Effects of tyrosine kinase inhibitor E7080 and eNOS inhibitor L-NIO on colorectal cancer alone and in combination

Abstract

Objective: To investigate the effects of E7080 and N (5)-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO) on colorectal cancer alone and in combination.

Methods: HT29 colorectal cancer cell line from Sap Institute was used. Real-time cell analysis (xCELLigence system) was performed to determine the effects of E7080 and L-NIO on colorectal cell proliferation. While apoptosis was determined with Annexin V staining, and the effect of agents on angiogenesis was determined with chorioallantoic membrane (CAM) model.

Results: We found that E7080 has a strong antiproliferative effect with an half maximum inhibition of concentration (IC50) value of 5.60×10(-8) mol/L. Also it has been observed that E7080 showed antiangiogenic and apoptotic effects on HT29 colorectal cancer cells. Antiangiogenic scores of E7080 were 1.2, 1.0 and 0.6 for 100, 10 and 1 nmol/L E7080 concentrations, respectively. Furthermore, apoptosis has been detected in 71% of HT29 colorectal cancer cells after administration of 100 nmol/L E7080 which may indicate strong apoptotic effect. Meanwhile administration of L-NIO alone did not show any effect, but the combination of E7080 with L-NIO increased the antiproliferative, antiangiogenic and apoptotic effects of E7080.

Conclusions: Results of this study indicate that E7080 may be a good choice in treatment of colorectal tumors. Furthermore the increased effects of E7080 when combined with L-NIO raise the possibility to use a lower dose of E7080 and therefore avoid/minimize the side effects observed with E7080.

Keywords: E7080; N5-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO); colorectal cancer; tyrosine kinase (TK); xCELLigence system.

Figure 1

CAM assay. (A) Albumen is removed with a syringe; (B) The appearance of the CAM through a window on the egg shell (×8); (C) The placement of the pellet on the CAM (×8); (D) Inhibition of the capillaries on the CAM (score: 1) by the drug (×8).

Figure 2

Dynamic monitoring of cell proliferation using the xCELLigence system. HT29 cells seeded at a density of 100,000, 50,000, 25,000, 12,500, 6,250, 3,125 and 1,562 cells/well in E-plate 16 were observed during 24 h.

Figure 3

Real-time monitoring of cytotoxic effect on HT29 cells using RTCA. (A) E7080; (B) L-NIO; (C) E7080-L-NIO combination.

Figure 4

Calculation of IC₅₀ of E7080 (IC₅₀ =5.60×10^–8 mol/L, square R =0.998).

Figure 5

The antiangiogenic scores of E7080 and L-NIO alone and in combination. (A) E7080 + L-NIO, 50+50, 5+5, 0.5+0.5 nmol/L; (B) E7080 100 nmol/L, L-NIO 100 nmol/L, E7080 + L-NIO 50+50 nmol/L; (C) E7080 10 nmol/L, L-NIO 10 nmol/L, E7080 + L-NIO 5+5 nmol/L; (D) E7080 1 nmol/L, L-NIO 1 nmol/L, E7080 + L-NIO 0.5+0.5 nmol/L. Beva (bevacizumab) is a positive control as mentioned in method.

Figure 6

Dot plot distribution. (A) Dot plot distribution of live, preapoptotic and apoptotic cells after administration of E7080 alone; (B) Dot plot distribution of live, preapoptotic and apoptotic cells after administration of L-NIO alone; (C) Dot plot distribution of live, preapoptotic and apoptotic cells after administration of E7080 and L-NIO in combination.

Figure 7

Cell distributions. (A) Distribution of live, preapoptotic and apoptotic cells after administration of E7080 alone; (B) Distribution of live, preapoptotic and apoptotic cells after administration of L-NIO alone; (C) Distribution of live, preapoptotic and apoptotic cells after administration of E7080 and L-NIO in combination; (D) Comparison of apoptotic cell percentage of alone and combination administrations.