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Apoptosis induction by different pathways with methylene blue derivative and light from mitochondrial sites in V79
B B Noodt1, G H Rodal, M Wainwright
1Department of Pathology, the Norwegian Radium Hospital, Oslo. barbara.noodt@dnr.uio.no
Abstract:
The importance of mitochondria for the induction of apoptosis by photodynamic therapy (PDT) was studied with a new photosensitizing dye, methylene blue derivative (MBD), and light. By using fluorescence microscopy and by measuring the MBD-PDT-induced inhibition of specifically subcellularly localized marker enzymes, we show that MBD is localized in mitochondria and not in lysosomes, endoplasmic reticulum or Golgi apparatus of V79 Chinese hamster fibroblasts. Cellular uptake kinetics and fluorescence properties of the dye in cells were characterized. Cell death was studied by a cell survival assay and by flow cytometry of cells stained using the terminal deoxynucleotidyl transferase (TdT) assay. MBD with light induced cell death by apoptosis via 2 different pathways, one rapid and one delayed, depending on the amount of dye in the cells. Cells treated with an MBD concentration higher than 0.05 microg/ml died by apoptosis within 3 hr after light exposure. At a concentration of 0.05 microg/ml MBD, cell death was induced slowly, and apoptotic cells appeared increasingly from the second day after PDT. Combination studies with 2-deoxyglucose (2-DOG) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), inhibitors of glycolysis and oxidative phosphorylation, respectively, indicated that MBD and light inhibited mitochondrial oxidative phosphorylation. Abolishment of both energy sources led to cell death by necrosis within 6 hr. Inhibition of glycolysis alone induced apoptosis between 3 and 6 hr, while inhibition of mitochondrial oxidative phosphorylation alone led to delayed apoptosis within days.
Insights
Methylene blue derivative (MBD) photodynamic therapy induces apoptosis by targeting mitochondria. The dye
Area of Science:
- Cell Biology
- Biochemistry
- Photochemistry
Background:
- Photodynamic therapy (PDT) is a treatment modality that uses photosensitizers and light to induce cell death.
- Mitochondria play a crucial role in apoptosis, the programmed cell death pathway.
- Understanding the subcellular localization and mechanism of action of photosensitizers is key to optimizing PDT efficacy.
Purpose of the Study:
- To investigate the role of mitochondria in apoptosis induction by a novel photosensitizing dye, methylene blue derivative (MBD), in photodynamic therapy (PDT).
- To determine the subcellular localization of MBD and its effects on cellular energy metabolism.
- To elucidate the pathways and kinetics of MBD-PDT-induced cell death.
Main Methods:
- Fluorescence microscopy to determine MBD subcellular localization.
- Measurement of subcellularly localized marker enzymes to assess MBD effects.
- Cellular uptake kinetics and fluorescence property characterization of MBD.
- Cell survival assays and flow cytometry (TdT assay) to study cell death.
- Combination studies with glycolysis (2-deoxyglucose) and oxidative phosphorylation (CCCP) inhibitors.
Main Results:
- MBD was localized in mitochondria, not in lysosomes, endoplasmic reticulum, or Golgi apparatus.
- MBD-PDT induced apoptosis via rapid and delayed pathways, dependent on MBD concentration.
- High MBD concentrations (>0.05 microg/ml) led to apoptosis within 3 hours.
- Low MBD concentrations (0.05 microg/ml) induced delayed apoptosis, appearing from day two.
- MBD-PDT inhibited mitochondrial oxidative phosphorylation.
- Inhibition of glycolysis alone induced apoptosis (3-6 hours); inhibition of oxidative phosphorylation alone induced delayed apoptosis.
- Abolishment of both energy sources resulted in necrosis within 6 hours.
Conclusions:
- Mitochondria are critical targets for MBD-PDT-induced apoptosis.
- MBD-PDT triggers distinct apoptotic pathways based on dye concentration and cellular energy status.
- MBD-PDT offers a potential therapeutic strategy by selectively targeting mitochondrial function.