Role of heat shock proteins in oxygen radical-induced gastric apoptosis.

BACKGROUND: The generation of reactive oxygen species (ROS) and their resultant oxidative damage is a common pathway for gastric mucosal injury. Developing strategies to protect the gastric epithelium against oxygen free radical damage is of profound pathophysiological interest. We have previously shown caspase-mediated apoptosis as a major cause of ROS-induced cell death in gastric mucosa. Because heat shock proteins (Hsps) confer protection against many cytotoxic agents, this study was undertaken to determine whether modulation of Hsps was protective against oxidative damage. MATERIALS AND METHODS: AGS cells (human gastric mucosal cell line) received either no pretreatment, heat shock pretreatment (1 h at 42 ± 1°C), or pretreatment with an Hsp modulating drug (geldanamycin or quercetin). Cells were then exposed to hydrogen peroxide (H2O2), a representative ROS (1 mM, a physiologically relevant concentration), for 24 h. Caspase-3 activation and Poly ADP Ribose Polymerase (PARP) inactivation, as well as DNA-histone complex formation were used as measures of apoptosis. Inducible Hsps (Hsp70 and Hsp90) were detected using Western blot analysis. RESULTS: Results showed heat shock pretreatment induced increased expression of Hsp70 without change in Hsp90. In response to H2O2 exposure alone, there was significant increase in DNA-histone complex formation as well as caspase-3 activation and PARP cleavage in gastric epithelium. Heat shock pretreatment resulted in statistically significant prevention in these measures of apoptosis. Geldanamycin increased Hsp70, but elicited cleavage of Hsp90 and subsequently resulted in an increase in H2O2-induced apoptosis. Quercetin decreased Hsp70 and resulted again in increased H2O2-induced apoptosis. CONCLUSIONS: These findings indicate that heat shock pretreatment protects gastric mucosal cells against H2O2-induced apoptosis and that Hsp70 and Hsp90 may play key roles in this process. These results further suggest that perturbations in Hsp metabolism may induce mucosal injury in response to oxygen free radicals.

Preconditioning with cobalt protoporphyrin protects human gastric mucosal cells from deoxycholate-induced apoptosis.

In this study, a known inducer of heme oxygenase-1 (HO-1) expression, cobalt protoporphyrin, and the introduction of a recombinant plasmid expressing HO-1 were examined for their ability to protect gastric epithelial cells from deoxycholate-induced injury. Physiologic levels of the secondary bile salt induce apoptosis in a human gastric adenocarcinoma mucosal cell line. Cobalt protoporphyrin induced expression of HO-1 protein with maximal levels attaining a plateau at 48 hours. Pretreatment with cobalt protoporphyrin before challenge with 200 µM deoxycholate inhibited cell death, DNA fragmentation, the appearance of cytosolic nucleosomes, and cleavage of caspase-3, caspase-9, and poly-(ADP-ribose) polymerase 1. Similarly, expression of HO-1 by introduction of a recombinant plasmid also showed a resistance to deoxycholate-induced apoptosis. These results implicate a possible role for HO-1 in modulating apoptosis in gastric epithelial cells.

Targeting PI3K/Akt/HSP90 signaling sensitizes gastric cancer cells to deoxycholate-induced apoptosis.

