Thomas A. Miller

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.

Prostaglandins Protect Human Intestinal Cells Against Ethanol Injury by Stabilizing Microtubules (Role of Protein Kinase C and Enhanced Calcium Efflux)

Prostaglandins (PG) protect gastrointestinalcells against damage induced by ethanol (EtOH) and othernoxious agents, a process termed cytoprotection. Thepresent study investigated the relationships between microtubule (MT) stability, protein kinase C(PKC) activation, and calcium efflux as a possiblemechanism of PGs protective action using a humancolonic cell line (Caco-2) exposed to known damagingconcentrations of EtOH (7.5% and 10% ). Preincubation ofCaco-2 cells with 16,16-dimethyl-PGE2 (PG,2.6 μM) significantly increased PKC activity in thesecells. Pretreatment of Caco-2 cells with 50 μM OAG (asynthetic diacylglycerol and PKC activator) or 30 nM TPA(a direct PKC activator) prior to exposure to 7.5% or10% EtOH for 5 min significantly reduced cell injury, asdetermined by trypan blue exclusion, and increased MT stability, as confirmed by confocalmicroscopy. Pretreatment of Caco-2 cells with 4alpha-PDD (an inactive phorbol ester, 20 nM) failed toprevent cell injury and disruption of the MTcytoskeleton. Preincubation with staurosporine (a PKC inhibitor, 3 nM)abolished the protective effects of PG in cells exposedto 7.5% and 10% EtOH. Incubation of Caco-2 cells withA23187 (a Ca2+ ionophore), similar to 10%EtOH, caused a significant reduction in cell viability andMT stability. Preincubation with A23187 in combinationwith PG or OAG prior to subsequent exposure to EtOHsignificantly abolished the protective effects of PG or OAG pretreatment. Finally, pretreatmentwith OAG, TPA, or PG resulted in significant increasesin calcium-45 efflux, which correlated with increasedstability of the MT cytoskeleton. These data suggest that PG possesses direct protective effectsagainst EtOH injury in Caco-2 cells and may act bystabilizing MT through the PKC signal transductionpathway and/or stimulation of calcium efflux from thecells.

Role of calcium homeostasis in gastric mucosal injury and protection

Using a human gastric mucosal cell line, known as AGS cells, we determined the role that perturbations in intracellular Ca2+ concentration [Ca2+]i might play in cellular injury induced by various damaging agents. For deoxycholate (CD) and ethanol (EtOH) induced damage, a concentration related increase in [Ca2+]i was noted that preceded and closely paralleled the magnitude of injury. Thus, the higher the concentration of DC or EtOH, the more profound were the changes in [Ca2+]i and the resultant degree of cellular injury. Pretreatment with a low concentration of DC (50 microM; called a mild irritant) that was not damaging by itself attenuated injury induced by a damaging concentration (i.e. 250 microM) of DC, and appeared to elicit this protective action through mechanisms that resisted intracellular Ca2+ accumulation. Additional studies indicated that the mechanism of aspirin damage may be similar and that other protective agents such as prostaglandins and growth factors appear to mediate their protective properties through prevention of intracellular Ca2+ alterations. We propose that agents that prevent mucosal injury mediate this activity through a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.

Targeting PI3K/Akt/HSP90 Signaling Sensitizes Gastric Cancer Cells to Deoxycholate-Induced Apoptosis

BackgroundThe 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.MethodsAGS cells in the presence of Akt inhibitors (LY294002 and wortmannin), or HSP90 inhibitor (geldanamycin, GA) for 30xa0min or 18xa0h, respectively, were treated with DC (50xa0µM). 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.ResultsThe 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 (50xa0µM) slightly inhibited the expression of pAkt and cleaved HSP90 to 55xa0KDa fragment, no significant effect on apoptosis induction, up to 4xa0h (as assessed by caspase-3 activation) was observed. The higher concentrations of DC (100xa0µM-300xa0µM) 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 (50xa0µM) 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 (50xa0µM) was observed in cells with depleted Akt protein.ConclusionsThese results demonstrate that down-regulation of PI3K/Akt pathway with specific cleavage of HSP90 to 45xa0KDa 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 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 (α and βII) that translocated from the cytosol into the cell membrane fraction after ASA (40xa0mM) stimulation. PKC βI 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 (α) and (β) isoforms and that PKC signaling operates upstream of the caspase cascade, which when activated elicits its downstream effects on DNA degradation.

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 (H2O2), 0.5–2xa0mM, from 6 to 24xa0h. 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 1xa0h with 50–100xa0μM z-VAD, a pan-caspase inhibitor, and were then treated with 1–2xa0mM H2O2. With high concentrations of H2O2, 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 H2O2 induced cell death is predominantly a caspase-mediated apoptosis.

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 (βI) isoform expression increased with translocation into the cell membrane fraction after DC (300 μ M) stimulation. In contrast, PKCε 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 (βI) and (ε ɛ 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 (ɛ) 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 (βI) and PKC (ɛ) isoforms being particularly involved in this process. The processing of PKC (βI and ε) 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.

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 30xa0min. Cells were then treated with 40xa0mM aspirin for 2–4xa0h. 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 40xa0mM ASA at 4xa0h, 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.

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

BACKGROUNDnThe 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.nnnMATERIALS AND METHODSnAGS cells (human gastric mucosal cell line) received either no pretreatment, heat shock pretreatment (1xa0h at 42xa0±xa01°C), or pretreatment with an Hsp modulating drug (geldanamycin or quercetin). Cells were then exposed to hydrogen peroxide (H2O2), a representative ROS (1xa0mM, a physiologically relevant concentration), for 24xa0h. 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.nnnRESULTSnResults 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.nnnCONCLUSIONSnThese 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.