Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line

Banu Aydın; Aysın Tulunay; Emel Ekşioğlu-demiralp; Beki Kan; Hulya Cabadak

doi:10.5472/marumj.518983

Research Article

İnsan kronik miyeloid lösemi K562 hücrelerinde karbakolün apoptoza etkisi

Year 2019, Volume: 32 Issue: 1, 38 - 43, 29.01.2019

Banu Aydın Aysın Tulunay Emel Ekşioğlu-demiralp Beki Kan Hulya Cabadak

https://doi.org/10.5472/marumj.518983

Cited By: 5

Abstract

Amaç: Muskarinik reseptörler merkezi sinir sisteminde

asetilkolinin çeşitli etkilerine aracılık ettiği gibi, parasempatik sinir

sistemi ile etkileşen sinirsel olmayan dokulara da aracılık ederler.

Çalışmamızda, M3 muskarinik reseptör alttipinin K562 kanser

hücre çoğalması ve ölümündeki rolünü belirlemeye çalıştık.

Gereçler ve Yöntemler: Hücre çoğalması bromodeoksiüridin

(BrDU) yöntemi ile belirlenmiştir. Hücre ölümü, erken ve geç

apoptoz Anneksin V ve propidyum iodid (PI) varlığında akış

sitometrisi yöntemi ile gösterilmiştir. Nuklear dış sinyal düzenleyici

kinaz (ERK/fosfo-ERK) ekspresyonu western emdirimi yöntemi

ile belirlenmiştir.

Bulgular: Çalışmamızda 48 saat karbakol(CCh) ile muamele

edilen K562 hücre sayısında azalma belirlenmiştir. Hücreler

24 saat CCh ile muamele edildiklerinde erken apoptotik hücre

sayısında azalma gözlemlenirken geç apoptoz ve nekrotik hücre

sayısında değişim olmamıştır. Bununla birlikte, 48 saat boyunca

CCh ile muamele olan hücrelerde, erken ve geç apoptotik hücre

sayısı azalırken, nekrotik hücrelerin sayısı artmıştır. CCh ile

muamele edilen hücrelerde nüklear ERK ekspresyonu artarken

bu etki 1,1-dimetil-4-difenilasetoksipiperidinium iodid (4DAMP)

ile geri çevrilmiştir. Aynı koşullarda CCh ile muamele edilen

hücrelerde nuklear pERK ekspresyonu azalmış, bu etki 4DAMP

ile geri çevrilmemiştir.

Sonuç: Bulgularımız, K562 hücre proliferasyonundaki

kolinerjik etkinin yalnızca muskarinik mekanizma ile değil

diğer kolinerjik reseptörlerin de katkısıyla gerçekleştiğini

düşündürmektedir.

