Recombinant snake venom cystatin inhibits the growth, invasion and metastasis of B16F10 cells and MHCC97H cells in vitro and in vivo

Studies have shown that expression of snake venom cystatin (sv-cystatin) in mouse melanoma cells and human gastric carcinoma cells can inhibit their invasion and metastasis. To advance the research into the biological features and pharmaceutical applications of sv-cystatin, we investigated the expression of recombinant sv-cystatin in an optimized Pichia pastoris system. Approximately 5 mg/L of bioactive sv-cystatin was obtained with a purity of 95.08%. Kinetic analyses of recombinant sv-cystatin revealed highly effective inhibitory efficiency against papain (Ki = 2.67 nM). We further investigated the effects of recombinant sv-cystatin on the invasion and metastasis of B16F10 cells and MHCC97H cells in vitro and in vivo. Matrigel invasion assays showed significant inhibition of recombinant sv-cystatin on the tumor cells in vitro. For experimental lung colonization assays, C57BL/6 mice inoculated in the lateral tail vein with B16F10 cells were treated with three i.v. injections of recombinant sv-cystatin (25 and 50 mg/kg) 24 h before cell inoculation, and 2 h and 24 h after cell inoculation. Administration of recombinant sv-cystatin significantly suppressed the formation of lung tumor colonies. For spontaneous metastasis assays, MHCC97H cells were inoculated s.c. into nude mice. After 24 h, recombinant sv-cystatin was administered by i.p. injections at 25, 50 or 100 mg/kg once daily for 5 days. Administration of recombinant sv-cystatin significantly decreased the formation of lung tumor colonies. Taken together, recombinant sv-cystatin inhibits the invasion and metastasis of tumor cells in vitro and in vivo. These results may facilitate the future evaluation of the pharmaceutical applications of sv-cystatin.