ABSTRACT
@#[Abstract] Objective: To construct recombinant plasmid Egr1-XPO4 and evaluate its synergic inhibition with 5-FU against hepatocarcinoma SK-Hep1 cells. Methods: The XPO4 gene was inserted into vector carrying promoter Egr1 to construct a new recombinant vector, Egr1-XPO4, which was then transfected into human hepatocarcinoma cell line SK-Hep1 and sensitized with chemotherapeutic drug 5-FU. Western blotting was adopted to examine the protein expression of XPO4; CCK assay was used to detect SK-Hep1 cell proliferation after transfection, and Flow Cytometry with Annexin V-FITC/PI staining was used to detect the apoptosis of SK-Hep1 cells. SKHep1 cell xenograft model was constructed on nude mice, and the effect of Egr1-XPO4 in combination with 5-FU on the growth of xenograft was observed. Results: The recombinant plasmid Egr1-XPO4 was successfully constructed.With the sensitization of 5-FU, the expression of XPO4 protein in SK-Hep1 cells was significantly elevated after Egr1-XPO4 transfection, and the evlevation was in a 5FU dose-depend manner.The combined treatment of Egr1-XPO4 and 5-FU produced a significantly stronger inhibition against SKHep1 cell proliferation and greatly promoted apoptosis of SK-Hep1cells compared with 5-FU or pEgr-XPO4 mono-treatment group (all P<0.05). And in vivo antitumor experiment showed that the tumor volume in Egr1-XPO4+5-FU treatment group was significantly smaller than that of Egr1-XPO4 or 5-FU mono-treatment group (P<0.05). Conclusion: The recombinant plasmid Egr1-XPO4 in combination with 5-FU could exertsynergic inhibitionagainst hepatocarcinomaSK-Hep1 cells.
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Objective:To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR)promoter.Methods:The synergistic transcriptional effect,subcellular localization,and protein-protein interaction for IRF4 and MITF were observed by luciferase assay,immunofluorescence,GST-pull down,and co-immunoprecipitation,respectively.Results:IRF4 and MITF proteins were co-expressed in the cell nucleus.IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter,but with no effect on other MITF-specific target promoters.IRF4 alone did not affect TYR promoter significantly.No direct interaction between the two proteins was noted.Conclusion:IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF.This synergistic effect is mainly regulated by MITF;DNA might be involved in the interaction between the two proteins.
ABSTRACT
Objective To investigate the synergic ratio of sorafenib (SO) and sulforaphane (SF) against the hepatocellu‐lar carcinoma cell line HepG2 in vitro .Methods The synergic effect of SO combined with SF against HepG2 cells was deter‐mined by the CCK8 assay (the synergic effect was determined by combination index (CI) value:CI>1 .1 ,antagonistic;0 .9<CI<1 .1 ,additive;CI<0 .9 ,synergic) .The effectiveness of the synergic effect of the synergic ratio was confirmed by the An‐nexinV‐FITC apoptosis experiment and colony formation assay .Results The CCK‐8 assay showed that SO combined with SF at the molar rate of 1∶1 ,1∶10 and 1∶20 exhibited strong antagonism (CI>1 .1) ,whereas 20∶1 ,5∶1 and 1∶5 ratio resul‐ted in synergistic activity (CI<0 .9) .Furthermore ,10∶1 ratio acted as an additive effective (0 .9<CI<1 .1) .The number of colony formation (14 .66 2 .08) of the group treated with SO combined with SF at 5∶1 synergistic ratio was significantly lower than that of the group treated with after SO combined with SF at 1∶1 antagonistic ratio (31 .33 4 .16) (P<0.01) .The early and late apoptosis rate of HepG2 cells (21 .71 ± 6 .06) of the group treated with SO combined with SF at 5∶1 synergistic ratio was significantly higher than that of the group treated with SO combined with SF at 1∶1 antagonistic ratio(6 .64 ± 0 .311)(P<0.01) .Conclusion SO combined with SF at the molar rate of 5∶1 can synergistically inhibit the growth of hepatocellular car‐cinoma cell line HepG2 in v itro .