| Part1The effect of CC1007on survival, cell cycle, and apoptosis of human hepatoma cells and its anti-tumour activity in vivoObjective:To study the effect of CC1007on survival, cell cycle, and apoptosis of human hepatoma cells and its anti-tumour activity in vivoMethods:Human hepatoma cells and fibroblasts were treated with different concentrations of CC1007for the indicated time. Cell survival/viability was tested by using the MTT assay, cell cycle distribution was detected by flow cytometry, and apoptosis was examined by Hoechst staining and flow cytometry using annexin V/propidium iodide (PI) staining. Furthermore, in vivo anti-tumour activity was examined in a nude mice model.Results:1.CC1007significantly decreased the survival rate of hepatoma cells in a dose-and time-dependent manner (P<0.05) while it had little effect on fibroblast cells. After treatment with40μM CC1007for72h, the survival rates of HepG2, PLC/PRF/5, Hep3B, and Huh7cells were 8.2±4.04%,2.3±1.78%,12.5±7.25%, and6.6±5.03%, respectively. The survival rate of fibroblasts was61.1±4.19%(P<0.05, versus hepatoma cell lines). The50%inhibitory concentrations after treatment for72h of HepG2, PLC/PRF/5, Hep3B, Huh7, and fibroblasts were27.1μM,2.6μM,12.12μM,5.6μM, and>40μM, respectively.2. After treatment with CC1007for24h, cell cycle analysis showed an increase in the G0/G1phase, which was accompanied with a reduction in the S phase. At concentrations of0μM,10μM,20uM, and40μM, the percentages of cells in the G0/G1phase were61.1%,79.4%,80.2%, and77.8%, respectively. The percentages of cells in the S phase were28.5%,12.1%,12.6%, and19.3%, respectively.3.Hoechst staining revealed typical apoptotic characteristics in HepG2cells treated with CC1007for48h. Flow cytometry using annexin V/PI staining showed that the cell apoptosis rates were increased with increasing drug concentrations in all4hepatoma cell lines. At a concentration of40μM, the apoptosis rates of HepG2, PLC/PRF/5, Huh7, and Hep3B cells were41.43±1.7%(P<0.05, versus control),70.46±10.48%(P<0.05, versus control),23.63±2.68%(P<0.05, versus control), and20.23±2.97%(P<0.05, versus control), respectively.4. In the in vivo study, the tumour volume was55.4%smaller in mice treated with50mg/kg per day of CC1007than in control, untreated mice (n=5,P<0.05). Body weight reduction was not observed in the treatment group.Conclusion:1. CC1007selectively kills human hepatoma cells in a dose-and time-dependent manner and showed little cytotoxic effect on fibroblast cells.2. CC1007treatment resulted in partial G1cell cycle arrest, at a concentration as low as10μM, in HepG2cells.3. CC1007can induce apoptosis in human hepatoma cells.4. CC1007can suppress tumour growth in a xenograft model. Part2Exploring the mechanisms of anti-hepatoma activity of CC1007Objective:To exploring the mechanisms of anti-hepatoma activity of CC1007Methods:Hepatoma cells were treated with CC1007for the indicated time. Acetylated histone3(Ace-H3) and4(Ace-H4) were detected by using western blot. The expression of P21and Nur77was detected by using reverse transcription-polymerase chain reaction and western blot. Caspase activity was tested by using the caspase3/7assay and western blot.Results:1. Western blot analysis showed that incubation with CC1007for24h resulted in the accumulation of Ace-H3and Ace-H4in4hepatoma cell lines.2. CC1007induced the expression of p21in HepG2cells, both at the mRNA and protein levels.3. After treatment with40μM CC1007for30h, the results of the caspase3/7assay suggested that caspase3/7was activated in HepG2and PLC/PRF/5cells. Western blot analysis revealed that caspases3and9were activated, while caspase8was not activated.4. CC1007induced the expression of Nur77in HepG2and PLC/PRF/5cells, both at the mRNA and protein levels. Conclusion:1. CC1007increases the expression of Ace-H3and Ace-H4in hepatoma cells.2. CC1007induces the expression of p21both at the mRNA and protein levels. Thus, p21may participate in the cell cycle arrest caused by CC1007.3. An intrinsic apoptotic pathway is activated by CC1007in hepatoma cells.4. The expression of apoptosis-related factor Nur77is induced by CC1007, both at the mRNA and protein levels, which indicates that Nur77may participate in the apoptosis caused by CC1007. Part3The role of Nur77in CC1007-induced apoptosisObjective:To exploring the role of Nur77in CC1007-induced apoptosisMethods:After treatment with40μM CC1007for the indicated time, the distribution pattern of Nur77in HepG2cells was detected by immunofluorescence and confocal microscopy. Western blot was used to detect the expression of Nur77in the nuclear and cytoplasmic fractions. Furthermore, we generated stable HepG2cell lines carrying Nur77short hairpin RNA (Nur77shRNA) or an empty shRNA vector (control) and compared the effect of CC1007on those2cell lines by using the MTT assay, Hoechst staining, and flow cytometry.Result:1. Immunofluorescence and confocal microscopy results suggested that the Nur77level was increased and that Nur77was confined to the nucleus after treatment with CC1007for24h. However, after treatment for30h, cytoplasmic localization of Nur77could be seen in most treated cells and the distribution patterns for Nur77and mitochondria overlapped extensively.2. Western blot analysis revealed that the expression of Nur77was increased in the nuclear fraction and constant in the cytoplasmic fraction after treatment with40μM CC1007for24h. After treatment for30h, Nur77was down-regulated in the nuclear fraction and up-regulated in the cytoplasmic fraction.3. Stable HepG2cell lines, overexpressing the shNur77vector or an empty shRNA vector (control) were treated with40μM CC1007for30h. The MTT assay revealed that the viability of shNur77and control groups were77.4±2.7%and63.4±0.8%(P<0.05), respectively. Hoechst staining showed that the apoptosis rates were43.1±1.8%and52.13±0.5%(P<0.05), respectively. Propidium iodide staining and flow cytometry revealed that the percentages of cells in the sub-G1phase were24.04±2.8%and30.4±2.0%(P<0.05), respectively.Conclusion:1. CC1007increases Nur77expression in the nucleus and subsequently induces the nuclear export of Nur77and mitochondrial targeting.2. Stable knock down of Nur77reduces the susceptibility of HepG2cells to CC1007-induced apoptosis.3. Nur77induced by CC1007participates in apoptosis. |
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