Prostaglandins (PGs) are known to play important roles in the proliferation of various types of cancer cells. PGs are produced by the action of cyclooxygenase (COX) enzymes, and two forms of COX, COX-1 and COX-2, have been described. Previous studies have demonstrated that overexpression of COX-2 is associated with colon carcinogenesis, tumor invasion and metastatic potential of colon cancer. In this study, the role of COX-2 on proliferation of squamous cell carcinoma cell lines was investigated. NS-398, a selective COX-2 inhibitor, inhibited proliferation of NA cells, a squamous cell caricinoma cell line that constitutively expresses COX-2 mRNA. NS-398 suppressed the spontaneous production of PGE2 by NA cells, and the antiproliferative effect of NS-398 was abolished by addition of PGE2. Similar results were obtained from experiments using COX-2 antisense oligonucleotide. These results suggest that specific inhibition of COX-2 inhibits proliferation of cancer cells expressing COX-2 mRNA via suppression of PGE2 production.

Cisplatin (CDDP) and 5-fluorouracil (5-FU) are common anti-tumour agents, and the anti-tumour effect of CDDP and 5-FU are synergistically enhanced by combined treatment. To clarify the mechanisms of this synergism, we examined the effect of CDDP and 5-FU on the expression of cell adhesion molecules involved in recognition of cancer cells by T lymphocytes. When NA cells, a squamous cell carcinoma cell line, were exposed to CDDP and 5-FU for 18 h, the expression of intercellular adhesion molecule-1 (ICAM-1) was synergistically induced, whereas CDDP or 5-FU alone did not induce the expression of ICAM-1, as determined by flow cytometry. Expression of ICAM-2 and ICAM-3, which are recognized by the same counter receptor on T-cells, were not up-regulated by CDDP and 5-FU. RT-PCR analysis showed that the induction of ICAM-1 on NA cells might be due to transcriptional induction of ICAM-1 mRNA. Treatment with genistein, a protein tyrosine kinase (PTK) inhibitor, inhibited the induction of ICAM-1 on NA cells by CDDP and 5-FU, whereas staurosporin, a protein kinase C inhibitor, did not. Although CDDP and 5-FU induced binding at the nuclear factor kappa B (NF-κB) site in the ICAM-1 promoter, pretreatment with genistein did not prevent CDDP and 5-FU-induced binding at the NF-κB site. Moreover, a NF-κB nuclear translocation inhibitor did not inhibit the induction of ICAM-1 expression by treatment with CDDP and 5-FU. The synergistic effect of CDDP and 5-FU was not specific to NA cells, since ICAM-1 was synergistically induced by CDDP and 5-FU on HSC-4 cells, a squamous cell carcinoma cell line. These findings indicate that treatment with CDDP and 5-FU induces ICAM-1 expression by a NF-κB independent regulatory mechanism involving PTK. © 1999 Cancer Research Campaign

Cyclooxygenase-2 is the rate-limiting enzyme in synthesis of prostaglandins and other eicosanoids. Prior reports have shown that inhibition of cyclooxygenase-2 activity, either by selective inhibitors or by antisense oligonucleotide, results in suppression of growth of squamous cell carcinoma cell lines which express high cyclooxygenase-2 levels, such as NA, a cell line established from a squamous cell carcinoma of the tongue. To investigate the mechanisms by which cyclooxygenase-2 inhibitors suppressed growth of these cells, the effects of NS-398, the selective cyclooxygenase-2 inhibitor, on cell-cycle distribution were examined. NS-398 induced G0/G1 cell-cycle arrest in NA cells which expressed cyclooxygenase-2. G0/G1 arrest induced by NS-398 was accompanied by up-regulation of cyclin-dependent kinase inhibitor p21, but not by up-regulation of the other cyclin-dependent kinase inhibitors. Transfection with p21 antisense oligonucleotide inhibited cell-cycle arrest induced by NS-398. Accumulation in G0/G1 was also observed in NA cells transfected with cyclooxygenase-2 antisense oligonucleotide. On the other hand, NS-398-treated NA cells showed a loss of plasma membrane asymmetry, a marker of early events in apoptosis. However, NS-398 did not induce other morphological and biochemical changes related to apoptotic cell death. These results suggest that cyclooxygenase-2 inhibitor induces G0/G1 cell-cycle arrest in NA cells by upregulation of p21. Our results also suggest that NS-398 is not sufficient to complete the whole process of apoptosis in NA cells, although it induces an early event in apoptosis.

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