Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library
Ming-Szu Hung
0
1
2
Zhidong Xu
2
Yu-Ching Lin
0
1
Jian-Hua Mao
4
Cheng- Ta Yang
1
3
Pey-Jium Chang
0
David M Jablons
2
Liang You
2
0
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University
,
Taoyuan, Taiwan
,
R.O.C.
1
Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital
,
Chiayi, Taiwan
,
R.O.C.
2
Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California
,
San Francisco, CA 94115
,
USA
3
Department of Respiratory Care, College of Medicine, Chang Gung University
,
Taoyuan
,
Taiwan, R.O.C
4
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California
,
Berkeley, CA
,
USA
Background: Casein kinase 2 (CK2) is dysregulated in various human cancers and is a promising target for cancer therapy. To date, there is no small molecular CK2 inhibitor in clinical trial yet. With the aim to identify novel CK2 inhibitors, we screened a natural product library. Methods: We adopted cell-based proliferation and CK2 kinase assays to screen CK2 inhibitors from a natural compound library. Dose-dependent response of CK2 inhibitors in vitro was determined by a radioisotope kinase assay. Western blot analysis was used to evaluate down stream Akt phosphorylation and apoptosis. Apoptosis was also evaluated by annexin-V/propidium iodide (PI) labeling method using flow cytometry. Inhibition effects of CK2 inhibitors on the growth of cancer and normal cells were evaluated by cell proliferation and viability assays. Results: Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases. It appears to be an ATP non-competitive and partially reversible CK2 inhibitor with an IC50 value of 0.55 M. In addition, hematein inhibited cancer cell growth partially through downregulation of Akt phosphorylation and induced apoptosis in these cells. Furthermore, hematein exerted stronger inhibition effects on the growth of cancer cells than in normal cells. Conclusion: In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor. Hematein showed stronger growth inhibition effects to cancer cells when compared to normal cells. This compound may represent a promising class of CK2 inhibitors.
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Background
CK2 is a serine/threonine protein kinase composed of 2
catalytic subunits (, '' or ') and 2 regulatory
subunits (). CK2 is ubiquitously expressed and highly
conserved in cells and plays multiple roles in cellular
processes, including gene expression, protein synthesis,
cell proliferation and apoptosis[1]. So far, CK2 is known
to phosphorylate more than 300 proteins in cells and is
also an important regulator of intracellular signalling
pathways[2]. For example, CK2 promotes survival by
increasing survivin expression via
beta-catenin-Tcf/Lefmediated transcription[3]. CK2 also constitutively
phosphorylates and upregulates Akt/PKB Ser129 in vitro and in
vivo, which may be required for maximal activation of
Akt/PKB[4].
Dysregulation of CK2 in association with other proteins
also increases oncogenic potential of cells[5]. In
transgenic mouse study, expression of CK2 subunits in
lymphocyte induces lymphoma, and the coexpression of
cmyc protein results in neonatal leukemia[6].
Overexpression of CK2 in the mammary gland of transgenic mouse
induces mammary hyperplasia, dysplasia, and eventually
adenocarcinomas[7]. In primary embryo fibroblasts,
coexpression of CK2' and H-Ras induces
transformation[8]. Overexpression of CK2 has been noted in a
variety of human cancers, including acute myeloid
leukaemia[9], mammary gland[7], prostate[10], lung[11],
head and neck[12], and kidney cancer[13], and also
correlates with metastatic potential, undifferentiated
histological type and poor clinical outcome in human cancers.
As a result, CK2 is a potential candidate of targeted
therapy for cancers[1]. Although CK2 inhibitors like TBB
(4,5,6,7 tetrabrome benzotriazole)[14] and its
derivatives[1,15] have been shown to induce apoptosis in
human cancer cells, more selective CK2 inhibitors are
needed, since to our knowledge there is still not one CK2
small molecule inhibitor in clinical trials for cancer
treatment yet.
In this study, we screened for potential CK2 inhibitors
from a natural compound library via cell based
proliferation and kinase assays. Through these assays, hematein
was identified as a novel CK2 inhibitor. We further
evaluated the dose dependent inhibition response of hematein
on CK2 kinase activity in vitro and in cancer cells. Effects
of hematein on apoptosis and cell growth were also
evaluated in cancer and normal cells.
Methods
Cell culture
HeLa (CCL-2), HCT116 (CCL-247), A549 (CCL-185),
A427 (HTB-53), WI-38 (CCL-75) and CCL-211 cell lines
were purchased from American Type Culture Collection
(Manassas, VA). Cells were grown in complete growth
medium (Dulbecco's modified Eagle's medium for HeLa,
A549 and CCL-211; Eagle's Minimum E (...truncated)