| Background and ObjectiveTumor has been a significant threat to human health.Operation,radiotherapy and chemotherapy are widely used as routine methods in tumor treatment,while normal tissues can be damaged at the same time.So to explore more effective treatment method is particularly importment.5-aminolevulic acid-mediated photodynamic therapy（5-ALA-PDT）has been a relatively new modality in the treatment of tumor.As a new generation photosensitizer,5-ALA is the biochemical precusor of hemoglobin,can be taken up by various cancer cells and accumulated a certain period of time in a specific manner.Through a series of metabolic enzymes,5-ALA is metabolically converted to protoporphyrin IX（PpIX）,which has good fluorescent and photosensitizing properties.PpIX can be activated by blue light irradiation in the presence of oxygen and generates singlet oxygen and other reactive oxygen species,which can damage biomolecules such as nucleic acids and proteins,resulting in apoptosis or necrosis within these cells to reach the therapeutic purposes.However,influenced by some factors,PDT efficiency in the treatment of clinical is not ideal completely.Knowledge of the 5-ALA-PDT mechanism is essential to improve PDT efficacy and requires further studies.However,PDT efficiency can be influenced by many factors,in addition to light and oxygen,the accumulation of high concentrations photosensitizer is an important premise.The ideal photosensitizer should be able to have better organization specificity.Further to explore the mechanism of tumor selective accumulation is important for improving the photosensitizer concentrations and enhancing PDT efficacy.Multiple studies have recently shown that 5-ALA is absorbed by active transportation in many cells,and the uptake systems differ among cell types.5-ALA is incorporated into the cells by GABA transporters in neurons and other cells.Protein 4.1R,as one of the important members of membrane skeleton,can play an important role in maintaining cell mechanical stability,cell division and signal transduction of red cell by interacting with spectrin-actin network and transmembrane proteins.In the previous study,we have found that in 4.1R-/-MEF cells,the efficacy of 5-ALA-PDT and the accumulation volume of PpIX showed a significant decline phenomenon,but the 5-ALA metabolism process hasn’t been affected,which suggested that the lack of protein 4.1R reduced the efficacy of 5-ALA-PDT by influencing the transport of 5-ALA.In this study,we explored the transporter protein of 5-ALA in 4.1R+/+ and 4.1R-/-MEF cells and the interaction between 4.1R and transporter proteins.In addition,4.1R+/+ and 4.1R-/-P815 cells were used to investigate the effects of protein 4.1R on photodynamic therapy in tumor cells,which could be the research target to improve the PDT efficiency in the treatment of tumor.Methods1.4.1R+/+ and 4.1R-/-MEF cells were incubated with 5-ALA and various concentrations of GABA transporters GAT1-3 specific inhibitors.Cell viability was tested by CCK-8 assay followed PDT treatment.2.4.1R+/+ and 4.1R-/-MEF cells were incubated with 5-ALA and optimal concentrations of GAT1-3 specific inhibitors.The PpⅨ contents were detected using a fluorescence spectrometer and the ROS contents were detected using a fluorometric imaging plate reader.3.Real time PCR and Western blot were used to detect the expression of GAT1-3 in 4.1R+/+ and 4.1R-/-MEF cells.The localization of GAT1 and GAT2 was detected by immunofluorescence.4.The interaction of 4.1R with GAT1 and GAT2 was tested using co-immunoprecipitation.5.4.1R+/+ and 4.1R-/-P815 cells were treated with 5-ALA at different concentrations,incubation time and different doses of light.Cell viability after PDT was tested using CCK-8 assay.6.Fluorescence spectrometer was used to detect the accumulation of PpⅨ in 4.1R+/+ and 4.1R-/-P815 cells after 5-ALA incubation.Fluorometric imaging plate reader was applied to detect the ROS contents in two cells after PDT.7.Fluorescence spectrometer was used to detect the accumulation of PpⅨ in 4.1R+/+ and 4.1R-/-P815 cells after ATP inhibitors incubation.Results1.The CCK-8 results showed that cell survival rates in 4.1R+/+ and 4.1R-/-MEF cells were increased notably with Tiagabine and NNC 05-2090 incubation.The optimum inhibition concentration of Tiagabine and NNC 05-2090 were 7.5μM and 2μM respectively,while the GAT3 inhibitor（S）-SNAP-5114 had no effect on the photodamage.2.It showed that the PpⅨ accumulation and ROS contents were much lower by NNC 05-2090 inhibition compared to by Tiagabine inhibition,while inhibiting GAT3 by（S）-SNAP 5114 at 5 μM did not show any effect on PpIX fluorescence intensity and ROS contents.3.The mRNA expression levels of GAT1 and GAT2 in 4.1R+/+ MEF cells were significantly higher than that in the 4.1R-/-MEF cells,while there was no significant change for the mRNA level of GAT3 in 4.1R+/+ MEF cells relative to that in the 4.1R-/-MEF cells.Compared with the 4.1R-/-MEF cells,Western blot showed that protein levels of GAT1 and GAT2 were significantly increased in 4.1R+/+ MEF cells.GAT1 and GAT2 located in cell membrane surrounding.4.Co-immunoprecipitation showed that 4.1R could bind to GAT1 and GAT2 in situ.5.The CCK-8 results showed that the cell survival rates in 4.1R+/+ and 4.1R-/-P815 cells were depended on 5-ALA concentration,incubation time and the doses of light.When cells were incubated with 0.7mM ALA for 3 h,and then exposed to light at an intensity of 90mJ/cm2,the survival rate in 4.1R-/-P815 cells was higher relative to that in 4.1R+/+ P815 cells（51.1%±3.3vs22.4%±3.1,P<0.001）.6.The fluorescence spectrometer results showed that PpⅨ fluorescence intensity in 4.1R+/+ and 4.1R-/-P815 cells was increased along with the increasing concentration of 5-ALA,and the intensity was higher in 4.1R+/+ P815 cells than that in 4.1R-/-P815 cells at 0.7mM（P<0.001）.Fluorometric imaging plate reader results showed that the ROS contents in 4.1R+/+ P815 cells were higher relative to that in 4.1R-/-P815 cells.7.Fuorescence spectrometer results showed that PpⅨ fluorescence intensity in 4.1R+/+ and 4.1R-/-P815 was decreased after ATP inhibitors incubation（P<0.001）,which indicated that 5-ALA was absorbed by active transportation in P815 cells.Conclusion1.5-ALA was transported through GAT1 and GAT2 in MEF cells,mainly by GAT2.2.By interacting with GAT1 and GAT2,4.1R affected the transport of 5-ALA,which impacted the efficacy of 5-ALA-PDT.3.The lack of protein 4.1R in P815 cells reduced the efficacy of 5-ALA-PDT. |
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