Stimulation of Eryptosis by Afatinib

Abstract

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at least partially effective by triggering suicidal tumor cell death. Side effects of afatinib treatment include anemia. At least in theory, afatinib induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ), induction of oxidative stress, and increase of ceramide abundance. The present study explored, whether afatinib induces eryptosis and, if so, whether its effect involves Ca 2+ entry, oxidative stress, and/or ceramide. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca 2+ ] i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to afatinib (≥ 4 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Afatinib significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance. The effect of afatinib on annexin-V-binding and forward scatter was significantly blunted by removal of extracellular Ca 2+ . Conclusions: Afatinib triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca 2+ entry, oxidative stress, and ceramide.

Molecular Mechanisms and Pathophysiological Significance of Eryptosis

“…The characteristics of eryptosis involves dysfunctional ion exchange, cell shrinkage, ceramide accumulation, cell membrane vesiculation due to cytosolic calcium overload, and membrane phospholipid scrambling with alteration of the cell membrane asymmetry and phosphatidylserine (PS) exposure at the cell surface [ 9 , 10 ]. Ca 2+ influx opens Ca 2+ -sensitive K + channels, or Gardos channels, leading to subsequent loss of KCl, hyperpolarization of the membrane, loss of water, and cell shrinkage [ 11 , 12 ].…”

“…The process of eryptosis regulation is complex, implicating a multitude of cellular machinery, and there are various triggers and inhibitors. Several kinases, including cyclin-dependent kinase 4, p38 mitogen-activated kinase, casein kinase 1α (CK1α), mitogen- and stress-activated kinase MSK1/2, and Janus-activated kinase JAK3, have been shown to participate in the stimulation or inhibition of eryptosis [ 10 , 19 ]. Many food-derived phytochemicals and natural compounds, including phenolic compounds and alkaloids, with antioxidant and anti-inflammatory properties have been reported to prevent eryptosis-driven cell death and associated clinical conditions [ 20 ].…”

Liraglutide protects renal mesangial cells against hyperglycemia‑mediated mitochondrial apoptosis by activating the ERK‑Yap signaling pathway and upregulating Sirt3 expression

“…Experiments were then performed to determine the upstream regulatory factors for Sirt3 upregulation in response to liraglutide treatment. Previous studies have identified the ERK-Yap axis as a novel signaling pathway that modifies mitochondrial function (36)(37)(38). Therefore, the present study investigated whether the ERK-Yap signaling pathway was involved in liraglutide-mediated Sirt3 activation.…”

<p>Mst1-Hippo pathway triggers breast cancer apoptosis via inducing mitochondrial fragmentation in a manner dependent on JNK&ndash;Drp1 axis</p>

“…Ample evidence has identified Mst1 as an activator of caspases to initiate the apoptotic signaling pathway 4. The anticancer effects of Mst1 have also been reported in several tumors such as colorectal cancer, prostate cancer, lung cancer and liver cancer 5. Notably, previous studies have found that Mst1 is increased in HER2-positive and triple-negative breast cancer patients treated with neoadjuvant therapy.…”