Autophagic and Apoptotic Effects of Tyrosine Kinase Inhibitors in Myeloid Leukemia: Comparison of Three Generation
Authors: Kayabasi Cagla , Avci Cigir Biray , Susluer Sunde Yilmaz , Balci Tugce , Baran Yusuf , Saydam Guray , Gunduz Cumhur 
Published: October 2019

Abstract
Abstract 4915
The aim of the study was to evaluate the differences in cytotoxicity, apoptosis and autophagy levels in myeloid leukemia cell lines treated with tyrosine kinase inhibitors compared to cell line resistant to imatinib and control group. Chronic myeloid leukemia model was created by using cell lines as K-562 cell line for Ph+ chronic myeloid leukemia model, HL-60 cell line for acute promyelocytic Ph- leukemia model. NCI-BL2171 normal cell line was used as a control group while K562/ima3 cell line was used as an imatinib resistant model. Imatinib (STI571), Dasatinib (BMS-354825), Ponatinib (AP24534) were used as tyrosine kinase inhibitors in this study.
Cytotoxicity analysis was conducted by WST-1 analysis. Apoptotis was evaluated by AnnexinV-enhanced green fluorescent protein (EGFP) and by Mitoprobe JC-1 for Mitochondrial Potential Detection. Autophagy was analyzed by The Premo Autophagy Tb/GFP TR-FRET LC3B assay which measures autophagy in cells expressing green fluorescent protein (GFP)-tagged LC3B using a Tb-based TR-FRET immunoassay approach.
By using IC50 doses of tyrosine kinase inhibitors, autophagic effect of these drugs on cell lines were examined at 24th hours. Cells not treated with the active substance or chloroquine were considered as control groups. Chloroquine-treated cells were used as positive control for autophagy. LC3B-II increase is an indicator of autophagic suppression.
Cells treated with chloroquine were compared with cells treated with active substances and concentrations of BacMam that displayed the highest LC3B-II increase were selected. Autophagic suppression ratio of the drugs was evaluated among the control group.
Cytotoxicity, apoptosis and autophagy analysis results were provided in Table. Compared to control group, 30 μM chloroquine repressed autophagy 1. 93, 1. 48, 2. 74 and 1. 54 fold in K562, HL-60, K562/ima3 and NCI-BL 2171 cells, respectively. In HL-60 cells while Imatinib represented 0. 77 fold autophagy, it repressed autophagy 1. 77 and 3. 49 fold in K562 and K562/ima3 cells respectively. Dasatinib repressed autophagy 2. 11, 1. 95 and 4. 62 fold and Ponatinib repressed autophagy 2. 09, 1. 60 and 9. 15 fold in K562, HL-60, K562/ima3 cells respectively. Imatinib, Dasatinib and Ponatinib did not repressed autophagy in NCI-BL 2171 cells.
In conclusion, apoptosis and autophagy paradox was illuminated in myeloid leukemia cells via tyrosine kinase inhibitors and autophagy may be a new strategy for targeted therapy in myeloid leukemia after clarifying responsible genes and proteins in signal transduction pathways.
Cytotoxicity Apoptosis Autophagy WST-1 IC50 (nM) Annexin V JC-1 Premo Autophagy Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib K562 24th hour 1.70 3.65 3.05 3.07 1.37 1.35 1.43 1.77 2.11 2.09 48th hour 650.00 0.24 2.67 2.51 2.32 2.03 2.35 2.06 72nd hour 4.53 4.81 3.00 2.97 3.07 2.50 HL-60 24th hour 1.33 1.26 1.32 1.29 1.22 1.34 0.77 1.95 1.60 48th hour 18000.00 1.39 1.23 1.41 1.61 1.92 1.96 72nd hour 896.00 607.00 2.21 1.80 2.82 1.58 1.73 2.23 K562/ima3 24th hour 1.33 0.76 1.69 1.51 1.36 1.59 3.49 4.62 9.15 48th hour 18350.00 1830.00 9.87 1.80 1.94 2.03 2.82 1.22 1.40 72nd hour 1.34 1.44 1.41 2.61 1.40 1.56 NCI-BL 2171 24th hour 48.00 2.48 2.79 2.62 3.99 4.04 4.25 1.01 0.88 0.90 48th hour 274.00 30.00 4.11 4.33 4.15 5.05 2.75 3.11 72nd hour 6.14 6.04 6.03 8.27 3.71 3.95

Disclosures:
No relevant conflicts of interest to declare.