Nobiletin Promotes Megakaryocytic Differentiation through the MAPK/ERK-Dependent EGR1 Expression and Exerts Anti-Leukemic Effects in Human Chronic Myeloid Leukemia (CML) K562 Cells.

Jui-Hung Yen,Ching-Yen Lin,Chin-Hsien Chuang,Pei-Yi Chen,Ming-Jiuan Wu,Hsien-Kuo Chin

doi:10.3390/cells9040877

Jui-Hung Yen, Ching-Yen Lin + Show 4 more

Open Access

https://doi.org/10.3390/cells9040877

Copy DOI

Abstract

Differentiation therapy is an alternative strategy used to induce the differentiation of blast cells toward mature cells and to inhibit tumor cell proliferation for cancer treatment. Nobiletin (NOB), a polymethoxyflavone phytochemical, is present abundantly in citrus peels and has been reported to possess anti-cancer activity. In this study, we investigated the anti-leukemic effects of NOB on cell differentiation and its underlying mechanisms in human chronic myeloid leukemia (CML) K562 cells. NOB (100 μM) treatment for 24 and 48 h significantly decreased viability of K562 cells to 54.4 ± 5.3% and 46.2 ± 9.9%, respectively. NOB (10–100 μM) significantly inhibited cell growth in K562 cells. Flow cytometry analysis and immunoblotting data showed that NOB (40 and 80 μM) could modulate the cell cycle regulators including p21, p27, and cyclin D2, and induce G1 phase arrest. NOB also increased the messenger RNA (mRNA) and protein expression of megakaryocytic differentiation markers, such as CD61, CD41, and CD42 as well as the formation of large cells with multi-lobulated nuclei in K562 cells. These results suggested that NOB facilitated K562 cells toward megakaryocytic differentiation. Furthermore, microarray analysis showed that expression of EGR1, a gene associated with promotion of megakaryocytic differentiation, was markedly elevated in NOB-treated K562 cells. The knockdown of EGR1 expression by small interference RNA (siRNA) could significantly attenuate NOB-mediated cell differentiation. We further elucidated that NOB induced EGR1 expression and CD61 expression through increases in MAPK/ERK phosphorylation in K562 cells. These results indicate that NOB promotes megakaryocytic differentiation through the MAPK/ERK pathway-dependent EGR1 expression in human CML cells. In addition, NOB when combined with imatinib could synergistically reduce the viability of K562 cells. Our findings suggest that NOB may serve as a beneficial anti-leukemic agent for differentiation therapy.

Highlights

Chronic myeloid leukemia (CML) is a type of myeloproliferative neoplasm characterized by generating Bcr-Abl fusion oncogene, which constitutively translates into an active tyrosine kinase and leads to a massive influx of immature myeloid cells into the circulation [1,2]
We found that NOB exerted its anti-leukemic activities by inhibiting cell growth, modulating cell cycle progression and inducing K562 cells toward megakaryocyte differentiation
The K562 cell line is a well-known model of human chronic myeloid leukemia (CML)

Summary

Introduction

Chronic myeloid leukemia (CML) is a type of myeloproliferative neoplasm characterized by generating Bcr-Abl fusion oncogene, which constitutively translates into an active tyrosine kinase and leads to a massive influx of immature myeloid cells into the circulation [1,2]. Small molecule tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, and bosutinib, are used as frontline therapy [3]. Differentiation therapy is to induce the differentiation of blast cells toward a mature type and to inhibit tumor cell proliferation. This approach may devoid general cytotoxic effects, especially for the treatment of leukemia patients who cannot tolerate intensive chemotherapy or bone marrow transplantation [7]. The best example is the induction therapy of acute promyelocytic leukemia (APL)

Methods

Results

Conclusion