The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells.
To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein ("ZEBRA") in this system, as demonstrated by induction of the BZLF1 protein-responsive DR promoter upstream of the luciferase gene. Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin. Semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor-beta (TGF-beta) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells. TPA treatment also induced luciferase mRNA with similar kinetics. Curcumin was found to be highly effective in decreasing TPA-, butyrate-, and TGF-beta-induced levels of BZLF1 mRNA, and of TPA-induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin. Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP-1) binding to a cognate AP-1 sequence was detected at 6 h and could be blocked by curcumin. Protein binding to the complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin. Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody-induced supershift analysis. The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR-CAT cells (carrying DR-dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription.
Hergenhahn
Manfred
M
Division of Genetic Alterations in Carcinogenesis, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Soto
Ubaldo
U
Weninger
Annette
A
Polack
Axel
A
Hsu
Chih-Hung
CH
Cheng
Ann-Lii
AL
Rösl
Frank
F
D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D002273:Carcinogens / Q000037:antagonists & inhibitors; D003474:Curcumin / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D017930:Genes, Reporter; D004854:Herpesvirus 4, Human / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000037:antagonists & inhibitors; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014764:Viral Proteins; D017735:Virus Latency
D016588:Anticarcinogenic Agents; C063676:BZLF1 protein, Herpesvirus 4, Human; D002273:Carcinogens; D004268:DNA-Binding Proteins; D012367:RNA, Viral; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; D008156:Luciferases; D003474:Curcumin; D013755:Tetradecanoylphorbol Acetate