A hyaluronan synthase suppressor, 4‐methylumbelliferone, inhibits the tumor invasion associated with N‐cadherin decreasement

To the Editor: 4-Methylumbelliferone (MU) suppresses a hyaluronan (HA) synthase and inhibits HA synthesis in cultured human skin fibroblasts. In malignant melanoma, MU suppresses cell adhesion and locomotion, and inhibits liver metastasis in vivo. These findings indicate that MU inhibits tumor cell invasion, probably by modulating adhesion molecules. However, little is known about the involvement of these proteins in the effects of MU treatment. The cadherins are a family of transmembrane receptors that function in promoting calcium-dependent cell–cell adhesion. The main subtypes of classical cadherins are E-cadherin and N-cadherin. Loss of E-cadherin accompanied by expression of N-cadherin, i.e. the epithelial-mesenchymal transition (EMT), is thought to play a fundamental role during the steps of invasion and metastasis of tumor cells. In particular, N-cadherin plays an important role in tumor cell invasion. Here, we investigated the MU induced N-cadherin alteration of a HA producing lymphoma cell line, OHK, by real time polymerase chain reaction (PCR), immunohistochemical and immunoblotting analyses. The primers of real time PCR used were F:5′tgcacagatgtggacaggattg3′ and R:5′atcccggcgtttcatccata3′ (GenBank accession no. NM 001792). The antibodies against N-cadherin, clone 13A9 (Upstate, Lake Placid, NY, USA) and clone EPR1791-4 (Epitomics, Burlingame, CA, USA) were used for immunohistochemistry and immunoblotting, respectively. The invasion assay was performed using a cell invasion assay kit (Chemicon, Temecula, CA, USA). Statistical analyses of differences among values were performed with Student’s t-test, and values of P < 0.05 were considered to indicate statistical significance. Quiescent subconfluent cells were washed with phosphate-buffered saline and then incubated in the presence of 1 mM MU (Wako Pure Chemical Industries, Osaka, Japan) for 1,3,5,7 or 9 days. Untreated cells were examined as controls. Real time PCR analysis showed that N-cadherin expression was suppressed at days 7 and 9 of MU treatment (Fig. 1a). Immunohistochemically, N-cadherin was present on the cell membrane of control cells, including the cell–cell contact sites. Its expression was decreased and specifically localized at cell–cell contact sites at days 7 and 9 of MU treated cells (Fig. 1b). By immunoblotting analyses, N-cadherin was decreased after day 5 of MU treatment (Fig. 1c). The translation disturbance of N-cadherin may be present at day 5. Cells treated with MU exhibited reduced invasion by more than 70%, compared with the control cells at day 7 (P < 0.05, Fig. 2). 4-Methylumbelliferone suppresses HA synthesis, and inhibits the invasion and metastasis of tumor cells. The mechanisms involved in the inhibition of metastasis are thought to be that: (i) HA-poor matrices reduce tumor cell migration and infrequently lead to metastasis; (ii) MU suppresses tumor cell adhesion and locomotion; and (iii) MU reduces matrix metalloproteinase-9 activation. In the present study, we found that MU decreases the N-cadherin. The Eto N-cadherin transition, which is seen in the EMT, has been implicated in the progression of malignant cells. In particular, N-cadherin is correlated with motility, invasiveness and poor prognosis in many kinds of cancers. The adhesion of tumor cells to stromal fibroblasts, smooth muscle cells and vascular endothelial cells through N-cadherin homophilic interactions seems to induce cell invasion. Also in hematopoietic cells, the interactions could underlie tumor cell frequent invasion. N-cadherin reduction by MU may be involved in the suppression of tumor invasion through inhibiting the interaction between tumor cells and stromal cells. Although the mechanisms by which MU decreases N-cadherin are unclear, HA concentration may be associated with cadherin alteration as seen in the cutaneous spongiosis formation. N-cadherin has recently been considered as a potential therapeutic target in cancer, and a peptide N-cadherin antagonist, ADH-1, has been developed. In addition to the N-cadherin antagonist, MU may have potential as an anticancer drug by mediating a decrease in N-cadherin expression.