Abstract
Background: Mushroom tyrosinase, a copper containing enzyme, modifies growth and survival of tumor cells. Mushroom tyrosinase may foster apoptosis, an effect in part due to interference with mitochondrial function. Erythrocytes lack mitochondria but are able to undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and activation of sphingomyelinase with subsequent formation of ceramide. The present study explored, whether tyrosinase stimulates eryptosis. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 24 h exposure to mushroom tyrosinase (7 U/mL) was followed by a significant increase of [Ca2+]i, a significant increase of ceramide abundance, and a significant increase of annexin-V-binding. The annexin-V-binding following tyrosinase treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca2+. Tyrosinase did not significantly modify forward scatter. Conclusions: Tyrosinase triggers cell membrane scrambling, an effect, at least partially, due to entry of extracellular Ca2+ and ceramide formation.
Highlights
Mushroom tyrosinase has been suggested for the use in malignancy [1]
The present study addressed the effect of tyrosinase on eryptosis
A hallmark of eryptosis is the breakdown of phosphatidylserine asymmetry of the erythrocyte cell membrane, which increases the phosphatidylserine abundance at the cell surface
Summary
Mushroom tyrosinase has been suggested for the use in malignancy [1]. When applied with appropriate substrates, it may generate cytostatic products effective in vivo [2,3,4]. Mushroom tyrosinase generates products leading to mutagenesis and carcinogenesis [5,6,7,8,9,10]. Even though lacking mitochondria and nuclei, erythrocytes are still able to undergo apoptosis-like suicidal death or eryptosis [12]. An increase of [Ca2+]i may shrink erythrocytes due to activation of Ca2+-sensitive K+ channels leading to. Increased [Ca2+]i further stimulates cell membrane scrambling with translocation of phosphatidylserine to the erythrocyte surface [12]. The Ca2+ sensitivity of cell membrane scrambling is increased by ceramide [12]. The present study explored, whether tyrosinase influences [Ca2+]i, cell volume and phosphatidylserine translocation to the erythrocyte surface. The observations disclose that exposure to tyrosinase stimulates erythrocyte cell membrane scrambling, an effect paralleled by and at least in part secondary to increase of [Ca2+]i
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