蜂毒素对人肝癌BEL-7402细胞裸鼠皮下移植瘤生长及肿瘤血管生成的影响

背景与目的:蜂毒素体外抗骨肉瘤、白血病及宫颈癌的研究已有报道.我们先前的实验发现蜂毒素能够抑制人肝癌细胞株BEL-7402细胞增殖,并诱导其凋亡.本研究拟探讨蜂毒素在裸小鼠体内的抗肝癌作用及其对肿瘤血管生成的影响.方法:建立人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,随机分为5组:生理盐水对照组(10 ml/kg)、阳性对照组(沙利度胺,200 mg/kg)、蜂毒素低剂量组(40 μg/kg)、蜂毒素中剂量组(60 μg/kg)和蜂毒素高剂量组(80 μg/kg).测量各组裸鼠肿瘤体积,观察蜂毒素的体内抗肿瘤作用.光镜下观察肿瘤组织及瘤内血管形态.采用免疫组织化学SABC法检测微血管密度(microvessel density,MVD)及血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和核转录因子κB(nuclear factor κB,NF-κB)蛋白表达.应用实时荧光定量PCR法检测BEL-7402细胞VEGF mRNA和bFGF mRNA的表达.结果:蜂毒素低、中、高剂量组裸鼠肿瘤相对体积(V/V0)分别为4.42±0.58、3.47±0.97和3.06±1.23,与生理盐水对照组(V/V0为9.06±1.45)相比,蜂毒素处理组肿瘤体积明显缩小(P＜0.01).与生理盐水对照组MVD(16.50±2.35)比较,蜂毒素低、中、高剂量组肿瘤组织MVD(11.33±1.86、9.17±1.17和6.67±1.21)明显降低(P＜0.01).显微镜下可见蜂毒素各剂量组肿瘤组织呈片状坏死,肿瘤间质内可见肿瘤血管,并有血管破坏现象.蜂毒素低、中、高剂量组肿瘤组织VEGF(2.59±0.27、2.61±0.17和1.55±0.22)、bFGF(2.45±0.78、2.27±0.36和2.10±0.27)及NF-κB(2.79±0.29、2.71±0.66和2.26±0.56)阳性表达指数均低于生理盐水对照组(3.80±0.60、4.43±0.34和4.98±0.63)(P＜0.01).实时荧光定量PCR检测显示,蜂毒素能够抑制BEL-7402细胞VEGF mRNA和bFGF mRNA的表达.结论:蜂毒素能够明显抑制人肝癌BEL-7402细胞裸鼠移植瘤的生长,影响NF-κB表达、下调VEGF和bFGF的表达、抑制肝癌血管生成可能是其抗肿瘤作用的重要机理之一.

Effects of melittin on growth and angiogenesis of human hepatocellular carcinoma BEL-7402 cell xenografts in nude mice

BACKGROUND & OBJECTIVE: Melittin has antitumor effects on osteosarcoma, leukemia, and cervical cancer in vitro. Our previous experiments showed that melittin could inhibit proliferation and induce apoptosis of human hepatocellular carcinoma BEL-7402 cells. This study was to examine the effects of melittin on the growth and angiogenesis of BEL-7402 cell xenografts in nude mice. METHODS: The xenografts derived from BEL-7402 cells were established in BALB/C nude mice. Inoculated mice were randomly divided into normal saline (NS, 10 ml/kg) group, positive control (thalidomide, TLD, 200 mg/kg) group, low dose melittin (40 microg/kg) group, moderate dose melittin (60 microg/kg) group and high dose melittin (80 microg/kg) group. Tumor volume was measured. Tumor tissue was observed under microscope. Microvessel density (MVD) and the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and nuclear factor kappaB (NF-kappaB) were detected by SABC immunohistochemistry. The mRNA levels of VEGF and bFGF were analyzed by real-time fluorescent quantitative polymerase chain reaction. RESULTS: The relative tumor volume (V/V0) and MVD were significantly lower in low, moderate and high dose melittin groups than in NS group (4.42+/-0.58, 3.47+/-0.97, and 3.06+/-1.23 vs. 9.06+/-1.45, P<0.01; 11.33+/-1.86, 9.17+/-1.17, and 6.67+/-1.21 vs. 16.50+/-2.35, P<0.01). Tumor tissue necrosis was observed in melittin-treated groups and tumor vessels were destroyed by melittin. The positive expression indexes of VEGF (2.59+/-0.27, 2.61+/-0.17, 1.55+/-0.22 vs. 3.80+/-0.60, P<0.01), bFGF (2.45+/-0.78, 2.27+/-0.36, 2.10+/-0.27 vs. 4.43+/-0.34, P<0.01) and NF-kappaB (2.79+/-0.29, 2.71+/-0.66, 2.26+/-0.56 vs. 4.98+/-0.63, P<0.01) were significantly lower in low, moderate and high dose melittin groups than in NS group. The mRNA levels of VEGF and bFGF were also significantly lower in melittin-treated groups than in NS group. CONCLUSIONS: Melittin could inhibit the growth of BEL-7402 cell xenografts in nude mice. The down-regulation of VEGF, b-FGF and NF-kappaB expression and the inhibition of angiogenesis might play key roles in the antitumor effect of melittin.