The effects of cystatin C construct clones on B16F10 in vitro cell behavior

Background: Metastasis is the cause of most cancer related morbidity. A naturally occurring cysteine protease inhibitor, cystatin C, has been reported to inhibit tumor cell metastasis for several different cancer types, however, the mechanism is still unknown. Our study focused on determining which region of cystatin C is responsible for anti-metastatic action by characterizing specific constructs of cystatin C in melanoma cells. In one construct, the N-terminal peptide amino acids 1-10, required for cysteine protease inhibition, were deleted. In the second construct the conserved motif QLVA was altered to GGGG. Methods and Results: Net proliferation, migration and invasion of the cystatin C constructs were assessed. The modified Boyden chamber revealed 75% reduced invasion of N-truncated clones compared to control B16F10, similar to cystatin C overexpression. A scratch migration assay also showed a three-fold reduction in migration rate. The QLVA sequence was found to be required for inhibition of B16F10 invasion and migration. Net cell proliferation remained constant between clones. Conclusions: Overexpression of cystatin C with or without cysteine protease inhibitor activity inhibits the migration and invasion, but not proliferation, of B16F10 melanoma cells. The conserved cystatin sequence, QLVA, is required for cystatin actions on B16 melanoma cells