BACKGROUND: The heat shock protein 90 (HSP90) plays a crucial role in the stability of several proteins that are essential for cell survival and for malignant transformation. The binding of HSP90 with pro-survival kinase Akt prevents proteosomal degradation of Akt and contributes to the functional stabilization of PI3K/Akt signaling and cell survival. Akt kinase and HSP90 are therefore highly over-expressed in a large panel of cancer cell lines and are present in multi-chaperoning complexes. In this paper, we investigated whether targeting both Akt and HSP90 would inhibit the survival pathway in AGS cells (human gastric mucosal cells), and how Akt/HSP90 inhibition modulates the deoxycholate (DC)-induced apoptosis. METHODS: AGS cells in the presence of Akt inhibitors (LY294002 and wortmannin), or HSP90 inhibitor (geldanamycin, GA) for 30 min or 18 h, respectively, were treated with DC (50 µM). Activation of PI3K/Akt signaling was evaluated by measuring the Akt and PTEN phosphorylation. HSP90, caspase-3 and caspase-9 were detected in whole lysates by Western blot analysis. AGS cells, transiently transfected with Akt siRNA, were treated with DC, and apoptosis was measured by caspase-3 activation. Apoptotic-positive cells were counted according to changes of cell morphology by Hoechst staining and fluorescence microscopy. RESULTS: The intrinsic level of phospho-Akt (pAkt; active form), phospho-PTEN (pPTEN; inactive enzyme) and HSP90 were highly expressed in AGS cells indicating the active PI3K/Akt/HSP90 signaling. Although, deoxycholate at low concentration (50 µM) slightly inhibited the expression of pAkt and cleaved HSP90 to 55 KDa fragment, no significant effect on apoptosis induction, up to 4 h (as assessed by caspase-3 activation) was observed. The higher concentrations of DC (100 µM-300 µM) resulted in progressive inhibition of pAkt, activation of PTEN, and specific cleavage of HSP90 to approximately 45 KDa fragments with significant induction of apoptosis. Although DC (50 µM) had no profound effect on Akt/HSP90 and did not induce apoptosis, it became an inducer of apoptosis when cells were pretreated with LY294002, wortmannin, or geldanamycin. Consistent with these findings, significant activation of apoptosis in response to DC (50 µM) was observed in cells with depleted Akt protein. CONCLUSIONS: These results demonstrate that down-regulation of PI3K/Akt pathway with specific cleavage of HSP90 to 45 KDa modulates the pro-apoptotic effects of DC in gastric cells. They further indicate the importance of stable Akt/HSP90 complex in regulation of survival/death responses.

Aspirin-induced mucosal cell death in human gastric cells: role of a caspase-independent mechanism.

Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used for the treatment of pain and inflammation. Their use may result in gastroduodenal side effects, such as gastric irritation and ulcer formation. Although various strategies have been employed to minimize these adverse effects induced by NSAIDs, effective therapeutic targeting of this problem has been prevented by an incomplete understanding of the mechanisms underlying their pathogenesis. This study was undertaken to determine the role that non-caspase-mediated apoptosis plays in inducing cellular injury and death in gastric mucosa exposed to aspirin. We proposed that the responsible mechanism was through mitochondrial failure, increased mitochondrial membrane permeability, and translocation of the intramitochondrial protein apoptosis-inducing factor (AIF). Human gastric adenocarcinoma mucosal cells (AGS cells) received no pretreatment or were preincubated with caspase inhibitors for 30 min. Cells were then treated with 40 mM aspirin for 2-4 h. Apoptosis was assessed by measuring the DNA-histone complex formation. Cell viability was determined by an acridine orange-ethidium bromide (EtBr) assay. The activation of AIF was evaluated by both Western blotting of the cytosol and mitochondrial extracts as well as by visualization and staining using fluorescence microscopy. Results showed that caspase inhibitor preincubation decreased DNA-histone complex formation when compared to aspirin treatment alone. Based on light microscope visualization, however, we determined that caspase inhibitor preincubation was unable to prevent AGS cell damage and death. These findings were confirmed by the acridine orange-EtBr test, which showed decreased cell viability with caspase inhibitor preincubation and aspirin treatment. We then tested whether non-caspase-mediated cell death occurred through an AIF mitochondrial pathway using Western blotting and fluorescence microscopy to determine AIF activation. The results showed that untreated cells had AIF localized to the mitochondria and cytosol. With 40 mM ASA at 4 h, translocation of AIF from the mitochondria to the nucleus occurred, showing activation. Caspase inhibition with z-VAD was unable to prevent AIF localization to the nucleus and subsequently unable to prevent cell death. Our results indicate that ASA in the presence of caspase inhibitors causes gastric mucosal cell death through a caspase-independent pathway suggestive of apoptosis-like programmed cell death. Effective therapeutic targeting of aspirin-induced apoptosis likely requires inhibition of both mitochondrial and caspase-mediated pathways.

Oxygen radical induced gastric mucosal cell death: apoptosis or necrosis?

