OBJECTIVE

To investigate the effects of norcantharidin (NCTD) on angiogenesis of human gallbladder carcinoma and its anti-angiogenic mechanisms.

METHODS

Human gallbladder carcinoma cells of the line GBC-SD were cultured. BALB/c nude mice were inoculated subcutaneously with the GBC-SD cells and then randomly divided into 6 groups: NCTD group, injected intraperitoneally with 1/5 of the LD(50) of NCTD twice a week for 6 weeks; 5-fluorouracil (5-FU) group, injected intraperitoneally with 1/5 of the LD(50) of 5-FU twice a week for 6 weeks; endostatin (ES) group, intraperitoneally with ES; NCTD + 5-FU group, injected intraperitoneally with 1/5 of the LD(50) of NCTD and 1/5 of the LD(50) of 5-FU twice a week for 6 weeks; NCTD + ES group, injected intraperitoneally with 1/5 of the LD(50) of NCTD and ES twice a week for 6 weeks; and normal saline (NS) group (control group), injected with NS. The mice were killed in the 7th week. The tumors were taken out to measure their volumes and undergo microscopy. SABC method of immunohistochemistry was used to measure the microvessel density (MVD) and the protein expression of the angiogenesis-related factors: proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), angiopoietin (Ang)-2, thrombospondin (TSP), and tissue inhibitor of metalloprotease (TIMP)(2). Suspension of single tumor cell was prepared to examine the cell apoptosis by flow cytometry. RT-PCR was used to examine the mRNA expression of PCNA, VEGF, Ang-2, TSP, and TIMP2.

RESULTS

(1) The MVD of the NCTD group was 4.12 +/- 1.4, significantly lower than those of the 5-FU group (15.8 +/- 5.9) and control group (17.6 +/- 3.2) (both P < 0.01), but not significantly different from those of the NCTD + 5-FU group (3.8 +/- 1.7), ES group (4.5 +/- 2.1), and NCTD + ES group (2.9 +/- 1.5) (all P > 0.05). The mice treated with NCTD showed significantly smaller tumor volume, lower PCNA protein expression, higher apoptotic rate, and higher PCNA/apoptosis ratio (P < 0.05 or P < 0.01), and significant correlation between MVD and tumor volume and between MVD and PCNA/apoptosis ratio (both P < 0.05). (2) The protein expression of VEGF and of Ang-2 of the NCTD group were both significantly lower than those of the control and 5-FU groups (all P < 0.01), however, not significantly different from those of the ES, NCTD + 5-FU, and NCTD + ES groups; and the protein expression of TSP and of TIMP2 of the NCTD group were both significantly higher than those of the control and 5-FU groups (all P < 0.01), however, not significantly different from those of the ES, NCTD + 5-FU, and NCTD + ES groups. MVD was positively correlated with VEGF and Ang-2 expression and negatively correlated with the expression of TSP and TIMP2 (all P < 0.05). (3) In comparison with the control group, the mRNA expression of VEGF and of Ang-2 of the tumor cells of the NCTD group were both significantly lower and the mRNA expression of TIMP2 was significantly higher.

CONCLUSION

NCTD down-regulates the expression of the angiogenic factors, such as VEF|GF and Ang-2, and up-regulates the expression of the anti-angiogenic factors, such as TDP and TIMP2, thus inhibiting the angiogenesis in tumor, such as human gallbladder carcinoma, and further inhibiting the growth of tumor.