Keywords

Muskarinik reseptör, K562 hücreleri, Karbakol, Kolinerjik sistem

References

1. Pierce KL, Premont RT, Lefkowitz RJ. Seven transmembrane receptors. Nat Rev Mol Cell Biol 2002;3:639-50. doi: 10.1038/nrm908
2. Haga T. Molecular properties of muscarinic acetylcholine receptors. Proc Jpn Acad Ser B Phys Biol Sci 2013;89:226- 56. doi: 10.2183/pjab.89.226
3. Gutkind JS, Novotny EA, Brann MR, Robbins KC. Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes. Proc Natl Acad Sci USA 1991;88:4703– 07.
4. Rayford W, Noble MJ, Austenfeld MA, Weigel J, Mebust WK, Shah GV. Muscarinic cholinergic receptors promote growth of human prostate cancer cells. Prostate 1997; 30:160-6. doi: 10.1002/(SICI)1097-0045(19970215)30:3<160::AID – PROS3>3.0.CO;2-Q
5. Luthin GR, Wang P, Zhou H, Dhanasekaran D, Ruggieri MR. Role of m1 receptor-G protein coupling in cell proliferation in the prostate. Life Sci 1997;60:963-8. doi: 10.1016/S0024- 3205(97)00035-0
6. Wang L, Jiang R, Song SD, Hua ZS, Wang JW, Wang YP. Angelica sinensis polysaccharide induces erythroid differentiation of human chronic myelogenous leukemia k562 cells. Asian Pac J Cancer Prev 2015;16:3715-21.
7. Taverna S, Giallombardo M, Pucci M, et al. Curcumin inhibits in vitro and in vivo chronic myelogenous leukemia cells growth: a possible role for exosomal disposal of miR-21. Oncotarget 2015;6:21918-33. doi: 10.18632/ oncotarget.4204
8. Klein E, Ben-Bassat H, Neumann H, et al. Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia. Int J Can 1976;18:421-31. doi: 10.1002/ ijc.291.018.0405
9. 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 Transd Res 2011;31:26-32. doi: 10.3109/10799.893.2010.506484
10. Fleisher TA. Apoptosis. Ann Allergy Asthma Immunol 1997;78:245-9. doi: 10.1016/S1081-1206(10)63176-6
11. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Science 1998; 281:1305-8. doi: 10.1126/ science.281.5381.1305
12. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-12. doi: 10.1126/science.281.5381.1309
13. Hughes D, Mehmet H. Cell proliferation and apoptosis. Advanced Methods. Oxford: Bios Scientific Publishers Limited, 2003.
14. Wyllie AH, Moms RG, Smith AL, Dunlop D. Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 1984;142: 67. doi: 10.1002/path.171.142.0112.
15. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251-306.
16. Rosenblum K, Futter M, Jones M, E. Hulme C, Bliss T V P, “ERKI/II regulation by the muscarinic acetylcholine receptors in neurons,” J Neurosci 2000;20:977-85. doi: 10.1523/JNEUROSCI.20-03-00977.2000
17. Yu H, Xia H, Tang Q, et al. Acetylcholine acts through M3 muscarinic receptor to activate the EGFR signaling and promotes gastric cancer cell proliferation. Sci Rep 2017;7:40802. doi: 10.1038/srep40802
18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.
19. Aydın B, Kan B, Cabadak H. The role of intracellular pathways in the proliferation of human K562 cells that is mediated by muscarinic receptors. Leukemia Res 2013;37: 1144-9. doi: 10.1016/j.leukres.2013.05.018
20. Schuller HM. Cell type specific, receptor-mediated modulation of growth kinetics in human lung cancer cell lines by nicotine and tobacco-related nitrosamines. Biochem Pharmacol 1989;38:3439-42. doi: 10.1016/0006- 2952(89)90112-3
21. Maneckjee R, Minna JD. Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines. Proc Natl Acad Sci USA 1990;87:3294-8. doi: 10.1073/pnas.87.9.3294.
22. Cheng K, Zimniak P, Raufman JP. Transactivation of the epidermal growth factor receptor mediates cholinergic agonist-induced proliferation of H508 human colon cancer cells. Cancer Res 2003;63:6744–50.
23. Shant J, Cheng K, Marasa BS, Wang JY, Raufman JP. Aktdependent NF-kappaB activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis. Exp Cell Res 2009;315:432-50. doi: 10.1016/j.yexcr.2008.11.003
24. Peng Z, Heath J, Drachenberg C, Raufman JP, Xie G. Cholinergic muscarinic receptor activation augments murine intestinal epithelial cell proliferation and tumorigenesis. BMC Cancer 2013;13:204. doi: 10.1186/1471-2407-13-204
25. Raufman JP, Shant J, Xie G, et al. Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice. Carcinogenesis 2011;32:1396-402. doi: 10.1093/ carcin/bgr118
26. Raufman JP, Samimi R, Shah N,et al. Genetic ablation of M3 muscarinic receptors attenuates murine colon epithelial cell proliferation and neoplasia. Cancer Res 2008;68:3573-8. doi: 10.1158/0008-5472.CAN-07-6810
27. Cheng K, Shang AC, Drachenberg CB, Zhan M, Raufman J P. Differential expression of M3 muscarinic receptors in progressive colon neoplasia and metastasis. Oncotarget 2017;8: 21106-14. doi: 10.18632/oncotarget.15500
28. Cabadak H, Kücükibrahimoğlu E, Aydin B, Kan B, Gören MZ. 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
29. Cabadak H, Aydin B, Kan B. Muscarinic receptor mediated cAMP response in human K562 chronic myelogenous leukemia cells. Turk J Biochem 2011;36:18-92.
30. Kodaira M, Kajimura M, Takeuchi K, Lin S, Hanai H, Kaneko E. Functional muscarinic m3 receptor expressed in gastric cancer cells stimulates tyrosine phosphorylation and MAP kinase. J Gastroenterol 1999;34: 163-71. doi: 10.1007/ s005.350.050238
31. Fujii T, Tsuchiya T, Yamada S, et al. Localization and synthesis of acetylcholine in human leukemic T cell lines. J Neurosci Res 1996;44:66-72. doi: 10.1002/(SICI)1097- 4547(19960401)44:1<66::AID-JNR9>3.0.CO;2-G
32. Kawashima K, Fujii T. Extraneuronal cholinergic system in lymphocytes. Pharmacol Ther 2000;86:29-48. doi: 10.1016/ S0163-7258(99)00071-6.
33. Shah N, Khurana S, Cheng K, Raufman JP. Muscarinic receptors and ligands in cancer. Am J Physiol Cell Physiol 2009;296:C221-32. doi: 10.1152/ajpcell.00514.2008
34. Guizetti M, Moore NH, Van DeMark KL, Giordano G, Costa LG. Muscarinic receptor-activated signal transduction pathways involved in the neuritogenic effect of astrocytes in hippocampal neurons. Eur J Pharmacol 2011;659:102-7. doi: 10.1016/j.ejphar.2011.03.019

Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line

Year 2019, Volume: 32 Issue: 1, 38 - 43, 29.01.2019

Banu Aydın Aysın Tulunay Emel Ekşioğlu-demiralp Beki Kan Hulya Cabadak

https://doi.org/10.5472/marumj.518983

Cited By: 5

Abstract

Objectives: Muscarinic receptors mediate diverse actions of

acetylcholine in the central nervous system and in non-nervous

tissues innervated by the parasympathetic nervous system.

Our study aims to evaluate the potential association of the

M3 muscarinic receptor with K562 cell proliferation and death.

Materials and Methods: Cell proliferation was evaluated

by bromodeoxyuridine (BrDU) incorporation. To show early,

late apoptosis and cell death, cells were labelled with Annexin

V, propidium iodide (PI) and analyzed by flow cytometry. Nuclear

extracellular signal-regulated kinase (ERK/pERK) expression

was measured by western blot analysis.

Results: Treatment with carbachol (CCh) for 48h decreased cell

number. Exposing K562 cells to CCh for 24h decreased the number of

early apoptotic cells but did not change the number of late apoptotic and

necrotic cells. CCh treatment for 48h increased the number of necrotic

cells, but decreased the number of early and late apoptotic cells. In

response to CCh, nuclear ERK expression was increased and this effect

was reversed by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide

(4DAMP). Nuclear pERK expression was decreased in CCh treated

cells, 4DAMP did not reverse the effect.

Conclusion: Our data suggest that cholinergic agonist CCh

affects cell proliferation in K562 cells not only through muscarinic

receptors but also through other cholinergic receptors.

