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.