Qu Hongjie, Hu Bin, Wang Cheng, Tao Jingchao, Zhang Yunxiao, Cui Jinquan
Department of Obstetrics and Gynecology, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China.
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Zhonghua Fu Chan Ke Za Zhi. 2015 Jul;50(7):529-36.
To provide the theoretical supportting for targeted heparanase (HPA) inhibition of cervical cancer through observing the anti-proliferative effect of the HPA inhibitor on HeLa cell line of cervical cancer.
The two series of 13 kinds of novel HPA inhibitors were synthesized and optimized. Heparan degrading enzyme assay kit was used to test the effect of the inhibitors on the inhibition of HPA enzyme activity. Methyl thiazolyl tetrazolium (MTT) method and scratch test were used to observe the anti-proliferative and the migration effect of the inhibitors on HeLa cells. Flow cytometry was performed to determine the cell cycles and apoptosis. The expression of HPA was evaluated by reverse transcription (RT)-PCR, western blot and immunocytochemistry.
All tested inhibitors could inhibit the activity of HPA enzyme [the range of 50% inhibiting concentration (IC50) values from 4.47 to 47.19 µmol/L] and the growth of HeLa cells (the range of IC50 values from 48.16 to 96.64 µmol/L). Among them, No.16 compound exhibits the strongest inhibition against the growth of HeLa, which could arrest the cell into G0/G1 and G2/M phases. The rate of cell apoptosis in the group treated with 50 µmol/L No.16 for 48 hours [(11.9 ± 1.2)%] was significantly higher than that [(6.6 ± 1.8)%] in untreated group (P = 0.013). Real time PCR and western blot showed that expression levels of HPA mRNA (1.23 ± 0.46) and protein (0.46 ± 0.31) significantly decreased in the treated group as compared with the levels of HPA mRNA (3.43 ± 0.45) and protein (1.30 ± 0.58) in the untreated group (both P < 0.05). Immunocytochemistry also showed that the treatment of No.16 significantly reduced the average optical density (0.39 ± 0.04) of HPA immuostaining signal compared with that in the control group (0.50 ± 0.09; P = 0.026).
Novel 1,3-O,N spiroheterocyclic HPA inhibitors could inhibit the proliferation of HeLa cells, inhibit the HPA enzyme activity in different degree, and downregulate the expression of HPA protein.
通过观察乙酰肝素酶（HPA）抑制剂对宫颈癌HeLa细胞系的抗增殖作用，为靶向抑制HPA治疗宫颈癌提供理论依据。
合成并优化了两个系列的13种新型HPA抑制剂。采用乙酰肝素降解酶检测试剂盒检测抑制剂对HPA酶活性的抑制作用。采用甲基噻唑基四氮唑（MTT）法和划痕试验观察抑制剂对HeLa细胞的抗增殖和迁移作用。采用流式细胞术检测细胞周期和凋亡情况。通过逆转录（RT）-PCR、蛋白质印迹法和免疫细胞化学法评估HPA的表达。
所有测试的抑制剂均能抑制HPA酶的活性[半数抑制浓度（IC50）值范围为4.47至47.19μmol/L]和HeLa细胞的生长（IC50值范围为48.16至96.64μmol/L）。其中，16号化合物对HeLa细胞生长的抑制作用最强，可使细胞停滞于G0/G1期和G2/M期。50μmol/L 16号化合物处理48小时组的细胞凋亡率[（11.9±1.2）%]明显高于未处理组[（6.6±1.8）%]（P = 0.013）。实时PCR和蛋白质印迹法显示，与未处理组HPA mRNA水平（3.43±0.45）和蛋白质水平（1.30±0.58）相比，处理组HPA mRNA水平（1.23±0.46）和蛋白质水平（0.46±0.31）均显著降低（均P < 0.05）。免疫细胞化学也显示，与对照组相比，16号化合物处理显著降低了HPA免疫染色信号的平均光密度（0.39±0.04）（对照组为0.50±0.09；P = 0.026）。
新型1,3 - O,N螺杂环HPA抑制剂可抑制HeLa细胞增殖，不同程度抑制HPA酶活性，并下调HPA蛋白表达。
