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Casein kinase 2 (CK2) is a protein kinase which is frequently activated in cancer. The Hedgehog (Hh) signaling pathway is involved in the stimulation of cancer stem cell growth. Its aberrant activation has been validated in several types of cancer, including ovarian cancer. In the present study, the sphere-forming cells (SFCs) of the human ovarian cancer SKOV3 cell line were observed to have self-renewal capacity, indicating the possession of ovarian cancer stem-like cell properties. SKOV3-derived SFCs had higher levels of CK2α and glioma-associated oncogene 1 (Gli1) proteins compared with those of parental cells. Apigenin, a common flavonoid, significantly inhibited the self-renewal capacity and the protein expression of CK2α and Gli1 proteins in the SKOV3-derived SFCs, which occurred in a concentration-dependent manner. In addition, CK2α small interfering RNA downregulated the protein expression of CK2α and Gli1 and synergistically inhibited the self-renewal capacity of the SKOV3-derived SFCs with apigenin. However, forced overexpression of CK2α resulted in an increase in the expression of CK2α and Gli1 and attenuated the apigenin-inhibited self-renewal effect in the SKOV3-derived SFCs. These results suggested that apigenin inhibited the self-renewal capacity of SKOV3-derived SFCs and was involved in downregulating the expression of Gli1 by the inhibition of CK2α.
The protein kinase casein kinase 2 (CK2) is a highly conserved serine/threonine kinase with a broad spectrum of substrates (
Sonic hedgehog (Shh), a member of the Hh family of proteins, consists of secreted signaling molecules and has several functions during vertebrate development (
Apigenin is chemically known as 4′,5,7,-trihydroxyflavone and has the molecular formula C15H10O5, with a molecular weight of 270 g/mol. Apigenin has been observed to have marked effects in inhibiting cancer cell growth in cell culture systems and in
The present study investigated the self-renewal capacity, a main feature for identifying OCSLCs, of sphere-forming cells (SFCs) of the human ovarian cancer cell line SKOV3. The effect of apigenin on the self-renewal capacity of SKOV3-derived SFCs and the involvement of CK2α and the Hedgehog signaling pathway in this process were studied. The results provide important evidence for the potential benefits of CK2 inhibitors, including apigenin, in the treatment of ovarian cancer.
Apigenin was obtained from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in dimethyl sulfoxide to a final concentration of 0.1% in media without causing cytotoxicity. The following reagents were purchased: anti-CK2α (Millipore, Billerica, MA, USA), anti-Gli1 (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-β-actin (Sigma-Aldrich), Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) and fetal bovine serum (Invitrogen Life Technologies). All other chemicals were obtained from Sigma-Aldrich.
The human ovarian cancer SKOV3 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in DMEM supplemented with 10% fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and incubated at 37°C in a humidified atmosphere of 5% CO2.
For the tumorsphere assay, single-cell suspensions were prepared at a density of 5,000 cells/ml in condition-medium comprising serum-free DMEM/F12 supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml human recombinant epidermal growth factor, 10 ng/ml human recombinant basic fibroblast growth factor, 2% B27 supplement without vitamin A and 1% N2 supplement (Invitrogen Life Technologies), which were then seeded into ultra low attachment six-well plates (Corning Inc., Corning, NY, USA) at a density of 3,000 cells/ml. The suspension cultures were continued for six days until the formation of tumor spheres. To propagate the spheres
To investigate the percentage of single cells able to regenerate new spheres, the cells were seeded at a density of 1,000 cells/ml in a six-well plate to obtain new spheres. The total number of tumor spheres was counted after six days of culture. The efficiency of sphere formation was calculated using the following formula: Total number of spheres formed/total number of live cells seeded × 100.
