The chemopreventive compound curcumin is an efficient inhibitor of Epstein‐Barr virus BZLF1 transcription in Raji DR‐LUC cells*

Abstract

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.

Alveolar epithelial cell injury with Epstein-Barr virus upregulates TGFβ1 expression

“…VAAK and V-AEC2 cells were exposed in different experiments to 30 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA), 3 mM sodium butyrate (BA), 15 M curcumin, 10 g/ml ganciclovir, and 10 M SB-431542 at several time points. Curcumin has been utilized as an inducer of apoptosis by reducing AP-1 transcription factor activity in EBV-infected cells, as previously described (17). We used SB-431542, an inhibitor of ALK4/5/7 via TGF␤RI, to inhibit autocrine TGF␤1-induced apoptosis, as previously described (20).…”

Inhibition of Epstein-Barr virus reactivation by the flavonoid apigenin

“…In addition to apigenin, curcumin has been shown to have an inhibitory effect on Zp to block the EBV lytic cycle in B cells [22]. Using a reporter assay, retinoic acid was also found to have potent anti-EBV activity [20].…”

“…Using a reporter assay, retinoic acid was also found to have potent anti-EBV activity [20]. Compared to some other natural compounds affecting only Zp [22] or Rp [44], apigenin is able to interfere with both Zp and Rp activities, suggesting it may have a broader spectrum for application.…”

Cancer and diet: How are they related?

“…Curcumin, a polyphenol derived from turmeric, has been shown to inhibit TPA-induced AP-1 activation in HL-60 cells and Raji cells [86,87]. Curcumin treatment has also been shown to abrogate constitutive AP-1 activity in prostate cancer cell lines [88,89].…”