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. 2016 Jun;11(6):4167-4176.
doi: 10.3892/ol.2016.4522. Epub 2016 May 5.

Embelin prevents LMP1-induced TRAIL resistance via inhibition of XIAP in nasopharyngeal carcinoma cells

Shu Yang  1 , Shi-Sheng Li  1 , Xin-Ming Yang  1 , Dan-Hui Yin  1 , Lin Wang  1
Affiliations

Embelin prevents LMP1-induced TRAIL resistance via inhibition of XIAP in nasopharyngeal carcinoma cells

Shu Yang et al. Oncol Lett. 2016 Jun.

Abstract

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in the majority of tumor cells, whilst sparing normal cells. However, the potential use of TRAIL in the treatment of cancer is limited by the inevitable emergence of drug resistance. The present study reports the upregulation of latent membrane protein 1 (LMP1)-induced TRAIL resistance via the enhanced expression of X-linked inhibitor of apoptosis protein (XIAP) in nasopharyngeal carcinoma (NPC) cells. LMP1-positive NPC cells were indicated to be more sensitive to TRAIL compared with LMP1-negative NPC cells in three NPC cell lines. CNE-1 is a LMP1-negative NPC cell line that was transfected with pGL6-LMP1; following which, sensitivity to TRAIL decreased. LMP1-induced TRAIL resistance was associated with the decreased cleavage of caspase-8,-3 and -9, BH3 interacting domain death agonist (Bid) and mitochondrial depolarization, without any effects on the expression of the death receptors, B-cell lymphoma (Bcl)-2 and Bcl-extra long. Knockdown of XIAP with small interfering RNA increased caspase-3 and -9 and Bid cleavage, and prevented LMP1-induced TRAIL resistance. Furthermore, embelin, the inhibitor of XIAP, prevented LMP1-induced TRAIL resistance in the Epstein-Barr virus (EBV)-positive CNE-1-LMP1 and C666-1 NPC cell lines. However, embelin did not enhance TRAIL-induced apoptosis in NP-69, which was used as a benign nasopharyngeal epithelial cell line. These data show that LMP1 inhibits TRAIL-mediated apoptosis by upregulation of XIAP. Embelin may be used in an efficacious and safe manner to prevent LMP1-induced TRAIL resistance. The present study may have implications for the development and validation of novel strategies to prevent TRAIL resistance in EBV-positive NPC.

Keywords: X-linked inhibitor of apoptosis protein; apoptosis; embelin; latent membrane protein 1; nasopharyngeal carcinoma; tumor necrosis factor-related apoptosis-inducing ligand.

