Treatment of human peritoneal metastasis in gastric cancer remains a major clinical problem [
19]. Metastasis is associated with the process of cancer cells from a primary site invading surrounding tissues [
20,
21]. Here, we clearly show that blockade of the u-PA with amiloride suppresses the development of peritoneal metastasis in gastric cancer.
It is reported that amiloride, an inhibitor of Na
+ transport, competitively inhibits the catalytic activity of u-PA, without decreasing those of t-PA, or plasmin [
22]. Thus, amiloride may be a useful model compound for the development of u-PA-specific protease inhibitors [
23]. u-PA is a pivotal proteolytic kinase known to regulate the process of cancer metastasis [
24] . Lokman
et al. have observed that u-PA knockdown could inhibit the proliferation of cancer cells in peritoneal metastasis [
25]. Jankun
et al. reported that treatment of amiloride could not only reduce the size of prostate cancer xenografts in severe combined immunodeficient mice, but also help survival [
26]. To further study the inhibition effects of u-PA on gastric cancer peritoneal metastasis, we investigated the effects and explored the anti-tumor mechanisms of amiloride, a selective u-PA inhibitor, on a panel of gastric cancer cell lines and in a mouse model of human gastric cancer MKN45. Previous research found that amiloride participated in the transcriptional and post-transcriptional regulation of u-PA gene expression in colon cancer cells [
27]. Ogura
et al. also agreed that amiloride played a specific role in inhibiting u-PA activity [
28]. We analyzed the effects of amiloride on mRNA and protein production and activity of u-PA in MKN45 gastric cancer cells
in vitro. In accordance to previous reports, the results showed that amiloride not only decreased both mRNA and protein production of u-PA in MKN45 gastric cancer cells, but also reduced the u-PA activity of MKN45 cell line. Amiloride inhibited MKN45-derived tumor growth and prolonged the survival of the tumor-bearing mice. Consequently, it might be concluded that the inhibition of u-PA by amiloride could suppress peritoneal metastasis in gastric cancer. Furthermore, activation of Na+/H+ exchange activity is found as a ubiquitous response to early growth factors, such as u-PA [
29]. uPA/uPAR-mediated tumor progression and metastasis requires Na+/H+ exchange [
30]. Amiloride is reported as a Na+/H+ exchange inhibitor to inhibit cancer cell invasion and motility [
31‐
33]. Taken together, it is believable that amiloride may first inhibit u-PA expression and then affect Na+/H+ exchange activity, ultimately resulting in suppression of peritoneal metastasis in gastric cancer.
In a recent study on breast cancer cells
in vitro, Tuck
et al. had found that the same as anti-u-PA antibody and anti-u-PAR antibody, amiloride significantly inhibited migration and invasion of breast cancer cells [
34]. Evans
et al. observed that oral amiloride inhibited lung metastasis in pulmonary metastasis in the rat mammary cancer model and this effect was positively correlated with time- and dose-dependence [
35]. In SCID mice subcutaneously injected with prostate cancer cells, JanKun
et al. found that oral amiloride could potently inhibit tumor growth and prolong the survival of tumor bearing mice [
26]. Evans
et al. reported that amiloride could dose- and time-dependently inhibit cancer cell metastasis [
36]. In the adhesion test between MKN45 cells and mesothelial cells, we found the inhibition of adhesion by amiloride was correlated with inhibition of the growth of MKN45 cells. In addition, at the time point of 24 h, 0.01 mM amiloride had lost the inhibitive role of adhesion, which might be related to the inhibition of u-PA activity caused by long-time stimulation of amiloride at a low concentration.