The Effects Of 5-FU Combined With Epigenetics Drug FK228 On Growth Of Human Hepatoma Line HepG2

Objective:To investigate the effects of 5-FU combined with FK228 on the proliferation,apoptosis and cell cycle arrest of HepG2.Methods:The HepG2 cells in the logarithmic growth period were incubated with FK228 and 5-FU in alone or combining manner at different doses for 72 hours.There were five groups in this experiment including untreated group;FK228 treated group;5-FU treated group;5-FU combined with FK228 group(after exposed to FK228 24h then exposing to 5-FU for 48h group);and cells exposed to FK228 and 5-FU at same time.The capacity of proliferation of HepG2 cells was assessed by tetrazolium salt colorimetry assay(MTT assay).The flow cytometry(FCM) was used to measure apoptosis rate and cell cycle distribution and reverse transcriptase PCR(RT-PCR)was used to detect the mRNA expression levels ofp21,p16 and Fas.Results:1.The effect of the proliferation of HepG2 cells treated by 5-FU or FK228.MTT assay showed that FK228 or 5-FU that can inhibit the growth of the HepG2 cells in comcentration-dependent and in time-dependent manner.2.The effect of the proliferation of HepG2 cells incubated by 5-FU combined with FK228.The MTT assay that combination FK228 with 5-FU for 72 hours.The can inhibit the growth of HepG2 cell in concentration dependent and time dependent manner.After exposing to(4μg/L,8μg/L)FK228 for 24h then followed exposing to(0.01,0.1,1.0,10μmol/L)5-FU for 48h,There were significant increasing of the growth inhibiting degrees between 5-FU treated group and 5-FU combined with FK228 group(p＜0.05).FK228 show interaction effect with 5-FU(p＜0.05.).The results show 5-FU combined with FK228 have good synergistic effect of proliferation of HepG2 cells.3.The effects of apoptosis and cell cycle arrest of HepG2 cells incubated by 5-FU combined with FK228.3.1 The flow cytometry showed that:FK228(4μg/L)can induce G0/G1 phase arresting and 5-FU(1.0μmol/L)can induce S phase arresting in HepG2 cells.The ratio of cell cycle arrested 5-FU combined with FK228 group at G0/G1 phase is higher than that of FK228 and 5-FU alone treated group(p＜0.05).3.2 The flow cytometry profiles showed that:the apotosis rate of HepG2 cells treated with FK228(4μg/L)treated group and 5-FU(1.0μmol/L)group was(4.05±0.30)%and(5.81±0.32)%respectively. The apotosis rate of HepG2 cells treated with 5-FU combined with FK228 group is(16.9±0.67)%and is higher than that FK228 or 5-FU treated group(p＜0.05).4.The effects of mRNA expression levels of p21and p16 and fas of HepG2 cells incubated by 5-FU combined with FK228.The results of RT-PCR of the p21 and p16 mRNA expression levels of HepG2 cells incubated y by FK228 and 5-FU alone or combining for 72 hours are following.The p21 mRNA expression levels of 5-FU combined with FK228 group are higher than that of FK228 and 5-FU alone treated groups(p＜0.05).However,The p16 mRNA expression levels of 5-FU combined with FK228 group was similar to that the FK228 and 5-FU alone treated group(p＞0.05).Conclusions:1.The capacities of proliferation of HepG2 cells incubated by 5-FU and FK228 for 24 hours,48 hours and 72 hours are inhibited by FK228 or 5-FU at the doses and the growth inhibiting effects become stronger along with dose increasing and time lengthening.2.Low dose FK228 pretreatment can increase the sensitivity of 5-FU,5-Fu combined with FK228 can synergistically inhibit HepG2 cell proliferation.3.Low dose FK228 pretreatment can increase the 5-FU apotosis and cell cycle arrest in HepG2 cell.4.FK228 and 5-FU synergistic inhibit HepG2 cell growth might be associated mostly with up-regulating the expression of p21 mRNA and Fas mRNA...