Promotion of hematogenous metastatic potentials in human KB carcinoma cells with overexpression of cyclooxygenase-2

Abstract

Abstract.To understand the role of cyclooxygenase (COX)-2 in metastatic potential of oral cancer, COX-2 overexpressing KB/COX-2 cells were inoculated orthotopically into the masseter muscle or injected into the left cardiac ventricle of nude mice. KB/COX-2 showed about 4-fold increase of COX-2 protein expression as compared to KB/Neo which was a mock transfected control. In orthotopic inoculation, metastasis to the regional lymph nodes occurred in 2 out of 15 mice, and metastasis to the lung in 3 out of 15 mice. On the other hand, in intra-cardiac injection, hematogenous metastasis to the lung and bone occurred in 8 out of 10 mice in KB/COX-2, but no metastasis occurred except for only one metastasis to the femur bone out of 10 mice in KB/Neo. Treatment of KB/COX-2 with COX-2 small interfering RNA (siRNA) inhibited the colony formation but not cell growth in vitro, and suppressed tumorigenicity and hematogenous metastasis in nude mice. When expression of adhesion molecules such as E-cadherin, ·-catenin, ß-catenin and CD44 was examined, there was no difference in ·-and ß-catenin between the cells. However, expression of E-cadherin was detected in KB/Neo, but not in KB/COX-2. In contrast, expression of CD44 was markedly increased in KB/COX-2 as compared to KB/Neo. Treatment with COX-2 siRNA resulted in suppression of CD44 expression and detectable expression of E-cadherin in KB/COX-2. These findings suggested that overexpression of COX-2 increased hematogenous metastasis, at least in KB cells, via down-regulating E-cadherin and up-regulating CD44 expression. IntroductionCyclooxygenase (COX)-2 is an enzyme induced by mitogens, cytokines and growth factors of epithelial cells and plays an important role in prostaglandin (PG) production. Overexpression of COX-2 has been reported in a variety of cancers (1-4) including head and neck cancer (5-8). Recently, a human colon cancer cell line transfected with a COX-2 expression vector acquired increased invasiveness and metastatic potential with activation of matrix metalloproteinase (MMP) (9). A correlation between COX-2 overexpression and hematogenous metastasis of colorectal cancer was also demonstrated (10). These findings suggest that COX-2 overexpression is involved in colon cancer metastasis. In head and neck cancer, however, the significance of COX-2 overexpression in metastasis is poorly understood.We have already shown that KB/COX-2, which was established by stable transfection with full length COX-2 cDNA to human KB carcinoma cells, showed elevated PGE 2 production, cell migration and invasion as compared to mock transfected control, KB/Neo. Furthermore, it was found that KB/COX-2 increased tumorigenicity, tumor growth and local tumor invasion in nude mice. These effects were apparently modulated by up-regulation of MMPs and Rho family small guanosine triphosphatases (GTPases) and down-regulation of tissue inhibitor of metalloproteinase (TIMP) activities (11).The aim of this study was to investigate the metastatic potential in nude mice using KB/COX-2. In additio...