A Comprehensive Two-Decade Analysis of Lymphoma Incidence Patterns in Saudi Arabia.

Background: Lymphomas account for approximately 10% of all cancer cases among the Saudi population. Even when separated, Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) are in the top ten most commonly diagnosed cancers among Saudi men and women. Despite the substantial cost of HL and NHL to public health, the resources to assess their impact are insufficient. This study provides a two-decade detailed assessment of lymphoma incidence trends in the Saudi population. Methods: Analysis of the Saudi Cancer Registry (SCR) data for various incidence metrics from 2001 to 2020 was conducted. Joinpoint regression analysis was further performed to investigate temporal trends globally and by age group, gender, and administrative region. Results: HL cases grew by 174.1%, whereas NHL cases increased by only 80% for that time period. The HL overall Age-Standardized Incidence Rate (ASR) increased by 100% for both genders combined but remained unchanged for NHL. The median age at diagnosis for HL (20-30 years) and NHL (46-57 years) was lower than in many other nations. Our model identified increasing trends for HL with annual percentage changes (APCs) of 2.94% (CI: 2.2-3.7) and 3.67% (CI: 2.6-4.7) for males and females, respectively. The rise was mainly among young groups under 40. On the contrary, the NHL cohort revealed notable declining tendencies. We discovered alarming rates of HL in Saudi Arabia's APC (2.23% for males and 3.88% for females) and ASR compared to other Western countries. Overall, the majority of the patients presented with advanced-stage disease at a younger age and with slight male predominance. Conclusions: The overall incidence of lymphoma (especially HL) has been rising among Saudis. Implementation of secondary and tertiary prevention measures, as well as management of modifiable risk factors, is warranted.

Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase.

Ample evidence indicates that bufalin (BFN), a cardiotonic steroid in Bufo toad toxin, possesses a potent anticancer activity mainly by stimulating apoptosis in cancer cells. Human red blood cells (RBCs) undergo eryptosis which contributes to a plethora of pathological conditions. No reports, however, have examined the potential toxicity of BFN to RBCs. This study aims to characterize the biochemical mechanisms governing the influence of BFN on the physiology and lifespan of RBCs. Isolated RBCs from healthy volunteers were exposed to anticancer concentrations of commercially available BFN from the skin of Bufo gargarizans (10-200 µM) for 24 h at 37 °C. Photometric assays were used to estimate hemolysis and hemolytic markers, and flow cytometry was used to detect eryptotic markers. Phosphatidylserine externalization was captured by fluorescein isothiocyante-labeled annexin V, cellular dimensions by light scatter patterns, and intracellular Ca2+ and reactive oxygen species (ROS) by fluorogenic dyes Fluo4/AM and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. BFN caused Ca2+-independent hemolysis and release of LDH, AST, CK, and K+, and increased annexin V-bound cells, cytosolic Ca2+, cell shrinkage, and ROS levels. BFN also disrupted Na+ and Mg2+ trafficking, and was sensitive to PEG 8000, sucrose, SB203580, and NSC 23766. In whole blood, BFN depleted hemoglobin stores, increased fragmented RBCs, and was selectively toxic to reticulocytes, lymphocytes, and platelets. In conclusion, BFN elicits premature RBC death, subject to regulation by p38 MAPK and Rac1 GTPase, and is detrimental to other peripheral blood cells. Altogether, these novel findings prompt cautious consideration of the toxin in anticancer therapy.

Eriocitrin Disrupts Erythrocyte Membrane Asymmetry through Oxidative Stress and Calcium Signaling and the Activation of Casein Kinase 1α and Rac1 GTPase.

