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

Background: Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms. Methods: RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10–800 μM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell size by FSC, and oxidative stress by H2DCFDA. Ions and pH were assessed by an ion-selective electrode, while B12 was detected by chemiluminescence. Results: RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca2+ removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B12 content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000. Conclusions: RA stimulates Ca2+-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca2+ accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.


Introduction
Chemotherapy-associated anemia (CAA) is a major obstacle in cancer treatment, with a prevalence of at least 75% in patients undergoing therapy [1].Although complex, the underlying mechanisms of CAA involve decreased RBC production due to myelosuppression and the premature death of circulating cells by either hemolysis or eryptosis [2].Suicidal RBC death is characterized by phospholipid scrambling of the cell membrane, loss of ionic homeostasis, reduced cellular volume, oxidative stress, ceramide accumulation, and metabolic deprivation.Hemolysis and eryptosis are regulated by caspase, p38 MAPK, casein kinase 1α (CK1α), protein kinase C (PKC), Rac1 GTPase, and JAK3, among other kinases.Loss of membrane asymmetry results in phosphatidylserine (PS) exposure to the outer membrane leaflet, which serves as a binding site for recognition and engulfment by phagocytes, which predisposes to anemia [3].
Although RA has promising anticancer potential, no study has thus far examined its toxicity to human RBCs.Our aim is thus to investigate the interaction of RA with RBCs and identify the underlying molecular mechanisms.

RA Leads to Cell Shrinkage
A significant increase in the percentage of cells that underwent shrinkage was observed at 800 μM of RA (3.89 ± 0.61% to 6.24 ± 2.16%), as shown in Figure 4d.Accordingly, at the same concentration, efflux of K + increased from 6.35 ± 0.19 mmol/L to 7.18 ± 0.69 mmol/L (Figure 4f), and that of Cl − increased from 136.3 ± 3.65 mmol/L to 144.3 ± 3.65 mmol/L (Figure 4g).Interestingly, the hemolytic effect of RA was significantly increased in the presence of extracellular 125 mM of KCl when compared to standard Ringer solution (11.53 ± 4.41% to 23.69 ± 6.95%, Figure 4i), while eryptosis was not appreciably affected (Figure 4k).

RA Leads to Cell Shrinkage
A significant increase in the percentage of cells that underwent shrinkage was observed at 800 µM of RA (3.89 ± 0.61% to 6.24 ± 2.16%), as shown in Figure 4d.Accordingly, at the same concentration, efflux of K + increased from 6.35 ± 0.19 mmol/L to 7.18 ± 0.69 mmol/L (Figure 4f), and that of Cl − increased from 136.3 ± 3.65 mmol/L to 144.3 ± 3.65 mmol/L (Figure 4g).Interestingly, the hemolytic effect of RA was significantly increased in the presence of extracellular 125 mM of KCl when compared to standard Ringer solution (11.53 ± 4.41% to 23.69 ± 6.95%, Figure 4i), while eryptosis was not appreciably affected (Figure 4k).

RA Causes of Morphological Alterations
Treatment of cells with 800 µM of RA resulted in the formation of schistocytes and acanthocytes, as revealed by Giemsa staining (Figure 5a) and SEM analysis (Figure 5b).The ESR of treated cells was also significantly elevated (5.33 ± 0.58 mm/h to 7.33 ± 0.58 mm/h, Figure 5c).Treatment of cells with 800 μM of RA resulted in the formation of schistocytes and acanthocytes, as revealed by Giemsa staining (Figure 5a) and SEM analysis (Figure 5b).The ESR of treated cells was also significantly elevated (5.33 ± 0.58 mm/h to 7.33 ± 0.58 mm/h, Figure 5c).

Chemicals and Reagents
All chemicals were of analytical grade and were obtained from Solarbio Life Science (Beijing, China).RA (CAS #20283-92-5) was purchased as a pure compound extracted and purified from the Rosmarinus officinalis shrub (purity ≥ 98%).To prepare a 50 mM stock solution of RA, 10 mg were dissolved in 555 µL of DMSO, and aliquots were stored at −80 • C. Standard Ringer solution was composed of 125 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 32 mM HEPES, 5 mM glucose, and 1 mM CaCl 2 with a pH of 7.4.Several experiments were conducted to manipulate the extracellular environment by eliminating extracellular Ca 2+ , substituting NaCl and KCl with 125 mM KCl, substituting NaCl with 250 mM sucrose, or adding 10% w/v PEG 8000 [15].