Keywords

Muscarinic receptors, K562 cells, Carbachol, Cholinergic system

References

1. Pierce KL, Premont RT, Lefkowitz RJ. Seven transmembrane receptors. Nat Rev Mol Cell Biol 2002;3:639-50. doi: 10.1038/nrm908
2. Haga T. Molecular properties of muscarinic acetylcholine receptors. Proc Jpn Acad Ser B Phys Biol Sci 2013;89:226- 56. doi: 10.2183/pjab.89.226
3. Gutkind JS, Novotny EA, Brann MR, Robbins KC. Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes. Proc Natl Acad Sci USA 1991;88:4703– 07.
4. Rayford W, Noble MJ, Austenfeld MA, Weigel J, Mebust WK, Shah GV. Muscarinic cholinergic receptors promote growth of human prostate cancer cells. Prostate 1997; 30:160-6. doi: 10.1002/(SICI)1097-0045(19970215)30:3<160::AID – PROS3>3.0.CO;2-Q
5. Luthin GR, Wang P, Zhou H, Dhanasekaran D, Ruggieri MR. Role of m1 receptor-G protein coupling in cell proliferation in the prostate. Life Sci 1997;60:963-8. doi: 10.1016/S0024- 3205(97)00035-0
6. Wang L, Jiang R, Song SD, Hua ZS, Wang JW, Wang YP. Angelica sinensis polysaccharide induces erythroid differentiation of human chronic myelogenous leukemia k562 cells. Asian Pac J Cancer Prev 2015;16:3715-21.
7. Taverna S, Giallombardo M, Pucci M, et al. Curcumin inhibits in vitro and in vivo chronic myelogenous leukemia cells growth: a possible role for exosomal disposal of miR-21. Oncotarget 2015;6:21918-33. doi: 10.18632/ oncotarget.4204
8. Klein E, Ben-Bassat H, Neumann H, et al. Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia. Int J Can 1976;18:421-31. doi: 10.1002/ ijc.291.018.0405
9. 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 Transd Res 2011;31:26-32. doi: 10.3109/10799.893.2010.506484
10. Fleisher TA. Apoptosis. Ann Allergy Asthma Immunol 1997;78:245-9. doi: 10.1016/S1081-1206(10)63176-6
11. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Science 1998; 281:1305-8. doi: 10.1126/ science.281.5381.1305
12. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-12. doi: 10.1126/science.281.5381.1309
13. Hughes D, Mehmet H. Cell proliferation and apoptosis. Advanced Methods. Oxford: Bios Scientific Publishers Limited, 2003.
14. Wyllie AH, Moms RG, Smith AL, Dunlop D. Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 1984;142: 67. doi: 10.1002/path.171.142.0112.
15. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251-306.
16. Rosenblum K, Futter M, Jones M, E. Hulme C, Bliss T V P, “ERKI/II regulation by the muscarinic acetylcholine receptors in neurons,” J Neurosci 2000;20:977-85. doi: 10.1523/JNEUROSCI.20-03-00977.2000
17. Yu H, Xia H, Tang Q, et al. Acetylcholine acts through M3 muscarinic receptor to activate the EGFR signaling and promotes gastric cancer cell proliferation. Sci Rep 2017;7:40802. doi: 10.1038/srep40802
18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.
19. Aydın B, Kan B, Cabadak H. The role of intracellular pathways in the proliferation of human K562 cells that is mediated by muscarinic receptors. Leukemia Res 2013;37: 1144-9. doi: 10.1016/j.leukres.2013.05.018
20. Schuller HM. Cell type specific, receptor-mediated modulation of growth kinetics in human lung cancer cell lines by nicotine and tobacco-related nitrosamines. Biochem Pharmacol 1989;38:3439-42. doi: 10.1016/0006- 2952(89)90112-3
21. Maneckjee R, Minna JD. Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines. Proc Natl Acad Sci USA 1990;87:3294-8. doi: 10.1073/pnas.87.9.3294.
22. Cheng K, Zimniak P, Raufman JP. Transactivation of the epidermal growth factor receptor mediates cholinergic agonist-induced proliferation of H508 human colon cancer cells. Cancer Res 2003;63:6744–50.
23. Shant J, Cheng K, Marasa BS, Wang JY, Raufman JP. Aktdependent NF-kappaB activation is required for bile acids to rescue colon cancer cells from stress-induced apoptosis. Exp Cell Res 2009;315:432-50. doi: 10.1016/j.yexcr.2008.11.003
24. Peng Z, Heath J, Drachenberg C, Raufman JP, Xie G. Cholinergic muscarinic receptor activation augments murine intestinal epithelial cell proliferation and tumorigenesis. BMC Cancer 2013;13:204. doi: 10.1186/1471-2407-13-204
25. Raufman JP, Shant J, Xie G, et al. Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice. Carcinogenesis 2011;32:1396-402. doi: 10.1093/ carcin/bgr118
26. Raufman JP, Samimi R, Shah N,et al. Genetic ablation of M3 muscarinic receptors attenuates murine colon epithelial cell proliferation and neoplasia. Cancer Res 2008;68:3573-8. doi: 10.1158/0008-5472.CAN-07-6810
27. Cheng K, Shang AC, Drachenberg CB, Zhan M, Raufman J P. Differential expression of M3 muscarinic receptors in progressive colon neoplasia and metastasis. Oncotarget 2017;8: 21106-14. doi: 10.18632/oncotarget.15500
28. Cabadak H, Kücükibrahimoğlu E, Aydin B, Kan B, Gören MZ. 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
29. Cabadak H, Aydin B, Kan B. Muscarinic receptor mediated cAMP response in human K562 chronic myelogenous leukemia cells. Turk J Biochem 2011;36:18-92.
30. Kodaira M, Kajimura M, Takeuchi K, Lin S, Hanai H, Kaneko E. Functional muscarinic m3 receptor expressed in gastric cancer cells stimulates tyrosine phosphorylation and MAP kinase. J Gastroenterol 1999;34: 163-71. doi: 10.1007/ s005.350.050238
31. Fujii T, Tsuchiya T, Yamada S, et al. Localization and synthesis of acetylcholine in human leukemic T cell lines. J Neurosci Res 1996;44:66-72. doi: 10.1002/(SICI)1097- 4547(19960401)44:1<66::AID-JNR9>3.0.CO;2-G
32. Kawashima K, Fujii T. Extraneuronal cholinergic system in lymphocytes. Pharmacol Ther 2000;86:29-48. doi: 10.1016/ S0163-7258(99)00071-6.
33. Shah N, Khurana S, Cheng K, Raufman JP. Muscarinic receptors and ligands in cancer. Am J Physiol Cell Physiol 2009;296:C221-32. doi: 10.1152/ajpcell.00514.2008
34. Guizetti M, Moore NH, Van DeMark KL, Giordano G, Costa LG. Muscarinic receptor-activated signal transduction pathways involved in the neuritogenic effect of astrocytes in hippocampal neurons. Eur J Pharmacol 2011;659:102-7. doi: 10.1016/j.ejphar.2011.03.019

