Recombinant Sv-cystatin Inhibits Tumor Invasion And Metastasis

Snake venom cystatin (sv-cystatin), whose structure is similar to family 2 cystatins of human (e.g. cystatin M), is a low molecular protein isolated from the venom of naja naja atra. The sv-cystatin possesses a potential for anticancer invasion and metastasis. The earlier study in our laboratory suggests that sv-cystain might be one of candidate drug for anticancer invasion and metastasis with several target contribution. Based on the earlier studies, the recombinant sv-cystatin was purified to obtain high-purity and high-activity and the function and mechanism of anticancer invasion and metastasis both in vitro and in vivo was investigated.Expression, separation and purification of sv-cystatin in Pichia pastoris expression systemIn order to evaluate the function of sv-cystatin anticancer invasion and metastasis overall, recombinant sv-cystatin with high-yield, high-purity and high-activity must be produced. GS115-cystatin transformants were induced and expressed in optimized Pichia pastoris expression system, and the secretion expression protein was purified using IMAC affinity chromatograph tag purification, ultrafiltrating and desalting methods, then sv-cystatin inhibitory activity was analyzed by Papain Assays. The maximum yield was about 18 mg/L in shaking-flask fermentation. The purity was 95.08%. And approximately 5 mg/L pure bioactive sv-cystatin of 14-kDa was obtained. Values of Ki of the inhibition of papain were 2.67 nM, which showed highly effective inhibition activity.Recombinant sv-cystatin inhibits tumor invasion and metastasis in vitro and in vivoTo determine whether recombinant sv-cystatin affected the ability of tumor cells to invade in vitro, we performed Matrigel invasion assays. Invasion of B16F10 cells was significantly lesser (P<0.01) by recombinant sv-cystatin of five different concentration, and the inhibitory rate were 17.05%, 27.13%, 31.01%, 37.98% and 47.29% respectively. Invasion of MHCC97H cells was significantly lesser (P<0.01) by recombinant sv-cystatin of the other four different concentration except for 10μg/ml of recombinant sv-cystatin, and the inhibitory rate were 19.7%, 21.21%, 30.3% and 40.91% respectively. It has been shown that the recombinant sv-cystatin protein could significantly inhibit invasion and metastasis of tumor cells in vitro.To test the in vivo effect of recombinant sv-cystatin on metastasis, experimental melanoma pulmonary metastases in C57BL/6 Mice and spontaneous pulmonary metastases in BALB/cA-nude model was formed. The number of metastatic lesions and lung weights in C57BL/6 mice of treatment group at 25mg/kg and 50mg/kg were less than those control group mice (P<0.01), and the inhibitory rate were 34.90% and 41.58% respectively. The number of metastatic lesions in BALB/cA-nude of treatment group at 25 mg/kg, 50 mg/kg and 100 mg/kg were less than those control group mice (P<0.05), and the inhibitory rate were 25.71%, 32.80% and 35.66% respectively. These results demonstrate that recombinant sv-cystatin might have a significant effect on metastasis in vivo. Moreover, recombinant sv-cystatin also might inhibit tumor growth.Recombinant sv-cystatin induces apoptosis of tumor cellsTo determine the effect of treatment with recombinant sv-cystatin on tumor cells apoptosis, FCM and TUNEL methods were used and ultrastructural features of apoptosis tumor cells were observed. The activity of caspase-2 and caspase-3 was determined by chromatometry, the content of NO was detected by Griess reagent method, and the expression of Bcl-2, Bax, Bcl-w was detected by real time PCR and (or) Western Blot. It showed obvious apoptosis in B16F10 and MHCC97H cells treated with sv-cystatin by FCM, TUNEL assay and electronic microscope, increased activity of caspase-2 and caspase-3, increased production of NO, up-regulated expression of Bax and Cyto C, meanwhile down-regulated expression of Bcl-2 and Bcl-w, which indicated that recombinant sv-cystatin results in cell apoptosis involved in mitochondrium path.Recombinant sv-cystatin inhibits neovascularizationTo test whether neovascularization was affected by recombinant sv-cystatin, biological function of HUVEC was observed with the treatment of recombinant sv-cystatin of five different concentrations, MVD was detected in experimental melanoma pulmonary metastases of C57BL/6 Mice and spontaneous pulmonary metastases of BALB/cA-nude model cured by recombinant sv-cystatin. Adhesion of B16F10 and MHCC97H cells treated by recombinant sv-cystatin to HUVECs was significantly decreased (P<0.05), and tubule-like structure (TLS) of HUVECs was also reduced. We also observed a significantly lower microvessel density (MVD) of lung colonization in C57BL/6 Mice and tumor in situ of BALB/cA-nude. The expression of VEGF-A, bFGF and Flt-1 was down-regulated by real time PCR and Western Blot assay. These data demonstrate that recombinant sv-cystatin inhibits neovascularization involved in VEGF/Flt-1/RTK system.In summary, recombinant sv-cystatin in Pichia pastoris expression system after purification could not only inhibit tumor invasion and metastasis both in vitro and in vivo, but also induce cell apoptosis and inhibit neovascularization, which offered the theory foundation and procedure condition for development and application of anticancer invasion and metastasis drug.