Potent Antitumor Activity Of MONCPT, A Novel Camptothecin Analogue

Objective:To evaluate the effect of the novel anti-tumor compound MONCPT to inhibit the TOPO1 activity in vitro, its anti-proliferation activity and the therapeutical effect on A549 and PC-3 xenografted athymic mice. To explore the mechanisms by which MONCPT induced cell cycle arrest and exerted tumor angiogenesis inhibition activity.Methods:1. Anti-tumor property of MONCPT in vitro and in vivo:(1) DNA Relaxation assay was used to investigate Topo I inhibitory acitivity of MONCPT. (2) MTT method was used to detect cytotoxicity of MONCPT on nine tumor cell lines and calculate the median inhibitory concentration (IC50 value). (3) A549 and PC-3 xenografted athymic mice model was established to evaluate anti-tumor activity of MONCPT in vivo.2. MONCPT inhibit tumor growth by cell cycle arrest:(1) Flow cytometry assay was used to detect the cell cycle distributions of PC-3 and A549 cells treated with MONCPT. (2) Western-blot assay was used to investigate the expression of the protein related to cell cycle in A549 cells treated with MONCPT. (3) Flow cytometry assay was used to detect the cell cycle distributions of P53-knockdown or P38-knowdown A549 cells. (4) Western-blot assay was used to investigate the expression of protein related to cell cycle in P53-knockdown or P38-knowdown A549 cells.3. Anti-angiogenesis of MONCPT:(1) MTT method was used to detect cytotoxicity of MONCPT on EA.hy926 cells and calculate the median inhibitory concentration (IC50 value). (2)AO/EB staining was used to evaluat the apoptosis rate of EA.hy926 cells after the MONCPT treatment. (3) Flow cytometry assay was used to evaluate the apoptosis rate of EA.hy926 cells after the MONCPT treatment. (4) CAM assay was used to test the antiangiogenic activity of MONCPT. (5) HUVEC migration assay was used to evaluat the antiangiogenic activity of MONCPT. (6) HUVEC tube formation assay was used to evaluat the antiangiogenic activity of MONCPT.Results:1. MONCPT showed broad anti-tumor spectrum and effective anti-tumor activity in vitro and in vivo:(1)DNA Relaxation assay showed that MONCPT exerted high potency as Topo I inhibitor. (2)Cytotoxicity assay demonstrated that MONCPT was a potential and high efficient anti-tumor compound with IC50 values of 1.0×10-10 M-2.3×10-7 M in 9 tumor cell lines. (3)In A549 xenografts model, the mice were sacrificed after MONCPT administration for 15 days. The tumor inhibition rate (%) of MONCPT 20 mg/kg, MONCPT 10 mg/kg and MONCPT 5 mg/kg group were 98.0, 78.5, and 32.0, respectively, and the corresponding T/C (%) values were 5.5,30.9, and 70, respectively. In PC-3 xenografts model, the mice were sacrificed after MONCPT administration for 17 days. The tumor inhibition rate (%)of MONCPT 20 mg/kg,MONCPT 10 mg/kg and MONCPT 5 mg/kg group were 16.7,37.7,71.3, respectively, and T/C (%) values were 77.7,51.8, and 29.6, respectively.2. MONCPT induced the G2-M phase arrest by regulation of cell cycle related proteins:(1) MONCPT 100 nM could block the A549 cells in the G2/M phase in a time-dependent manner. G0/G1 phase arrest was caused by MONCPT 1000 nM in PC3 cells, whereas MONCPT 10 nM and 100 nM could induce cell cycle arrest in G2/M phase. (2) Downregulation of CDK2, CDK4 and cyclin D1 protein expression was observed in PC3 cells after treated with MONCPT 1000 nM, while upregulation of CDK7. CDK1, and cyclin B1 protein expression was observed in PC3 cells treated with MONCPT 10.0 nM and MONCPT 100.0 nM. (2) Downregulation of CDK7 and cyclin H protein expression was observed in A549 cells treated with MONCPT 100.0 nM for 12-48h. (3) Furthermore, upregulation of p53 and p21 protein expression in A549 cells treated with MONCPT was observed in a time-dependent manner, indicating that MONCPT was a p53 co-activator to increase the transcription of p21, which induced the down-regulation of the CDK1 protein, and finally suppressed the kinase activity of Cdc2/cyclinB complexes, leading to G2/M phase arrest of the A549 cells. (4) The MAPK and P13K/Akt signaling pathways play an important role in the cell cycle control, and our results showed that the down-regulation of the p-ERK, JNK and p-AKT protein expression induced by the MONCPT was involved in its regulation of the G2/M phase arrest.3. MONCPT showed high anti-angiogenesis activity:(1) MONCPT exhibited high antiproliferation action in human EA.hy926 endothelial cells, and the IC50 value was 0.13±0.04μM. (2) With AO/EB stain, apoptosis induced by MONCPT was observed in EA.hy926 cells in a dose-dependent manner. (3) MONCPT-mediated apoptosis was observed in EA.hy926 cells in a dose-dependent manner by flow cytometry assay. (4) In Chick embryo chorioallantoic membrane (CAM) assay, MONCPT exhibited anti-angiogenesis activity in a dose-dependent manner. (5) MONCPT 100 nM and MONCPT 200 nM could obviously inhibit the migration of the HUVEC endothelia cells. (6) MONCPT 50 nM, MONCPT 100 nM and MONCPT 200 nM significantly inhibited chemotactic-migration invasion on gelatin and tube formation on Matrigel of HUVECs in a dose-dependent manner.Conclusion:(1) MONCPT possessed high antitumor activity in vitro and in vivo.(2) MONCPT induced the cell cycle G2-M arrest by regulation of AKT, p53 and MAPK expression.(3) MONCPT can inhibit the cancer angiogenesis, and enhance the anti-tumor activity.