蟾毒灵对裸鼠大肠癌原位移植瘤的抗肿瘤作用及其对凋亡相关基因Bcl-x_L、Bax表达的影响

Anticancer Activities and Effects of Bufalin on Apoptosis-regulate Gene Bcl-x_L,Bax in Orthotopic Transplantation Model of Human Colorectal Cancer in Nude Mice

目的探讨蟾毒灵对裸鼠大肠癌原位移植瘤的抗肿瘤作用及其可能诱导凋亡的机制。方法将60只人结肠癌细胞株HCT-116建立的裸鼠原位移植瘤模型随机分为生理盐水(NS)组、5-Fu组、蟾毒灵低(BL)、中(BM)、高(BH)5组,每组12只,腹腔注射给药持续7d。用药结束后第24天每组分别处死6只裸鼠,完整取出肿瘤,测量肿瘤大小,计算肿瘤抑制率;剩余裸鼠观察带瘤生存期;TUNEL法检测原位移植瘤细胞的凋亡指数;实时荧光定量PCR法和Western blot法检测凋亡相关基因Bcl-xL、Bax mR-NA和蛋白的表达。结果 5-Fu组、BL组、BM组、BH组的肿瘤抑制率分别为69.6%、45.6%、56.2%、58.5%,肿瘤体积与NS组比较明显缩小(P<0.01);BL组、BM组带瘤生存期与NS组相比较明显延长(P<0.05)。TUNEL结果显示蟾毒灵用药组的凋亡指数明显高于NS组(P<0.01);荧光定量PCR和Western结果显示蟾毒灵可抑制Bcl-xL基因表达,促进Bax基因表达(P<0.05)。结论蟾毒灵对裸鼠大肠癌原位移植瘤有显著的抗肿瘤作用,能够抑制Bcl-xL基因和促进Bax基因表达,诱导肿瘤细胞凋亡可能是蟾毒灵抗肿瘤的重要机制之一。

Objective To investigate the anticancer activity and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation model of human colorectal cancer in nude mice. Methods HCT-116 cells of human colorectal cancer were implanted into the colon to establish orthotopic transplantation tumor models of human colorectal cancer in nude mice. Sixty mice were randomly divided into five groups： NS group, 5-Fu group, Low dose bufalin group（BL）, Medium dose bufalin group（BM） and High dose bufalin group（BH）, with 12 mice in each group. Each group was injected intraperitoneally for 7days, respectively. Six mice in each group were killed at d24 and survival time in each group was calculated. The volume of the tumor were measured and the tumor inhibiting rate were calculated. The apoptotic rate measured by TUNEL staining method, and the expression of apoptosis regulated genes Bcl-xL and Bax were detected by real-time fluorogenic quantitative polymerase chain reaction（RFQ-PCR） and Western blot in tumor tissues. Result The tumor volume of 5 Fu group and each Bufalin group were reduced significantly corn pared with NS group （P〈0. 01 ）,and the tumor inhibiting rate were 69. 6% ,45.6% ,56. 2% and 58. 5% for group of 5-Fu,BL,BM and BH respectively. The survival time were prolonged in group BL and BM （P〈0.05）. The apoptotic rate in each group of bufalin were increased significantly compared with NS group（P〈0.05）. The result of RFQ-PCR and Western blot staining showed that the expression of Bcl-Xc was decreased both in mRNA and protein level, while the expression of Bax was increased. Conclusion Bufalin has significant anticancer activity in the orthotopic transnplantation model of human colorectal cancer in nude mice and it can induce apoptosis of transplanted tumor cells. The mechanism may be asso- ciated with the down-regulation of Bcl-xc and up-regulation of Bax in tumor cells.