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Figures

Figure 1.
Figure 1.
Expression of LMP1 was associated with the TRAIL sensitivity of NPC cell lines. (A) CNE-1, CNE-2 and C666-1 cells were treated with escalating doses of TRAIL for 24 h and 100 ng/ml TRAIL for various time periods, and cell viability was determined by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl tetrazolium bromide assay. (B) Cells were treated with escalating doses of TRAIL for 24 h and 100 ng/ml TRAIL for various time periods, and apoptotic rate was determined by flow cytometric analysis. Apoptosis rate included early apoptosis (Annexin-V positive) and late apoptosis (propidium iodide positive). (C) Western blot analysis was used to detected LMP1 protein expression in three NPC cell-lines. *P<0.05 compared with CNE-1 cells. LMP1, latent membrane protein 1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; NPC, nasopharyngeal carcinoma.
Figure 2.
Figure 2.
Overexpression of LMP1 protein induced TRAIL resistance in nasopharyngeal carcinoma cells. (A) Expression level of LMP1 was assessed by reverse transcription-polymerase chain reaction and western blot analysis, following transfection of pGL-6-LMP1 to CNE-1. (B) CNE-1, CNE-1-pGL6 and CNE-1-LMP1 were treated with escalating doses of TRAIL for 24 h or escalating times with 100 ng/ml TRAIL, respectively, and cell viability was determined by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl tetrazolium bromide assay. (C) The cell apoptotic rate was determined by flow cytometry analysis. Apoptosis rate included early apoptosis (Annexin V positive) and late apoptosis (propidium iodide positive). *P<0.05 compared with CNE-1-LMP1 cells; **P<0.01 compared with CNE-1-LMP1 cells. LMP1, latent membrane protein 1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 3.
Figure 3.
LMP1 induced TRAIL resistance via inhibition of mitochondrion-dependent apoptotic pathway. (A) Expressions of DR4 and DR5 were measured by flow cytometry analysis. (B) Mitochondrial depolarization was measured by DiOC6(3) fluorescence during TRAIL treatment (100 ng/ml) for various periods of time. (C) Bcl-2 and Bcl-XL expression were assessed by western blot analysis during TRAIL treatment (100 ng/ml) for various periods of time. **P<0.01 compared with CNE-1-LMP1 cells. LMP1, latent membrane protein 1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; DR4, death receptor 4; DR5, death receptor 5; Bcl-2, B-cell lymphoma-2; Bcl-XL, B-cell lymphoma-extra long; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide.
Figure 4.
Figure 4.
LMP1 induced TRAIL resistance via inhibition of caspase and Bid cleavage. Cleavage of caspases-3, −8 and −9 and Bid was assessed by western blot analysis during TRAIL treatment (100 ng/ml) for various periods of time. Caspase-3, p32-proform, p17, p10-cleavage fragments; caspase-8, p55/53-proform, p43/41, p18-cleavage fragments; caspase-9: p47-proform, p35-cleavage fragments. LMP1, latent membrane protein 1 TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Bid, BH3 interacting domain death agonist.
Figure 5.
Figure 5.
Upregulation of XIAP was involved in LMP1-induced TRAIL resistance. (A) Expressions of XIAP were assessed by western blot analysis. (B) Upon treatment with TRAIL (100 ng/ml) for various periods of time, apoptosis in CNE-1-LMP1 transfected with XIAP-siRNA was determined by flow cytometric analysis. (C) Upon treatment with TRAIL (100 ng/ml) for various periods of time, caspase-3, −8 and −9 and Bid cleavage was assessed by western blot analysis in CNE-1-LMP1 cells transfected with XIAP-siRNA. (D) Upon treatment with TRAIL (100 ng/ml) for various periods of time, mitochondrial depolarization was measured by DiOC6(3) fluorescence in CNE-1-LMP1 cells transfected with XIAP-siRNA. siRNA-, CNE-1-LMP1 transfected with negative control siRNA; XIAP-siRNA, CNE-1-LMP1 transfected with XIAP-siRNA. *P<0.05 compared to CNE-1-LMP1 transfected with XIAP-siRNA; **P<0.01 compared to CNE-1-LMP1 cells transfected with XIAP-siRNA. XIAP, X-linked inhibitor of apoptosis protein; LMP1, latent membrane protein 1 TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; siRNA, small interfering RNA; Bid, BH3 interacting domain death agonist; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide.
Figure 6.
Figure 6.
XIAP special inhibitor embelin inhibited XIAP expression and prevented LMP1-induced TRAIL resistance. (A) Upon treatment with various does of embelin for 24 h, XIAP expression in CNE-1-LMP1 cells was assessed by western blot analysis. (B) Apoptotic rate was determined by flow cytometry analysis. (C) XIAP expression in four cell-lines (CNE-1, CNE-2, C666-1 and NP-69) was assessed by western blot analysis. (D) Upon treatment with 25 µM embelin for 24 h, XIAP expression in C666-1 cells was assessed by western blot analysis and apoptotic rate was determined by flow cytometric analysis following treatment with TRAIL. (E) Upon treatment with 25 µM embelin for 24 h, XIAP expression in NP-69 cells was assessed by western blot analysis and apoptotic rate was determined by flow cytometric analysis following treatment with TRAIL. *P<0.05 compared with cells treated with embelin; **P<0.01 compared to cells treated with embelin. XIAP, X-linked inhibitor of apoptosis protein; LMP1, latent membrane protein 1 TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
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