Immortalized Human Retinal Pigment Epithelial Cells

Effects of SFN on Mitochondrial Membrane Potential, Mass, Matrix Redox Status, and Mitophagy

Sulforaphane (SFN) from cruciferous vegetables shows anti-cancer, anti-microbial, and anti-oxidant effects, similar to dietary restriction benefits. Plafker et al. tested whether SFN acts as a fasting/caloric restriction mimetic by examining its effects on nutrient-sensing pathways in human retinal pigment epithelial cells.

They first compared SFN effects versus nutrient deprivations on mitochondrial mass and membrane potential in immortalized human retinal pigment epithelial cells (RPE-1 cells) using MitoTracker Green (MTG) and TMRE labeling with flow cytometry. FCCP served as positive control for membrane potential loss (Fig. 1A). SFN caused modest membrane potential decrease at 4h but preserved potential and increased mitochondrial mass at 24h, closely resembling amino acid (AA) deprivation. Glucose deprivation reduced membrane potential, while serum deprivation increased mass but reduced potential (Fig. 1A). Using mito-roGFP to assess mitochondrial matrix oxidation, SFN suppressed oxidized mitochondria at 4h (Fig. 1B). Both AA and glucose starvation also decreased oxidation, though less than SFN, with AA starvation most similar to SFN. Given SFN's resistance to mitochondrial oxidation, they tested protection against exogenous oxidative challenge. SFN pre-treatment prevented H₂O₂-induced mitochondrial oxidation, unlike nutrient deprivations (Fig. 1C). Using mtKeima-parkin RPE-1 cells to measure mitophagy, SFN, AA starvation, and serum deprivation increased mitophagic flux, while glucose deprivation showed no effect (Fig. 1D). Together, SFN preserves mitochondrial membrane potential, limits matrix oxidation, and promotes homeostatic mitophagy, most closely mirroring AA starvation.