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...
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| Formáid: | Alt |
| Teanga: | English |
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MDPI AG
2023-12-01
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| Sraith: | Molecules |
| Ábhair: | |
| Rochtain ar líne: | https://www.mdpi.com/1420-3049/28/24/8053 |
| _version_ | 1827574019718643712 |
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| author | Sumiah A. Alghareeb Mohammad A. Alfhili Jawaher Alsughayyir |
| author_facet | Sumiah A. Alghareeb Mohammad A. Alfhili Jawaher Alsughayyir |
| author_sort | Sumiah A. Alghareeb |
| collection | DOAJ |
| description | 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 Ca<sup>2+</sup> by Fluo4/AM, cell size by FSC, and oxidative stress by H<sub>2</sub>DCFDA. Ions and pH were assessed by an ion-selective electrode, while B<sub>12</sub> 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 Ca<sup>2+</sup> removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B<sub>12</sub> 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 Ca<sup>2+</sup>-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca<sup>2+</sup> 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. |
| first_indexed | 2024-03-08T20:30:30Z |
| format | Article |
| id | doaj.art-fa641fb55ce14964823c4aa4ac268d92 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-08T20:30:30Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-fa641fb55ce14964823c4aa4ac268d922023-12-22T14:27:33ZengMDPI AGMolecules1420-30492023-12-012824805310.3390/molecules28248053Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKCSumiah A. Alghareeb0Mohammad A. Alfhili1Jawaher Alsughayyir2Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh 12372, Saudi ArabiaDepartment of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh 12372, Saudi ArabiaDepartment of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh 12372, Saudi ArabiaBackground: 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 Ca<sup>2+</sup> by Fluo4/AM, cell size by FSC, and oxidative stress by H<sub>2</sub>DCFDA. Ions and pH were assessed by an ion-selective electrode, while B<sub>12</sub> 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 Ca<sup>2+</sup> removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B<sub>12</sub> 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 Ca<sup>2+</sup>-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca<sup>2+</sup> 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.https://www.mdpi.com/1420-3049/28/24/8053eryptosishemolysisrosmarinic acidchemotherapy |
| spellingShingle | Sumiah A. Alghareeb Mohammad A. Alfhili Jawaher Alsughayyir Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC Molecules eryptosis hemolysis rosmarinic acid chemotherapy |
| title | Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC |
| title_full | Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC |
| title_fullStr | Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC |
| title_full_unstemmed | Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC |
| title_short | Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC |
| title_sort | rosmarinic acid elicits calcium dependent and sucrose sensitive eryptosis and hemolysis through p38 mapk ck1α and pkc |
| topic | eryptosis hemolysis rosmarinic acid chemotherapy |
| url | https://www.mdpi.com/1420-3049/28/24/8053 |
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