In vitro anti-cancer activities in Caco-2 and HCT-116 cells of recombinant cystatin C prepared by a Pichia expression system.

Caco-2 and HCT-116 cells were used to access growth-inhibition and anti-invasion activity of recombinant cystatin C expressed in Pichia pastoris X33, G12W/H86V. The mutant G12W/H86V prepared by a pilot plant production system showed more than 10% growth inhibition of Caco-2 cells at 0.56-56 nM concentrations. Growth-inhibited cells had lower cathepsin L activity than the control cells that were not treated with the inhibitor. Conversely, the cathepsin B activity was not changed by treatment with G12W/H86V. The in vitro anti-invasion test using HCT-116 cells showed that G12W/H86V suppressed the cell invasion by 15%, while its wild-type cystatin, aspartic protease inhibitor pepstatin A, and matrix metalloproteinase (MMP) inhibitor MMP-2/MMP-9 inhibitor III did not suppress cell invasion. These results indicate that the recombinant cystatin C with higher protease inhibitory activity effectively retards the growth and invasiveness of human colon carcinoma cells.