Shingo Tsuji

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.

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.

Does Cyclooxygenase-2 Down-Regulate Gastric Inflammation?

This paper updates concepts on the role of cyclooxygenase (COX) in tissue inflammation. An inducible isoform of cyclooxygenase is expressed in various cell types by inflammatory promoters, mitogens, cytokines, their receptors, and pathogenic factors such as Helicobacter. Overexpressed COX-2 has been believed to serve as a proinflammatory enzyme that increases prostaglandin production and causes swelling, pain, and hyperemia. Recent data indicate that certain prostaglandins suppress immune responses and inflammatory cell infiltration. To test the hypothesis that COX-2 acts as antiinflammatory enzyme in a certain aspect, we examined the effects of specific and nonspecific COX-2 inhibitors on gastric mucosa. The results demonstrate that COX-2 inhibition activates inflammatory cell infiltration. Thus, the inducible isoform of cyclooxygenase may have antiinflammatory properties in the stomach.

Roles of Reactive Nitrogen Species in Helicobacter-Related Carcinogenesis

Although data indicate an association between Helicobacter infection and gastric carcinogenesis, the precise mechanism for Helicobacter-related carcinogenesis are not yet clarified. An extract of Helicobacter stimulated inflammatory production of nitric oxide (NO) and other reactive nitrogen species. Nitrotyrosine, an adduct of active nitrogen species, increased in gastric epithelia in subjects with the infection. Studies show that reactive nitrogen species cause point mutations at CpG sites; and that such mutations in the p53 gene are frequently found in cancerous and precancerous lesions in the stomach. Thus, reactive nitrogen species including inflammatory NO might play important roles in Helicobacter-induced gastric carcinogenesis.

Flow cytometric analysis of cell proliferation kinetics during duodenal ulcer healing

Cell proliferation in the gastroduodenal mucosa of patients with duodenal ulcers was evaluated using flow cytometry. Forty patients with duodenal ulcers and 12 normal subjects were investigated. Biopsy samples were obtained during endoscopic examination and subjected to DNA analysis by flow cytometry. Thirty patients with duodenal ulcers were healed within 3 months with H2 blockers (tractable or responsive ulcers), whereas 10 patients did not respond to treatment (intractable ulcers). The percentage of cells at the DNA‐synthetic phase, an index of cell proliferation, was constant in the adjacent duodenal mucosa 2cm from ulcer margin and antral mucosa during duodenal ulcer healing. The index at the margin of tractable ulcers was elevated during the active stage (12.9 ± 1.3), peaked during the healing stage (15.4 ± 2.8) and returned to the same level at the scarring stage (10.9 ± 2.0) as normal controls (10.3 ± 1.7). However, the index was not elevated in intractable ulcers (10.3 ± 1.7 in the healing stage) and was smaller than in tractable ulcers. These data indicate that augmented mucosal cell proliferation at the ulcer margin plays an important role in duodenal ulcer healing and intractable ulcers are characterized by an abnormal failure to accelerate DNA synthesis to achieve ulcer repair.