The blots were first incubated with blocking buffer (10 mM Tris-HCl, pH 8.0, 0.9% NaCl, and 4% skim milk) for 1 h and then reacted with the antibodies for 1 h at room temperature. inhibits the tumorigenic properties induced by repeated EBV reactivation, including micronucleus formation, cell proliferation, migration, and matrigel invasiveness. Emodin administration also represses the tumor growth in mice which is usually induced by EBV activation. Taken together, our results provide a potential chemopreventive agent in restricting EBV reactivation and NPC recurrence. 0.01; HONE1 vs. HA: = 0.06). Based on these results, we chose 1 to 50 M of emodin as our working concentrations for further studies. Open in a separate window Physique 1 Epstein-Barr virus (EBV) positive nasopharyngeal carcinoma (NPC) cells are more resistant to emodin. (a) The chemical structure of emodin. (b) NPC cell lines (TW01, HONE-1) and their EBV infected counterparts (NA, HA) were treated with indicated concentrations of emodin for 48 h, followed by cell viability assay and CC50 calculation (top of each panel). The values are means SD from at least three impartial experiments. (* 0.05, ** 0.01, *** 0.001 compared to the group of 0 M). 2.2. Emodin Inhibits EBV Lytic Protein Expression in NPC Cells In our hands, EBV lytic replication can be efficiently induced by treating NA Mouse monoclonal to HAUSP or HA cells with 40 ng/mL 12- 0.05, ** 0.01, *** 0.001 compared to the TS group). Taken together, the results above indicate that emodin can repress Ledipasvir acetone EBV lytic protein expression and attenuate virion production, clearly suggesting its ability to inhibit EBV reactivation. 2.4. The Repression of Zta Promoter (Zp) and Rta Promoter (Rp) Transcriptional Activities by Emodin Zta and Rta are two important immediate-early (IE) proteins involved in the initiation of EBV lytic reactivation. To access whether emodin exerts its anti-EBV activity through interfering with IE gene promoters, a luciferase reporting assay was performed to detect promoter activities (Zp and Rp, respectively) in the presence or absence of emodin. Both EBV-positive (NA) and -unfavorable (TW01) NPC cells were used in this study. As shown in Physique 5a,b, while TPA+SB significantly increased Zp and Rp activities in both NA and TW01 cells, addition of emodin decreased both promoter activities in a dose-dependent manner. Of note, promoter activities detected in NA cells are higher than in TW01 cells because the EBV harboring in NA cells creates an autocrine regulation to amplify the Zp and Rp activities under simulation. Next, in addition to TPA + SB, we asked whether emodin also inhibits Zta or Rta mediated EBV reactivation. To this end, Zta- or Rta-expressing plasmids were co-transfected with Zp or Rp reporter plasmids, respectively, followed by emodin treatment for 24 h. As expected, ectopic Zta activated both Zp Ledipasvir acetone and Rp, Ledipasvir acetone whereas co-treatment of emodin significantly reduced both promoter activities in a dose-dependent manner (Physique 5c,d). Similarly, over-expression of Rta resulted in Zp and Rp activation; addition of emodin reversed this phenomenon (Physique 5e,f). Thus, these results suggest that emodin is able to inhibit both chemical and Zta/Rta-induced EBV lytic reactivation via repressing IE gene promoter activation. Open in a separate window Open in a separate window Physique 5 The activities of Zp and Rp are repressed by emodin treatment of NA cells. (a,b) NA and its parental EBV unfavorable cell line, TW01, were transfected with luciferase reporters made up of Zp or Rp, followed by emodin (E) and TPA + SB (TS) treatments. After TS induction Ledipasvir acetone for 24 h, cell lysates were collected for measurement of luciferase activity. Data are means SD from at least two impartial experiments. (c,d) Zta-expressing plasmid (Z) was co-transfected with Zp or Rp luciferase reporters into NA (c) or TW01 (d) cells, with same emodin and TPA+SB treatments depicted in (a,b). (e,f) same to (c,d), except Rta-expressing plasmid (R) was used. (* 0.05, ** 0.01, *** 0.001 compared to the groups of TS, Z or R, respectively). 2.5. Identification of Emodin Responsive Element in Zp Because Zta is the first protein expressed in the EBV lytic cycle, we wished to know which elements in Zp are essential for emodin inhibition. To this end, luciferase-reporting plasmids driven by a series of 5-deletion mutants of Zp were constructed. These mutants were respectively transfected into TW01 cells, followed by aforementioned compound treatment and luciferase-reporting assay. As depicted in Physique 6a, compared to wild type, promoter Zp-99 retained only half of the.