The Effects Of Vitamin D₃ Analogue ED-71 On Liver Carcinoma Prevention

ObjectivesTo explore the effects of vitamin D₃ analogue ED-71 on human liver carcinoma and its possible mechanisms.1. To explore the effects of ED-71 which is vitamin D₃ analogue on proliferation and hase growth arrest in human liver carcinoma cell lines2. To explore the mechanism of ED-71 on restraining proliferation and inducing apoptosis in human liver carcinoma cell line. To seek the relationships between differential antioncogene and ED-71. To argue the route of apoptosis in human liver carcinoma cell line induced by ED-71.3. To investigate the prevention of hepatocarcinogenesis by ED-71 and mechanisms of the effect.Methods1. Carcinoma cells HepG₂ were treated with ED-71 at different concentration. MTT assay was adopted to describe the proliferation of carcinoma cells. Light microscopy was used to find morphological changes in carcinoma cells. Cell cycle and early apotosis was determined by flow cytometry. Electron microscope was employed to observe morphological changes of apotosis in carcinoma cells. 2. Carcinoma cells HepG₂ were treated with Ed-71 at different concentration. Expression of P^21WAF1/CIP1 , P^27kip1 andIGFBP-3 was determined by RT-PCR. The level of protein including P^21WAF1/CIP1 , P^27kip1 and IGFBP-3 was determined byWestern-blot.3. Made rats models of hepatocellular carcinoma (HCC) with diethylnitrosamine (DEN). Dissected rats and detected the number of liver tumor of rats with or without ED-71. Tested the effects of ED-71 on positive rates of GST-P and PCNA cells in liver tissues with immunohistochemistry. Detected the effect of ED-71 on inducing apoptosis of liver cells in rats with TUNEL test. Analysed the effect of ED-71 on cell cycle of hepatocyte in rats with flow cytometry. Detected the levels of Ca⁺⁺, P in blood of rats.Results1. ED-71 treatment led to morphological changes in carcinoma cells HepG₂2. ED-71 treatment caused a decline in the fraction of S-phase cells and an increase in G₀/G₁ cells. Ed-71 increased the apoptosis rate of HepG₂.3. ED-71 treatment caused a time- and dose-dependent inhibition in carcinoma cells HepG₂ proliferation.4. ED-71 treatment led to increasing mRNA expression of P^21WAF1/CIP1 , P^27kip1 and IGFBP-3 in human liver carcinoma cellsHepG₂.5. ED-71 treatment inhanced synthsizing protein of P^21WAF1/CIP1 , P^27kip1 and IGFBp-3 in human liver carcinoma cellsHepG₂. 6. The results of animal experiment indicated that, ED-71 could significantly reduce the number of liver tumors and the positive rates of GST-P and PCNA cells in liver tissues of rats (P<0.05) , increase the apoptosis rates and arrest the cell cycle to G₀/G₁ phase. ED-71 had no effect on the levels of Ca⁺⁺, P in blood of rats.Conclusion1. ED-71 treatment could inhibit proliferation of human liver carcinoma cells HepG₂, cause partial differentiation and led to partial apoptosis. Suggesting ED-71 might play a role in treatment of liver tumor.2. ED-71 treatment could inhibit proliferation of human liver carcinoma cells HepG₂ , cause partial differentiation and led to partial apoptosis through inducing the expression ofP^21WAF1/CIP1 , P^27kip1 and IGFBp-33. ED-71 could inhibit proliferation and induce apoptosis of liver cells of experimental rats.ED-71 could prevent the hepatocarcinogenesis in experimental rats. ED-71 could reduce the cancer cells in the blood of experimental rats.