BACKGROUND: Hemolysis and eryptosis result in the premature elimination of circulating erythrocytes and thus contribute to chemotherapy-related anemia, which is extremely prevalent in cancer patients. Eriocitrin (ERN), a flavanone glycoside in citrus fruits, has shown great promise as an anticancer agent, but the potential toxicity of ERN to human erythrocytes remains unstudied. METHODS: Erythrocytes were exposed to anticancer concentrations of ERN (10-100 µM) for 24 h at 37 °C, and hemolysis and associated markers were quantified using colorimetric assays. Eryptosis was assessed by flow cytometric analysis to detect phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ using Fluo4/AM, and oxidative stress with 2-,7-dichlorodihydrofluorescin diacetate (H2DCFDA). ERN was also tested against specific signaling inhibitors and anti-hemolytic agents. RESULTS: ERN caused significant, concentration-dependent hemolysis at 20-100 µM. ERN also significantly increased the percentage of eryptotic cells characterized by Ca2+ elevation and oxidative stress. Furthermore, the hemolytic activity of ERN was significantly ameliorated in the presence of D4476, NSC23766, isosmotic urea and sucrose, and polyethylene glycol 8000 (PEG). In whole blood, ERN significantly elevated MCV and ESR, with no appreciable effects on other peripheral blood cells. CONCLUSIONS: ERN promotes premature erythrocyte death through hemolysis and eryptosis characterized by PS externalization, Ca2+ accumulation, membrane blebbing, loss of cellular volume, and oxidative stress. These toxic effects, mediated through casein kinase 1α and Rac1 GTPase, can be ameliorated by urea, sucrose, and PEG. Altogether, these novel findings are relevant to the further development of ERN as an anticancer therapeutic.

Stimulation of Hemolysis and Eryptosis by ß-Caryophyllene Oxide.

BACKGROUND: Eryptosis stimulated by anticancer drugs can lead to anemia in patients. ß-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. METHODS: 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 (H2DCFDA). 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. RESULTS: CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca2+ 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. CONCLUSIONS: CPO stimulates hemolysis and eryptosis through energy depletion, Ca2+ 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, Ca2+ channel blockers, and antioxidants.

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.

Comprehensive Retrospective Analysis of Colorectal Cancer Incidence Patterns in Saudi Arabia.

Colorectal cancer (CRC) is the commonest cancer in Saudi males and the third most common in Saudi females. Although CRC represents a major public health challenge, the resources to evaluate its burden are inadequate. This study aims to elucidate the magnitude of CRC incidence trends in the Saudi population by age, gender, and administrative region. Data for multiple incidence measures were analyzed from the Saudi Cancer Registry (SCR) retrospectively from 2001 to 2018. Temporal trends were further analyzed by age group, gender, administrative region, and globally using joinpoint regression analysis. The number of CRC cases climbed by 335.6% and the disease increased by 56.4% to comprise 12.2% of all cancers cases. The age-standardized incidence rate (ASR) increased by 152% overall, and the median age at diagnosis peaked at 60 and 58 years for males and females, respectively. Riyadh and the Eastern Region had the highest ASR for both genders, peaking at 21.8 and 19.2 for males and 17.4 and 16.5 for females per 100 K population. Our prediction model identified growing trends with annual percentage changes (APCs) of 4.59% in males (CI: 3.1-6.1) and 3.91% among females (CI: 2.4-5.5). Males above 75 years had the highest APC (7.9%, CI: 5.3-10.7), whereas the highest APC among females was found in the age group 70-74 (5.4%, CI: 2.8-8). Globally, APC was the highest for both genders compared to selected countries. CRC incidence is increasing alarmingly in Saudi Arabia and is projected to continue. There is a need for better screening strategies, preventative measures, and awareness-building.

Tamoxifen induces eryptosis through calcium accumulation and oxidative stress.

