Study On Lipid Rafts Modulatory Role In Human Nasopharyngeal Carcinoma Cells Apoptosis With Quantitative Fluorescence Resonance Energy Transfer

Nasopharyngeal carcinoma(NPC)is a malignancy,which is prevalent in Southern China and Southeast Asia.It is important to study the pathogenesis and progression mechanism of NPC.Advanced biomedical photonic technologies,especially the quantitative fluorescence resonance energy transfer(FRET)technology,have been applied to investigate the protein-protein interaction in physiological condition,which provide novel and powerful methods for solving the key problems of tumor pathogenesis and progression.In this paper,a novel quantitative FRET measurement method was developed to acquire the optical images and characteristic data related to NPC cells apoptosis and protein-protein interaction associated with latent membrane protein 1(LMP1),which were based on fluorescence imaging and spectra detection.Furthermore,the roles of lipid rafts and LMP1 during NPC cells apoptosis were investigated at the cellular and molecular level.Firstly,a novel quantitative FRET measurement method(Single control and Double channels-FRET,SD-FRET)was developed to detect the protein phosphorylation.Moreover,a FRET measurement platform based on fluorescence imaging and spectra detection was built.The measurement accuracy and stability of FRET measurement platform and SD-FRET method were tested using FRET-standard constructs and intracellular calcium concentration probe.Furthermore,the applicable conditions of SD-FRET method were discussed.These results showed that the FRET measurement platform can be applied in quantitative FRET measurement in live cells.The SD-FRET measurement method can be applied in the quantitative measurement of protein phosphorylation and the qualitative analysis of protease activity.These works laid a solid foundation for detection of apoptosis and protein-protein interaction.Secondly,the roles of lipid rafts in apoptosis and metastasis of NPC cells were studied by FRET method with lipid rafts disruption induced by M?CD.High-resolution and dynamic images of cysteinyl aspartate specific proteinases(caspases)activation related to apoptosis and protein phosphorylation were acquired by FRET methods.Moreover,the concentration of intracellular reactive oxygen species(ROS)and calcium ions were measured in real-time.Importantly,the optical characteristics of NPC cells apoptosis were acquired at the cellular and molecular level.These results showed that lipid rafts disruption can inhibit migration of NPC cells effectively and induce apoptosis of NPC cells by activating the caspases cascade.Thirdly,the fluorescent probes of LMP1,Phosphoinositide 3-kinase(PI3K)and Vimentin expressing in live cells were constructed.Quantitative FRET measurement and spectra detection were used to monitor the intensity(FRET efficiency between fluorescent probes)and distribution of LMP1-LMP1 interaction,LMP1-PI3 K interaction and LMP1-Vimentin interaction in NPC cells.These results showed that the majority of LMP1-LMP1 interaction localized to internal perinuclear,the interactions of LMP1-PI3 K and LMP1-Vimentin were modulated by LMP1,and the distribution of LMP1-PI3 K interaction and LMP1-Vimentin interaction was consistent with LMP1 basically.Finally,the constructed fluorescent probes were used to acquire high-resolution and dynamic optical images of protein-protein interaction associated with LMP1 during lipid rafts disruption.These results showed that lipid rafts disruption can enhance the protein-protein interaction associated with LMP1.Furtherly,the effect of LMP1 on caspase-3 and NF-?B signaling pathways was investigated.These results showed that LMP1 can enhance the activation of NF-?B signaling pathways,and inhibit the activity of.caspase-3 in NPC cells.These works partially revealed the association in lipid rafts,LMP1 and apoptosis of NPC cells from the view of photonics.Disruption of lipid rafts induced apoptosis of NPC cells in caspases-dependent pathways.On the other hand,LMP1 can resist apoptosis induced by lipid rafts disruption,at least partially through enhancing protein-protein interaction associated with LMP1,enhancing the activation of NF-?B signaling pathways,and inhibiting the activity of caspase-3 in NPC cells.In this paper,the FRET measurement platform based on fluorescence imaging and spectra detection can acquire the optical characteristics of NPC cells apoptosis and protein-protein interaction at the cellular and molecular level effectively.These results have important academic value for delving deeper into pathogenesis and development mechanism of tumors.