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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 study the mechanism of anti-tumor invasion and metastasis and angiogenesis of APN inhibitor CIP-13F and lay the experimental foundation for drug development and clinical treatment of malignant tumors. Methods: Human ovarian clear cell carcinoma ES-2 and human fibrosarcoma cell HT-1080 were cultured in vitro. The inhibitory effect of CIP-13F on APN activity was evaluated by L-leucyl-p-nitroanilide substrate method. The inhibitory effect of CIP-13F on the growth of tumor cells was evaluated. Transwell cham-ber method was used to observe the inhibitory effect of CIP-13F on the migration ability of ES-2 cells. The model of spontaneous lung metastasis of Lewis lung carcinoma was established in vivo. Tumor tissues and lung tissues were taken after administration. The expression of APN in Lewis lung cancer tissues was detected by immunohistochemistry. The expression of CD34 was detected by immunohistochemistry in order to evaluate the microvessel density (MVD). Results: The effects of CIP-13F on ES-2 and HT-1080 cells were dose-dependent and the effect of HT- APN and growth inhibition in 1080 cells is weak. In addition, different concentrations of CIP-13F and ES-2 cells penetrated the artificial basement membrane in a dose-dependent manner. The inhibitory rates of 100 and 500 μmol / L CIP-13F on the migration of tumor cells through Matrigel were 42.0% and 72.1%, respectively, with P values of 0.003 and 0.001, respectively. In vivo tests showed that the tumor growth inhibition rates of Bestatin 100mg / kg, CIP-13F50 and 100mg / kg were 33.2%, 29.4% and 45.8% respectively (P values were 0.013,0.020 and 0.005, respectively) The inhibition rates of lung metastases of Lewis lung carcinoma with 50 and 100 mg / kg CIP-13F were 52.2% and 81.3%, respectively (P values were 0.001 and 0.000, respectively) compared to the control group. In addition, the MVD values of Lewis lung cancer tissues were 18.6 and 14.9 under the action of 50 and 100 mg / kg CIP-13F, respectively. Compared with the negative control group, the MVD values were significantly decreased, with P values of 0.000 and 0.001, respectively. Conclusion: The cyclic imide peptide CIP-13F can inhibit the proliferation and metastasis of tumor cells by inhibiting the activity of APN, and then inhibit the growth of primary tumor in the axillary tumor and reduce the number of secondary metastases in the lung. CIP-13F for the treatment of APN-positive tumors have a good prospect.