Snake Venom Cystatin Inhibits B16F1 Melanoma Invasion And Metastasis Both In Vitro And In Vivo

Cystatins are endogenous reversible inhibitors of cysteine proteases. A number of studies have demonstrated that impaired regulation of expression/activity of cystatin was closely associated with cancer invasion and metastasis. Snake venom cystatin (sv-cystatin) is a low molecular protein isolated and purified from the venom of naja naja atra, which structure is similar to human family 2 of cystatins (e.g. cystatin M), suggesting potential of sv-cystatin anticancer invasion and metastasis . In the present study, anti-B16F1 melanoma invision and metastasis of sv-cystatin both in vitro and in vivo was investigated.1. Cloning of a synthetic gene coding for sv-cystatinBy using of gene recombinant method, sv-cystatin gene were artificially synthesized based on its amino acid sequence and the biased codon usage of Pichia pastoris. Slowly annealing PCR methods were conducted to obtain the full length of sv-cystatin cDNA. Sv-cystatin gene was cloned into pUC18 vector and pUC18-cystatin plasmid constructed for the producing of recombinant sv-cystatin.2. Effects of the recombinant sv-cystatin produced in Pichia Pastoris expression system on the invasion and metatasis of B16F1 melanoma cellsDNA fragment of sv-cystatin encoding gene was amplified by PCR from pUC18-cystatin plasmid and then inserted into pPICZ α A vector down the a -factorsignal sequence to construct an expression plasmid pPICZ α A-cystatin. The constructed pPICZ α A-cystatin was linearized by Sac 1 and transformed into yeast Pichia pastoris, strain GS115 by the electrophoration. The positive clones were identified by the phenotype and PCR analysis. GS115-cystatin transformants were induced in 1.0% methanol and the secretion expression protein was examined by SDS-PAGE, Western blot analysis. The molecular mass of experssion product was about 14KD. Approximately 16 mg/L of bioactive sv-cystatin was produced from one of GS115-cystatin transformants and about 5 mg/L pure sv-cystatin was obtained after purified by ProBond protein purification system. Purified sv-cystatin protein was assayed for biological activity and the result indicated that sv-cystatin could inhibit the activity of papain.The ability of B16F1 cells treated with recombinant sv-cystatin to invade the reconstituted basement membrane decreased significantly (P<0.01), and the inhibitory rate were 36% and 51 % for recombinant sv-cystatin at 0.25 mg/ml and 0.5 mg/ml, respectively. It has been shown that the recombinant sv-cystatin protein could significantly inhibit invasion and metastasis of B16F1 cells in vitro.3. Effects of sv-cystatin expression on B16F1 melanoma cell invasion and metastasisRecombinant expression plasmid pcDNA3.1-cystatin was constructed and transfected into the murine melanoma cell lines B16F1 by use of lipofectamine gene transfer technique. High expression of sv-cystatin clones were screened by G418 and identified via RT-PCR and Western blot assay. The ability of sv-cystatin transfected B16F1 cells to invade the reconstituted basement membrane, Matregel was attenuated as compared to nontransfected cells or cells transfected with vector alone. The date indicated that sv-cystatin expression in B16F1 cells might suppress the cell Matrigel invasion, but no effect on cell growth, adhension and migration.To test the in vivo effect of sv-cystatin expression on metastasis, 2× 105 cells of the sv-cystatin transfected, mock transfected and nontransfected cell clones were injected into C57BL/6 mice via the tail lateral vein and experimental pulmonary metastasis model was formed. Mice were sacrificed at 3 weeks after vein injection of cell clones, and histological examination of metastatic lesions in the lungs wereperformed. All mice had metastasis in lungs (100%). The number of metastatic lesions and lung weights in B16Fl/sv-cystatin group mice were less than those Bl6Fl/pcDNA3.1 transfected and nontransfected control (P<0.01). These results demonstrate that sv-cystatin expression in B16F1 cells might have a strongly effect on metastasis to the lungs.In conclusion, this study provides the first evidence that sv-cystatin could not only inhibit papain activity, but also suppress tumor invasion and metastasis both in vitro and in vivo, and offered the theory foundation and preceeding condition for development and application of anti-tumor invasion and metastasis drug.