Objective: To explore the effects of membrane skeleton protein 4.1R on the efficiency of photodynamic therapy (PDT).
Methods: 4.1R gene knockout and wild-type mouse embryonic fibroblasts (MEFs) were incubated with various concentrations of 5-aminolevulinic acid (5-ALA) (0.25, 0.50, 1.00, 1.50 and 2.00 mmol/L), followed by exposure to 450 nm light at a dose of 72, 96, 120, 180, 240 mJ/cm(2). Cell counting kit 8 (CCK-8) assay was used to assess the survival rate after PDT treatment. Laser confocal microscopy was used to observe the location of photo-sensitizer protoporphyrin and fluorescence spectrophotometer for detecting the fluorescent intensity of intracellular protoporphyrin. The protein levels of rate-limiting enzyme of protoporphyrin synthesis, ferrochelatase (FECH) and hydroxymethylbilane synthase (HMBS) were determined by Western blot.
Results: Both cell lines were killed after 5-aminolevulinic acid (5-ALA)-PDT and its efficacy was dependent on 5-ALA concentration, incubation duration and light dose. The cell survival rates of 4.1R(-/-) MEF were significantly higher than those of 4.1R(+/+) MEF (46.9% ± 7.1% vs 12.5% ± 2.1%, P < 0.001) after PDT treatment with a light dose of 120 mJ/cm(2) mediated by 5-ALA 1.00 mmol/L. After incubation with 1.00 mmol/L 5-ALA, protoporphyrin was distributed throughout cytoplasm in both cell lines while the fluorescent intensity of 4.1R(+/+) MEF was higher than that of 4.1R(-/-) MEF (124.2 ± 3.5 vs 34.6 ± 3.8, P < 0.001). Western blot showed that no difference of FECH and HMBS protein level was found in two cell lines.
Conclusions: A lack of protein 4.1R may attenuate the intracellular protoporphyrin level and the photo-cytotoxicity of PDT. No cellular change of ALA metabolic activity is found. Protein 4.1R may be involved in the ALA uptake in MEF cells so that the cellular level of protoporphyrin ultimately affects the PDT efficiency.