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Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM - Redox Biol (2015)

Bottom Line: Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear.A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence.Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, California University of Science & Medicine, Colton, CA 92324, USA; Department of Basic Sciences, The Commonwealth Medical College (TCMC), Scranton, PA 18509, USA. Electronic address: atamnah@calmedu.org.

No MeSH data available.


Related in: MedlinePlus

Time-dependent build-up of cytochrome c oxidase (Complex IV) activity in response to MB. IMR90 cell cultures were treated with 100 nM MB for increasing intervals (up to 6 days). At the end of each specific time point, the cells were harvested, the lysates were prepared, and complex IV activity was determined as described in Section 2. The assay buffer contained 10 mM Tris HCl/120 mM KCl, 0.3 µM n-Dodecyl beta-D-maltoside, pH 7. The rate of oxidation of the substrate ferrocytochrome c to ferricytochrome c was measured at 550 nm and used to calculate the enzymatic activity with the help of the millimolar extinction coefficient of 21.84. The data are Mean±sem of five independent experiments. **P<0.001, One-way ANOVA, Dunn's multiple comparisons test.
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f0025: Time-dependent build-up of cytochrome c oxidase (Complex IV) activity in response to MB. IMR90 cell cultures were treated with 100 nM MB for increasing intervals (up to 6 days). At the end of each specific time point, the cells were harvested, the lysates were prepared, and complex IV activity was determined as described in Section 2. The assay buffer contained 10 mM Tris HCl/120 mM KCl, 0.3 µM n-Dodecyl beta-D-maltoside, pH 7. The rate of oxidation of the substrate ferrocytochrome c to ferricytochrome c was measured at 550 nm and used to calculate the enzymatic activity with the help of the millimolar extinction coefficient of 21.84. The data are Mean±sem of five independent experiments. **P<0.001, One-way ANOVA, Dunn's multiple comparisons test.

Mentions: The induction of PGC1α by MB suggests a molecular connection to mitochondrial biogenesis. We further investigated for a direct connection to complex IV by studying the effect of MB on SURF1, a key factor in the biogenesis of active complex IV. MB gradually induced SURF1 over time (Fig. 4A and B) and subsequently the enzymatic activity of complex IV also increased (Fig. 5). Since complex IV resides only in the mitochondrial inner membrane, we decided to examine if the increase in complex IV is accompanied by an increase in the mitochondrial inner membrane. NAO is a fluorescent dye that binds cardiolipin, a specialized lipid found only in the mitochondrial inner membrane. An increase in the mass of the mitochondrial inner membrane was observed in MB-treated cells as determined by the increase in NAO fluorescence. NAO fluorescence gradually increased in MB-treated cells in a time line that was consistent with the increase in complex IV (Fig. 6). Intracellular incorporation of NAO reached plateau at fluorescence level 30% above that of the control (Fig. 6).


Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

Atamna H, Atamna W, Al-Eyd G, Shanower G, Dhahbi JM - Redox Biol (2015)

Time-dependent build-up of cytochrome c oxidase (Complex IV) activity in response to MB. IMR90 cell cultures were treated with 100 nM MB for increasing intervals (up to 6 days). At the end of each specific time point, the cells were harvested, the lysates were prepared, and complex IV activity was determined as described in Section 2. The assay buffer contained 10 mM Tris HCl/120 mM KCl, 0.3 µM n-Dodecyl beta-D-maltoside, pH 7. The rate of oxidation of the substrate ferrocytochrome c to ferricytochrome c was measured at 550 nm and used to calculate the enzymatic activity with the help of the millimolar extinction coefficient of 21.84. The data are Mean±sem of five independent experiments. **P<0.001, One-way ANOVA, Dunn's multiple comparisons test.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588422&req=5

f0025: Time-dependent build-up of cytochrome c oxidase (Complex IV) activity in response to MB. IMR90 cell cultures were treated with 100 nM MB for increasing intervals (up to 6 days). At the end of each specific time point, the cells were harvested, the lysates were prepared, and complex IV activity was determined as described in Section 2. The assay buffer contained 10 mM Tris HCl/120 mM KCl, 0.3 µM n-Dodecyl beta-D-maltoside, pH 7. The rate of oxidation of the substrate ferrocytochrome c to ferricytochrome c was measured at 550 nm and used to calculate the enzymatic activity with the help of the millimolar extinction coefficient of 21.84. The data are Mean±sem of five independent experiments. **P<0.001, One-way ANOVA, Dunn's multiple comparisons test.
Mentions: The induction of PGC1α by MB suggests a molecular connection to mitochondrial biogenesis. We further investigated for a direct connection to complex IV by studying the effect of MB on SURF1, a key factor in the biogenesis of active complex IV. MB gradually induced SURF1 over time (Fig. 4A and B) and subsequently the enzymatic activity of complex IV also increased (Fig. 5). Since complex IV resides only in the mitochondrial inner membrane, we decided to examine if the increase in complex IV is accompanied by an increase in the mitochondrial inner membrane. NAO is a fluorescent dye that binds cardiolipin, a specialized lipid found only in the mitochondrial inner membrane. An increase in the mass of the mitochondrial inner membrane was observed in MB-treated cells as determined by the increase in NAO fluorescence. NAO fluorescence gradually increased in MB-treated cells in a time line that was consistent with the increase in complex IV (Fig. 6). Intracellular incorporation of NAO reached plateau at fluorescence level 30% above that of the control (Fig. 6).

Bottom Line: Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear.A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence.Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, California University of Science & Medicine, Colton, CA 92324, USA; Department of Basic Sciences, The Commonwealth Medical College (TCMC), Scranton, PA 18509, USA. Electronic address: atamnah@calmedu.org.

No MeSH data available.


Related in: MedlinePlus