Background: Previous studies showed that lysophosphatidic acid (LPA) promotes proMMP-2 activation and MMP-dependent invasion in epithelial ovarian cancer (EOC) cells. Recently, we found that matrilysin (MMP-7) is over-expressed in EOC cell lines and surgical tissue specimens and it is able to activate proMMP-2 in vitro. In this study, we examined the correlation between matrilysin expression and EOC cellular invasiveness and the effect of matrilysin gene down-regulation on LPA-induced EOC invasion. Methods: Gelatin zymograhy was used to detect the secretion and activity of matrilysin. ELISA was used to quantify the secretion of promatrilysin in EOC cell conditioned medium. DNA plasmids containing matrilysin sense (P47-M7-GFP) or antisense (P47-M7AS-GFP) gene were transfected into ovarian cancer DOV13 cells using FuGene6 transfection reagent. Positive clones were selected and proliferated with culture medium containing G418. Matrilysin mRNA expression in sense and antisense gene stably transfected DOV13 clones was evaluated by RT-PCR and real-time PCR. The invasiveness of DOV13 clone that was stably transfected with sense or antisense matrilysin gene was determined by in vitro Matrigel invasion assay. Results: In EOC OVCA429, OVCAR3 and R182 cell conditioned medium, LPA (20-80 μM) significantly increased the secretion of promatrilysin by a range of 10% to 400%. LPA moderately stimulated the secretion and activity of matrilysin in DOV13 cells, as shown by gelatin zymography. In P47-M7-GFP positive DOV13 clone (P47-M7-GFP6), the expression of matrilysin mRNA is approximately increased to 2 fold of that in vector control. While in P47-M7AS-GFP positive clone (P47-M7AS-3), the expression of matrilysin mRNA is down regulated by 91% compared to vector control, as calculated by real-time PCR. In DOV13 clone P47-M7-GFP6, the number of cells invaded through the artificial basement membrane significantly increased from 754±158/well (vector control) to 1750±228/well (P<0.01) (∼2-fold increase); while in DOV13 clone P47-M7AS-3, the number of cells invaded through the membrane significantly decreased from 754±158/well (vector control) to 101±3 cells/well (P<0.05)(∼7-fold decrease). In vector-transfected DOV13 cells, LPA treatment, at 20 μM and 40 μM, significantly increased their in vitro invasiveness by 10.6 fold and 15.5 fold (P<0.05). While in DOV13 clone P47-M7AS-3, LPA treatment at 20 μM and 40 μM minimally increased their invasiveness by 110% and 2%, without showing a significant difference as compared to vector control(P>0.05). Conclusions: Our results showed that matrilysin expression is positively correlated with the invasiveness of DOV13 cells. Down-regulation of matrilysin expression by antisense gene transfection could, at least in part, block LPA-induced EOC cellular invasion, suggesting that matrilysin plays an important role in LPA-induced EOC invasion.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]