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

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-β-cyclodextrin (MβCD) can disrupt lipid rafts through cholesterol depletion. In this study, we revealed that MβCD 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 MβCD 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 MβCD treatment. MTT assays showed that MβCD 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 MβCD. 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 MβCD can induce apoptosis of NPC cells, but enhancing of LMP1/LMP1 interaction may likely resist apoptosis through inhibiting cleavage of caspase-3.

Keywords

• fluorescence resonance energy transfer (fret)
• nasopharyngeal carcinoma (npc)
• latent membrane protein 1 (lmp1)
• lipid rafts