Search

Keloids exhibit cancer-like properties without spontaneous regression and usually recur post excision. Although photodynamic therapy (PDT) is a promising treatment, details of the mechanisms remain to be elucidated. In this study, we investigated mechanisms involved in 5-Aminolevulinic Acid (5-ALA)–based PDT against keloid. Found that 5-ALA-PDT induced superoxide anion-dependent autophagic cell death. Application of autophagy inhibitor 3-Methyladenine (3-MA) significantly prevented the effect that 5-ALA-PDT induced keloid–derived fibroblasts death, but Z-VAK-FMK (apoptotic inhibitor) did not. Interestingly, 5-ALA-PDT promoted the SIRT3 protein expression and the activity of mitochondrial superoxide dismutase 2 (SOD2), but SIRT1 protein expression level was decreased. SOD2 as a key enzyme can decrease mitochondrial ROS (mROS) level, Deacetylation of SOD2 by SIRT3 regulates SOD2 enzymatic activity has been identified. Then we explored SOD2 acetylation level with immunoprecipitation, found that 5-ALA-PDT significantly increased the acetylation levels of SOD2. In order to confirm deacetylation of SOD2 regulated by SIRT3, 3-TYP (SIRT3 inhibitor) was used. Found that inhibition of SIRT3 by 3-TYP significantly increased the level of SOD2 acetylation level compared with control group or 5-ALA-PDT group. To explore the connection of SIRT1 and SIRT3, cells were treated with EX527(SIRT1 inhibitor) or SRT1720 (SIRT1 activator), and EX527 increased SIRT3 protein level, however, SRT1720 displayed the opposite effect in the present or absence of 5-ALA-PDT. Moreover SIRT1-inhibited cells are more resistant to 5-ALA-PDT and showing decreased ROS accumulation. These results may demonstrate that 5-ALA-PDT induced SIRT1 protein level decreased, which promoted the effect of SIRT3 increased activity of SOD2 that can reduce mROS level, and then compromised 5-ALA-PDT induced autophagic cell death. Keywords: Keloid, 5-aminolevulinic acid, SIRT1, SIRT3, Autophagy

Rare pleiotropic genetic disorders, Ataxia-telangiectasia (A-T), Bloom syndrome (BS) and Nijmegen breakage syndrome (NBS) are characterised by immunodeficiency, extreme radiosensitivity, higher cancer susceptibility, premature aging, neurodegeneration and insulin resistance. Some of these functional abnormalities can be explained by aberrant DNA damage response and chromosomal instability. It has been suggested that one possible common denominator of these conditions could be chronic oxidative stress caused by endogenous ROS overproduction and impairment of mitochondrial homeostasis. Recent studies indicate new, alternative sources of oxidative stress in A-T, BS and NBS cells, including NADPH oxidase 4 (NOX4), oxidised low-density lipoprotein (ox-LDL) or Poly (ADP-ribose) polymerases (PARP). Mitochondrial abnormalities such as changes in the ultrastructure and function of mitochondria, excess mROS production as well as mitochondrial damage have also been reported in A-T, BS and NBS cells. A-T, BS and NBS cells are inextricably linked to high levels of reactive oxygen species (ROS), and thereby, chronic oxidative stress may be a major phenotypic hallmark in these diseases. Due to the presence of mitochondrial disturbances, A-T, BS and NBS may be considered mitochondrial diseases. Excess activity of antioxidant enzymes and an insufficient amount of low molecular weight antioxidants indicate new pharmacological strategies for patients suffering from the aforementioned diseases. However, at the current stage of research we are unable to ascertain if antioxidants and free radical scavengers can improve the condition or prolong the survival time of A-T, BS and NBS patients. Therefore, it is necessary to conduct experimental studies in a human model., Graphical abstract fx1

Our aim was to elucidate the relationship between the rate of mitochondrial reactive oxygen species (mROS) formation and the reduction level of the mitochondrial coenzyme Q (mQ) pool under various levels of engagement of the mQ-reducing pathway (succinate dehydrogenase, complex II) and mQH2-oxidizing pathways (the cytochrome pathway and alternative oxidase pathway, (AOX)) in mitochondria isolated from the amoeba Acanthamoeba castellanii. The mQ pool was shifted to a more reduced state by inhibition of mQH2-oxidizing pathways (complex III and complex IV of the cytochrome pathway, and AOX) and the oxidative phosphorylation system. The mQ reduction level was lowered by decreasing the electron supply from succinate dehydrogenase and by stimulating the activity of the cytochrome or AOX pathways. The results indicate a direct dependence of mROS formation on the reduction level of the mQ pool for both mQH2-oxidizing pathways. A higher mQ reduction level leads to a higher mROS formation. For the cytochrome pathway, mROS generation depends nonlinearly upon the mQ reduction level, with a stronger dependency observed at values higher than the mQ reduction level of the phosphorylating state (~ 35%). AOX becomes more engaged at higher mQ pool reduction levels (above 40%), when mROS production via the cytochrome pathway increases. We propose that the mQ pool reduction level (endogenous mQ redox state) could be a useful endogenous reporter that allows indirect assessment of overall mROS production in mitochondria., Graphical abstract fx1, Highlights • mROS generation depends on the reduction level of the endogenous mQ pool. • A stronger dependency is observed above mQ reduction level of phosphorylating state. • The mQ reduction level can be an endogenous reporter of overall mROS production.

Highlights • Autophagy was augmented in RPE cells in the presence of a lipofuscin pigment, A2E. • RPE cell death was induced in the presence of A2E with an autophagy inhibitor. • Inhibition of autophagy resulted in the accumulation of abnormal mitochondria. • Autophagy in RPE cells prevents the accumulation of damaged cellular molecules., In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.