Blood Collection and Experimental Design
Ethical approval was obtained from the Ethics Committee of King Saud University Medical City (E-23-7764).Blood samples were obtained from 27 healthy individuals (18 males and 9 females) aged 26-42 years with a BMI of <25, normal CBC results, and no history of chronic disease.All participants signed informed consent according to the Declaration of Helsinki.Heparinized blood samples were collected to isolate RBCs (2500 RPM, 20 min, RT), and, following washing two times in PBS, cells were stored in PBS or Ca 2+ -free Ringer solution at 4 • C for a maximum of 48 h.Exposure to 10-800 µM of RA was carried out at 5% hematocrit (11.0 × 10 5 cells/µL) in PBS or Ringer solutions for 24 h at 37 • C. In some experiments, cells were cotreated with 400 µM of RA in the presence or absence of 1 µM of PKC inhibitor staurosporin (STSP), 100 µM of p38 inhibitor SB203580, 20 µM of CK1a inhibitor D4476, or 500 µM of ATP.Additionally, whole blood in EDTA was diluted 1:2 with PBS and exposed to 800 µM RA for 24 h at 37 • C. Control and experimental cells from the same subject were used in distinct experiments to account for potential individual variation [16].

Hemolysis and Hemolytic Markers
Supernatants of control and experimental cells were harvested by centrifugation (13,000× g, 1 min, RT) to measure hemoglobin (Hb) at 405 nm using a LMPR-A14 microplate reader (Labtron Equipment Ltd., Surrey, UK).The percentage of hemolysis was derived relative to cells suspended in distilled water [17].AST and LDH activities were detected in the supernatants by the BS-240Pro clinical chemistry analyzer (Mindray Medical International Limited, Shenzhen, China).

Eryptosis
Control and experimental cells were labeled with 1% annexin-V-FITC for 10 min at RT in the dark and analyzed using a Northern Lights TM flow cytometer (Cytek Biosciences, Fremont, CA, USA).FITC was excited by the blue laser at 488 nm, and the emitted green light was captured at 512 nm for a total of 10,000 events.Forward scatter (FSC) and side scatter (SSC) were used as indicators of cell volume and complexity, respectively [18].

Intracellular Ca 2+
Control and experimental cells were stained with 5 µM Fluo4/AM (Ex/Em = 488/520 nm) for 30 min at RT away from light, and 10,000 events were analyzed by flow cytometry [19].

Intracellular B 12
Control and experimental hemolysates were assayed for B 12 content using Mindray's CL-1200i chemiluminescence analyzer.In this competitive binding immunoenzymatic assay, B 12 in the sample competes with paramagnetic microparticles coated with biotinylated B 12 for binding to alkaline phosphatase-labeled intrinsic factor.Microparticles are then magnetically captured, while other unbound substances are removed by washing.The chemiluminescent signal generated upon addition of a substrate is inversely proportional to the concentration of B 12 in the sample, which is determined from a calibration curve.

Systemic Toxicity
A CBC of control and experimental whole blood samples was performed using Mindray's BC-6200 hematology analyzer [22].

Erythrocyte Sedimentation Rate (ESR)
The rate of vertical sedimentation (mm/h) of control and experimental RBCs in whole blood was recorded using Westergren tubes [23].

Statistics
Results are presented as means ± SD of three independent experiments.Flow cytometric data were analyzed by FlowJo TM v10.7.2 (Becton, Dickinson, and Company, Ashland, OR, USA), and all statistical analyses were performed by GraphPad Prism v9.2.0 (Graph-Pad Software, Inc., San Diego, CA, USA).Two experimental groups were analyzed by the unpaired, two-tailed Student's t-test, while three or more groups were analyzed by a one-way ANOVA with Dunnett's correction.Statistical significance was defined by a p value of <0.05.