Reactive oxygen species (ROS) and resultant oxidative damage is a common pathway for gastric mucosal injury. This study was undertaken to determine whether apoptosis or necrosis was responsible for hydrogen peroxide (a representative ROS)-induced gastric mucosal death and whether caspase cascade blockade could prevent this process. AGS cells (human gastric adenocarcinoma cells) were exposed to hydrogen peroxide (H(2)O(2)), 0.5-2 mM, from 6 to 24 h. Lactic dehydrogenase (LDH) measured necrosis, whereas Caspase-3 and PARP activation and DNA-histone complex formation measured apoptosis. In addition, AGS cells received no pretreatment or preincubation for 1 h with 50-100 microM z-VAD, a pan-caspase inhibitor, and were then treated with 1-2 mM H(2)O(2). With high concentrations of H(2)O(2), cell death was predominantly necrotic, whereas lower concentrations evoked time and concentration dependent apoptosis. Furthermore, z-VAD pretreatment prevented oxidant induced apoptosis and necrosis. Since caspase cascade blockade prevents both processes, our results support the hypothesis that H(2)O(2) induced cell death is predominantly a caspase-mediated apoptosis.

Prevention of deoxycholate-induced gastric apoptosis by aspirin: roles of NF-kappaB and PKC signaling.

BACKGROUND: Apoptosis is a major mechanism of gastric cell death induced by deoxycholate (DC) and aspirin (ASA), and the caspase cascade and protein kinase C (PKC) signaling play key roles in this process. The transcription factor kappa B (NF-kappaB) has been shown to modulate apoptosis by regulating the transcription of numerous pro- and anti-apoptotic genes. The aim of this study was to investigate the effect of DC and ASA on NF-kappaB signaling, and determine its role in programmed cell death in a human gastric carcinoma cell line. METHODS: Cells were incubated with DC in the presence or absence of ASA or proteasome inhibitors (PI- I, lactacystin, and MG-132). Cell lysates were evaluated by Western blotting. NF-kappaB (p65) was measured in the cytosol and nuclear fractions. RESULTS: DC induced a translocation of NF-kappaB into the nuclear compartment that was completely blocked by proteasome inhibitors. Although, ASA itself had no effect on the NF-kappaB pathway, nor did it reduce DC-induced NF-kappaB translocation, it did prevent DC-induced caspase-3, -6 and -9 activation, poly (ADP-ribose) polymerase and lamin A processing, DNA degradation, and PKC signaling, all indices of apoptosis. In contrast, proteasome inhibitors had no effect on DC-induced apoptosis. CONCLUSIONS: Deoxycholate activates NF-kappaB at the same time that it induces apoptosis in gastric epithelial cells. Prevention of NF-kappaB activation does not alter DC-induced apoptosis, indicating that in our experimental conditions, NF-kappaB is not essential for apoptosis to proceed. In contrast, the ability of aspirin to restore the alterations in PKC isoforms induced by DC and at the same time prevent caspase cascade activation suggests the importance of the PKC signaling system in this process.

Aspirin-induced apoptosis in human gastric cancer epithelial cells: relationship with protein kinase C signaling.

This study examined the relationship of protein kinase C (PKC) signaling with apoptosis induced by aspirin (ASA) in gastric surface cancer cells (AGS cell line). We found increased expression of two PKC isoforms (alpha and betaII) that translocated from the cytosol into the cell membrane fraction after ASA (40 mM) stimulation. PKC betaI expression markedly decreased in response to ASA treatment. This process was independent of caspase activation because no caspase inhibitors used (i.e., inhibitors to caspase 3, 6, 7, 8, and total caspase activity) significantly changed PKC processing, although inhibition of caspase cascade activity markedly attenuated the apoptosis induced by ASA as measured by DNA-histone complex formation. Upstream PKC signaling induced by ASA seems to play an important role in the regulation of apoptosis because PKC inhibitors significantly reduced the magnitude of DNA-histone complex formation. We conclude that ASA-induced apoptosis in gastric cancer cells is mediated, at least in part, through a PKC mechanism involving the (alpha) and (beta) isoforms and that PKC signaling operates upstream of the caspase cascade, which when activated elicits its downstream effects on DNA degradation.

Protein kinase C involvement in deoxycholate-induced apoptosis in human gastric cells.

