Camalexin-Induced Cell Membrane Scrambling and Cell Shrinkage in Human Erythrocytes

Abstract

Background/Aims: The thaliana phytoalexin Camalexin has been proposed for the treatment of malignancy. Camalexin counteracts tumor growth in part by stimulation of suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms contributing to the complex machinery executing eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, protein kinase C and caspases. The present study explored, whether Camalexin induces eryptosis and, if so, to shed light on mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo-3 fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Camalexin significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥ 5 µg/ml) and significantly increased Fluo-3-fluorescence (≥ 10 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Camalexin on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺, by kinase inhibitors staurosporine (1 µM) and chelerythrine (10 µM), as well as by caspase inhibitors zVAD (10 µM) and zIETD-fmk (50 µM). Conclusions: Camalexin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part depending on Ca²⁺ entry, as well as staurosporine and chelerythrine sensitive kinase(s) as well as zVAD and zIETD-fmk sensitive caspase(s).

Physcion Induces Hemolysis and Premature Phosphatidylserine Externalization in Human Erythrocytes

“…27) In erythrocytes, PKC inhibition has been shown to be protective against energy exhaustion 28) as well as some toxic agents. 29,30) CK1α similarly mediates eryptosis secondary to energy exhaustion, oxidative stress, and calcium loading. 31) Moreover, this kinase also participates in eryptosis elicited by various compounds as reported by us and others.…”

Camalexin Induces Apoptosis via the ROS‐ER Stress‐Mitochondrial Apoptosis Pathway in AML Cells

“…According to previous studies, camalexin could induce ROS in prostate cancer and leukemia cells [ 6 , 7 ]. Interestingly, camalexin was found unable to induce ROS in erythrocytes in a recent study [ 27 ]. This discrepancy may be due to different cell types.…”

Anticancer Potential of Indole Phytoalexins and Their Analogues

“…This discrepancy might arise from variations in cell types. The author hypothesizes that camalexin could potentially trigger ROS in cancer cells while sparing normal cells [79].…”

“…Camalexin-induced cell membrane scrambling also involves protein kinase C and caspase activation. Notably, camalexin induces these effects without increasing ceramide levels or inducing oxidative stress [79], which are commonly associated with eryptosis triggers [83]. The potential clinical implications of camalexin use warrant careful consideration.…”

“…The potential clinical implications of camalexin use warrant careful consideration. While it demonstrates neuroprotective and antioxidant properties, its eryptotic effects on erythrocytes could lead to side effects such as anemia and an elevated risk of thrombosis if effective concentrations are achieved in patients' plasma [79]. This complex interplay of beneficial and potentially adverse effects underscores the need for a thorough investigation before considering camalexin for therapeutic applications.…”