Lupeol is one of active components in the extract of Chrysanthemum indicum Linne that inhibits LMP1-induced NF-κB activation

Abstract

We have previously reported that seventy percent ethanol extract of Chrysanthemum indicum Linne (CIE) strongly reduces Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) survival by inhibiting virus-encoded latent infection membrane protein 1 (LMP1)-induced NF-κB activation. To identify an active compound(s) in CIE that inhibits LMP1-induced NF-κB activation, activity-guided fractionation was employed. The CH2Cl2 fraction of CIE strongly reduced LMP1-induced NF-κB activation and LCL viability with relatively low cytotoxic effects on primary human foreskin fibroblast (HFF), HeLa or Burkitt's lymphoma (BL41) cells. Furthermore, lupeol, a pentacyclic triterpene, was identified in the CH2Cl2 fraction of CIE to attenuate LMP1-induced NF-κB activation and LCL viability. This study demonstrates that lupeol is one of active compounds in the CH2Cl2 fraction of CIE that inhibits LMP1-induced NF-κB activation and reduces NF-κB-dependent LCL viability.

Figure 1. Fractionation scheme for the CIE.

Figure 2. The CH₂Cl₂ fraction of CIE inhibits LMP1-induced NF-κB activation.

(A) HEK293 cells were co-transfected with pSG5 (lanes 1, 3, 5, 7, 9, and 11) or pSG5-FLAG-LMP1 (lanes 2, 4, 6, 8, 10 and 12) plus NF-κB dependent firefly luciferase and control Renilla luciferase plasmids. Cells were treated with DMSO (lanes 1 and 2) or ddH₂O (lanes 3 and 4), n-Hexane (lanes 5 and 6), CH₂Cl₂ (lanes 7 and 8), EtOAc (lanes 9 and 10) or n-BuOH (lanes 11 and 12) fraction of CIE at 100µg/ml, and luciferase activity was measured using a dual luciferase assay system. (B) HEK293 cells were co-transfected with pSG5 (lanes 1 and 2), pSG5-FLAG-LMP1 WT (lanes 3 and 4), pSG5-FLAG-LMP1 1-231 (lanes 5 and 6) or pSG5-FLAG-LMP1 Δ187-351 (lanes 7 and 8) plus NF-κB dependent firefly luciferase and control Renilla luciferase plasmids. Cells were treated with either DMSO (lanes 1, 3, 5 and 7) or the CH₂Cl₂ fraction of CIE (lanes 2, 4, 6 and 8) at 100µg/ml, and luciferase activity was measured using a dual luciferase assay system. NF-κB dependent luciferase activity was expressed in RLU by normalizing firefly luciferase activity with constitutive Renilla luciferase activity. To calculate relative luciferase activity, LMP1-induced luciferase activities in the presence of DMSO was set 100%. Significant difference between lanes 5 and 6 was determined by the P value of a two-sample t test (P < 0.0001). Luciferase data shown here represent three independent experiments. (RLU, relative luciferase unit).

Figure 3. The CH₂Cl₂ fraction of CIE inhibits IKK activation.

(A) Parental BL41 cells and their counterparts expressing LMP1 were treated with either DMSO or CH₂Cl₂ fraction of CIE at 100µg/ml for 24hr, and equal amounts of cell extracts were subjected to Western blot analysis with anti-p100/p52, anti-phopho-IκBα, anti-IκBα, anti-LMP1 or anti-tubulin antibody. (B) Raw 264.7 cells were pre-treated with either DMSO or CH₂Cl₂ fraction of CIE at 100µg/ml for 3hr and stimulated with LPS at 1µg/ml. At 0, 15, 30, 45 and 60 min after LPS treatment, equal amounts of cell extracts were subjected to Western blot analysis with anti-IκBα or anti-tubulin antibody. (C) HeLa cells were pre-treated with either DMSO or CH₂Cl₂ fraction of CIE at 100µg/ml for 3hr and stimulated with IL-1β at 20ng/ml. At 0, 15, 30, 45 and 60 min after IL-1β treatment, equal amounts of cell extracts were subjected to Western blot analysis with anti-IκBα or anti-tubulin antibody.

Figure 4. The CH₂Cl₂ fraction of CIE reduces LCL viability.

LCLs were treated with either DMSO or n-Hexane, CH₂Cl₂, EtOAc or n-BuOH fraction of CIE at 100µg/ml, and cell viability was determined at 0, 3, 6, 9, 12 or 24hr after treatment using CellTiter-Glo Luminescent Cell Viability Assay. A score of 1.0 indicates that there is no difference in viability between DMSO or CIE fraction treated cells. Luciferase data shown here represent three independent experiments.

Figure 5. The CH₂Cl₂ fraction of CIE is more cytotoxic toward LCLs than HFF, HeLa or BL41 cells.

(A) LCLs, (B) HFF, (C) HeLa or (D) BL41 cells were treated with either DMSO or CH₂Cl₂ fraction of CIE at 6.25, 12.5, 25, 50 or 100µg/ml, and cell viability was determined at 24, 48 or 72hr after treatment using CellTiter-Glo Luminescent Cell Viability Assay. A score of 1.0 indicates that there is no difference in viability between DMSO or CH₂Cl₂ fraction of CIE treated cells. Luciferase data shown here represent three independent experiments.

Figure 6. The CH₂Cl₂ fraction of CIE induces apoptosis in LCLs.

LCLs were treated with 100µg/ml of either DMSO or the CH₂Cl₂ fraction of CIE, and PARP cleavage was determined by Western blot analysis at 0, 1, 3, 6, 9, 12 or 24hr after treatment.

Figure 7. Sub-fractionation and isolation scheme for lupeol from the CH₂Cl₂ fraction of CIE.

Figure 8. Lupeol inhibits LMP1-induced NF-κB activation.

(A) The lupeol structure. (B) HEK293 cells were co-transfected with pSG5 (lanes 1, 3, 5, 7, 9, and 11) or pSG5-FLAG-LMP1 (lanes 2, 4, 6, 8, 10 and 12) plus NF-κB dependent firefly luciferase and control Renilla luciferase plasmids. Cells were treated with lupeol at 0, 3.125, 6.25, 12.5, 25 or 50µg/ml, and luciferase activity was measured using a dual luciferase assay system. NF-κB dependent luciferase activity was expressed in RLU by normalizing firefly luciferase activity with constitutive Renilla luciferase activity. To calculate relative luciferase activity, LMP1-induced luciferase activities in the presence of lupeol at 0µg/ml was set 100%. Luciferase data shown here represent three independent experiments.

Figure 9. Lupeol is more cytotoxic toward LCLs than HFF, HeLa or BL41 cells.

(A) LCLs, (B) HFF, (C) HeLa or (D) BL41 cells were treated with either DMSO or lupeol at 3.125, 6.25, 12.5, 25 or 50µg/ml, and cell viability was determined at 0, 3, 6, 9, 12, 24, 48 or 72hr after treatment using CellTiter-Glo Luminescent Cell Viability Assay. A score of 1.0 indicates that there is no difference in viability between DMSO or lupeol treated cells.

Figure 10. Lupeol induces apoptosis in LCLs.

LCLs were treated with 50µg/ml of either DMSO or lupeol, and PARP cleavage was determined by Western blot analysis at 0, 1, 3, 6, 9, 12 or 24hr after treatment.