The machinery of programmed cell death.

Apoptosis or programmed cell death is an essential physiological process that plays a critical role in development and tissue homeostasis. However, apoptosis is also involved in a wide range of pathological conditions. Apoptotic cells may be characterized by specific morphological and biochemical changes, including cell shrinkage, chromatin condensation, and internucleosomal cleavage of genomic DNA. At the molecular level, apoptosis is tightly regulated and is mainly orchestrated by the activation of the aspartate-specific cysteine protease (caspase) cascade. There are two main pathways leading to the activation of caspases. The first of these depends upon the participation of mitochondria (receptor-independent) and the second involves the interaction of a death receptor with its ligand. Pro- and anti-apoptotic members of the Bcl-2 family regulate the mitochondrial pathway. Cellular stress induces pro-apoptotic Bcl-2 family members to translocate from the cytosol to the mitochondria, where they induce the release of cytochrome c, while the anti-apoptotic Bcl-2 proteins work to prevent cytochrome c release from mitochondria, and thereby preserve cell survival. Once in the cytoplasm, cytochrome c catalyzes the oligomerization of apoptotic protease activating factor-1, thereby promoting the activation of procaspase-9, which then activates procaspase-3. Alternatively, ligation of death receptors, like the tumor necrosis factor receptor-1 and the Fas receptor, causes the activation of procaspase-8. The mature caspase may now either directly activate procaspase-3 or cleave the pro-apoptotic Bcl-2 homology 3-only protein Bid, which then subsequently induces cytochrome c release. Nevertheless, the end result of either pathway is caspase activation and the cleavage of specific cellular substrates, resulting in the morphological and biochemical changes associated with the apoptotic phenotype.

How cells die: apoptosis pathways.

Generally speaking, there are 2 types of cell death: apoptosis and necrosis. Necrotic cell death is considered an accidental type of death, caused by gross cell injury, and results in the death of groups of cells within a tissue. In contrast, apoptotic cell death may be induced or is preprogrammed into the cell (eg, during development) and results in the death of the individual cells. Apoptotic cells may be characterized by specific morphologic and biochemical changes orchestrated by a family of cysteine proteases known as caspases. At the molecular level, apoptosis is tightly regulated. There are 2 main pathways to apoptotic cell death. One involves the interaction of a death receptor, such as the TNF receptor-1 or the Fas receptor with its ligand, and the second pathway depends on the participation of mitochondria. Proapoptotic and antiapoptotic members of the Bcl-2 family regulate the mitochondrial pathway. The end result of either pathway is caspase activation and the cleavage of specific cellular substrates, resulting in the morphologic and biochemical changes associated with the apoptotic phenotype.

Aspirin induces apoptosis through release of cytochrome c from mitochondria.

Nonsteroidal anti-inflammatory drugs (NSAID) reduce the risk for cancer, due to their antiproliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and mitochondrial release of cytochrome into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

Cyclooxygenase-2 expression in human esophageal carcinoma.

On the basis of epidemiological observations that nonsteroidal antiinflammatory drugs reduce the risk of esophageal carcinoma, we studied the expression of cyclooxygenase-2 (COX-2) in esophageal squamous cell carcinomas (SCCs; n = 172) and in esophageal adenocarcinomas (ADCs; n = 27). Using immunohistochemistry, we observed COX-2 expression in 91% of the SCCs and in 78% of the ADCs. Western blot analysis showed enhanced expression of the COX-2 protein in some tumors as compared with normal esophageal squamous epithelium, whereas similar amounts of the COX-1 protein were found in normal and cancerous tissues. COX expression was also studied in two esophageal cancer cell lines (OSC-1 and OSC-2) to evaluate the functional relevance of COX-2-derived prostaglandins (PGs). OSC-2 cells expressed COX-2 but not COX-1, whereas OSC-1 cells expressed high levels of COX-1 but showed only a very weak COX-2 expression. Accordingly, PGE2 synthesis was 600 times higher in the OSC-2 cells as compared with the OSC-1 cells. Treatment of OSC-2 cells with the selective COX-2 inhibitors flosulide and NS-398 concentration dependently suppressed PGE2 synthesis and proliferation and also induced apoptosis. In contrast, no effect of the COX-2 inhibitors was seen in OSC-1 cells. Our data demonstrate that COX-2 is expressed in the majority of esophageal SCCs and ADCs and that COX-2-derived PGs play an important role in the regulation of proliferation and apoptosis of esophageal tumor cells. It is concluded that inhibition of COX-2 may be useful in the therapy of esophageal cancer.

Augmented myocardial ischaemia by nicotine--mechanisms and their possible significance.

1. To study the effect of nicotine on the severity of experimental myocardial ischaemia, Langendorff hearts of rabbits (n=7-12 per group) were subjected to 2 h of low-flow ischaemia followed by 1 h of reperfusion. 2. Infusion of nicotine (100 ng ml(-1)) caused only minor changes in non-ischaemic conditions but a significant (P<0.05) increase in end-diastolic pressure (LVEDP), loss of creatine kinase (CK) and troponin (TnT) as well as increase in noradrenaline (NA) overflow in reperfused ischaemic hearts. 3. RT PCR was done on total RNA for mRNA expression of the constitutive (COX-1) and inducible cyclooxygenase (COX-2). There was no COX-2 in non-ischaemic hearts but a significant expression in ischaemia (n=5) which was further increased by nicotine. These data were confirmed at the protein level by Western blotting and additionally shown that COX-1 remained unchanged. 4. There was a marked increase in prostacyclin (PGI2) and a 2 fold increase in NA overflow which were both stimulated by nicotine. 5. The aggravating effects of nicotine on myocardial ischaemia (CK release) as well as the expression of COX-2 mRNA were prevented by pretreatment with the beta-blocker pindolol (1 microM). 6. The data demonstrate marked deleterious actions of nicotine in reperfused ischaemic hearts. These actions are probably related to the increase in catecholamine overflow, are beta-receptor-mediated and involve enhanced gene expression of COX-2.

Constitutive cyclooxygenase-2 expression in healthy human and rabbit gastric mucosa.

Selective cyclooxygenase (COX)-2 inhibitors are expected to cause fewer gastric side effects because of sparing of COX-1-dependent prostaglandin (PG) synthesis in the gastric mucosa. However, the possible contribution of COX-2 to overall gastric PG biosynthesis is not known. This study demonstrates constitutive expression of COX-2 mRNA and protein in apparently healthy human and rabbit gastric mucosa. This basal expression of COX-2 protein in human gastric mucosa was increased by lipopolysaccharide and phorbol ester, indicating its up-regulation in response to appropriate stimuli. The functional significance of COX-2-dependent PG formation was studied in terms of PGE2 generation in the rabbit mucosa and its inhibition by the COX-2-selective inhibitor flosulide. There was concentration-dependent (IC50 = 107 +/- 55 nM) and ultimately complete inhibition of PGE2 generation by flosulide. In addition, gastric mucosa generated 15-hydroxyeicosatetraenoic acid upon treatment with acetylsalicylic acid. The data suggest an important role for COX-2-dependent PG production in apparently healthy gastric mucosa and raise the issue of whether selective COX-2 inhibitors might also interfere with physiological PG formation and actions in the stomach.