Effect Of EEF1A1 On The Enrichment Of Photosensitizer PpIX In Tumor Cells During Photodynamic Therapy

5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence for PDT in tumors has gained increasing recognition during the last decade. Detectable amounts of5-ALA-induced PpIX accumulate within the cells are the basis for use of PpIX as imaging marker and photosensitizer in diagnostic and PDT. But the exact mechanism of PpIX selective accumulation in tumor cell is not clear. The efficacy and safety of PDT depend much on selective accumulation of photosensitizer. To aid PDT development, a better understanding of how these PpIX accumulate themselves within the cells is required, as this will significantly improve the therapeutic effect of photosensitizer.In our previous studies, we successfully screened a protein binding to protoporphyrin IX (PpIX) which is a widely used photosensitizer in PDT, from phage displayed cDNA library. The protein was proved to be eukaryotic translation elongation factor1alphal1(eEFlAl) by DNA sequencing. We expressed eEF1A1in E. coli BL21and artificially synthesized its C terminal LysM. With ELISA and Biacore assay, we demonstrated that eEF1A1protein and its C terminal LysM can bind to the photosensitizer PpIX directly in vitro respectively with high affinity, which is close to or exceeds the affinity between antigen and antibody(105-107M-1).In the present study, the interaction between eEF1A1and PpIX in cell was further studied by immunocytofluorescent assays. Co-localizations of PpIX and eEFlA1in HepG2cells on subcellular level were showed by confocal microscopy and structured illumination microscopy (SIM) with higher resolution. To study intracellular roles of eEF1A1, we measured the accumulation of PpIX in both tumor (HepG2) and normal(LO2) cell line, since it has been proposed that eEF1A1is over-expressed in tumor cells. In addition, we measured the amount of PpIX in HepG2cells transfected with GFP-fusion protein expression plasmid. The eEF1A1high expression tumor cells by adding5-ALA, were measured more PpIX accumulation by M5. The results showed that eEF1A1affect the alterations of PpIX levels.In addition, we access the possible mechanism of tumor-selective PpIX accumulation after exogenous intake of5-ALA. We measured PpIX concentration in cell lysates after exogenous eEF1A1and PpIX were added. With the addition of eEF1A1, the fluorescence intensity of PpIX showed slower decrease than the control group, which cell lysates were mixed with PBS and PpIX. Therefore we speculate that the binding of PpIX to eEF1A1may inhibit the degradation of PpIX, which can lead to PpIX accumulation in the cells.All the results above proved that eEF1A1acted as a intracellular receptor of the photosensitizer PpIX which is a new mechanism of PpIX accumulation in tumor cells and supplied basis for further studies on improving the effect of PDT.