Chemotherapy-related anemia is a major obstacle in anticancer therapy. Tamoxifen (TAM) is an antiestrogen prescribed for breast cancer patients with hemolytic potential and apoptotic properties in nucleated cells. However, the eryptotic activity of TAM has hitherto escaped the efforts of investigators. RBCs from apparently healthy volunteers were treated with 1-50 µM of TAM for 24 h at 37 °C. Hemoglobin leakage and LDH, AST, and AChE activities were photometrically determined while K+, Na+, and Mg2+ were detected by ion-selective electrode. Flow cytometry was used to identify eryptotic cells by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, sell size and morphology by FSC and SSC signals, respectively, and oxidative stress by H2DCFDA. Whole blood was also exposed to 30 µM of TAM for 24 h at 37 °C to examine the toxicity of TAM to WBCs and platelets. TAM caused Ca2+-independent, dose-responsive hemolysis accompanied by K+, LDH, and AST leakage without improving the mechanical stability of RBCs in hypotonic environments. TAM treatment also increased the proportion of cells positive for annexin-V-FITC, Fluo4, and DCF, along with diminished FSC and SSC signals and AChE activity. Notably, TAM toxicity was aggravated by sucrose but abrogated by vitamin C, PEG 8000, and urea. Moreover, TAM exhibited distinct cytotoxic profiles against leukocytes and platelets. TAM-induced eryptosis is characterized by breakdown of membrane asymmetry, inhibition of AChE activity, Ca2+ accumulation, cell shrinkage, and oxidative stress. Vitamin C, PEG 8000, and urea may hold promise to subvert the undesirable toxic effects of TAM on RBCs.

Stimulation of Hemolysis and Eryptosis by α-Mangostin through Rac1 GTPase and Oxidative Injury in Human Red Blood Cells.

BACKGROUND: Chemotherapy-related anemia is prevalent in up to 75% of patients, which may arise due to hemolysis and eryptosis. Alpha-mangostin (α-MG) is a polyphenolic xanthonoid found in the mangosteen tree (Garcinia mangostana) whose antitumor medicinal properties are well-established. Nevertheless, the potential toxic effects of α-MG on red blood cells (RBCs) have, as of yet, not been as well studied. METHODS: RBCs were exposed to 1-40 µM of α-MG for 24 h at 37 °C. Hemolysis and related markers were measured using colorimetric assays, eryptotic cells were identified through Annexin-V-FITC, Ca2+ was detected with Fluo4/AM, and oxidative stress was assessed through H2DCFDA using flow cytometry. The toxicity of α-MG was also examined in the presence of specific signal transduction inhibitors and in whole blood. RESULTS: α-MG at 10-40 µM caused dose-dependent hemolysis with concurrent significant elevation in K+, Mg2+, and LDH leakage, but at 2.5 µM it significantly increased the osmotic resistance of cells. A significant increase was also noted in Annexin-V-binding cells, along with intracellular Ca2+, oxidative stress, and cell shrinkage. Moreover, acetylcholinesterase activity was significantly inhibited by α-MG, whose hemolytic potential was significantly ameliorated by the presence of BAPTA-AM, vitamin C, NSC23766, and isosmotic sucrose but not urea. In whole blood, α-MG significantly depleted intracellular hemoglobin stores and was selectively toxic to platelets and monocytes. CONCLUSIONS: α-MG possesses hemolytic and eryptotic activities mediated through Ca2+ signaling, Rac1 GTPase activity, and oxidative injury. Also, α-MG leads to accelerated cellular aging and specifically targets platelet and monocyte populations in a whole blood milieu.

Metabolic exhaustion and casein kinase 1α drive deguelin-induced premature red blood cell death.

1. Deguelin (DGN), a retinoid isolated from many plants, exhibits a potent anticancer activity against a wide spectrum of tumour cells. There is a dearth of evidence, however, regarding the toxicity of DGN to red blood cells (RBCs). This is relevant given the prevalent chemotherapy-associated anaemia observed in cancer patients.2. RBCs were exposed to 1-100 µM of DGN for 24 h at 37 °C. Haemolysis and related markers were photometrically measured while flow cytometry was employed to detect phosphatidylserine exposure through Annexin-V-FITC binding and light scatter properties. Additionally, cytosolic Ca2+ and reactive oxygen species were quantified using Fluo4/AM and H2DCFDA, respectively. DGN was also tested against specific signalling inhibitors in addition to vitamin C and ATP.3. DGN caused a significant increase in Annexin-V-positive cells which was accompanied by cell shrinkage without Ca2+ elevation or oxidative stress. DGN also elicited dose-responsive haemolysis which was ameliorated by preventing KCl efflux and in the presence of sucrose, D4476, and ATP. In whole blood, DGN significantly reduced the reticulocyte count and increased platelet distribution width and large cell count.4. DGN triggers premature RBC eryptosis and haemolysis through casein kinase 1α and ATP depletion, and exhibits a specific toxicity towards reticulocytes and platelets.

