The antitumorigenic activity of nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase (COX) inhibitors, is well established, but responsible molecular mechanisms are not fully understood. NSAIDs stimulate apoptosis by COX dependent and independent mechanisms in colorectal cells in culture. Identification of genes regulated by COX inhibitors could lead to a better understanding of their proapoptotic and anti-neoplastic activities. Using subtractive hybridization, a cDNA which was designated as NSAID activated gene (NAG-1) was identified from NSAID-treated HCT-116, human colorectal cells. NAG-1 has an identical sequence with a novel member of the TGF-beta superfamily that has 5 different names. In the HCT-116 cells, NAG-1 expression is increased and apoptosis is induced by treatment with some NSAIDs in a concentration and time-dependent manner. NAG-1 transfected cells exhibited increased basal apoptosis, increased response to NSAIDs and reduced soft agar cloning efficiency. Furthermore, transplantable tumors derived from NAG-1 transfected HCT-116 cells showed reduced tumorigenicity in athymic nude mice compared with vector-transfected HCT-116 cells. The increased NAG-1 expression by NSAIDs provides a suitable explanation for COX-independent apoptotic effects of NSAIDs in cultured cells. These data demonstrate that NAG-1 is an antitumorigenic and proapoptotic protein, and its regulation by COX inhibitors may provide new clues for explaining their proapoptotic and antitumorigenic activities.

The neutrophil NADPH-oxidase can be activated by protein kinase C (PKC) agonists such as phorbol myristate acetate (PMA), resulting in superoxide anion release. This superoxide release is independent of phosphatidylinositol 3-kinase (PI 3-kinase) because the inhibitor wortmannin does not affect the response. In this study, PMA is shown to also induce a wortmannin-sensitive NADPHoxidase activation, however, not resulting in release of superoxide but in intracellular production of the radical. This indicates that two pools of NADPH-oxidase, one localized in the plasma membrane and the other in the granule membranes, are separately regulated and the signal transduction pathways leading to activation of these pools differ regarding involvement of PI 3-kinase. Activation of both pools was dependent on ERK/MAPK kinase (MEK) activity and protein phosphatase 1 and/or 2A. As the two oxidase responses were differently affected by the inhibitor Gö -6850, different PKC isozymes are suggested to take part in the two signal transduction pathways. J. Leukoc. Biol. 67: 396-404; 2000.

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The effect of overexpression of 15-lipoxygenase-1 (15-LO-1) was studied in the human prostate cancer cell line, PC-3. Stable PC-3 cell lines were generated by transfection with 15-LO-1-sense (15-LOS), 15-LO-1-antisense (15-LOAS) or vector (Zeo) and selection with Zeocin. After characterization by RT-PCR, western and HPLC, a PC3-15LOS clone was selected that possessed 10-fold 15-LO-1 enzyme activity compared with parental PC-3 cells. The PC3-15LOAS clone displayed little or no 15-LO-1 activity. These PC-3 cell lines were characterized for properties of tumorigenesis. The proliferation rates of the cell lines were as follows: PC3-15LOS > PC-3 = PC3-Zeo > PC3-15LOAS. Addition of a specific 15-LO-1 inhibitor, PD146176, caused a dose-dependent inhibition of proliferation in vitro. Overexpression of 15-LO-1 also caused [(3)H]thymidine incorporation to increase by 4.0-fold (P < 0.01). Compared with parental and PC-3-Zeo cells, PC3-15LOS enhanced whereas PC3-15LOAS reduced the ability of PC-3 cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. These data suggested a pro-tumorigenic role for 15-LO-1 in PC-3 cells in vitro. Therefore, to clarify the role of 15-LO-1 in vivo, the effect of 15-LO-1 expression on the growth of tumors in nude mice was investigated. The PC-3 cell lines were inoculated subcutaneously into athymic nude mice. The frequency of tumor formation was increased and the sizes of the tumors formed were much larger in the PC3-15LOS compared with PC3-15LOAS, parental PC-3 and PC-3-Zeo cells. Immunohistochemistry for 15-LO-1 confirmed expression throughout the duration of the experiment. The expression of factor VIII, an angiogenesis marker, in tumor sections was increased in tumors derived from PC3-15LOS cells and decreased in those from PC3-15LOAS cells compared with tumors from parental or Zeo cells. These data further supported the evaluation by ELISA of vascular endothelial growth factor (VEGF) secretion by PC-3 cells in culture. Secretion of this angiogenic factor was elevated in PC3-15LOS cells compared with the other cell lines. These results support a role for 15-LO-1 in a novel growth-promoting pathway in the prostate.

Garlic is appealing as an anti-carcinogenic agent due to its ability to induce apoptosis in vitro and inhibit the formation and growth of tumors in animals in vivo. Diallyl disulfide (DADS) is a constituent of garlic that suppresses neoplastic cell growth and induces apoptosis. We examined the effects of DADS on various cancer cell lines to better understand its effect on apoptosis and apoptosis-related genes. The nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) has proapoptotic and antitumorigenic activities and is upregulated by anticancer agents such as NSAIDs. In this study, human colorectal HCT-116 (wild-type p53), HCT-15 (p53 mutant) and human prostate PC-3 (p53 mutant) cells were exposed to DADS. DADS inhibited cell proliferation in all cell lines albeit to a lesser extent in HCT-15 and PC-3 cells at 11.5 and 23 micromol/L. In HCT-116 cells, DADS induced p53 and NAG-1 in a dose-dependent manner and the induction of p53 preceded that of NAG-1. In HCT-116 cells, NAG-1 protein expression was increased 2.4-fold +/- 0.6 at 4.6 micromol/L and 6.1-fold +/- 1.7 at 23 micromol/L DADS, whereas p53 was induced 1.5-fold +/- 0.1 and 2.3-fold +/- 0.4. DADS did not induce NAG-1 or p53 in p53 mutant cell lines; however, NAG-1 expression was induced by sulindac sulfide. HCT-116 cells treated with 4.6 and 23 micromol/L DADS resulted in a 1.9- and 2.9-fold increase in apoptosis, respectively. In contrast, 23 micromol/L DADS induced apoptosis only 1.8-fold in HCT-15 cells and not at all in PC-3 cells. Thus, DADS-induced apoptosis and NAG-1 protein expression appear to occur via p53.

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