Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB090

Poster Communications

Regulation of acethylcholineesterase activity, CXCL8 production and proliferation
by pilocarpine in hHuman K562 eErythroleukemia cells

Z. Kanli1, B. Aydin1, H. Cabadak1

1. Biophysics, Marmara University School of Medicine, Istanbul, Turkey.

  • Proliferation Assay Results

  • Acetylcholine Esterase Activity of Different Doses of Pilocarpine

Muscarinic receptors are found neuronal and nonneuronal cells (1,2). Different studies suggest that muscarinic receptors mediate some cellular events in hematopoietic cells (3). Muscarinic agonist, pilocarpine binds to M1/M3 muscarinic receptors (4). We previously showed that carbachol (CCh), treatment leads to changes in muscarinic M2, M3 and M4 receptor transcripts in hHuman K562 eErythroleukemia cell line and muscarinic activation leads to eErythroleukemia cell proliferation dependent on the presence of fetal bovine serum (5). TNF-a, cytokine, induce apoptosis different tumor cells. But, different researcher also reported that TNF -a also stimulate cell proliferation in leukemia cells . The present study to investigate effects of the muscarinic agonist, pilocarpine, TNF -a on CXCL8 release and AChE activity to determine their function in human erythroleukemia K562 cell proliferation. Human erythroleukemia K562 cells were incubated with the M1/M3R agonist pilocarpine, the M3R antagonist DAMP or TNF-a to investigate the role of M3 receptor in human erythro leukemia K562 cells. AChE activity was determined by QuantiChrom TM Acethylcholine Assay kit. CXCL8 release was detected by using the colorimetric ELISA kit. Cell proliferation was analyzed by measuring BrdU incorporation during DNA synthesis in proliferating cells. Western blot analyses for M3 muscarinic receptor protein was performed in the homogenates of human erythroleukemia cells treated with pilocarpine. All data show means ± S.E.M.of at least six independent experiments. Statistically significant differences were determined by using the one-way analysis of variance followed by Bonferonni post tests. Our experiments showed that pilocarpine caused a decrease in DNA synthesis in K562 cells supplemented with 0% or 1% fetal bovine serum after starvation. There was a significant inhibitory effect in pilocarpine group whereas 4DAMP treatment not reversed this effect in K562 cell proliferation (P<0.0001)(Table 1). Pilocarpine (1 and 10 uM) induced ACHE activity in human erythroleukemia K562 cell (P<0.0001) (Table 2). Pilocarpine (10 and 100uM) alone inhibited CXCL8 release however, pilocarpin 1 and 10 uM and TNF-a together were caused 6 fold increased CXCL8 release in human erythroleukemia K562 cell (***P<0.0004). In conclusion, the findings of the current study clearly showed beneficial effects of pilocarpine on inhibition of human erythroleukemia K562 cell proliferation.

Where applicable, experiments conform with Society ethical requirements