Molecular Mechanisms and Pathophysiological Significance of Eryptosis.

Despite lacking the central apoptotic machinery, senescent or damaged RBCs can undergo an unusual apoptosis-like cell death, termed eryptosis. This premature death can be caused by, or a symptom of, a wide range of diseases. However, various adverse conditions, xenobiotics, and endogenous mediators have also been recognized as triggers and inhibitors of eryptosis. Eukaryotic RBCs are unique among their cell membrane distribution of phospholipids. The change in the RBC membrane composition of the outer leaflet occurs in a variety of diseases, including sickle cell disease, renal diseases, leukemia, Parkinson's disease, and diabetes. Eryptotic erythrocytes exhibit various morphological alterations such as shrinkage, swelling, and increased granulation. Biochemical changes include cytosolic Ca2+ increase, oxidative stress, stimulation of caspases, metabolic exhaustion, and ceramide accumulation. Eryptosis is an effective mechanism for the elimination of dysfunctional erythrocytes due to senescence, infection, or injury to prevent hemolysis. Nevertheless, excessive eryptosis is associated with multiple pathologies, most notably anemia, abnormal microcirculation, and prothrombotic risk; all of which contribute to the pathogenesis of several diseases. In this review, we provide an overview of the molecular mechanisms, physiological and pathophysiological relevance of eryptosis, as well as the potential role of natural and synthetic compounds in modulating RBC survival and death.

An Insight into the Impact of Serum Tellurium, Thallium, Osmium and Antimony on the Antioxidant/Redox Status of PCOS Patients: A Comprehensive Study.

Humans exploit heavy metals for various industrial and economic reasons. Although some heavy metals are essential for normal physiology, others such as Tellurium (Te), Thallium (TI), antimony (Sb), and Osmium (Os) are highly toxic and can lead to Polycystic Ovarian Syndrome (PCOS), a common female factor of infertility. The current study was undertaken to determine levels of the heavy metals TI, Te, Sb and Os in serum of PCOS females (n = 50) compared to healthy non-PCOS controls (n = 56), and to relate such levels with Total Antioxidant Capacity (TAC), activity of key antioxidant enzymes, oxidative stress marker levels and redox status. PCOS serum samples demonstrated significantly higher levels of TI, Te, Sb and Os and diminished TAC compared to control (p < 0.001). Furthermore, there was significant inhibition of SOD, CAT and several glutathione-related enzyme activities in sera of PCOS patients with concurrent elevations in superoxide anions, hydrogen and lipid peroxides, and protein carbonyls, along with disrupted glutathione homeostasis compared to those of controls (p < 0.001 for all parameters). Additionally, a significant negative correlation was found between the elevated levels of heavy metals and TAC, indicative of the role of metal-induced oxidative stress as a prominent phenomenon associated with the pathophysiology of the underlying PCOS. Data obtained in the study suggest toxic metals as risk factors causing PCOS, and thus protective measures should be considered to minimize exposure to prevent such reproductive anomalies.

Apoptosis-mediated anti-proliferative activity of Calligonum comosum against human breast cancer cells, and molecular docking of its major polyphenolics to Caspase-3.

