Advances in Clinical and Experimental Medicine

Adv Clin Exp Med
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ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
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Advances in Clinical and Experimental Medicine

2015, vol. 24, nr 2, March-April, p. 195–202

doi: 10.17219/acem/32934

Publication type: original article

Language: English

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Creative Commons BY-NC-ND 3.0 Open Access

Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines

Joanna Szymańska1,A,D,F, Piotr Smolewski2,A,C,D,F, Agata Majchrzak2,B,F, Barbara Cebula-Obrzut2,B,F, Krzysztof Chojnowski3,C,E,F, Jacek Treliński3,A,C,D,E,F

1 Laboratory of Clinical and Transplant Immunology and Genetics, Copernicus Memorial Hospital, Łódź, Poland

2 Department of Experimental Hematology, Medical University of Lodz, Poland

3 Department of Hematology, Medical University of Lodz, Poland

Abstract

Background. The JAK2V617F mutation plays a crucial role in the pathogenesis of myeloproliferative neoplasms (MPNs). Inhibition of JAK2 activity by ruxolitinib (RX) results in growth inhibition and apoptosis of cells carrying the JAK2V617F mutation however the exact mechanisms regulating apoptosis have not been fully elucidated.
Objectives. This study assessed the potential cytotoxicity of RX against JAK2-positive human cell lines (SET-2 and HEL), either alone or in combination with hydroxyurea, busulphan, rapamycin or LY294002.
Material and Methods. Cell viability, the apoptosis rate (annexin-V staining), drop of mitochondrial transmembrane potential (Δψm) and caspase activation, were measured using flow cytometry. Additionally, the expression of several apoptosis-regulating proteins was evaluated.
Results. RX showed cytotoxicity against both SET-2 and HEL cell lines. The main mechanism of this action was apoptosis, with significant drop of Δψm, caspase-3 and -9 activation, and moderate activation of caspase-8 (only for SET-2 cells). Corresponding to enhanced apoptosis, the expression levels of some apoptosis-regulating proteins were changed, the most pronounced in both cell lines being up-regulation of Bax and down-regulation of Bcl-2 proteins. Additionally, up-regulation of Bak and Bad (SET-2) and down-regulation of Mcl-1 (HEL) were observed. Of the studied compounds, a combination of RX + LY294002 induced the greatest cytotoxicity in both SET-2 and HEL cell lines, and rapamycin the least.
Conclusion. . This study shows that the combination of RX and a PI3K kinase inhibitor provokes a significant pro-apoptotic effect in JAK2V617F mutated cells, which may justify the beginning of clinical trials based on the combination of these drugs.

Key words

ruxolitinib, hydroxyurea, busulphan, rapamycin, LY294002.

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