Alghareeb Sumiah A, Alfhili Mohammad A, Alsughayyir Jawaher
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh 12372, Saudi Arabia.
Life (Basel). 2023 Dec 4;13(12):2299. doi: 10.3390/life13122299.
Eryptosis stimulated by anticancer drugs can lead to anemia in patients. β-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however, its effect on the structure and function of human red blood cells (RBCs) remains unexplored. The aim of this study was to investigate the hemolytic and eryptotic activities and underlying molecular mechanisms of CPO in human RBCs.
Cells were treated with 10-100 μM of CPO for 24 h at 37 °C, and hemolysis, LDH, AST, and AChE activities were photometrically assayed. Flow cytometry was employed to determine changes in cell volume from FSC, phosphatidylserine (PS) externalization by annexin-V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (HDCFDA). Cells were also cotreated with CPO and specific signaling inhibitors and antihemolytic agents. Furthermore, whole blood was exposed to CPO to assess its toxicity to other peripheral blood cells.
CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca accumulation, oxidative stress, and reduced AChE activity. The toxicity of CPO was ameliorated by D4476, staurosporin, and necrosulfonamide. ATP and PEG 8000 protected the cells from hemolysis, while urea and isotonic sucrose had opposite effects.
CPO stimulates hemolysis and eryptosis through energy depletion, Ca buildup, oxidative stress, and the signaling mediators casein kinase 1α, protein kinase C, and mixed lineage kinase domain-like pseudokinase. Development of CPO as an anticancer therapeutic must be approached with prudence to mitigate adverse effects on RBCs using eryptosis inhibitors, Ca channel blockers, and antioxidants.
抗癌药物刺激的红细胞凋亡可导致患者贫血。β-石竹烯氧化物（CPO）是一种存在于多种植物中的抗癌倍半萜；然而，其对人类红细胞（RBC）结构和功能的影响仍未得到探索。本研究的目的是研究CPO在人类红细胞中的溶血和红细胞凋亡活性及其潜在分子机制。
细胞在37℃下用10 - 100μM的CPO处理24小时，通过光度法测定溶血、乳酸脱氢酶（LDH）、天冬氨酸转氨酶（AST）和乙酰胆碱酯酶（AChE）活性。采用流式细胞术通过前向散射光（FSC）测定细胞体积变化、用膜联蛋白-V-异硫氰酸荧光素（annexin-V-FITC）测定磷脂酰丝氨酸（PS）外化、用Fluo4/AM测定细胞内钙以及用2',7'-二氯二氢荧光素二乙酸酯（HDCFDA）测定氧化应激。细胞还与CPO和特定信号抑制剂及抗溶血剂共同处理。此外，将全血暴露于CPO以评估其对其他外周血细胞的毒性。
CPO诱导浓度依赖性溶血，伴有LDH和AST泄漏，此外还有PS暴露、细胞收缩、钙积累、氧化应激和AChE活性降低。D4476、星形孢菌素和坏死磺酰胺可改善CPO的毒性。三磷酸腺苷（ATP）和聚乙二醇8000（PEG 8000）可保护细胞免受溶血，而尿素和等渗蔗糖则有相反作用。
CPO通过能量耗竭、钙积累、氧化应激以及信号介质酪蛋白激酶1α、蛋白激酶C和混合谱系激酶结构域样假激酶刺激溶血和红细胞凋亡。必须谨慎开发CPO作为抗癌治疗药物，使用红细胞凋亡抑制剂、钙通道阻滞剂和抗氧化剂来减轻对红细胞的不良影响。
