Lipofuscin-mediated photodynamic stress induces adverse changes in nanomechanical properties of retinal pigment epithelium cells

Abstract Retinal pigment epithelium (RPE) is an important part of the blood-retina barrier (BRB) that separates the retina from the choroid. Although melanin granules contribute to the mechanical stability of the BRB complex, it is unknown if the age pigment lipofuscin affects mechanical properties of the tissue. To address this issue the effect of sub-lethal photic stress mediated by phagocytized lipofuscin granules, isolated from RPE of human donors, on morphology and mechanical properties of ARPE-19 cells was investigated. Nanomechanical analysis using atomic force spectroscopy revealed that irradiation of cells containing lipofuscin granules with blue light induced significant softening of the cells, which was accompanied by substantial reorganization of the cell cytoskeleton due to peroxidation of cellular proteins. Our results indicate that lipofuscin-mediated photic stress can cause significant modification of the RPE cells with the potential to disturb biological function of the BRB complex

The effect of antioxidants on photoreactivity and phototoxic potential of RPE melanolipofuscin granules from human donors of different age

One of the most prominent age-related changes of retinal pigment epithelium (RPE) is the accumulation of melanolipofuscin granules, which could contribute to oxidative stress in the retina. The purpose of this study was to determine the ability of melanolipofuscin granules from younger and older donors to photogenerate reactive oxygen species, and to examine if natural antioxidants could modify the phototoxic potential of this age pigment. Electron paramagnetic resonance (EPR) oximetry, EPR-spin trapping, and time-resolved detection of near-infrared phosphorescence were employed for measuring photogeneration of superoxide anion and singlet oxygen by melanolipofuscin isolated from younger and older human donors. Phototoxicity mediated by internalized melanolipofuscin granules with and without supplementation with zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells by determining cell survival, oxidation of cellular proteins, organization of the cell cytoskeleton, and the cell specific phagocytic activity. Supplementation with antioxidants reduced aerobic photoreactivity and phototoxicity of melanolipofuscin granules. The effect was particularly noticeable for melanolipofuscin mediated inhibition of the cell phagocytic activity. Antioxidants decreased the extent of melanolipofuscin-dependent oxidation of cellular proteins and disruption of the cell cytoskeleton. Although melanolipofuscin might be involved in chronic phototoxicity of the aging RPE, natural antioxidants could partially ameliorate these harmful effects

Invasive Cx43^{high} sub-line of human prostate DU145 cells displays increased nanomechanical deformability

Connexin(Cx)43high cells are preferentially recruited to the invasive front of prostate cancer in vitro and in vivo. To address the involvement of Cx43 in the regulation of human prostate cancer DU145 cell invasiveness, we have analysed the nanoelasticity of invasive Cx43high sub-sets of DU145 cells by atomic force microscopy (AFM). The Cx43high DU145 cells displayed considerably higher susceptibility to mechanical distortions than the wild type DU145 cells. Transient Cx43 silencing had no effect on their elastic properties. Our data confirm the relationship between the invasive potential, Cx43 expression and nanoelasticity of the DU145 cells. However, they also show that Cx43 is not directly involved in the maintenance of DU145 invasive phenotype

Photoactivated gallium porphyrin reduces Staphylococcus aureus colonization on the skin and suppresses its ability to produce enterotoxin C and TSST-1

Staphylococcus aureus is a key pathogen in atopic dermatitis (AD) pathogenicity. Over half of AD patients are carriers of S. aureus. Clinical isolates derived from AD patients produce various staphylococcal enterotoxins, such as staphylococcal enterotoxin C or toxic shock syndrome toxin. The production of these virulence factors is correlated with more severe AD. In this study, we propose cationic heme-mimetic gallium porphyrin ($Ga^{3+}CHP$), a novel gallium metalloporphyrin, as an anti-staphylococcal agent that functions through dual mechanisms: a light-dependent mechanism (antimicrobial photodynamic inactivation, aPDI) and a light-independent mechanism (suppressing iron metabolism). $Ga^{3+}CHP$ has two additive quaternary ammonium groups that increase its water solubility. Furthermore, $Ga^{3+}CHP$ is an efficient generator of singlet oxygen and can be recognized by heme-target systems such as Isd, which improves the intracellular accumulation of this compound. $Ga^{3+}CHP$ activated with green light effectively reduced the survival of clinical S. aureus isolates derived from AD patients (>5 $log_{10}$ CFU/mL) and affected their enterotoxin gene expression. Additionally, there was a decrease in the biological functionality of studied toxins regarding their superantigenicity. In aPDI conditions, there was no pronounced toxicity in HaCaT keratinocytes with both normal and suppressed filaggrin gene expression, which occurs in ∼50% of AD patients. Additionally, no mutagenic activity was observed. Green light-activated gallium metalloporphyrins may be a promising chemotherapeutic to reduce S. aureus colonization on the skin of AD patients

Nanomechanical phenotype of melanoma cells depends solely on the amount of endogenous pigment in the cells

Cancer cells have unique nanomechanical properties, i.e., they behave as if they were elastic. This property of cancer cells is believed to be one of the main reasons for their facilitated ability to spread and metastasize. Thus, the so-called nanomechanical phenotype of cancer cells is viewed as an important indicator of the cells’ metastatic behavior. One of the most highly metastatic cancer cells are melanoma cells, which have a very unusual property: they can synthesize the pigment melanin in large amounts, becoming heavily pigmented. So far, the role of melanin in melanoma remains unclear, particularly the impact of the pigment on metastatic behavior of melanoma cells. Importantly, until recently the potential mechanical role of melanin in melanoma metastasis was completely ignored. In this work, we examined melanoma cells isolated from hamster tumors containing endogenous melanin pigment. Applying an array of advanced microscopy and spectroscopy techniques, we determined that melanin is the dominating factor responsible for the mechanical properties of melanoma cells. Our results indicate that the nanomechanical phenotype of melanoma cells may be a reliable marker of the cells’ metastatic behavior and point to the important mechanical role of melanin in the process of metastasis of melanoma