Due to poor diagnosis breast cancer in women has emerged as the most common cause of death disease in developing countries. Medicinal plants have been used for thousands of years and can be useful in healthcare, especially in developing countries. Ethanol extracts of leaves of fire bush or arta (Calligonum comosum; EECC), exhibited significant anticancer potencies against two breast cancer cell lines, MCF-7 and MDA 231. These in vitro effects of EECC indicated potential anticancer activities that were determined to be specific since minimal toxicity was recorded against MCF-12, a non-cancerous breast cell line used as a reference. EECC also induced cell cycle arrest in MCF-7 and MDA 231 as revealed by the increased proportions of sub-G1 cells. Fluorescence-activated cell sorter analysis (FACS), utilizing double staining by annexin V-FITC/propidium iodide, revealed that the observed cytotoxic effects were mediated via apoptosis and necrosis. FACS measurement of thegreater in fluorescence intensity, linked with oxidation of DCFH to DCF, revealed that apoptosis was attributable to production of free radicals. EECC-mediated apoptosis was further validated by observation of up-regulation in the "executioner" enzyme, caspase 3. The current findings reveal that EECC exhibits significant, selective cytotoxicity to breast cancer cells, that proceeds via the generation of ROS, which culminates in apoptosis. The anti-proliferative effects of EECC weres further verified by use of a structure-based, virtual screening between its major bioactive polyphenolic constituents and the apoptosis executioner marker enzyme, caspase-3. Based on their glide score values against the active site of caspase 3, some phyto-constituents present in EECC, such as DL-alpha-tocopherol and campesterol, exhibited distinctive, drug-like potential with no predicted toxicity to non-target cells. Taken together, the usefulness of natural phenolic and flavonoid compounds contained in Calligonum comosum were suggested to be potent anticancer agents.

Antioxidant, Anti-Proliferative Activity and Chemical Fingerprinting of Centaurea calcitrapa against Breast Cancer Cells and Molecular Docking of Caspase-3.

Centaurea calcitrapa has been intensively utilized in ethnomedicinal practices as a natural therapeutic recipe to cure various ailments. The current study aimed to chemically characterize ethanolic extract of C. calcitrapa (EECC) aerial parts (leaves and shoots) by use of gas chromatography-mass spectrometry analyses (GC-MS) and investigate its antioxidant and in vitro anticancer activities, elucidating the underlying molecular mechanism by use of flow cytometry-based fluorescence-activated cell sorting (FACS) and conducting in silico assessment of binding inhibitory activities of EECC major compounds docked to caspase-3. CG-MS profiling of EECC identified a total of 26 major flavonoids and polyphenolic compounds. DPPH and ABTS assays revealed that EECC exhibits potent antioxidant activity comparable to standard reducing agents. Results of the proliferation assay revealed that EECC exhibit potent, dose-dependent cytotoxic activities against triple-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell models, with IC50 values of 1.3 × 102 and 8.7 × 101 µg/mL, respectively. The observed cytotoxic effect was specific to studied cancer cells since EECC exhibited minimal (~<10%) cytotoxicity against MCF-12, a normal breast cell line. FACS analysis employing annexin V-FITC/propidium iodide double labeling demonstrated that the observed anti-proliferative activity against MCF-7 and MDA-MB-231 was mediated via apoptotic as well as necrotic signaling transduction processes. The increase in fluorescence intensity associated with DCFH oxidation to DCF, as reported by FACS, indicated that apoptosis is caused by generation of ROS. The use of caspase-3-specific fluorogenic substrate revealed a dose-dependent elevation in caspase-3 substrate-cleavage activity, which further supports EECC-mediated apoptosis in MCF-7 cells. The major EECC compounds were examined for their inhibitory activity against caspase-3 receptor (1HD2) using molecular docking. Three compounds exhibited the highest glide score energy of −5.156, −4.691 and −4.551 kcal/mol, respectively. Phenol, 2,6-dimethoxy established strong binding in caspase-3 receptor of hydrogenic type, with residue ARG 207 and of PI-PI stacking type with residue HIS 121. By contract, hexadecenoic acid showed 3 H-bond with the following residues: ASN 615, ASN 616a and THR 646. Taken together, the current findings reveal that EECC exhibits significant and specific cytotoxicity against breast cancer cells mediated by the generation of ROS and culminating into necrosis and apoptosis. Further investigations of the phytoconstituents-rich C. calcitrapa are therefore warranted against breast as well as other human cancer cell models.

Biochemical and molecular assessment of selenium forms for the alleviation of oxidative stress in senescent human fibroblasts.

