Neamine inhibits oral cancer progression by suppressing angiogenin-mediated angiogenesis and cancer cell proliferation

Abstract

Background: Angiogenin undergoes nuclear translocation and stimulates ribosomal RNA transcription in both endothelial and cancer cells. Consequently, angiogenin has a dual effect on cancer progression by inducing both angiogenesis and cancer cell proliferation. The aim of this study was to assess whether neamine, a blocker of nuclear translocation of angiogenin, possesses antitumor activity toward oral cancer.

Materials and methods: The antitumor effect of neamine on oral cancer cells was examined both in vitro and in vivo.

Results: Neamine inhibited the proliferation of HSC-2, but not that of SAS oral cancer cells in vitro. Treatment with neamine effectively inhibited growth of HSC-2 and SAS cell xenografts in athymic mice. Neamine treatment resulted in a significant decrease in tumor angiogenesis, accompanied by a decrease in angiogenin- and proliferating cell nuclear antigen-positive cancer cells, especially of HSC-2 tumors.

Conclusion: Neamine effectively inhibits oral cancer progression through inhibition of tumor angiogenesis. Neamine also directly inhibits proliferation of certain types of oral cancer cells. Therefore, neamine has potential as a lead compound for oral cancer therapy.

Keywords: Neamine; angiogenesis; angiogenin; cell proliferation; oral cancer.

Figure 1

Chemical structure of neomycin and neamine. The amino group in neomycin and neamine shown in red is essential for blocking nuclear translocation of angiogenin.

Figure 2

Inhibition of nuclear translocation of angiogenin in HSC-2 and SAS cells by neamine. Cells were incubated with 1 μg/ml angiogenin in the presence of 100 μM neomycin, neamine or paromomycin at 37°C for 30 min. Angiogenin was visualized by monoclonal antibody and Alexa 488-labeld goat anti-mouse IgG.

Figure 3

Effect of neamine on proliferation of HSC-2 and SAS cells. Cells were seeded at a density of 2.5×10⁴ cells per 35-mm dish and starved in serum-free DMEM/F12 for 24 h. They were then washed in PBS three times and cultured in serum-free DMEM/F12 in the presence of neamine or paromomycin for 48 h. Cells were subsequently detached by trypsinization and counted. The percentage of cell proliferation was calculated based on the cell number in the absence of the test compounds. All experiments were repeated three times. Data are presented as the means±SD of triplicates from a typical experiment. *p<0.05, **p<0.01.

Figure 4

Effect of neamine on xenograft growth of HSC-2 and SAS cells in athymic mice. HSC-2 or SAS cells, 5×10⁵ per mouse, were inoculated subcutaneously into the right dorsal region of each mouse. The animals were treated with local subcutaneous injections of PBS or neamine (30 mg/kg) three-times weekly. Five mice per group were used. A: Tumor size (as volume) was measured weekly and recorded in cubic millimeters (length × width²/2). B: Mice were sacrificed at day 35 for HSC-2 and day 21 for SAS cells. Tumor tissues were dissected, weighed, and photographed. C: Final tumor weights are shown as the means±SD. *p<0.05, **p<0.01.

Figure 5

IHC analyses of HSC-2 and SAS xenograft tumor specimens. IHC staining for angiogenin, PCNA, and neovessels in the tumors from control (PBS-treated) and neamine-treated groups. CD31-positive neovessels in each tumor were counted in five most vascularized areas at ×200 magnification and averaged. PCNA-positive and total numbers of cells were counted in five randomly selected areas at ×200 magnification. Images shown were from a representative animal of each group. Vessel density (vessels per field) and percentage of PCNA-positive cells are shown as the means±SD for each group. Bars, 100 μm. **p<0.01.

Figure 6

Effect of neamine on ribosome biogenesis and apoptosis of HSC-2 and SAS xenografts. A: NOR dots in both types of tumor cells were examined to obtain a quantitative assessment of the changes in ribosome biogenesis affected by neamine. Silver-stained NOR dots were counted in 60 randomly selected nuclei, and the numbers were averaged. Results are shown as the means±SD for each group. Inset panels show higher magnification views of NOR dots. Bars, 25 μm. **p<0.01. B: Apoptosis was examined by TUNEL staining. Nuclear staining with DAPI was carried out to quantify cell number. Five randomly selected areas at ×200 magnification were counted, and the percentage of apoptotic cells was calculated. Results are shown as the means±SD for each group. Bars, 100 μm. *p<0.05.