Examination of molecular biologic changes that occur result of in-vitro application of ruxolitinib in chronic myeloid leukemia treatment

Chronic myeloid leukemia is a clonal disorder of hematopoietic stem cells. Over-expression level of BCL-ABL fusion protein is the result of reciprocal (9;22) translocation known as Philadelphia chromosome is responsible for occurring leukemic phenotype. Ruxolitinib is the first agent approved for the treatment of myelofibrosis. Ruxolitinib is a selective inhibitor of Janus kinases (JAK) 1 and 2, which are involved in the signalling pathway of various cytokines and growth factors essential to haematopoiesis. Apoptosis and autophagy known as type I and type II programmed cell death, respectively. These two mechanisms determine cell survival or death via regulating important protein cascades as essential response of cell to stimulus. Raise of apoptotic and autophagic activity in cancers is a novel therapeutic target using different active substance. In our study we aimed to determine cytotoxic, apoptotic and autophagic changes in K-562 human chronic myeloid cell line and NCI-BL 2171 human normal B lymphocyts treated with potent JAK inhibitor ruxolitinib compared to untreated control group, and detect the expression changes of apoptosis, autophagy and leukemogesis related genes with the treatment of ruxolitinib. The cytotoxic effects of ruxolitinib on K-562 and NCI-BL 2171 cells were determined in time and dose dependent manner by using WST-1 analysis. Total RNA was isolated from K-562 and NCI-BL 2171 cells treated with ruxolitinib and untreated cells as control group. Reverse transcription procedure was performed for cDNA synthesis. Apoptotic effect of ruxolitinib was evaluated with JC-1 and Annexin-V methods, and gene expressions were shown by RT-qPCR. IC50 (48th hours) dose of ruxolitinib was determined as 20µM, 23,6µM for K-562 and NCI-BL 2171 cells, respectively. Ruxolitinib caused no apoptotic effect on K-562 and NCI-BL2171 cells. However, ruxolitinib induced autophagy in K-562 and NCI-BL 2171 cells 2,01 and 1,79 fold, respectively. We supposed that ruxolitinib realizes this cell death via down-regulating autophagy inhibitors mTOR, AKT and STATs. Ruxolitinib is thought to be potential treatment method in chronic myeloid leukemia by blocking JAK/STAT pathway known as BCR-ABL downstream target, but there has been no study correlated with autophagy. The most critical trouble caused by using BCR-ABL tyrosine kinase inhibitors in chronic myeloid leukemia treatment is drug resistance or loss of sensitiveness in patients. We suggest that a new perspective would be generated in treatment of chronic myeloid leukemia by clarifying of molecular mechanisms of ruxolitinib through autophagy induction.