Selenium enhances the cellular antioxidant capacity and alleviates oxidative stress. We investigated the transcriptional and enzymatic activities of selenium-dependent glutathione peroxidase 1 and thioredoxin reductase 1 (TrxR1), and levels of glutathione, hydrogen peroxide, lipid peroxides, and protein carbonyls in primary passage 5 (P5) and senescent passage 25 (P25) and 30 (P30) fibroblasts. Cells were incubated in either standard Dulbecco growth medium (CM1) containing normal plasma selenium levels (0.8 µmol/l), or in CM2, CM3, and CM4 containing 3 µmol/l (5 µmol/l for TrxR1) sodium selenite, L-hydroxyselenomethionine, or Se-methylselenocysteine, respectively. Gene transcripts and activities of both investigated enzymes as well as the levels of reduced glutathione were significantly increased in CM2-, CM3-, and CM4-incubated senescent P25 and P35 cells compared against those incubated in CM1. In congruence, although all oxidative stress parameters including oxidized glutathione were significantly lower in CM2-, CM3-, and CM4-incubated senescent cells compared against those incubated in CM1, such reductions were of significantly higher magnitude in CM3 and CM4 cells compared against those in CM2. In conclusion, organic L-hydroxyselenomethionine and Se-methylselenocysteine are equally more potent at alleviating oxidative stress in senescent cells than inorganic sodium selenite, and thus could be beneficial for use in elderly subjects and those with oxidative stress-associated disease.

Geraniin inhibits whole blood IFN-γ and IL-6 and promotes IL-1ß and IL-8, and stimulates calcium-dependent and sucrose-sensitive erythrocyte death.

Correlations between circulating cytokine levels and disease states are well established, and pharmacological modulation of the immune response is thus an important aspect of the assessment of investigational new drugs. Moreover, chemotherapy-related anemia is a major obstacle in cancer treatment. Geraniin (GRN), a tannin extracted from Geranium and other plants, possesses promising antitumor potential. However, the effect of GRN on whole blood (WB) cytokine response and RBC physiology remains unexplored. Heparinized blood from consented, healthy adults was challenged with 100 ng/mL of lipopolysaccharide (LPS) with and without pretreatment with 10 µM of GRN for 24 h at 37 °C, and tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, IL-8, and IL-10 were assayed by ELISA. Moreover, single-cell RBC suspensions were treated with 5-100 µM of GRN for 24 or 48 h at 37 °C and cytotoxicity and canonical eryptotic markers were examined by flow cytometry. It was revealed that GRN significantly attenuated LPS-induced IFN-γ levels, increased IL-1ß, decreased IL-6 only in absence of LPS, and aggravated LPS-induced IL-8 while together with LPS significantly diminished IL-10. Furthermore, GRN induced dose-responsive, Ca2+-dependent, and sucrose-sensitive hemolysis, along with phosphatidylserine exposure and Ca2+ accumulation with no appreciable cell shrinkage or oxidative damage. GRN was also selectively toxic to platelets, significantly delayed reticulocyte maturation, and significantly disrupted leukocyte proportions. In conclusion, GRN regulates the WB cytokine response and promotes premature hemolysis and eryptosis. This study provides insights into the therapeutic utility of GRN in a highly relevant cellular model system.

Induction of hemolysis and eryptosis by occupational pollutant nickel chloride is mediated through calcium influx and p38 MAP kinase signaling.

