CIP-13F抗肿瘤转移作用及机制的探讨

Inhibition effect of CIP-13F on metastasis and its mecgabusn

目的:研究APN抑制剂CIP-13F抗肿瘤侵袭转移及血管生成的作用机制,为药物开发和恶性肿瘤临床治疗奠定实验基础。方法:体外培养人卵巢透明细胞癌ES-2和人纤维肉瘤细胞HT-1080,应用L-亮氨酰对硝基苯胺底物法评价CIP-13F对APN酶活性的抑制作用,通过MTT比色评价CIP-13F对肿瘤细胞的生长抑制作用。以Transwell cham-ber法观察CIP-13F对ES-2细胞游走能力的抑制作用。体内建立Lewis肺癌自发性肺转移模型,分别给药后取肿瘤组织和肺组织,应用免疫组织化学法检测Lewis肺癌组织中APN的表达水平。以免疫组织化学法检测CD34表达水平评价肿瘤微血管密度(MVD)。结果:采用4、20、100和500μmol/L CIP-13F作用于ES-2和HT-1080细胞发现,对ES-2细胞,APN的表达和生长抑制作用呈现剂量-效应相关性,对HT-1080细胞中APN和生长抑制作用较弱。另外,不同浓度的CIP-13F与ES-2细胞穿过人工基底膜的能力呈剂量依赖性关系。100和500μmol/L的CIP-13F对肿瘤细胞穿过Matrigel的游走能力抑制率分别为42.0%和72.1%,P值分别为0.003和0.001。体内试验表明,Bestatin 100mg/kg组、CIP-13F50和100mg/kg剂量的肿瘤生长抑制率分别为33.2%、29.4%和45.8%(P值分别为0.013、0.020和0.005),同时,与阴性对照组相比,50和100mg/kg CIP-13F对Lewis肺癌肺转移抑制率分别为52.2%和81.3%(P值分别为0.001和0.000)。另外,50和100mg/kg CIP-13F作用下,Lewis肺癌组织的MVD值分别为18.6和14.9,与阴性对照组比较,MVD值均明显降低,P值分别为0.000和0.001。结论:环酰亚胺类肽化合物CIP-13F通过抑制APN活性,抑制肿瘤细胞的增殖和转移,进而抑制移植瘤鼠的腋部原发肿瘤灶的生长,降低肺继发转移灶的数目。CIP-13F用于治疗APN表达阳性的肿瘤有良好的应用前景。

OBJECTIVE： To evaluated the efficacy of CIP-13F as a novel APN inhibitor for treatment of cancers with positive APN expression. METHODS： Ovarian clear cells carcinoma ES-2 and fibrosareoma cells HT-1080 in vitro,the as- say of quantitating the enzymatic cleavage of the substrate L-Leucine p-nitroanilide was employed to evaluate APN activi- ty. MTT assay was employed to evaluate the efficacy of CIP-13F in vitro. The transwell chamber assay was used to meas- ure the inhibitory effect of CIP-13F on the invasion and migration of cancer cells penetrating the Matrigel. The in vivo ef- ficacy of CIP-13F was assessed in an Lewis lung carcinoma （LLC） implantation mouse model,and the assays of immuno- histochemical staining and western blotting were performed to estimate the expression of APN in LLC cells. Using immu- nohistochemical staining with CD34,the antiangiogenesis of CIPq3F was evaluated in LLC tissue sections. RESULTS： In vitro showed that CIP-13F inhibited ES-2 growth and migration via suppression of APN, whereas, CIP-13F had weak in- hibition on the growth and the expression of APN of HT-1080. The ability through artificial basement membrane of dif- ferent concentrations of CIP-13F exhibited a dose-dependent manner,the inhibition rates of 100 and 500 μmol/L CIP-13Fwere 42.0 % and 72.1% （P= 0. 003, P= 0. 001）. Further more to evaluated the inhibitory effect of CIP-13F in mice bear ing Lewis lung carcinoma implantation was evaluated too. Bestatin 100 mg/kg,CIP-13F 50 mg/kg and 100 mg/kg treat- ment resulted in a significant delay of LLC growth in anterior flank and the tumor inhibition rates were 33.2 %, 29.4 and 45.8% （P values were 0. 013,0. 020 and 0. 005）. Compared with the control,the inhition rates of Lewis lung metas- tasis of CIP-13F 50 mg/kg and 100 mg/kg treatment were 52.2% and 81.3% （P values were 0. 001 and 0. 000）. Further more the inhibition of angiogenesis in LLC tissues was determined, after treatment of 50 mg/kg and 100 mg/kg CIP-13F, the MVD values of Lewis lung tissue were 18.6 and 14.9. Compared with the negative control group, MVD values were significantly reduced（P values were 0. 000 and 0. 001）. CONCLUSIONS： CIP-13F is a novel cyclic-imide peptidomimetics with a great inhibition effect on the activity and expression of APN. CIP 13F can effectively inhibited LLC growth and pulmonary metastasis in mice and suggest that CIP-13F is a potential drug for the treatment of cancers with positive APN expression.