Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

Zhiwei Wu,Yimei Huang,Xiaoyan Wang,Qing Ye,Mengmeng Zheng,Juqiang Lin,Yating Lin,Shusen Xie

doi:10.1142/s1793545819500196

Abstract

Latent membrane protein 1 (LMP1) is known as an oncoprotein in nasopharyngeal carcinoma (NPC) cells, which is considered to have a strong association with growth, invasion and metastasis of NPC cells through lipid rafts. Methyl-[Formula: see text]-cyclodextrin (M[Formula: see text]CD) can disrupt lipid rafts through cholesterol depletion. In this study, we revealed that M[Formula: see text]CD induced apoptosis in two kinds of NPC cells including CNE1 cells, a LMP1-negative nasopharyngeal squamous carcinoma cell line, and CNE1-LMP1 cells, a LMP1-positive nasopharyngeal squamous carcinoma cell line. Furthermore, the impact of M[Formula: see text]CD on LMP1 was investigated by fluorescence resonance energy transfer (FRET) method in NPC cells. Synchronized tempo-spatial and spectral detection of LMP1/LMP1 interaction were performed using fluorescence microscope and spectrograph. FRET efficiency indicated that LMP1/LMP1 interaction gradually enhanced after M[Formula: see text]CD treatment. MTT assays showed that M[Formula: see text]CD caused strong cytotoxicity in CNE1 cells, but caused relatively weaker cytotoxicity in CNE1-LMP1 cells, which indicated that LMP1 may regulate sensitivity of NPC cells to M[Formula: see text]CD. Then, detection of cleaved caspase-3 in two kinds of NPC cells indicated that LMP1 may inhibit activation of caspase-3. These results strongly suggested that M[Formula: see text]CD can induce apoptosis of NPC cells, but enhancing of LMP1/LMP1 interaction may likely resist apoptosis through inhibiting cleavage of caspase-3.

Highlights

Nasopharyngeal carcinoma (NPC) is a head and neck cancer with high incidence of recurrence, especially in south-east Asia,[1] more than 50% of NPC patients experience recurrence after initial treatment.[2,3] Because of knockdowning latent membrane protein 1 (LMP1) gene can impair growth and inhibit transformation,[4] LMP1 is considered positively associated with NPC progression.[5]
We examined whether lipid rafts disruption can induce NPC cells apoptosis, and relationship between LMP1/LMP1 interaction and apoptosis induced by lipid rafts disruption
MCD showed strong cytotoxicity compared to control (Figs. 1(a) and 1(b)), and the cytotoxicity in CNE1 cells was stronger than that in CNE1-LMP1 cells (Fig. 1(c)). These results suggested that MCD can induce cell-death rather than promote cellgrowth, as well as LMP1 may likely mediate resistance of cytotoxicity caused by MCD in NPC cells

Summary

Introduction

Nasopharyngeal carcinoma (NPC) is a head and neck cancer with high incidence of recurrence, especially in south-east Asia,[1] more than 50% of NPC patients experience recurrence after initial treatment.[2,3] Because of knockdowning latent membrane protein 1 (LMP1) gene can impair growth and inhibit transformation,[4] LMP1 is considered positively associated with NPC progression.[5]. Lipid rafts act as platforms to regulate interaction of membrane receptor protein, activation of signaling pathway, and endocytosis and exocytosis of protein.[20] lipid rafts are reported to be associated with celldeath or proliferation through reorganizing membrane receptor molecules such as G-protein coupled receptors (GPCRs).[21,22] These studies suggested that self-aggregation of LMP1 was closely associated with cell proliferation through lipid rafts. Whether self-aggregation of LMP1 in lipid rafts can mediate LMP1/LMP1 interaction is not yet clear

Methods

Results

Discussion

Conclusion