OBJECTIVES: Nickel (Ni) is an abundant environmental hazard and an occupational pollutant. Exposure to Ni compounds is prevalent in electroplating workers and in the printing industry, among others. The toxicity of Ni manifests as dermatological, gastrointestinal, respiratory, allergic, and cardiovascular symptoms. In particular, hyperbilirubinemia and reticulocytosis have been detected in intoxicated subjects; an observation possibly implicating selective red blood cell (RBC) toxicity. Herein, the interaction of nickel chloride (NiCl2) with human RBCs and associated molecular mechanisms are described. MATERIAL AND METHODS: Cells from healthy donors were incubated for 24 h at 37°C in the presence or absence of 0.5‒10 mM of NiCl2, and cytotoxicity was determined through hemoglobin leakage by colorimetry under different experimental conditions. Eryptotic markers were also identified by flow cytofluorometry using Annexin-V-FITC tagging for phosphatidylserine (PS) exposure, light scatter properties for cellular dimensions, Fluo4/AM labeling for intracellular calcium, and H2DCFDA staining for reactive oxygen species (ROS). Additionally, small molecule inhibitors were used to probe the signaling pathways involved. RESULTS: It was found that NiCl2 at 10 mM caused profound intracellular calcium overload and significant calcium-dependent hemolysis. Also, NiCl2 reduced forward scatter and increased side scatter, Annexin- positive cells, and ROS levels. Importantly, NiCl2-induced hemolysis was significantly attenuated by the exclusion of extracellular calcium, and in the presence of p38 MAP kinase (MAPK) inhibitor SB203580. CONCLUSIONS: It is concluded that NiCl2 induces p38 MAPK-dependent hemolysis, and stimulates the canonical features of premature eryptosis. This report presents the first description of the molecular mechanisms underlying the hemolytic and eryptotic potential of NiCl2 and, thus, may explain changes in hematological parameters observed in poisoning victims. Int J Occup Med Environ Health. 2022;35(1):1-11.

Calcium-oxidative stress signaling axis and casein kinase 1α mediate eryptosis and hemolysis elicited by novel p53 agonist inauhzin.

Inauhzin (INZ) is a novel p53 agonist with antitumor activity. Anemia is a common side effect of chemotherapy and may arise from red blood cell (RBC) hemolysis or eryptosis. In this study, we investigate the mechanisms of INZ toxicity in human RBCs. RBCs were isolated from healthy donors and treated with antitumor concentrations of INZ (5-500 µM) for 24 h at 37 °C. Hemoglobin was photometrically measured, and cells were stained with Annexin-V-FITC for phosphatidylserine (PS), Fluo4/AM for calcium, and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for oxidative stress. INZ caused significant dose-responsive, calcium-dependent hemolysis starting at 40 µM. Furthermore, INZ significantly increased Annexin-positive cells and Fluo4 and DCF fluorescence. The cytotoxicity of INZ was also significantly mitigated in presence of D4476. INZ possesses hemolytic and eryptotic potential characterized by cell membrane scrambling, intracellular calcium overload, cell shrinkage, and oxidative stress secondary to calcium influx from the extracellular space.

Erythritol modulates the polarization of macrophages: Potential role of tumor necrosis factor-α and Akt pathway.

Low-calorie sweeteners are substitutes for sugar and frequently used by patients with cardiometabolic diseases. Erythritol, a natural low-calorie sugar alcohol, was linked to cardiometabolic diseases in several recent metabolomics studies. However, the characterization of its role in disease development is lacking. Macrophage polarization orchestrates the immune response in various inflammatory conditions, most notably cardiometabolic disease. Therefore, the physiological effects of Erythritol on THP-1 macrophages were investigated. We observed an increased cellular abundance of proinflammatory M1 macrophages, characterized by CD11c, TNF-α, CD64, CD38, and HLA-DR markers and decreased anti-inflammatory M2 macrophages, characterized by mannose receptor CD206. The, Erythritol increased ROS generation, and the activation of the AKT pathway, cytosolic calcium overload, and cell cycle arrest at the G1 phase. Concomitantly, an increased population of necroptotic macrophages was observed. In conclusion, we provide evidence that Erythritol induced the proinflammatory phenotype in THP-1 macrophages and this was associated with an increased population of necroptotic macrophages. PRACTICAL APPLICATIONS: This assessment provides evidence of the effects of Erythritol on macrophages, particularly THP-1-derived macrophages. Our results support the role of Erythritol in driving the inflammation that is associated with cardiometabolic diseases and provide insights in the role of Erythritol as an inducer of necroptosis in THP-1 derived macrophages that could be associated the disease.

Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis.

