Effects of herbal extract (GPO 1986) on tumor angiogenesis of hepatocellular carcinoma cells (HepG2) implanted in nude mice

Abstract

The objectives of this study were to examine effects of supplementation of herbal extract (GPO1986) on tumor angiogenesis and two kinds of angiogenic biomarkers (HIF-1[alpha] expression and serum VEGF level) in hepatocellular carcinoma cell (HepG2)-implanted nude mice. Male BALB/c-nude mice 8-10 weeks of age were used for this study. All animals were divided into two groups of HepG2 (HepG2) and control (Con). The animals were further separated into 3 subgroups of vehicle, treated with 640 and 3,200 mg/kg BW of GPO1986. In the nude mice model, a dorsal skin-fold chamber was used, in which HepG2 (American Type Culture Collection (ATCC)) was transplanted. On different time-points (2, 7 and 14 days) after HepG2 inoculation, the microcirculation within the chamber was observed using fluorescence videomicroscopy. Based on the recorded video images, capillary diameter and capillary density (CD) were determined to characterize tumor neovasculaization. VEGF expression was measured in blood withdrawn, using enzyme immunoassay while HIF-1[alpha] expression was measured on samples isolated from tumor inside the chamber, using immunohistochemistry. The experimental results revealed that the video images demonstrated dilation, hyperpermeability, tortousity of host microvessels 2 days after HepG2 inoculation. The tumor angiogenesis was initiated with endothelial sprouting from the host vessel on day 7 after HepG2 inoculation. Based on the recorded video images, the neocapillary density and diameter were measured using a digital image analysis (Global Lab image II software). The image analysis demonstrated that in the HepG2 group, the neocapillary density and diameter significantly increased on day 7 and 14 compared to the aged-matched controls (p<0.05). Supplementation of GPO1986 at high dose significantly attenuated the increase of tumor capillary density. However, at low dose of GPO1986, these parameters decreased significantly 14 day after HepG2 inoculation. The histological examination of tissue section within the dorsal skin-fold chamber showed remarkable decrease in tumor deposit with supplementation of GPO1986. The present VEGF measurement showed that in HepG2 group, the serum VEGF levels increased significantly up to 152 pg/ml on day 14 from 67 pg/ml on day 2. The VEGF levels on day 2 was not significantly different, compared with the control levels, but the serum VEGF levels on both 7 and 14 days were significantly higher than their control levels (p<0.05). Using the Pearson correlation, the association of serum VEGF with neocapillary density was significantly correlated (r=0.70, p<0.001). The HIF-1[alpha] staining showed that HIF-1[alpha] was expressed markedly 2 and 7 days after HepG2 inoculation, but its expression significantly decreased on day 14. The HIF-1[alpha] expression was not influenced by supplementation of GPO1986 at high and low dose any experimental periods after HepG2 inoculation. In conclusion, herbal extract (GPO1986) was able to suppress the growth of HepG2-implanted in nude mice, partly due to the inhibition of tumor angiogenesis. And this anti-angiogenesis might come from its inhibitory effect on VEGF expression, without any effects on HIF-1[alpha] expression.