Effects of curcumin on angiogenesis of hepatocellular carcinoma cells (HepG2)

Abstract

The objective of this study was to determine the effects of curcumin on angiogenesis in HepG2-implanted nude mice and to study the effects of curcumin on HepG2 angiogenic biomarkers, COX-2 and VEGF levels. Male BALB/c nude mice weighing 20-25 g were used. After the implantation of a dorsal skin-fold chamber, the Hepatocellular carcinoma cells (HepG2) (30 µl of 2 X 10⁶ cells) were then inoculated onto the upper layer of the skin within the chamber (HepG2). The control group (Con.) was received the normal saline instead. One day after the inoculation of HepG2, dimethyl sulfoxide solution (0.1%) and curcumin solution (300 or 3,000 mg/kgBW) were fed oral daily to both groups (Con and HepG2) and (Con-cur and HepG2-cur groups), respectively. On days 3, 7, and 14 post-inoculation, the tumor microvasculature was visualized by injecting 0.1 ml of 0.5% rhodamine B isothiocyanate-labeled dextran (RITC-dextran) intravenously, and observed under an intravital fluorescence videomicroscope. At the end of each experiment, serum VEGF and tissue COX-2 protein were determined by using VEGF ELISA kit and Western blot analysis, respectively. Moreover, the specimen from the tissue within dorsal skin chamber was collected for further histological examination using Hematoxylin&Eosin technique (H&E). The experimental results revealed that the videoimage demonstrated dilatation, tortuosity, hypermeability of host microvessels after 3 days of HepG2 inoculation. The tumor angiogenesis was observed with endothelial cells sprouting in the mother vessel on day 7 after the inoculation of HepG2. Based on the recorded videoimage, the tumor neocapillary density, diameters, and microvasculature were evaluated using a digital image analysis and correlated with tumor area. The image software analysis demonstrated that in the HepG2-group the neocapillary densities and diameters were significantly increased on day 7, and day 14, compared to the aged-matched control-group (p<0.001, p<0.05). This increment of neocapillary densities and diameters in HepG2 groups appeared to be correlated with increase in the tissue blood perfusion measured using a Laser Doppler Flowmeter. High dose treatment of curcumin significantly attenuated the increase of tumor neocapillary density, diameter and tissue blood perfusion after 7 days of the treatment. Low dose treatment of curcumin, these parameters were significantly decreased after 14 days of curcumin treatment. It is strongly supported by histological examination, the section of tissue within dorsal skin fold chamber of HepG2-cur 3,000 show remarkable decreased in tumor cells deposit. In both Con and Con-cur groups without tumor, the curcumin treatment did not any affect on the microcirculatory parameters during any periods. Western blot analysis data demonstrated overexpression of COX-2 since 3 days after the inoculation of HepG2. Moreover, the present study demonstrated the significantly relationship between COX-2 expression and neocapillary density in HepG2 groups (r² = 0.9607, p<0.01). Sera VEGF were markedly increased in 7 and 14 days HepG2 groups as compared to 3 days HepG2 group and aged- matched control groups (p<0.001). The tumor-induced overexpression of COX-2 was inhibited by both low and high dose of curcumin only after 3 days of the treatment. Elevations of serum VEGF in HepG2 groups were reduced by curcumin treatment at the dose of 300 mg/kgBW after 7 days of the treatment. A correlation between elevation of serum VEGF and NCD was lower than that between COX-2 and NCD (r² = 0.7681, p<0.32). No significant correlation was found between COX-2 expression and VEGF production (r² = 0.5829, p<0.447). In conclusion, the present study indicates that COX-2 is closely related to tumor angiogenesis in the HepG2-implanted nude mice model. COX-2 and VEGF may co-modulate angiogenesis in different pathway. Curcumin has anti-angiogenic activity in vivo. The activity of curcumin in inhibiting angiogenesis may be mediated in part through reduction of angiogenic stimulator or biomarkers production. Therefore, curcumin should be considered as a possible safe and non-toxic drug for treatment against cancer in the future.