutrophil infiltration, and decreases the production of proinflammatory cytokines by Kupffer cells. This latter effect is important in improving systemic disorders associated with hepatic ischemia/reperfusion. The signals triggering hepatic preconditioning have been partially characterized, showing that adenosine, nitric oxide, and reactive oxygen species can activate multiple protein kinase cascades involving, among others, protein kinase C and p38 mitogen-activated protein kinase. These observations, along with preliminary studies in humans, give a rationale to perform clinical trials aimed at verifying the possible application of hepatic preconditioning in preventing ischemia/reperfusion injury during liver surgery.

Animals↗
PubMed2000

Rapid induction of apoptosis in human gastric cancer cell lines by sorbitol.

Most solid tumors, including gastric cancers, respond poorly to non-surgical treatments which are expected to induce an apoptosis-dependent involution. We hypothesize that the apoptotic machinery in solid tumors is either defective or in a suppressed condition. Overcoming the ineffective induction of apoptosis may improve the responsiveness of solid tumors to non-surgical treatments. Recently, sorbitol, a kind of hexose, has been found to be an effective inducer of apoptosis in HEp-2 cells. Therefore, it is of particular interest to examine the effect of sorbitol-treatment on gastric cancer cells. in the present study, we selected 4 gastric cancer cell lines which have been reported to exhibit different abilities in regard to apoptosis induction, and examined the effect of sorbitol-treatment on apoptosis induction. Within 3 hr after sorbitol-treatment, apoptosis was induced comparably in all cell lines examined. Cell death in MKN-1, MKN-28 or MKN-74 proceeded in a biphasic manner, while cell death in KATO-III was monophasic. The cell death partially depended on caspase activity. Treatments with sorbitol in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly suppressed the apoptotic cell death, suggesting a role of protein kinase-C-dependent process. To our knowledge, this is the most rapid induction of apoptosis in human gastric cancer cells reported to date.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Inactivation of the apoptosis effector Apaf-1 in malignant melanoma.

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.

Antineoplastic Agents↗