Triggering of Eryptosis, the Suicidal Erythrocyte Death by Mammalian Target of Rapamycin (mTOR) inhibitor Temsirolimus

Abstract

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved. Methods: Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to temsirolimus (5 – 20 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca²⁺ and by addition of staurosporine (1 µM) or chelerythrine (10 µM) but not significantly modified by addition of SB203580 (2 µM), D4476 (10 µM), or zVAD (10 µM). Chelerythrine (10 µM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca²⁺ had no effect on temsirolimus induced ROS formation or ceramide abundance. Conclusions: Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca²⁺ entry, oxidative stress, ceramide and activation of staurosporine/Chelerythrine sensitive kinase(s).

Understanding the Link between Inflammasome and Apoptosis through the Response of THP-1 Cells against Drugs Using Droplet-Based Microfluidics

“…Temsirolimus triggers apoptosis in tumor cells via activation of caspases. 32 This drug causes the suspension of protein synthesis that manages the proliferation, growth, and survival of tumor cells. Cells exposed to temsirolimus encounter a cell cycle arrest in the G1 phase, and it also decreases the production of vascular endothelial growth factor (VEGF) resulting in the inhibition of tumor angiogenesis.…”

“…Temsirolimus triggers apoptosis in tumor cells via activation of caspases . This drug causes the suspension of protein synthesis that manages the proliferation, growth, and survival of tumor cells.…”

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC

“…Likewise, co-treatment of cells with RA and STSP (Figure 7) resulted in significantly less hemolysis than that observed in cells treated with RA alone. Previous reports have unequivocally demonstrated that PKC mediates RBC death induced by excessive Ca 2+ entry [36] and by eryptosis inducers costunolide [37] and temsirolimus [38], among others. Collectively, these studies indicate that PKC acts downstream of Ca 2+ accumulation but upstream of reactive oxygen species generation.…”

Eryptosis (quasi-apoptosis) the human red blood cells. Its role in medicinal therapy

“…Это делает слой менее текучим по сравнению с теми, которые находятся в составе фосфатидилэтаноламина и фосфатидилсерина внутренней поверхности. Во-вторых, отрицательно заряженный фосфатидилсерин, взаимодействующий с регуляторными и структурными белками, при нарушении асимметрии приводит к экспонированию фосфатидилсерина на наружной поверхности, что является сигналом к эриптозу [49,50,82,254], а также к изменению соотношения зарядов на внутренней и внешней сторонах бислоя мембраны эритроцита. Таким образом, aссимметрия фосфолипидов определяет текучесть мембраны (fragility) и вносит важный вклад в поддержание механических свойств мембраны [95,236].…”

“…Многочисленными исследованиями установлено, что эриптоз запускается прежде всего повышением внутриклеточной концентрации свободных ионов кальция, а также рядом веществ эндогенного и экзо генного происхождения: металлами и их соединениями, лекарственными препаратами и ксенобиотиками [26,27,51,90,91,267,268]. Воздействие этих веществ либо запускает механизмы, связанные с повышением внутриклеточной концентрации свободных ионов кальция и усилением выработки эндогенного церамида, либо активирует дополнительные механизмы, такие как энергетическое истощение по АТФ, окислительный стресс и/или активация каспазы 3.…”