Cardiovascular diseases (CVD) are a leading cause of mortality worldwide, and dietary habits represent a major risk factor for dyslipidemia; a hallmark of CVD. Saturated fatty acids contribute to CVD by aggravating dyslipidemia, and, in particular, lauric acid (LA) raises circulating cholesterol levels. The role of red blood cells (RBCs) in CVD is increasingly being appreciated, and eryptosis has recently been identified as a novel mechanism in CVD. However, the effect of LA on RBC physiology has not been thoroughly investigated. RBCs were isolated from heparin-anticoagulated whole blood (WB) and exposed to 50-250 µM of LA for 24 h at 37 °C. Hemoglobin was photometrically examined as an indicator of hemolysis, whereas eryptosis was assessed by Annexin V-FITC for phosphatidylserine (PS) exposure, Fluo4/AM for Ca2+, light scatter for cellular morphology, H2DCFDA for oxidative stress, and BODIPY 581/591 C11 for lipid peroxidation. WB was also examined for RBC, leukocyte, and platelet viability and indices. LA caused dose-responsive hemolysis, and Ca2+-dependent PS exposure, elevated erythrocyte sedimentation rate (ESR), cytosolic Ca2+ overload, cell shrinkage and granularity, oxidative stress, accumulation of lipid peroxides, and stimulation of casein kinase 1α (CK1α). In WB, LA disrupted leukocyte distribution with elevated neutrophil-lymphocyte ratio (NLR) due to selective toxicity to lymphocytes. In conclusion, this report provides the first evidence of the pro-eryptotic potential of LA and associated mechanisms, which informs dietary interventions aimed at CVD prevention and management.

Antiproliferative Wnt inhibitor wogonin prevents eryptosis following ionophoric challenge, hyperosmotic shock, oxidative stress, and metabolic deprivation.

Anemia is a common complication of chemotherapy and may arise due to premature or suicidal death of red blood cells (RBCs). Prevention of RBC death thus lends itself as a promising strategy to counteract anemia. Wogonin (WGN; 5,7-dihydroxy-8-methoxyflavone) is a Wnt inhibitor derived from Scutellaria baicalensis plant with potent cytotoxic and antitumor potential. However, the nature of interaction of WGN with human RBCs is unknown. RBCs from healthy participants were exposed to different hemolytic and eryptotic stimuli for 24 or 48 hr at 37°C in the presence and absence of 100 µM WGN. Calcium overload was induced by 2 µM ionomycin, hyperosmotic shock with excessive sucrose, oxidative stress by 2-phenethyl isothiocyanate (PEITC), and metabolic deprivation by exclusion of glucose. Hemolysis was estimated from extracellular hemoglobin, phosphatidylserine (PS) exposure by Annexin V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA). While WGN did not rescue the cells from the hemolytic activity of ionomycin, it reduced PS externalization by interfering with calcium influx under both ionomycin treatment and metabolic exhaustion. WGN also significantly inhibited PS exposure upon hyperosmotic shock, but the effect was independent of calcium entry. Moreover, WGN exhibited antihemolytic and anti-eryptotic activities against PEITC without appreciable reduction in ROS levels. Altogether, WGN prevents PEITC-induced hemolysis and suppresses eryptosis due to calcium accumulation, hyperosmotic shock, oxidative stress, and metabolic exhaustion. These novel insights may open new avenues into the therapeutic application of WGN for conditions in which anemia is commonly encountered, most notably cancer. PRACTICAL APPLICATIONS: The herbal supplement Sho-Saiko-To (Xiaochaihu-tang) is commonly prescribed to relieve symptoms of liver disease. Flavonoids from the herbal constituents of Sho-Saiko-To have demonstrated considerable anti-inflammatory, antioxidant, antimicrobial, antitumor, and immunomodulatory properties. In this work, we identify WGN, a major flavonoid in Sho-Saiko-To, as a novel inhibitor of hemolysis and eryptosis of human erythrocytes. Since inordinate erythrocyte death is a major obstacle in therapeutic drug development, our findings argue for the use of WGN as a natural alternative, either as a primary or an adjuvant drug, for a wide assortment of pathological conditions including cancer.