Effects of atropine on megakaryocytic differentiated K562 leukemia cells

Year 2025, Volume: 38 Issue: 3, 265 - 272, 10.10.2025

Hulya Cabadak , Zehra Kanlı , Banu Aydın

https://doi.org/10.5472/marumj.1800332

Abstract

Objective: Non-neuronal cholinergic system signaling pathways play a significant role in various malignancies, including leukemia,
lung, colon, brain, and breast cancer. This study aims to investigate the effect of atropine on megakaryocytic differentiated cells, as
well as to identify the apoptosis mechanisms. We also studied the effect of cholinergic drugs on muscarinic receptors and caspase gene
expressions, cell proliferation and caspase activities in differentiated cells.
Materials and Methods: K562 cells were induced into megakaryocytic differentiation using phorbol 12-myristate 13-acetate (PMA).
The effects of agonists/antagonists on differentiated K562 cells were examined using cell viability and 5-Bromo-2-deoxy-uridine
(BrdU) assays. Caspase activities were detected by the caspase assay kit. Protein expression levels were detected by western blotting.
Results: Atropine reversed the effects of carbachol (CCh) on increased megakaryocytic differentiated leukemia cell survival and
proliferation. The protein expression of M1 and M4 muscarinic receptors was upregulated by CCh, an effect that was reversed by
atropine. CCh alone did not significantly change levels of M2, M3, and M5 muscarinic receptor proteins in megakaryocytic differentiated
K562 leukemia cells.
Conclusion: M2, M3 muscarinic receptors and caspase 9 may have important functions in preventing the progression of leukemia and
may also make important contributions to targeted therapies in leukemia.

Keywords

Atropine , Leukemia , Megakaryocytic differentiation , Muscarinic receptors , Caspases

