[Effects of membrane skeleton protein 4.1R on the efficiency of photodynamic therapy]
Dandan Fan1, Yi Li, Jianhui Li
1School of Life Sciences, Zhengzhou University, Zhengzhou 450052, China.
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.
Insights
The absence of protein 4.1R reduces photodynamic therapy (PDT) effectiveness by lowering intracellular protoporphyrin levels. Protein 4.1R may influence 5-aminolevulinic acid uptake, impacting PDT efficacy.
Area of Science:
- Biochemistry
- Cell Biology
- Photomedicine
Background:
- Photodynamic therapy (PDT) is a treatment modality that utilizes photosensitizers to generate reactive oxygen species upon light activation.
- Protein 4.1R is a membrane skeleton protein implicated in various cellular functions, including cell structure and signaling.
- Understanding modulators of PDT efficiency is crucial for optimizing therapeutic outcomes.
Purpose of the Study:
- To investigate the role of membrane skeleton protein 4.1R in modulating the efficacy of photodynamic therapy (PDT).
- To determine if the presence or absence of protein 4.1R affects photosensitizer accumulation and phototoxicity.
Main Methods:
- Mouse embryonic fibroblasts (MEFs) with and without the 4.1R gene were treated with varying concentrations of 5-aminolevulinic acid (5-ALA) and exposed to specific light doses.
- Cell viability was assessed using the CCK-8 assay.
- Intracellular protoporphyrin localization and intensity were analyzed using laser confocal microscopy and fluorescence spectrophotometry.
- Western blot analysis was performed to quantify the protein levels of key enzymes in protoporphyrin synthesis (FECH and HMBS).
Main Results:
- PDT efficacy was dependent on 5-ALA concentration and light dose in both 4.1R knockout and wild-type MEFs.
- MEFs lacking protein 4.1R exhibited significantly higher survival rates post-PDT compared to wild-type MEFs.
- Intracellular protoporphyrin fluorescence intensity was markedly lower in 4.1R knockout MEFs than in wild-type MEFs.
- No significant differences in FECH and HMBS protein levels were observed between the two cell lines.
Conclusions:
- The absence of protein 4.1R attenuates intracellular protoporphyrin levels and reduces the photocytotoxicity of PDT.
- Protein 4.1R may play a role in 5-ALA uptake, thereby influencing the cellular protoporphyrin accumulation and ultimately affecting PDT efficiency.
- No alterations in 5-ALA metabolic activity were detected, suggesting a role for 4.1R in uptake rather than metabolism.
