To clarify the molecular mechanisms of the formation of peritoneal dissemination, a highly metastatic cell line, named MKN-45-P was established from gastric cancer cell line of MKN-45 by repeat intraperitoneal inoculation of free cancer cells in ascites. Intraperitoneal inoculation of 1O^7 cells of this cell line can reproduce peritoneal dissemination with bloody ascites. Screening of metastasis related genes, which are expressed from this cell line was performed using specific primers. Among more than 30 metastasis related genes, MKN-45-P expressed MMP-7, MMP-l1, urokinase type plasminogen activator, and its receptor, MTl-MMP, integrin alpha2, alpha3, betal, c-erbB-2 , c-met, mst-l, autoctine motility factor and its receptor. Among these gene products. MMP-7, integrin alpha2, alpha3, beta1, and c-erbB-2 are expressed in a large amount, as the metastatic potential increases. In the in vitro experiments, anti integrin antibodies against integrin alpha2, alpha3, beta1 inhibited the adhes
ion and invasion abilities of this cell line. Furthermore, when 1O^7 cells of this cell line after treatment of anti-integrin monoclonal antibody for 48 hours were intraperitoneally inoculated in nude mice, the survival of mice of Mab treated group was significantly longer than that of non treated group.
Antisense oligonucleotides of 16 mer DNA containing initiation codon of AUG of c-met, and AMFR mRNA completely suppressed the invasion ability of MKN-45-P through Matrigel coated chamber. Furthermore, antisense oligonucleotides against MMP-7 was tried whether this S-oligo. can inhibit the invasion and proliferation activity of MKN-45-P or not. Even by treating 10 muM of the antisense DNA for 48 hours, proliferative activity of MKN-45-P was not suppressed. In contrast, invasion ability of MKN-45-P was completely blocked by this antisense DNA.However, antisense DNA against MMP-7 did not improve the survival of nude mice bearing MKN-45-P, inoculated intraperitoneally.
MKN-45-P also expressed c-ets -l gene, and this gene product (Ets-l) is a transcription factor of several metastasis associated genes, such as MMP-l, -3, -9, MTl-MMP, urokinase, integrin subunites, and c-erbB-2. By treatment of MKN-45-P with antisense DNA against c-ets -l mRNA, expressions of urokinase, MMP-l , integrin a2, cx.3 and MMP-7 were dramatically reduced. As a results, adhesion and invasion ability of MKN-45-P were completely inhibited after treatment of antisense DNA agains c-ets -l mRNA.
In addition, the antisense DNA improved the survival of nude mice bearing intraperitoneal MKN45-P.From these results, this highly metastatic cell line acquires the overexpression of multiple metastasis related genes, such as adhesion molecules, motility factors and matrix digesting enzymes. The control of c-ets-1 gene may suppress several metastasis related genes simultaneously , and the strategy to control c-ets-I mRNA may be a hopeful one to treat peritoneal dissemination. Less