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Expression of RKIP in chronic myelogenous leukemia K562 cell and inhibits cell proliferation by regulating the ERK/MAPK pathway

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Tumor Biology

Abstract

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated the expression of RKIP in chronic myelogenous leukemia (CML) K562 cells and the effects of RKIP on the characteristics of K562 cells. The recombinant plasmid pcDNA3.1-RKIP was established and transfected into K562 cells with the help of Lipofectamine 2000. At the same time, the RKIP-siRNA was transfected into K562 cells in another group. The expressions of RKIP in all groups were assayed by Western blot after 48 h. MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to analyze the cell viability. Flow cytometry (FCM) was used to examine the cell cycle and cell apoptosis. Colony forming unit (CFU) assay was used to analyze the effect of RKIP on the clonogenic growth of CML cells. Western blot or luciferase reporter assay was used to detect the effect of RKIP on the level of phospho-ERK1/2 or the transcriptional activity of NF-κB. Western blot analysis showed that the plasmid pcDNA3.1-RKIP or RKIP-siRNA significantly enhanced or decreased RKIP expression (p < 0.01), respectively. In addition, MTT, FCM, and CFU assay indicated that the overexpression of RKIP significantly lowered the cell viability, cell proliferation and the clonogenic growth (p < 0.05), but improved cell apoptosis (p < 0.01). Western blot analysis or luciferase reporter assay showed that the level of phospho-ERK1/2 or the transcriptional activity of NF-κB was strongly inhibited by overexpression of RKIP. All these results could bring us a new perspective for biological therapy in myelogenous leukemia in the future.

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Acknowledgments

This study was supported by the Natural Science Foundation of Jiangsu Province, China (BK20131199).

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Authors and Affiliations

  1. Department of Paediatrics, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, JS, 226001, People’s Republic of China

    Bin Li, Baolan Sun, Nina Zhou, Zhiping Yang & Jianhui Gu

  2. Department of Orthopedics, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, JS, 226001, People’s Republic of China

    Jianwei Zhu

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  1. Bin Li
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Correspondence to Jianhui Gu.

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Li, B., Sun, B., Zhu, J. et al. Expression of RKIP in chronic myelogenous leukemia K562 cell and inhibits cell proliferation by regulating the ERK/MAPK pathway. Tumor Biol. 35, 10057–10066 (2014). https://doi.org/10.1007/s13277-014-2312-3

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  • DOI: https://doi.org/10.1007/s13277-014-2312-3

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