Discussion
CAA remains a challenging side effect of current chemotherapy, and investigating the toxicity of potential anticancer therapeutics to erythrocytes is therefore of utmost importance.In this work, we have shown for the first time that anticancer concentrations of RA (10-800 µM) stimulate hemolysis and eryptosis characterized by AST and LDH leakage, PS translocation, dysregulated ion trafficking, Ca 2+ accumulation, cell shrinkage, and B 12 entrapment.Both hemolysis and eryptosis were Ca 2+ -dependent and were sensitive to isosmotic sucrose.The toxicity of RA was blunted by energy replenishment and PEG 8000 and was mediated through p38 MAPK, CK1α, and PKC.
In contrast to a previous report showing that RA is not toxic to PBMCs [8], our findings indicate that RA exhibits a dual effect on RBCs (Figure 1), being hemolytic at 200-800 µM and antihemolytic at 50 µM under hypotonic conditions.In addition to its oxidative potential, circulating naked Hb undergoes glomerular filtration and precipitates in kidney tubules, giving rise to renal failure [24].Prevention of hypotonic lysis, on the other hand, suggests that RA intercalates into the lipid bilayer and expands the membrane, allowing the cell to withstand more water influx relative to untreated cells before hemolysis ensues.Counteracting hemolysis ostensibly by increasing cellular volume has previously been observed with Ginkgo biloba leaf extract [25] and mangostin [15].
The central finding in this study is the pro-eryptotic activity of RA (Figure 2).Translocation of PS to the outer membrane leaflet is observed in senescent, infected, and damaged cells.It serves as a binding site for phagocyte receptors to identify and engulf these cells to prevent their extended presence in the circulation, which could lead to intravascular hemolysis.Although suicidal RBC death is an effective defense mechanism that ensures the elimination of dysfunctional cells, premature eryptosis, as elicited by RA in this study, results in excessive removal of circulating cells, which outweighs the rate of erythropoiesis in the bone marrow, culminating in anemia [26].Importantly, the morphological alterations seen in eryptotic cells and elevated ESR (Figure 5) compromise their deformability, impede blood flow, and increase the risk of thromboembolic events [27].Also, patients with excessive eryptosis are prone to vaso-occlusive lesions because eryptotic cells adhere to the endothelial wall through CXCL16/SR-PSOX [28].
Of note, RA-induced eryptosis was found to be sensitive to the availability of isosmotic sucrose in the medium (Figure 2).Although the exact mechanism through which sucrose acts as an anti-eryptotic agent still eludes us, we propose the following three scenarios: First, sucrose may block Cl -efflux (Figure 4) and thus reduce fluid loss and cell shrinkage.Second, sucrose may prevent water influx through colloid osmotic pressure because it is a non-penetrating solute.Third, sucrose, being rich in hydrogen-accepting sites, may bind RA and reduce its activity [29].Further elucidation of these possible scenarios is warranted for future studies.
We have also found that RA elevates intracellular Ca 2+ (Figure 3), which is a characteristic sign of eryptotic cells.Ca 2+ regulates the activity of numerous enzymes that preserve the integrity of the cell membrane.Scramblases, flippases, and floppases maintain the orientation of phospholipids within the plasma membrane and are subject to regulation by Ca 2+ ions.Excessive buildup of Ca 2+ inside the cell thus leads to dysfunctional enzymatic activity, resulting in loss of membrane asymmetry and PS externalization (Figure 2).The physiological relevance of Ca 2+ extends to other functions, including signal transduction, motility, and transcriptional regulation [30].Interestingly, our data reveal that Ca 2+ are essential to the full hemolytic and eryptotic activities of RA (Figure 3), which indicates that Ca 2+ acts upstream of PS externalization and membrane rupture.However, since the nominal absence of Ca 2+ failed to fully abrogate the toxicity of RA, the involvement of other necessary mechanisms is strongly suggested.
Hyperactivity of Ca 2+ -sensitive K + channels secondary to Ca 2+ buildup leads to KCl efflux, followed by water exit, leading to severe loss of cellular volume and eventual cell shrinkage (Figure 4).Blocking KCl exit by increasing extracellular KCl to 125 mM aggravated the hemolytic effect of RA but failed to protect against its eryptotic effect.This observation suggests that preventing KCl loss either renders existing mechanisms more detrimental to membrane integrity or allows other additional mechanisms to be targeted.In the case of eryptosis, the result indicates that KCl efflux is not necessary for RA-induced PS exposure and Ca 2+ accumulation, thus activating other essential mechanisms.
