Many types of human tumor express trypsinogen-2, which may be a significant factor in the activation of pro-MMPs and the invasiveness of tumors. Prevention of trypsinogen-2 expression in cancer cells might be of benefit in cancer therapy. We describe here chemicals capable of down-regulating the expression of trypsinogen-2. Doxycycline (DOXY) and chemically modified tetracyclines (CMTs), previously known as inhibitors of the matrix metalloproteinase (MMP)-dependent proteinase cascade, down-regulated the mRNA and protein expression of trypsinogen-2 by COLO-205 human colon adenocarcinoma cells at therapeutically attainable concentrations (0.1 to 1.0 M). DOXY specifically inhibited the activation of pro-MMP-9 and cell migration induced by enteropeptidase, a specific activator of trypsinogen. Pro-MMP-9 activation and cell migration were also inhibited by tumor-associated trypsin inhibitor (TATI), which is a highly specific inhibitor of trypsin. CMT-3 as well as CMT-5 also inhibited cell migration, but an effect on the enteropeptidase-enhanced activation of pro-MMP-9 was not observed. Matrix metalloproteinases (MMPs) and various serine proteinases act in concert to mediate cancer progression and metastasis (Mignatti and Rifkin, 1993;Sorsa et al., 1997;Mazzieri et al., 1997; Romas-DeSimone et al., 1999). One of the serine proteinases potentially playing a role in tumor invasion is tumor-associated trypsinogen. Two isoenzymes, trypsinogen-1 (cationic) and especially trypsinogen-2 (anionic), are produced by several human tumors, such as ovarian carcinoma (Stenman, 1990) and cholangiocarcinoma (Terada et al., 1995), and by many tumor cell lines derived from other organs (Koivunen et al., 1991b). The production of trypsinogens by human gastric cancer cells correlates with their malignant phenotype, and over-expression of trypsinogen-1 by the cells increases their tumorigenicity in nude mice Miyata et al., 1998).Human trypsin-2 efficiently activates progelatinases, particularly pro-MMP-9 at very low concentrations . It is thus possible that even low concentrations of trypsin may suffice to initiate a proteinase cascade facilitating tumor invason. Expression of trypsinogen has also been observed in endothelial cells (Koshikawa et al., 1997), but whether trypsin plays a role in tumor-induced angiogenesis is unclear.Factors capable of modulating trypsinogen expression are of value for clarifying the biological function of trypsinogen. Doxycycline (DOXY) and chemically modified tetracyclines (CMTs) are chemical inhibitors of MMPs. They inhibit MMPs by mechanisms that are distinct from their anti-microbial activity (Golub et al., 1992). CMTs inhibit melanoma and prostate cancer cell invasion and metastasis (Seftor et al., 1998;Lokeshwar, 1999). We have studied whether CMTs and DOXY also down-regulate the expression of trypsinogen-2 and how they affect migration of COLO-205 colon adenocarcinoma cells.
MATERIAL AND METHODS
CMTsThe anti-microbial tetracycline DOXY and the non-anti-microbial CMT-1, CMT-3, CMT-5 and CMT-8 were provid...
