Effect of 1,3-O,N spiroheterocyclic inhibitors of heparanase on the growth of HeLa cells

Abstract

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.