Very few studies have examined the acidity of the extracellular milieu of erythrocytes subjected to chemical stress.In this work, we observed decreased extracellular pH in RA-treated cells (Figure 6), ostensibly due to the buildup of lactic acid from excessive glycolysis.Along those lines, our results suggest that energy replenishment rescues the cells from RA toxicity (Figure 7), an observation pointing at energy turnover being a target of RA in erythrocytes.It is worth mentioning that lactic acid accumulation has been observed in RBCs following exposure to methylglyoxal [31] and tashinone IIA [32], two other eryptotic compounds.
Erythroblasts require B 12 during their growth and differentiation for DNA synthesis, and a lack of it leads to halted erythropoiesis and the generation of macroerythrocytes characteristic of megaloblastic anemia.Exposure to RA caused significant entrapment of B 12 inside the cells (Figure 6), suggesting that RA interferes with cobalamin transport and trafficking.Earlier studies have demonstrated that B 12 uptake occurs in RBCs, and the process seems to be Ca 2+ -or Mg 2+ -dependent [33].It is plausible, then, to assume that the loss of ionic regulation, most importantly that of Ca 2+ (Figure 3), is related to the observed intracellular accumulation of B 12 (Figure 6).Further examination of this association is indeed warranted.
Signal transduction mediators are pivotal for the regulation of erythrocyte survival.Our results show that RA toxicity is significantly blunted in the presence of SB203580 (Figure 7), indicating the participation of p38 MAPK in membrane rupture.Similar to its role in nucleated cells, p38 in RBCs is activated by physical and chemical stress such as hyperosmotic shock [34] and tobacco extract [35].Notably, pharmacological inhibition of p38 activity also blocked Ca 2+ accumulation secondary to hyperosmotic shock [34], suggesting that p38 acts upstream of Ca 2+ signaling.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.However, since no appreciable oxidative stress was observed in RA-treated cells (Figure 6), PKC activation by RA more likely targets other mechanisms independent of oxidative damage.
Erythrocytes also express CK1α, which plays a significant role in cell differentiation, apoptosis, survival, and stress response [39].Zelenak et al. reported that CK1α mediates erythrocyte death upon energy deprivation and that D4476 reverses Ca 2+ accumulation and subsequent cell shrinkage [40], indicating that, like p38, CK1α operates upstream of Ca 2+ signaling.Since RA also exhausts the intracellular ATP pool (Figure 6), it is speculated that RA activates a CK1α/Ca 2+ /ATP molecular axis that eventually culminates in cell death.Congruently, we have recently reported that both CK1α and metabolic exhaustion, but not Ca 2+ signaling, are responsible for the toxicity of deguelin in RBCs [22].
Unlike sucrose, urea is a penetrating solute, and 300 mOsm in the presence of isotonic NaCl thus ensures equilibrium is reached inside and outside the cells.Other mechanisms suspected to mediate the protective effects of urea include inhibiting sphingomyelinase activity and modulating the Na + -K + -ATPase pump, KCl cotransport, or Na + -K + -2Cl − cotransport [41].However, among all identified RA inhibitors, PEG thoroughly abrogated the toxic effects of RA, which strongly suggests physical partitioning of either corpuscles or RA molecules, which is supported by its antihemolytic properties demonstrated against shear stress [42].It is also presumed that membrane pores formed by RA are smaller in size than the hydrodynamic radius of PEG.Future studies should probe whether RA-based PEGylated nanomedicines and pharmaceuticals retain their anticancer activities with lower hemolytic potential.
Our studies on whole blood have discerned that RA toxicity extends to reticulocytes, leukocytes, and platelets.Even in the presence of plasma proteins, mature RBCs were susceptible to RA, which also depleted Hb stores (Figure 8) and reduced corpuscular size (Figure 4).This indicates a lack of inhibitory effects of complement proteins on RA, unlike the susceptibility of sanguinarine [43].Alarmingly, RA was also cytotoxic to reticulocytes

Figure 4 .Figure 4 .
Figure 4. RA leads to cell shrinkage.(a) Representative dot plots of SSC and FSC signals.(b) Representative histograms of the FSC distribution of control (black line) and experimental (green line, 800 μM) cells.(c) Geomean of FSC,(d) percentage of shrunk cells, and (e) geomean of SSC.Leakage of (f) K + , (g) Cl − , and (h) Na + .(i) Effect of 125 mM of KCl on RA-induced hemolysis.(j) Representative histograms of annexin-V-FITC fluorescence in 5 mM of KCl (green line) and 125 mM of KCl (brown