蛇毒半胱氨酸蛋白酶抑制剂重组蛋白诱导肿瘤细胞体内外凋亡及其分子机制

Recombinant Snake Venom Cystatin Promotes Tumor Apoptosis in vitro and in vivo and its Molecular Mechanism

关键词:  蛇毒  半胱氨酸蛋白酶抑制剂  肝癌细胞  小鼠黑色素瘤细胞  凋亡

Key words:  snake venom  cystatin  hepatocellular carcinoma cell  melanoma cells  apoptosis

摘要:

目的 探讨蛇毒半胱氨酸蛋白酶抑制剂（snake venom cystatin，sv-cystatin）重组蛋白对肿瘤细胞凋亡的影响及其分子机制。方法 将sv-cystatin重组蛋白（10，25，50，100，200 mg·L^–1）作用于B16F10和MHCC97H细胞，C57BL/6小鼠尾静脉注射B16F10细胞建立黑色素细胞荷瘤模型并给予25，50 mg·kg^–1的sv-cystatin重组蛋白干预，建立MHCC97H细胞BALB/c–nude裸鼠腋下皮内成瘤模型并给予25，50，100 mg·kg^–1的sv-cystatin重组蛋白干预，分别检测体内外细胞凋亡，检测重组蛋白体外作用后肿瘤细胞Bcl-2、Bcl-w和Bax的基因与蛋白表达，检测Cyto C蛋白表达和caspase-2、caspase-3酶活性。结果 与对照组相比，25，50，100，200 mg·L^–1的sv-cystatin重组蛋白能够增强2种肿瘤细胞的体外凋亡能力（P<0.05）；C57BL/6小鼠肺转移结节中B16F10细胞（50 mg·kg^–1）和BALB/c-nude腋下皮内肿瘤组织中MHCC97H细胞（100 mg·kg^–1）的凋亡增强（P<0.05）；体外处理B16F10和MHCC97H细胞后Bcl-2和Bcl-w的基因与蛋白表达均下降，Bax表达则增强（P<0.05），Cyto C蛋白表达增强（P<0.05），caspase-2和caspase-3的活性上调（P<0.05）。结论 Sv-cystatin重组蛋白能够促进肿瘤细胞凋亡，从而发挥抗肿瘤作用。

Abstract:

OBJECTIVE To investigate the effect of recombinant snake venom cystatin(sv-cystatin) on apoptosis of tumor cells and its molecular mechanism. METHODS B16F10 and MHCC97H cells were incubated with recombinant sv-cystatin(10, 25, 50, 100, 200 mg·L^-1). Melanoma tumor-bearing mice model was established by inoculating in the lateral tail vein of C57BL/6 mice with B16F10 cells, and recombinant sv-cystatin was administered at 25, 50 mg·kg^-1. MHCC97H cells were inoculated subcutaneously(s.c.) into BALB/c-nude mice, and recombinant sv-cystatin was administered at 25, 50, 100 mg·kg^-1. TUNEL analysis was used to detected the apoptosis of B16F10 and MHCC97H cells. The mRNA and protein expression of Bcl-2, Bcl-w and Bax, and protein expression of Cyto C were measured, respectively. Activity of caspase-2 and caspase-3 were assessed. RESULTS Compared with control group, the apoptosis of B16F10 and MHCC97H cells was significantly upregulated by treatment with recombinant sv-cystatin at 25, 50, 100, 200 mg·L^-1(P<0.05). The apoptosis of B16F10 cells in the lung colonization of C57BL/6 mice(50 mg·kg^-1), and the apoptosis of MHCC97H cells in the primary tumor tissues of BALB/c-nude mice(100 mg·kg^-1) were higher than those in control mice(P<0.05). The mRNA and protein expression of Bcl-2 and Bcl-w in B16F10 and MHCC97H cells were decreased following treatment with recombinant sv-cystatin, compared to controls(P<0.05), while the expression of Bax and Cyto C was significantly raised(P<0.05). Activity of caspase-2 and caspase-3 was significantly increased(P<0.05). CONCLUSION Recombinant sv-cystatin can promote the apoptosis of tumor cells, which contribute to anti-tumor effect.