Yoshiyuki Takei

Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice

To clarify the role of mitogen-inducible cyclooxygenase (COX-2) in the development of malignant tumors, we investigated the effects of COX-2 inhibitors on the growth of gastric cancer xenografts in nude mice in vivo. MKN45 gastric cancer cells (5 x 10(6) cells/animal) that overexpress COX-2 were inoculated subcutaneously into athymic mice. NS-398, a specific COX-2 inhibitor, or indomethacin, a nonspecific COX-2 inhibitor, was administered orally to animals every day for 20 days. These drugs reduced the tumor volume significantly. Immunohistochemistry using bromodeoxyuridine, nick end labeling, and electron microscopy showed that NS-398 induced apoptosis in cancer cells in a dose-dependent manner and inhibited cancer cell replication slightly. Indomethacin also induced apoptosis and suppressed replication of tumor cells. There was a significant negative correlation between tumor volume and apoptotic cell number within the tumor. These results are consistent with the hypothesis that COX-2 inhibitors suppress growth of gastric cancer xenografts mainly by inducing apoptosis and suppressing replication of the neoplastic cells. It follows that COX-2 plays an important role in the development of gastric cancer.

RGM1, A CELL LINE DERIVED FROM NORMAL GASTRIC MUCOSA OF RAT

The epithelium of gastric mucosa undergoes constant renewal. However, the mechanisms of regulation of epithelial cell proliferation and differentiation in the gastric mucosa are poorly understood. Many investigators have used primary cultures of gastric mucosal cells to investigate cell physiology of gastric mucosal epithelium (5,8,14). Because this technique resulted in mixed cell populations of the stomach (5,15), the results may be influenced by complex cell-tocell interactions and are difficult to interpret. For this reason, a cell line made of a single type of cells is more ideal for precise investigation of proliferation or differentiation of gastric mucosal cells. However, cell lines from normal gastric mucosa have not been established. Recently, Matsui and Ohno (in preparation) established a new cell line, RGM1 (rat gastric mucosal cell first), from a rat glandular stomach by a method modified from Matsuoka et ah (7). In brief, the stomach was harvested from an anesthetized Wistar rat aged 4 wk and inverted. After washing the mucosa with phosphate-buffered saline (PBS) at 4 ~ C, the inverted pouch was immersed in a 0.2% pronase E solution at 37 ~ C. The solution was changed every 15 rain and centrifuged to collect the exfoliated gastric cells. Thereafter, the c.ells were washed twice with PBS, and cultivated in a 1:1 mixture of Dulbeccos modified Eagles medium (DMEM) and Hams F-12 medium supplemented with 20% fetal calf serum (FCS). When the cells were passaged to the 10th generation, this cell line was named RGM1. We examined character and usefulness of RGM1 cells using 3 0 4 0 times passaged cells. RGM1 cells are homogeneous epitheliallike cells with large oval nuclei and a polygonal shape (Fig. 1 A). They grow as monolayers of closely opposed cells, and many granules are visible in the cytoplasm. These cells were stained with periodic acid-Schiff (PAS) reagent and were negative for Bowie staining for chief cells (3) and succinic dehydrogenase activity for parietal cells (9). The cells were positive for cytokeratin in cytoplasm by immunohistochemistry. Transmission electron microscopy demonstrated that RGM1 cells had some microvilli on the surface. In the cytoplasm, a few homogeneous, dense cytoplasmic granules, presumably secretory granules, were observed. Junctional complexes were seen at the cell boundaries (Fig. 1 B and C). These data indicate that RGM 1 cells are epithelial in origin and like gastric mucous epithelial cells or mucous neck cells. RGM1 cells grew in an exponential manner with a population doubling time of 15.7 h, with saturation density of 1.97 + 0.38 X

Evidences for involvement of cyclooxygenase-2 in proliferation of two gastrointestinal cancer cell lines.

Cyclooxygenase (COX) consists of two isozymes, COX-1 and COX-2. The roles of these isozymes in the gastrointestinal tract are unknown. We investigated messenger RNA expression of the COX-1 and COX-2 genes in the gastrointestinal cancer cell lines MKN28, MKN45, KATO III CACO-2, DLD-1 and LoVo. These cell lines expressed comparable levels of COX-1 mRNA, although their expression of COX-2 varied. Therefore, we studied the effects of NS-398 and indomethacin, specific and non-specific inhibitors for COX-2, on proliferation of the cell lines. Both of the inhibitors suppressed proliferation of the two cell lines that highly expressed COX-2 (MKN45 and CACO-2). However, these inhibitors exerted minimal effects on proliferation of the other cell lines, which expressed significantly lower levels of COX-2. Therefore, it was proposed that COX-2 participates in proliferation of cancer cells because of over expression of the COX-2 gene.

