gms | German Medical Science

Objective: Due to their invasive behaviour, malignant gliomas are currently an incurable disease. Malignant gliomas are chiefly of oligodendroglial or astrocytic origin and display large genetic alterations. Previous histological findings have demonstrated higher TFPI-2 expression levels in low grade than in high grade gliomas. TFPI-2, a Kunitz-type serine protease, has been identified as a possible tumor suppressor gene. In our study we evaluated the effects of a TFPI-2 loss of expression in glioma cells on proliferation, their migratory and invasive potential and on the expression of MMPs.

Methods: TF expression was analyzed by RT-PCR. Inhibition of TFPI-2 dependent signaling was achieved via lentiviral-mediated knockdown of TFPI-2. Cell proliferation was measured by MTT-assays. Tumor cell migration and invasion were quantified by scratch-migration-assays and modified Boyden-chamber-assays, respectively. Specific inhibitors for MMP-1, MMP-2 and MMP-9 were employed at subtoxic concentrations.

Results: We analyzed a panel of 8 glioma cell lines of astrocytic origin (U87, U251, U343, U373, MZ-18, MZ-54, MZ-256, MZ-304) and one of oligodendroglial origin (Hs 683) for basic expression of TFPI-2. A stable knockdown of TFPI-2 was performed and cells with no measurable TFPI-2 expression were selected. MTT assays of empty vector transfected cells and TFPI-2 knockdown cells performed at several time points indicated increased proliferation of TFPI-2 KD cells. TFPI-2 knockdown cells also displayed enhanced migration and invasion when compared to empty vector transfected cells. To address the question inasmuch inhibition of MMPs might underlie the limiting effects of TFPI-2 on invasion, we employed specific inhibitors of MMP-1, MMP-2 and MMP-9. Interestingly, the MMP-1 and MMP-2 inhibitors were able to largely abrogate the potentiating effect of the TFPI-2 knockdown on invasion, whereas the MMP-9 inhibitor had only a moderately limiting effect in TFPI-2 knockdown cells.

Conclusions: Inhibition of TFPI-2 significantly enhanced the aggressive behaviour of glioma cells in vitro. Proliferation, migration and invasion of tumor cells of astrocytic and oligodendroglial origin were potently enhanced by knockdown of TFPI-2. Inhibition of MMP-1 and MMP-2 efficiently reduced the invasive behaviour of TFPI-2 knockdown cells, suggesting that TFPI-2 may limit invasion by inhibition of these two proteases. The results we obtained underline the importance of TFPI-2 as a tumor suppressor gene in gliomas.