Bile acids, such as deoxycholic acid (DC), are known to mediate some of their actions by differentially activating various protein kinase C (PKC) isoforms. This study confirms that DC induces apoptosis in gastric epithelial cells through PARP and caspase cascade activation, and examined the role of PKC in DC-induced apoptosis. We found increased activation of PKC in membrane fractions in response to DC that was concentration and time related. The PKC (beta(I)) isoform expression increased with translocation into the cell membrane fraction after DC (300 microM) stimulation. In contrast, PKCepsilon expression markedly decreased in response to DC treatment in a time- and concentration-dependent manner. In addition, this process was regulated by caspases, since the pan-caspase inhibitor z-VAD-fmk and caspase-3-, -6-, and -9-specific inhibitors prevented PKC (beta(I)) and (epsilon epsilon processing induced by DC. Treatment with the caspase-8-specific inhibitor, however, did not affect expression of either PKC isoform. No significant differences in the apoptotic response were observed when PKC (epsilon) overexpressed cells were exposed to DC in the presence of calcium-dependent conventional PKC inhibitors (Gö 6850 or Gö 6976). Our findings demonstrate that PKC is activated in gastric epithelial cells treated with DC with the PKC (beta(I)) and PKC (epsilon) isoforms being particularly involved in this process. The processing of PKC (beta(I) and epsilon) was shown to be closely regulated by caspases; however, modulations in PKC isoform concentrations by themselves have no effect on the apoptotic death of gastric mucosal cells induced by DC.

Role of mitochondria in aspirin-induced apoptosis in human gastric epithelial cells.

This study was undertaken to determine whether the Bcl-2 family proteins and Smac are regulators of aspirin-mediated apoptosis in a gastric mucosal cell line known as AGS cells. Cells were incubated with varying concentrations of acetylsalicylic acid (ASA; 2-40 mM), with or without preincubation of caspase inhibitors. Apoptosis was characterized by Hoechst staining and DNA-histone-associated complex formation. Antiapoptotic Bcl-2, proapoptotic Bax and Bid, Smac, and cytochrome-c oxidase (COX IV) were analyzed by Western blot analyses from cytosol and mitochondrial fractions. ASA downregulated Bcl-2 protein expression and induced Bax translocation into the mitochondria and cleavage of Bid. In contrast, expression of Smac was significantly decreased in mitochondrial fractions of ASA-treated cells. Bax and Bid involvement in apoptosis regulation was dependent on caspase activation, because caspase-8 inhibition suppressed Bax translocation and Bid processing. Caspase-9 inhibition prevented Smac release from mitochondria. Additionally, increased expression of the oxidative phosphorylation enzyme COX IV was observed in mitochondrial fractions exposed to ASA at concentrations >5 mM. Although caspase-8 inhibition had no effect on aspirin-induced apoptosis and DNA-histone complex formation, caspase-9 inhibition significantly decreased both of these events. We conclude that Bcl-2 protein family members and Smac regulate the apoptotic pathway in a caspase-dependent manner. Our results indicate also that mitochondrial integration and oxidative phosphorylation play a critical role in the pathogenesis of apoptosis in human gastric epithelial cells.

Aspirin-induced mucosal cell death in human gastric cells: evidence supporting an apoptotic mechanism.

This study was undertaken to define the role that apoptosis may play in inducing cellular injury and death in gastric mucosa exposed to aspirin. Apoptosis was characterized by DNA gel electrophoresis, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and DNA-histone-associated complex formation. A human gastric cell line (AGS) was exposed to physiologic concentrations (3 to 50 mM) of aspirin. Both time- and concentration-dependent effects on apoptosis were noted, which were effectively prevented by the caspase inhibitor z-VAD-fmk. Accordingly, the role of caspases in aspirin-induced apoptosis was also evaluated. Early activation of caspase-8 and caspase-9 was demonstrated, indicating a role for both receptor and mitochondrial pathways, respectively, in the apoptotic process. Corresponding activation of effector caspases-3, -6, and -7 was also evident, as was cleavage of PARP. We conclude that physiologically relevant concentrations of aspirin induces apoptosis in human gastric cells through a caspase-mediated mechanism.

Isolation and characterization of the human tRNA-(N1G37) methyltransferase (TRM5) and comparison to the Escherichia coli TrmD protein.