Liposome‐encapsulated dichloromethylene diphosphonate induces macrophage apoptosis in vivo and in vitro

Dichloromethylene diphosphonate (MDPCl2) encapsulated in multilamellar liposomes was selectively incorporated by macrophages, immediately transferred to lysosomes, then released from liposomes into lysosomes by enzymatic digestion of the liposomal lipid layers. From 4 h after ingesting liposome‐encapsulated MDPCl2 murine macrophages in vivo and in vitro acquired the ultrastructural features of apoptosis, such as condensed nuclear chromatin, nuclear fragmentation, cell shrinkage, and blebbing of the plasma membrane. Murine peritoneal macrophages and isolated rat Kupffer cells incubated in the medium containing liposome‐encapsulated MDPCl2 increased DNA fragmentation in a dose‐dependent manner. Electrophoretic analysis of extracted DNA from the isolated Kupffer cells showed DNA fragmentation. Another diphosphonate, Alendronate (4‐amino‐1‐hydroxybutylidene‐1,1‐diphosphonate) had less potent macrophage cytotoxicity. However, MDPCl2, Alendronate, and gadolinium chloride in solution were not cytotoxic to macrophages. These results implied that the intralysosomal accumulation of MDPCl2 generates signals to induce macrophage apoptosis. J. Leukoc. Biol. 60: 337–344; 1996.

Endogenous Nitric Oxide Modulates Ethanol-Induced Gastric Mucosal Injury in Rats

BACKGROUND/AIMS Endothelium-derived relaxing factor regulates vascular tone via vasodilation. The relative contribution of endogenous nitric oxide to the pathophysiology of ethanol-induced gastric mucosal microcirculatory disturbances was investigated in anesthetized rats. METHODS Macroscopic and microscopic gastric mucosal damage and gastric mucosal hemodynamics including blood flow and hemoglobin oxygen saturation (ISO2) were assessed by pretreatment with a specific NO synthase inhibitor, N omega-nitro-L-arginine (L-NNA), before and after intragastric administration of ethanol. RESULTS Pretreatment with L-NNA significantly increased macroscopic (7.7-fold) and microscopic damage caused by 30% ethanol. Concurrent administration of L-arginine, but not D-arginine, significantly reduced the increase in mucosal damage. Similar results were obtained with 60% ethanol. Pretreatment with L-NNA decreased both mucosal blood flow and ISO2 in the basal period and enhanced decreases in both mucosal blood flow (2.7-fold) and ISO2 (4.3-fold) induced by 30% ethanol compared with controls. Concurrent administration of L-arginine, but not D-arginine, significantly inhibited the effect of L-NNA on blood flow and ISO2 in the basal period as well as after intragastric administration of 30% ethanol. CONCLUSIONS Endogenous NO modulates ethanol-induced gastric mucosal injury through the regulation of gastric mucosal microcirculation.

Role of endothelin 1 in hemorrhagic shock-induced gastric mucosal injury in rats

BACKGROUND/AIMS Gastric microcirculatory disturbances are involved in the pathogenesis of stress ulcers; however, vasomodulators regulating this process are not fully understood. This study was conducted to investigate the role of endothelin 1 (ET-1) in hemorrhagic shock-induced gastric mucosal damage in rats. METHODS ET-1 contents in plasma and gastric mucosa were measured and gastric mucosal damage was evaluated during a control period, 60 minutes of ischemia, 15 minutes of reperfusion, and 30 minutes of postreperfusion. Next, effects of BQ-123, an endothelinA receptor antagonist, on the gastric mucosal damage and hemodynamics were studied. RESULTS Both plasma and mucosal ET-1 significantly increased after ischemia and reperfusion compared with the control values, but only mucosal ET-1 continued to increase after reperfusion, leading to the development of gastric mucosal damage. BQ-123, administered just before reperfusion, reduced mucosal damage in the postreperfusion period dose-dependently and improved mean gastric mucosal blood flow and mucosal hemoglobin oxygen saturation during the 30-minute postreperfusion period. CONCLUSIONS These results suggest that endogenous ET-1 plays an important role in the pathogenesis of hemorrhage shock-induced gastric mucosal damage through impairment of mucosal microcirculation. Further, endothelinA antagonists may have therapeutic benefits for shock-induced gastric mucosal damage.

Helicobacter pylori extract stimulates inflammatory nitric oxide production

This study examined whether an extract of Helicobacter pylori had the ability to stimulate an inflammatory synthesis of nitric oxide, a mutagen and precursor of nitrosocompounds. Macrophages and neutrophils were prepared from rat and incubated with the Helicobacter pylori extract. L-Arginine-dependent nitric oxide production in these cells was significantly stimulated by the co-incubation with the Helicobacter pylori extract. This ability of the extract was strongly attenuated by protease digestion or heating. These results indicate that Helicobacter pylori induces production of nitric oxide and participates in development of gastritis and gastric carcinogenesis.

