| BackgroundTumor diagnosis and treatment are the most demanding challenges facing cliniciansand scientists because of the lack of appropriate agents that can be used for early tumordetection and followed progression. Molecular imaging approaches could offer effectivetools for tumor detection and prognosis allowing visualization of tumors in real time andspace. Because of the lack of universal tumor specific targets, new highly sensitivemolecular probes are needed for precise detection of various tumor types. Recently, nearinfrared (NIR) dyes have been emerged as a potential tool for tumor imaging and targetedtherapy. These organic dyes are characterized by high extinction coefficients and relativelylarge Stokes’ shifts. With emission wavelength at700-900nm, their fluorescence can bereadily detected from deep tissues by commercially available imaging modalities. Due tothe excellent advantages of near infrared fluorescence imaging, including non-invasive,high sensitivity, deep tissue penetration and real-time image-guided property, many nearinfrared (NIR) probes have been developed and applied in clinical trials for tumor diagnosis,imaging and therapy. In our previous study, we have identified a NIR fluorescentheptamethine dye, IR-780, with excellent optical property for in vivo tumor imaging.However, the subcellular location and mechanism that IR-780selectively accumulates intumor cell has not been clarified. In this work, by confocal fluorescence imaging andquantitative flow cytometry, we investigated the subcellular location in tumor cells andstudied the role of energy metabolism, cellular endocytosis, membrane potential,ATP-binding cassette (ABC) transporters and organic anion transporter peptides (OATPs)for IR-780accumulation.Methods1. Study of the subcellular localization and binding property of IR-780in tumor cells1.1Study of the subcellular localization of IR-780in tumor cells in vitroWe observed the subcellular location of IR-780with combined incubation of subcellular organelle probes (mitochondria probe Mito-tracker Red or Rhodamine123,lysosome probe Lyso-tracker Red, Golgi body probe Golgi-tracker Red, endoplasmicreticulum probe ER-tracker Red) and IR-780. In addition, to investigate the subcellularlocation of IR-780at various concentrations, we observed the combined localization of10μM Rhodamine123and IR-780with incubation concentrations of1,5,10and20μM.1.2Study of the subcellular localization of IR-780in tumor cells in vivoTo investigate whether IR-780accumulated into the mitochondria of tumor cells invivo, we established subcutaneous xenograft animal models with rTDMC tumor cells withstable green fluorescent protein (GFP) expression. IR-780was administrated through tailinjection and then the tumor tissues were isolated at48h, digested by trysin and incubatedon Petri plates. Then we incubated the adherence cells with Mito-tracker Red and observedthe colocalization of IR-780, GFP protein and Mito-tracker Red.1.3Study of the binding property of IR-780in the mitochondria of tumor cellsTo detect whether IR-780could bind with proteins or DNAs in the mitochondria oftumor cell, we measured the change of absorption spectrum of IR-780labeled mitochondriawith or without lysis. To further observe the protein binding activity in tumor mitochondria,we studied the mitochondrial binding straps in IR-780labeled tumor cells in vitro and invivo with SDS-PAGE electrophoresis and NIR imaging.2. Study of the mechanism of IR-780for tumor targeting accumulation2.1The effect of energy metabolism on IR-780accumulation in tumor cellsTo investigate whether the uptake of IR-780in tumor cells was energy dependent ornot, we observed the accumulation of IR-780by ice treatment. Furthermore, we incubatedtumor cells with glycolysis inhibitor2-deoxy-D-glucose and oxidative phosphorylationinhibitor oligomycin to analyze the roles of the two energy supplied pathway for IR-780accumulation.2.2The role of plasma transporters for IR-780accumulation in tumor cellsTo investigate whether uptake and efflux transporters in tumor cellular membrane wereinvolved in the accumulation of IR-780, we treated tumor cells with inhibitors for varioussubtypes of organic anion transporting polypeptides (OATPs) and ATP binding cassettetransporters (ABCs) to observe the changes of IR-780accumulation. Moreover, wemeasured the mRNA expression level of OATP1B3in tumor cells and normal cells. 2.3The influence of plasma membrane potential, mitochondrial membrane potentialand cellular endocytosis on the uptake of IR-780by tumor cellsTo investigate the effect of cellular plasma membrane potential and mitochondrialmembrane potential of tumor cells on IR-780accumulation, we incubated tumor cells withhigh K+solution, gramicidin and ouabain to delocalize the plasma membrane potential andwith FCCP to delocalize mitochondrial membrane potential to observe the accumulation ofIR-780. In addition, we observed the fluorescence change of IR-780after treatment ofcellular endosytosis inhibitors to evaluate the influence of cellular entocytosis on IR-780uptake.Results1. IR-780selectively accumulated into the mitochondria of tumor cells and bound withmitochondrial proteinsThe colocalization observation of IR-780with subcellular organelle trackers in tumorcells indicated the mitochondrial location of this small molecule. And the subcellularlocation of IR-780in tumor cells was consistent at different incubation concentrations.More importantly, we verified the mitochondrial accumulating property by incubationMito-tracker Red in the GFP labeling tumor cells derived from the in vivo xenograftsreceiving IR-780injection. And measuring intact mitochondria and mitochondrial lysate oftumor cells with IR-780labeling, we found that the absorptive spectrum of IR-780andproteins were changed which indicated that IR-780bound proteins in the mitochondria oftumor cells. Furthermore, we collected tumor cells or tissues with IR-780incubation,separated mitochondrial proteins by SDS-PAGE electrophoresis and found70kD and40kDfluorescent straps detected by NIR imaging system.2. Glycolysis, plasma membrane potential and expression of OATP1B3were involvedin the IR-780selective accumulation in tumor cellsThe accumulation of IR-780in tumor cells was significantly inhibited after icetreatment indicated that the uptake of this small molecule was energy dependent.Furthermore, we found that the accumulation of IR-780in tumor cells was dependent oncellular glycolysis instead of oxidation phosphorylation. In addition, we also found thatcellular endocytosis and ABC transporters were not involved in the IR-780uptake in tumorcells. However, inhibitors of OATPs significantly suppressed the uptake of IR-780in cancer cells and high expression level of OATP1B3was recognized as the key factor for IR-780accumulation. Plasma membrane potential instead of mitochondrial membrane potential oftumor cells was involved in the uptake of IR-780.ConclusionsThis work studied the subcellular localization and mitochondrial binding character ofheptamethine cyanine fluorescent small molecule IR-780in tumor cells. Moreover, theeffective factors on the selective accumulation of IR-780in tumor cells were also analyzed.The main results were as followed:1. We observed the subcellular mitochondrial location of IR-780in tumor cells by invitro and in vivo experiments. And the mitochondria accumulation was consistent at variousincubation concentrations. We concluded that IR-780bound some proteins in tumormitochondria from the detection of IR-780and mitochondrial protein absorptive spectrums.Then we found that IR-780could bind with mitochondrial70kD and40kD proteins bySDS-PAGE electrophoresis and NIR imaging detection.2. The accumulation of IR-780in tumor cells was an energy dependent manner. Andthis process was much more dependent on glycolysis than oxidative phosphorylation.Moreover, OATP1B3and plasma membrane potential regulated the uptake of IR-780.However, oxidative phosphorylation, cellular endocytosis, ABC transporters andmitochondrial membrane potential was not involved in this process. |
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