A human TRM5 cDNA has been cloned and recombinant tRNA-N(1)G37 methyltransferase was produced. The recombinant enzyme methylates the N1 position of guanosine 37 (G37) in selected tRNA transcripts utilizing S-adenosyl methionine. The effects of RNA sequence and structure on the methylation reaction in comparison between the Escherichia coli TrmD and human TRM5 recombinant enzymes are presented. G37-methylation by TRM5 occurs regardless of the nature of the nucleotide at position 36. TRM5 also methylates inosine at position 37 unlike TrmD, which recognizes the G36pG37 motif preferentially and does not methylate inosine. New evidence is presented concerning TrmD showing that with some tRNA species, A at position 36 is also recognized. The TRM5 enzyme is sensitive to subtle changes in the tRNA-protein tertiary interaction leading to loss of activity. The TrmD enzyme is more tolerant of alterations in tRNA-protein tertiary interactions as long as the core tRNA structure and the G36pG37 are present. The TRM5 enzyme does not have an absolute requirement for magnesium ions, whereas TrmD requires magnesium to express activity. TRM5 demonstrates much higher affinity for substrates with K(m) values for tRNA that are nanomolar. TrmD has K(m) values for tRNA in the micromolar range. Recombinant TRM5 appears to function as a 60 772 Da monomer, while recombinant TrmD functions as a homodimer of 30 586 Da subunits. Bioinformatic analysis of the human TRM5 genomic locus (KIAA1393) have identified TRM5 homologues in eukaryotes and archaea; however, no significantly homologous regions were identified in any prokaryotes including the TrmD gene.

Insights into catalysis by a knotted TrmD tRNA methyltransferase.

The crystal structure of Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD) complexed with S-adenosyl homocysteine (AdoHcy) has been determined at 2.5A resolution. TrmD, which methylates G37 of tRNAs containing the sequence G36pG37, is a homo-dimer. Each monomer consists of a C-terminal domain connected by a flexible linker to an N-terminal AdoMet-binding domain. The two bound AdoHcy moieties are buried at the bottom of deep clefts. The dimer structure appears integral to the formation of the catalytic center of the enzyme and this arrangement strongly suggests that the anticodon loop of tRNA fits into one of these clefts for methyl transfer to occur. In addition, adjacent hydrophobic sites in the cleft delineate a defined pocket, which may accommodate the GpG sequence during catalysis. The dimer contains two deep trefoil peptide knots and a peptide loop extending from each knot embraces the AdoHcy adenine ring. Mutational analyses demonstrate that the knot is important for AdoMet binding and catalytic activity, and that the C-terminal domain is not only required for tRNA binding but plays a functional role in catalytic activity.

Apoptosis is a major mechanism of deoxycholate-induced gastric mucosal cell death.

This study was undertaken to determine whether necrosis or apoptosis was the predominant mechanism responsible for gastric mucosal cellular death using the cell line known as AGS cells. Cells were exposed to various concentrations of deoxycholate (DC; 50-500 muM) for periods ranging from 30 min to 24 h. Lactic dehydrogenase (LDH) activity was used as a marker for necrotic cell death, whereas apoptosis was characterized by 4',6-diamidino-2 phenylindole staining, DNA gel electrophoresis, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and DNA-histone-associated complex formation. When cells were bathed in Hank's balanced salt solution, DC-induced necrosis was the predominant mechanism of cell death. In contrast, when cells were bathed in Ham's F-12 solution (a more physiologically relevant medium), no evidence of cytotoxicity (by LDH assay) was discernible when cells were exposed to DC (50-300 muM) for periods as long as 8 h; instead, clear evidence of apoptosis was noted that was time and dose dependent. When cells were exposed for 24 h to these DC concentrations, cytotoxicity was also present, indicating necrosis as well. Furthermore, acidification of the ambient environment also evoked a necrotic response when exposed to DC. We demonstrated that apoptosis induced by DC shows early activation of caspase-3 that is dependent on both receptor and mitochondrial pathways. Our results indicate that physiological concentrations of DC (50-300 muM) primarily induce cellular death through an apoptotic process. Only after prolonged exposure to DC or acidification of the bathing solution does necrosis also occur.