Ethanol-induced disturbance of hepatic microcirculation and hepatic hypoxia.

The hypothesis was tested whether ingestion of ethanol might disturb the hepatic microcirculation with resulting hepatic hypoxia. Infusion of ethanol increased the portal pressure concentration-dependently in rat livers perfused with Krebs-Henseleit buffer at a constant flow rate (Emax = 11.5 cm H2O, EC50 = 90 mM). This increase in portal pressure was due to hepatic vasoconstriction, since it diminished in the presence of sodium nitroprusside, a direct acting vasodilator. The regional hepatic tissue hemoglobin concentration after perfusion with added erythrocyte suspension (hematocrit 1%), measured by tissue-reflectance spectrophotometry, was significantly diminished by the infusion of ethanol, indicating the impairment of the microcirculation of the superficial layer of the liver. When the absorption spectrum of the liver was examined by reflectance spectrophotometry, infusion of ethanol caused a parallel reduction of all the mitochondrial respiratory cytochromes in a concentration-dependent fashion, concomitant with the increase of portal pressure, indicating a marked reduction of oxygen concentration in superficial liver tissue. The reduction of the respiratory cytochromes was also associated with the decrease in oxygen consumption of the liver, indicating that the hepatic hypoxia was due to the reduction of oxygen delivery to hepatocytes rather than the increased oxygen consumption of the liver. The reduction of the respiratory cytochromes was correlated with the increase in portal pressure and was inhibited by sodium nitroprusside. These data indicate that the ethanol-induced hepatic vasoconstriction disturbs hepatic microcirculation, resulting in hepatic hypoxia and reduction of mitochondrial respiratory cytochromes.

Protection by Carolina rinse solution, acidotic pH, and glycine against lethal reperfusion injury to sinusoidal endothelial cells of rat livers stored for transplantation

The critical injury causing graft failure after prolonged liver storage involves reperfusion-induced killing of sinusoidal endothelial cells and activation of Kupffer cells. Treatment of stored livers with Carolina rinse solution (CRS) prevents endothelial cell killing, reduces Kupffer cell activation, and improves graft survival. Accordingly, our aim was to evaluate the components of CRS and other agents for protection against reperfusion injury to rat livers stored 24 hr in University of Wisconsin solution. CRS virtually abolished endothelial cell killing, prevented denudation of the sinusoidal lining, and decreased structural changes in Kupffer cells indicative of activation. The only component of CRS preventing endothelial cell killing was acidic pH of 6.5. However, when pH was subsequently increased to 7.4, antioxidants (allopurinol, deferoxamine mesylate, and glutathione), vasodilators (adenosine and nicardipine), and possibly energy substrates (fructose, glucose, and insulin) partially blocked pH-dependent cell killing (pH paradox). Na+/H+ exchange inhibition, protease inhibition, and Ca(2+)-free buffer did not decrease reperfusion injury, but the amino acid glycine protected strongly. Strychnine, which binds to glycine receptors in the central nervous system, protected equally well. Protection by glycine and CRS was synergistic, virtually.

Effects of loxoprofen sodium, a newly synthesized non‐steroidal anti‐inflammatory drug, and indomethacin on gastric mucosal haemodynamics in the human

Abstract Non‐steroidal anti‐inflammatory drugs (NSAID) are, and have been, frequently used for alleviation of pain in patients; however, they are known to cause gastric mucosal injury in experimental animals and in humans. A decrease in the gastric mucosal blood flow also plays an important role in the aetiology of acute gastric mucosal injury, as we previously reported. This study investigated the effect of a newly synthesized NSAID, loxoprofen sodium (sodium 2[p‐2 oxocyclopentylmethyl) phenyl]propionate dihydrate, on gastric mucosal haemodynamics using a reflectance spectrophotometry system. Both single and cross‐over methods were used in five volunteer subjects. Loxoprofen sodium 60 mg (one tablet) or indomethacin 25 mg (one tablet), was diluted in 10 mL water at 25°C and sprayed on the gastric mucosa via a polyethylene tube inserted into the biopsy channel of an endoscope. After drug administration, reflectance spectra were taken every 5 min for 30 min. The indices of mucosal haemoglobin content (IHb) and oxygen saturation of haemoglobin (ISO2) were determined by the method previously reported by the authors. Indomethacin administration produced a significant decrease in both IHb and ISO2 values, indication ischaemia. Loxoprofen sodium, however, showed no significant differences in either of the parameters. Haemorrhagic erosions were evident after indomethacin administration, but none were found after loxoprofen sodium administration. The conclusion reached on the basis of this evidence is that one‐time topical application of loxoprofen sodium is safer than indomethacin.