Vasculogenic Mimicry Of Human Hepatocellular Carcinoma Cell Line In Vitro

OBJECTIVETo investigate the plasticity of hepatoma cells and their ability to form vasculogenic mimicry, to illustrate mechanism of vasculogenic mimicry of hepatocellular carcinoma and to investigate the effect of angiogenesis inhibitor TNP-470 on the vasculogenic mimicry in human hepatocellular carcinoma cell line in vitro.METHODS(1) Human hepatocellular carcinoma cell lines ( HepG2, MHCC97-H and MHCC97-L) were tested for their ability to form tube-like networks in three-dimensional culture containing matrigel. The structures of the tubular networks were stained with periodic acid-schiff (PAS) and hematoxylin and eosin (HE), then observed by inverted microscope and scanning electron microscope.(2) Based on three-dimensional culture system developed by matrigel, human hepatocellular carcinoma cell lines were induced under condition of hypoxia, tubular network was observed by light microscopy. Relative expression of hypoxia-inducible factor (HIF) -1αmRNA and matrix metalloproteinases (MMP)-2mRNA was detected by RT-PCR simultaneously.(3) The effects of TNP-470 on proliferation and invasion of MHCC97-H cells were detected by MTT assay and invasion assay in vitro. MHCC97-H cells were tested for their ability to form vasculogenic mimicry and the effect of TNP-470 on vasculogenic mimicry formation in three-dimensional culture containing matrigel. The ultrastructrue of MHCC97-H cells was observed by scanning and transmission electron microscope. RESULTS(1) HepG2 cells linked each other to form wreaths and network structures after 72 hours based on three-dimensional culture. Vascular-like structures of HepG2 cells were stained positively with PAS, and network structures of HepG2 cells were stained with HE.Observation with scanning electron microscope showed that HepG2 cells stretched out thin and long processes to link each other, and formed tube-like structure when grown in three-dimensional culture. Whereas, HepG2 cells linked each other to form wreaths and network structures under condition of hypoxia after 24 hours.MHCC97-H cells (high metastastic potential) can form vasculogenic mimicry much more and faster than MHCC97-L cells (low metastastic potential) at different time.MHCC97-H cells had more surface microvillus and cell junctions than MHCC97-L cells by scanning and transmission electron microscope.(2)The expressions of HIF-1αmRNA and MMP-2mRNA were dramatically enchanced in 8-hour hypoxia group, then decreased in 12-hour,18-hour and 24-hour hypoxia group.The levels of HIF-1αmRNA and MMP-2mRNA expression of 8-hour hypoxia group were 1.043±0.019 and 1.256±0.024,which were significantly higher than those under no-hypoxia(0.220±0.005, 0.059±0.006;both p<0.01),and those of 24-hour hypoxia group(0.008±0.001, 0.219±0.009;both p<0.01).(3) MTT assay showed that the proliferation of MHCC97-H cells was not significantly affected by TNP-470 at 20,40,80,100μg/ml. MHCC97-H cell invasion could be significantly inhibited by TNP-470 (at 80,100μg/ml) with inhibitory rate of 66.7% and 77.8%(P<0.01).Both MHCC97-H cells in control group and TNP-470 group could form tube-like structure, but TNP-470at 100μg/ml) could significantly decrease tube-like formation compared with control group (48.6±9.61, 142.2±10.21, P<0.01).TNP-470 significantly decreased the surface microvillus and cell junction of MHCC97-H cells by scanning and transmission electron microscope. CONCLUSIONS(1) Hepatoma cells may generate vascular channels by self-metamorphosing and have the ability to form vasculogenic mimicry. MHCC97-H cells (high metastastic potential) can form vasculogenic mimicry much more and faster than MHCC97-L cells (low metastastic potential).(2)Hypoxia plays a key role in development of vasculogenic mimicry in hepatocellular carcinoma. Vasculogenic mimicry of human hepatocellular carcinoma cell line may be related to the expression of HIF-1αmRNA and MMP-2mRNA under condition of hypoxia.(3) TNP-470 can inhibit vasculogenic mimicry formation of human hepatocellular carcinoma cell line MHCC97-H by inhibiting invasion, decreasing the surface microvillus and cell junction in vitro.