Hitoshi Sawaoka

Cyclooxygenase Regulates Angiogenesis Induced by Colon Cancer Cells

To explore the role of cyclooxygenase (COX) in endothelial cell migration and angiogenesis, we have used two in vitro model systems involving coculture of endothelial cells with colon carcinoma cells. COX-2-overexpressing cells produce prostaglandins, proangiogenic factors, and stimulate both endothelial migration and tube formation, while control cells have little activity. The effect is inhibited by antibodies to combinations of angiogenic factors, by NS-398 (a selective COX-2 inhibitor), and by aspirin. NS-398 does not inhibit production of angiogenic factors or angiogenesis induced by COX-2-negative cells. Treatment of endothelial cells with aspirin or a COX-1 antisense oligonucleotide inhibits COX-1 activity/expression and suppresses tube formation. Cyclooxygenase regulates colon carcinoma-induced angiogenesis by two mechanisms: COX-2 can modulate production of angiogenic factors by colon cancer cells, while COX-1 regulates angiogenesis in endothelial cells.

Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma.

Objective:Recent epidemiological studies indicate that there is reduced risk of all digestive carcinomas in patients who regularly take nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin. Cyclooxygenase (COX) is a target enzyme for NSAIDs. We investigated the role of two isoforms, COX-1 and COX-2, in the development and metastasis of gastric carcinoma.Methods:Fifteen gastric carcinoma tissue specimens and accompanying adjacent mucosa specimens were obtained from surgical resections. COX-1 and COX-2 protein expression were evaluated using Western blotting analysis, and their relative band densities were semi quantified using standard densitometry scanning techniques.Results:Compared with paired noncancerous specimens, COX-2 was overexpressed in 10 of 15 carcinoma tissue specimens (66.7%). Overall, COX-2 levels in carcinoma tissue were significantly higher. Two early carcinomas (confined to the mucosa and submucosa) and three of 13 advanced carcinomas (extended below the submucosa into the muscular wall) had weak or similar COX-2 expression in paired tissue specimens. COX-2 overexpression in tumors significantly correlated with tumor invasion into the lymphatic vessels in the gastric wall and metastasis to the lymph nodes. Furthermore, the stage grouping in the TNM classification significantly correlated with COX-2 overexpression. In contrast, COX-2 overexpression did not correlate with histopathological grading, surface size, and venous vessel invasion of the tumors. COX-1 levels were similar between paired tissues.Conclusion:COX-2 overexpression might enhance lymphatic invasion and metastasis in patients with gastric carcinoma, implicating a poor prognosis. Therefore, the use of COX-2–specific inhibitor to suppress lymphatic metastasis in humans should be investigated.

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.

Helicobacter pylori infection induces cyclooxygenase-2 expression in human gastric mucosa

Recent studies indicate that expression of mitogen-inducible cyclooxygenase-2 (COX-2) occurs in gastrointestinal tumors. We investigated the effects of Helicobacter pylori (H. pylori) infection, a class I carcinogen for the human stomach, on gastric COX-2 expression using immunohistochemistry. Human subjects without macroscopic lesions, as determined by endoscopic screening, were biopsied for H. pylori infection. The biopsy samples were immunohistochemically examined for COX-2 expression. COX-2 was expressed in gastric epithelia and subepithelial inflammatory cells in all H. pylori-infected subjects. There was no expression of COX-2 in the gastric mucosa of H. pylori-negative subjects. COX-2 expression has been reported in gastrointestinal carcinomas, gastrointestinal cancer cell-lines, and in the gut after carcinogenic treatment. The present study demonstrates that H. pylori infection leads to gastric mucosal expression of COX-2, indicating that the enzyme is involved in H. pylori-related gastric pathology in humans.

Effects of NSAIDs on proliferation of gastric cancer cells in vitro : Possible implication of cyclooxygenase-2 in cancer development

The roles of cyclooxygenase-2 (COX-2) in the development of gastric cancer are unknown. We investigated the effects of nonsteroidal antiinflammatory drugs (NSAIDs), which are specific and nonspecific inhibitors of COX-2, on proliferation of the gastric cancer cell lines KATOIII, MKN28, and MKN45. The protein level of COX-2 was examined in these cell lines by Western analysis, and mRNA levels of COX-1/2 by Northern analysis. These cell lines expressed comparable levels of COX-1 mRNA. However, mRNA and protein expression of COX-2 in these cell lines was different. MKN45 expressed higher levels of COX-2 mRNA and protein than KATOIII and MKN28. We also examined the effects of NS-398 and indomethacin, specific and nonspecific inhibitors of COX-2, on the increase in cell number and [3H]thymidine uptake of these cell lines. NS-398 and indomethacin suppressed proliferation of MKN45 cells that overexpressed COX-2, although they exerted minimal effects on proliferation of KATOIII and MKN28, which expressed lower levels of COX-2. These results are consistent with the hypothesis that COX-2 is expressed in certain groups of gastric cancers and is related to their cell proliferation. It was proposed that COX-2 plays an important role in development of gastric cancer cells. Furthermore, NSAIDs may exert antiproliferative activity against gastric adenocarcinomas that overexpress COX-2.

Expression of the cyclooxygenase-2 gene in gastric epithelium.

This study examined the mRNA level of cyclooxygenase-2 (cox-2) in a rat gastric mucosal cell line after growth stimulation in vitro and in rat gastric mucosa before and after acid-induced injury in vivo. RGMI cells were stimulated with fetal calf serum in the in vitro study. Thereafter, the cox-2 mRNA level was examined by Northern analysis. Effects of NS-398, a specific inhibitor of cox-2, and indomethacin on prostaglandin production and cell proliferation were examined in RGM1 cells. In the in vivo study, rats were given 1 ml of 0.6 N hydrochloric acid into the stomach. The level of cox-2 mRNA was examined in rat gastric mucosa after acid administration. Cox-2 mRNA increased 20-60 min after growth stimulation in RGM1 cells. Both NS-398 and indomethacin suppressed prostaglandin production and cell proliferation after growth stimulation. Expression of cox-2 mRNA was observed 40 and 60 min after administration of 0.6 N HCl. Gastric lesions developed within 60 min after HCl administration and healed significantly within 48 h. The present study shows the expression of cox-2 in gastric epithelial cells in vitro and in gastric mucosa after injury in vivo. The results also suggest that cox-2 is involved in de novo synthesis of prostaglandins and cell proliferation in gastric epithelium.

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.

Cyclo-oxygenase-2 inhibitors suppress epithelial cell kinetics and delay gastric wound healing in rats

Background and Aims : The present study examined the effects of NS‐398, a specific cyclo‐oxygenase‐2 inhibitor, on gastric mucosal cell kinetics and gastric wound healing following acid‐induced injury.