EGCG Inhibited The Growth Of NP69-LMP1 Through Wnt-1/β-catenin Signal Transduction Pathway

Objective: Epstein Barr-virus (EBV) is a gamma herpesvirus that is hightly prevalent in human. EBV latent membrane protein1 (LMP1) was known expendingly for important oncogenic protein coded by EBV genome because it induced malignant transformation of epithelial cells. To investigate molecular mechanism of Epigallocatechin-3-gallate (EGCG) inhibited the growth of nasopharynx epithelium cells NP69 transformed by EBV-LMP1.Methods: Expression and allocation of LMP1 protein in NP69 cells transformed by EBV-LMP1 (NP69-LMP1) was detected by immunofluorescence. We drew cellular growth curve to observe the growth of NP69-LMP1. MTT method detected 50% inhibiting concentration (IC50) that EGCG inhibted the proliferation of NP69-LMP1 cells. The growth of NP69-LMP1 cell inhibited by EGCG was observed through the cellular growth curve, colony formation and soft agar formation. The cell cycle and apoptosis of EGCG affected NP69-LMP1 cells were checked by flow cytometry (FCM). The expression of Wnt^-1 gene and protein was respectively analyzed by RT-PCR and Western-blotting. Meantime, the expression ofβ-catenin protein and its phosphorylation level in NP69-LMP1 cells treated by EGCG were detected by Western-blotting.Results: 1.The expression of LMP1 protein in NP69-LMP1 cellular membrane and cytoplasm promoted to accelerate the cellular growth. 2. IC50 that EGCG inhibted NP69-LMP1 cells proliferation was 47.7 mg·L^-1. The results of the cellular growth curve, colony formation and soft agar formation displayed to raise inhibitory effct of NP69-LMP1 cellular growth with EGCG concentration increased and treatment time extended (n=3, P<0.05). 3. The result of flow cytometry (FCM) showed that G1 period rate of NP69-LMP1 cells gradually raised and S period obviously decreased with its concentration increased, and cellular apoptosis rate significantly increased (n=3, P<0.05). 4. The results of RT-PCR and Western-blotting discovered that the expression of Wnt-1 gene and protein in NP69-LMP1 cells both obviously degraded with its concentration increased. 5. The expression ofβ-catenin protein in NP69-LMP1 treated by EGCG gradually decreased, but its phosphorylation level successively raised with its concentration increased.Conclusion:1. EGCG can hold-up cell cycle of G1/S period and induce cell apoptosis to inhibit the growth of nasopharynx epithelium cell NP69 transformed by EBV-LMP1.2. The expression of Wnt-1 gene and protein in NP69-LMP1 cells were obviously depressed with EGCG concentration increased.3. EGCG induced the lower expression of Wnt-1 protein and affected expression ofβ-catenin protein and phosphorylation level ofβ-catenin protein to inhibited growth of NP69-LMP1.