References

• Kawashima K, Fujii T. Extraneuronal cholinergic system in lymphocytes, Pharmacol Ther 2000;86: 29-48. doi: 10.1016/ s0163-7258(99)00071-6.
• Tobin A B, Budd D C. The anti-apoptotic response of the Gq/11-coupled muscarinic receptor family. Biochem Soc Trans 2003; 31: 1182-5. doi: 10.1042/bst0311182.
• Wessler I, Kirkpatrick C J, Racke K. The cholinergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans. Clin Exp Pharmacol Physiol 1999; 26: 198- 205. doi: 10.1046/j.1440-1681.1999.03016.x
• Lev-Lehman E, Deutsch V, Eldor A, Soreq H. Immature human megakaryocytes produce nuclear-associated acetylcholinesterase. Blood 1997;89:3644-53.
• Costa P, Traver D J, Auger C B, Costa L G. Expression of cholinergic muscarinic receptor subtypes mRNA in rat blood mononuclear cells. Immunopharmacology 1994;28:113-23. doi: 10.1016/0162-3109(94)90027-2
• Fukamauchi F, Saunders P A, Hough C, Chuang D M. Agonist-induced down-regulation and antagonist-induced up-regulation of m2 – and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells. Mol Pharmacol 1993;44: 940-9. doi: 10.1111/j.1471- 4159.1991.tb08210.x
• Kawashima K, Fujii T, Moriwaki Y, Misawa H, Horiguchi K. Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function. Ann N Y Acad Sci 2012; 1261: 7-17. doi: 10.1111/j.1749- 6632.2012.06516.x
• Sato K Z, Fujii T, Watanabe Y, et al. Diversity of mRNA expression for muscarinic acetylcholine receptor subtypes and neuronal nicotinic acetylcholine receptor subunits in human mononuclear leukocytes and leukemic cell lines. Neurosci Lett 1999;266:17-20. doi: 10.1016/s0304-3940(99)00259-1
• Paleari L, Grozio A, Cesario A, Russo P. The cholinergic system and cancer. Semin Cancer Biol 2008;18: 211-7. doi: 10.1016/j.semcancer.2007.12.009
• Spindel E R. Muscarinic receptor agonists and antagonists: effects on cancer. Handb Exp Pharmacol 2012;208:451-68. doi: 10.1007/978-3-642-23274-9_19
• Campoy J F, Vidal C J, Munoz-Delgado E, Montenegro M F, Cabezas-Herrera J, S. Nieto-Ceron S. Cholinergic system and cell proliferation. Chem Biol Interact 2016;259:257-65. doi: 10.1016/j.cbi.2016.04.014.
• Wessler I, Kirkpatrick C J. Cholinergic signaling controls immune functions and promotes homeostasis, Int Immunopharmacol. 2020;83: 106345. doi: 10.1016/j. intimp.2020.106345
• Cabadak H, Aydin B, Kan B. Regulation of M2, M3, and M4 muscarinic receptor expression in K562 chronic myelogenous leukemic cells by carbachol. J Recept Signal Transduct Res 2011;31 26-32. doi: 10.3109/10799.893.2010.506484
• Onder Narin G, Aydin B, Cabadak H. Studies on the role of alpha 7 nicotinic acetylcholine receptors in K562 cell proliferation and signaling. Mol Biol Rep 2021;48:5045-55. doi: 10.1007/s11033.021.06498-4
• Jan R, Chaudhry G E. Understanding apoptosis and apoptotic pathways targeted cancer therapeutics. Adv Pharm Bull 2019;9: 205-18. doi: 10.15171/apb.2019.024
• Boice A, Bouchier-Hayes L. Targeting apoptotic caspases in cancer. Biochim Biophys Acta Mol Cell Res 2020;1867:118688. doi: 10.1016/j.bbamcr.2020.118688
• Costa L G, Guizzetti M, Oberdoerster J, et al. Modulation of DNA synthesis by muscarinic cholinergic receptors. Growth Factors 2001;18: 227-36. doi: 10.3109/089.771.90109029112
• Lanzafame AA, Christopoulos A, Mitchelson F. Cellular signaling mechanisms for muscarinic acetylcholine receptors. Recept Channels 2003;9:241-60.
• Nicke B, Detjen K, Logsdon C D. Muscarinic cholinergic receptors activate both inhibitory and stimulatory growth mechanisms in NIH3T3 cells. J Biol Chem 1999;274:21701-6. doi: 10.1074/jbc.274.31.21701
• Goldman J M, Melo J V. Chronic myeloid leukemia—advances in biology and new approaches to treatment. N Engl J Med 2003;349: 1451-64. doi: 10.1056/NEJMra020777
• Zhu H Q, Gao F H. Regulatory molecules and corresponding processes of BCR-ABL protein degradation. J Cancer 2019;10: 2488-500. doi: 10.7150/jca.29528
• Butler T M, Ziemiecki A, Friis R R, Megakaryocytic differentiation of K562 cells is associated with changes in the cytoskeletal organization and the pattern of chromatographically distinct forms of phosphotyrosylspecific protein phosphatases. Cancer Res 1990;50:6323- 9.
• Aydin B, Cabadak H, Goren M Z. Investigation of the roles of non-neuronal acetylcholine in chronic myeloid leukemic cells and their erythroid or megakaryocytic differentiated lines. Anticancer Agents Med Chem. 2018;18:1440-7. doi: 10.2174 /187.152.0618666.180.406123154
• Lowry O H, Rosebrough N J, Farr A L, Randall R J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.
• Lozzio C B, Lozzio B B. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 1975;45:321-34.
• Cabadak H, Kucukibrahimoglu E, Aydin B, Kan B, Zafer Goren M. Muscarinic receptor-mediated nitric oxide release in a K562 erythroleukaemia cell line. Auton Autacoid Pharmacol 2009;29: 109-15. doi: 10.1111/j.1474-8673.2009.00431.x
• Jabbour E, Cortes J E, Ghanem H, O’Brien S, Kantarjian H M. Targeted therapy in chronic myeloid leukemia. Expert Rev Anticancer Ther 2008;8: 99-110. doi: 10.1586/14737140.8.1.99
• Song P, Sekhon H S, Lu A, et al. M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion. Cancer Res 2007;67: 3936-44. doi: 10.1158/0008-5472.CAN-06-2484
• Ami A, Koga K, Fushiki H, Ueno Y, OginoY, Ohta H. Selective M3 muscarinic receptor antagonist inhibits small-cell lung carcinoma growth in a mouse orthotopic xenograft model. J Pharmacol Sci. 2011;116: 81-8. doi: 10.1254/jphs.10308fp
• Rosemond E, Rossi M, McMillin S M, Scarselli M, Donaldson J G, Wess J. Regulation of M(3) muscarinic receptor expression and function by transmembrane protein 147. Mol Pharmacol 2011;79: 251-61. doi: 10.1124/mol.110.067363
• Degirolamo C, Modica S, Palasciano G, Moschetta A. Bile acids and colon cancer: Solving the puzzle with nuclear receptors. Trends Mol Med 2011;17:564-72. doi: 10.1016/j. molmed.2011.05.010
• Gutkind J S, Novotny E A, Brann M R, Robbins K C. Muscarinic acetylcholine-receptor subtypes as agonistdependent oncogenes, P Natl Acad Sci 1991; 88: 4703-7. doi: 10.1073/pnas.88.11.4703
• Baumgold J, Dyer K. Muscarinic receptor-mediated inhibition of mitogenesis via a protein-kinase C-independent mechanism in M1-T-transfected A9 L-cells, Cell Signal 1994;6:103-8. doi: 10.1016/0898-6568(94)90065-5
• Renz B W, Tanaka T, Sunagawa M, et al. Cholinergic signaling via muscarinic receptors directly and indirectly suppresses pancreatic tumorigenesis and cancer stemness. Cancer Discov 2018;8: 1458-73. doi: 10.1158/2159-8290.CD-18-0046
• Bennett JA , Ture S K, Schmidt RA, et al. Acetylcholine inhibits platelet activation. Pharmacol Exp Ther 2019;369:182-7. doi: 10.1124/jpet.118.253583
• Reina S, Sterin-Borda L, Passafaro D, Borda E. Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells. J Cell Physiol 2010;222:640-7. doi: 10.1002/jcp.21981
• Ahmed E A, Alkuwayti M A, Ibrahim H M. Atropine is a suppressor of epithelial-mesenchymal transition (EMT) that reduces stemness in drug-resistant breast cancer cells. Int J Mol Sci 2022;23:9849. doi: 10.3390/ijms23179849