蟾毒灵对裸鼠结肠癌原位移植瘤凋亡基因Caspase-3表达的影响

Effects of bufalin on Caspase-3 expression in the nude mice with orthotopic transplantation of colon cancer

目的:探讨蟾毒灵治疗裸鼠结肠癌原位移植瘤的作用及机制。方法 :应用不同剂量蟾毒灵(0.5、1.0、1.5 mg/kg)处理人结肠癌细胞株HCT-116建立的裸鼠原位移植瘤模型,分别用生理盐水(NS)(0.2 mL/只)、5-FU(25 mg/kg)做阴性(NS组)和阳性对照(5-FU组),测量移植瘤的体积,观察蟾毒灵对移植瘤的肿瘤抑制率、裸鼠生存期的影响;HE染色观察移植瘤的坏死程度;TUNEL标记法检测肿瘤细胞的凋亡指数;荧光定量PCR方法检测基因Caspase-3 mRNA的表达,免疫组化法和Western印迹法检测Caspase-3蛋白的表达。结果:蟾毒灵各剂量组和5-FU组裸鼠原位移植瘤体积明显小于NS组(P<0.05);NS组,5-FU组和蟾毒灵0.5、1.0、1.5 mg/kg剂量组的裸鼠中位生存期分别为40、37和47、46、45 d,生存分析显示蟾毒灵各剂量组裸鼠的生存时间与NS组相比明显延长;1.0和1.5 mg/kg剂量组和5-FU组移植瘤的坏死程度增加(P<0.05);蟾毒灵各组移植瘤的凋亡指数与NS组相比明显增加(P<0.05),凋亡相关基因Caspase-3 mRNA和蛋白表达明显升高(P<0.05)。结论:蟾毒灵能够抑制裸鼠人结肠癌原位移植瘤的生长,促进肿瘤细胞凋亡,其机制可能与上调基因Caspase-3表达有关。

Objective To investigate the anticancer activities and mechanisms of bufalin in the orthotopic transplantation model of human colon cancer in nude mice. Methods Orthotopic transplantation tumor models of human colon cancer HCT-116 cells in nude mice were treated with different dose of bufalin （0.5,1.0 and 1.5 mg/kg）, using normal saline treatment （NS 0.2 mL each mouse） as negative control groups（NS group） and 5-FU（25 mg/kg） as the positive control groups （5-FU group）. The tumors were measured and the rates of tumor inhibition were calculated. The necrotic degree of orthotopic transplanted tumor was observed by hematoxylin-eosin staining. The apoptotic index of tumor cell was measured by TUNEL staining. The expression level of apoptosis-regulated gene Caspase-3 in tumor tissues was detected by real-time fluorogenic quantitative polymerase chain reaction（RFQ-PCR）, immunohistochemical method and Western-blot. Results The tumor volume of each bufalin group and the 5-FU group reduced significantly compared with that of NS group （P〈 0.05）. The median survival time of NS group, 5-FU group and three bufalin groups were 40, 37, 47, 46 and 45 days. The survival days of each bufalin group prolonged compared with that of NS group （P〈O.05）. The necrotic degree increased in 1.0 and 1.5 mg/kg of bufalin groups and the 5-FU group. The apoptotic index in each group of bufalin treatment increased significantly compared with NS group （P〈0.05）. The mRNA and protein expression of Caspase-3 increased in each bufalin group. Conclusions Bufalin has significant anticancer activities in the orthotopic transnplantation model of human colon cancer in nude mice. It induces apoptosis of transplanted tumor cells. The mechanism may be associated with the up-regulation of Caspase-3 in tumor cells.