Biological Evaluation Of Novel Dansyl Molecular Imaging Probes For Targeting Tumor Cells Apoptosis

Apoptosis is a phenomenon of programmed cell death, which occurs initiatively in particular time and space, tightly regulated by cellular mechanism. As we know, tumorigenesis is a complex biological process in which abnormal apoptosis leads to uncontrolled proliferation and high degree of malignancy. Selectively induce apoptosis of tumor cells has become one of the main strategies for the treatment of malignant tumors.Molecular imaging technique has been widely used in non-invasive and real-time imaging of physiological or pathological process of the organisms. It provides a new technologic platform for disease monitoring, gene therapy, drugs efficacy evaluation, and functional molecules tracer in vivo.The development of molecular imaging techniques paves a new way for monitoring apoptosis in vivo. The emergence of new molecular probes of high sensitivity and selectivity expands the scope of the apoptosis detection and benefits understanding of relationship between cell apoptosis and disease progress. Molecular probes serve as a bridge between medical imaging equipment and disease-characteristic molecules. In vivo apoptotic molecular imaging has close relationship with clinical practice, which can help accelerate early diagnosis, monitoring and evaluation of the treatment efficacy of diseases, also benefit developing new treatment strategies. We previously designed and synthesized a set of new molecular probes derived from the probe library of small organic molecules containing dansyl groups. Based on the structural modification and transformation of the dansyl groups, these molecular probes were found to have good fluorescence properties, selectivity and specificity for the identification and monitoring of apoptosis in breast cancer cell line MCF-7. Based on the tumor cell apoptotic model establishment by chemotherapy drug treatment and flow cytometry, we screened and found a novel molecular probe, Dansyl-8, which possesses the characteristics of targeting apoptotic cells. Furthermore, we adapted different apoptosis-inducing agents, simulating three classical apoptotic pathways respectively, to determine whether the molecular probe had selectivity of apoptotic pathways in tumor cells. Our data demonstrated the specific labelling of the molecular probe in apoptotic CNE1, MCF-7, A375and EC109tumor cells, suggesting that the probe acts without tumor cell type selectivity.By confocal microscopy, the subcellular localization in apoptotic cells of molecular probe Dansyl-8was investigated to explore the mechanism of it as a marker of apoptotic cells. Annexin V labeling is the gold standard for apoptotic detection. First, through Annexin V labeling, paclitaxel induced cells were sorted to positive and negative cells respectively by flow cytometry. Second, the parallel groups were labeled with Dansyl-8and sorted by flow cytometry. Then, the positive rate differences between the corresponding two groups were compared. According to our data, the uptake change of molecular probes Dansyl-8in CNEl cells represented the changes of apoptotic rate, which provided strong experimental evidence for the application of the molecular probe Dansyl-8.The molecular probe Dansyl-8belongs to a small molecule fluorescent probe and in the previous study it was found with a little coloring background without apoptotic inducing treatment. It suggested that the molecular probe was membrane-permeable. By changing the permeability of cell membranes we tried to determine its effect on Dansyl-8accumulating in apoptotic cells. The cellular uptake of molecular probes did not increase with the enhanced membrane permeability. We also observed that Dansyl-8could label cells at a greater rate in the presence of apoptosis-inducing agent, further supporting that the molecular probe Dansyl-8has specificity to the apoptotic cells.Due to the limit of detection spectrum, the in vivo studies only were carried out at the level of cells and tissues to evaluate the efficacy of the probe. The intake of molecular probe Dansyl-8significantly increased in paclitaxel treated tumor tissues, similar to the positive position of TUNEL assay detection. It suggested that the molecular probe has characteristics of targeting apoptotic cells in tissue sections and provides strong experimental evidence for in-depth study of the precursor synthesis multimode probe, also brings out new approach to transforming Dansyl-8into multi-modal apoptotic imaging probe.In summary, based on model of nasopharyngeal carcinoma cells CNE1, we designed and screened molecular probes targeting apoptosis in tumor cells. By study of different apoptotic pathways and tumor cells, we established apoptotic molecular imaging detection platform. In the meanwhile, we proved this probe labeling detection of apoptosis has no selectivity to apoptotic pathway and phases in cell and tissue levels. It provided meaningful experimental evidence for Dansyl-8as a novel apoptotic molecular probe.