There are 34 citations in total.

Details

Primary Language	English
Subjects	Clinical Sciences
Journal Section	Articles
Authors	Banu Aydın This is me Aysın Tulunay This is me Emel Ekşioğlu-demiralp This is me Beki Kan This is me Hulya Cabadak This is me
Publication Date	January 29, 2019
Published in Issue	Year 2019 Volume: 32 Issue: 1

Cite

APA	Aydın, B., Tulunay, A., Ekşioğlu-demiralp, E., Kan, B., et al. (2019). Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line. Marmara Medical Journal, 32(1), 38-43. https://doi.org/10.5472/marumj.518983
AMA	Aydın B, Tulunay A, Ekşioğlu-demiralp E, Kan B, Cabadak H. Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line. Marmara Med J. January 2019;32(1):38-43. doi:10.5472/marumj.518983
Chicago	Aydın, Banu, Aysın Tulunay, Emel Ekşioğlu-demiralp, Beki Kan, and Hulya Cabadak. “Effects of Carbachol on Apoptosis in Human Chronic Myelogenous Leukemic K562 Cell Line”. Marmara Medical Journal 32, no. 1 (January 2019): 38-43. https://doi.org/10.5472/marumj.518983.
EndNote	Aydın B, Tulunay A, Ekşioğlu-demiralp E, Kan B, Cabadak H (January 1, 2019) Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line. Marmara Medical Journal 32 1 38–43.
IEEE	B. Aydın, A. Tulunay, E. Ekşioğlu-demiralp, B. Kan, and H. Cabadak, “Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line”, Marmara Med J, vol. 32, no. 1, pp. 38–43, 2019, doi: 10.5472/marumj.518983.
ISNAD	Aydın, Banu et al. “Effects of Carbachol on Apoptosis in Human Chronic Myelogenous Leukemic K562 Cell Line”. Marmara Medical Journal 32/1 (January 2019), 38-43. https://doi.org/10.5472/marumj.518983.
JAMA	Aydın B, Tulunay A, Ekşioğlu-demiralp E, Kan B, Cabadak H. Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line. Marmara Med J. 2019;32:38–43.
MLA	Aydın, Banu et al. “Effects of Carbachol on Apoptosis in Human Chronic Myelogenous Leukemic K562 Cell Line”. Marmara Medical Journal, vol. 32, no. 1, 2019, pp. 38-43, doi:10.5472/marumj.518983.
Vancouver	Aydın B, Tulunay A, Ekşioğlu-demiralp E, Kan B, Cabadak H. Effects of carbachol on apoptosis in human chronic myelogenous leukemic K562 cell line. Marmara Med J. 2019;32(1):38-43.