gms | German Medical Science

Objective: Temozolomide (temodal, TMZ) is a standard chemotherapy treatment for high-grade gliomas. TMZ targets the DNA Damage Response (DDR) and leads to induction of DDR-responsive genes. It was reported earlier that upregulation of cell adhesion molecule L1-CAM and subsequent membrane cleavage confers resistance of glioma cells to DNA damage. We hypothesised that cleavage of membrane proteins such as L1-CAM by metalloproteases (MMPs and/or ADAMs) contributes to resistance against TMZ induced DNA damage and subsequent cell death.

Method: Cell death of U87MG cells treated with either TMZ alone or a combination of TMZ with the MMP inhibitor batimastat (BB-94) was assessed by MTT assays. Quantitative RT-PCR (qPCR) was applied to investigate the expression changes of metalloproteases at mRNA levels with and without TMZ incubation in U87MG cells for 5 days. Western blot, zymography and activity assays were employed to determine the corresponding distinction at protein and functional level. Matrigel invasion assays were performed to demonstrate the effect of MMP increase on the invasive behaviour of U87MG cells.

Results: QPCR analysis revealed that the expression levels of ADAM8, MMP1, MMP9 and MMP14 were significantly increased after incubation with 500 micromolar TMZ for 5 days compared to control. Using Western blot and zymography, we confirmed increased expression and activities of ADAM8, MMP-1, MMP-9, MMP-14. With respect to function, we observed sensitisation of U87MG cells to TMZ induced cell death when the broad-range MMP inhibitor Batimastat (BB-94) was used in conjunction with TMZ. Moreover using Matrigel invasion assays, increased numbers of U87MG cells migrated through Matrigel compared to controls implicating that TMZ promotes the invasive ability of normally non-invasive U87MG glioma cells via the induction of metalloproteases. The observed invasiveness of U87MG cells was inhibited by addition of BB-94.

Conclusions: The guiding notion of the role of metalloproteases in cancer is to promote proliferation and invasiveness by degradation of growth factors and extracellular matrix components, respectively. Our results showed that TMZ increases survival and enhances the invasive ability of glioma cells via the induction of metalloproteases, which could be instrumental in mediating chemoresistance of gliomas.