The CK2α-specific siRNA and control RNA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). In brief, the cells were seeded into six-well plates at 105 cells/well one day prior to transfection, with a target of 30–50% confluency at the time of transfection. The cells were transfected with 50 nmol/l siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies) according to the manufacturer’s instructions. Adequate inhibition of the siRNA-mediated-knockdown was confirmed using western blot analysis. The pcDNA3.1-CK2α or control pcDNA3.1-LacZ plasmid vectors were then transfected into the SKOV3 cells or SKOV3-derived SFCs (0.5 μg/ml in a 24-well plate) using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. The cells were harvested for western blot analysis and tumorsphere formation assay.
The cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1% (w/v) SDS, 0.5 mM phenylmethanesulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin and 1 mM dithiothreitol. The cell lysate, containing 50 μg protein, was separated by 12.5% SDS-PAGE and then blotted onto polyvinylidene difluoride membranes (Millipore). The murine monoclonal anti-CK2α immunoglobulin (Ig)G at 1:2,000 dilution (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), murine monoclonal anti-Gli1 IgG at 1:2,000 dilution (Santa Cruz Biotechnology, Inc.) and monoclonal anti-β-actin at 1:1,000 dilution (Sigma-Aldrich) antibodies were used as primary antibodies. Signals were detected using the enhanced chemiluminescence (ECL plus) detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Images were scanned by transmission scanner (E85 Plus; Unisplendour Corp., Ltd, Beijing, China) followed by densitometric analysis using Alphazmager 2200 software (Silk Scientific, Orem, UT, USA).
Data are expressed as the mean ± standard deviation for triplicate experiments and were analyzed using Student’s t-test (SPSS software, version 15.0 for Windows; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Self-renewal capacity is one of the main characteristics of cancer stem cells (CSCs) (
Protein kinase CK2 is frequently activated in various types of human cancer. The activation of CK2α is involved in the activation of the Hh/Gli1 pathway and is associated with the stemness maintenance of CSCs (
The Hh/Gli signaling pathway is important in the maintenance of stemness and tumorigenesis (
Since CK2α is highly expressed in CSCs compared with normal cells (
To investigate whether CK2 suppression had an effect on the Hh pathway, the expression of CK2 was silenced using CK2α-specific siRNAs. The results of western blot analysis confirmed that silencing of CK2α significantly inhibited the expression of Gli1 in the SKOV3-derived SFCs (
To confirm whether CK2α activity affected the expression of Gli1 and sphere-forming capability of the SKOV3 cells and its sphere forming capability, SKOV3 cells were transfected with either a pcDNA3.1-CK2α or a control pcDNA3.1-LacZ plasmid. Western blot analysis revealed that upregulation of
In addition, the overexpression of CK2α attenuated the apigenin-induced downregulation of CK2α and Gli1 protein expression (
The results of the present study suggested that apigenin inhibits the self-renewal capacity of SKOV3-derived SFCs via downregulation of Gli1 expression by the inhibition of CK2α. This was supported by several lines of evidence. Firstly, apigenin inhibited the sphere formation efficiency of SKOV3-derived SFCs, accompanied by a downregulation of the expression of CK2α and Gli1. Secondly, the inhibition of CK2α by siRNA acted synergistically with apigenin in downregulating the expression of CK2α and Gli1 as well as inhibiting the self-renewal capability of SKOV3-derived SFCs. Finally, the forced overexpression of CK2α resulted in an increase in the expression levels of CK2α and Gli1 and enhanced the percentage of sphere formation in the parental SKOV3 cells. This forced overexpression of CK2α also attenuated the apigenin-inhibited self-renewal capability of the SKOV3-derived SFCs.
There is no previous evidence for a correlation between CK2 and Hh/Gli signaling in human ovarian cancer cells, although CK2 has been suggested as a positive regulator of the Hh signal transduction pathway and led to the phosphorylation of two serine residues in Smo in
The Hh pathway may be important in the maintenance of CSCs; however, drug targeting of the Hh pathway is limited. CK2 provides an additional target for inhibition of Hh/Gli signaling. There has been little development of CK2 inhibitors as therapeutic agents, partially due to the adenosine triphosphate binding pocket of CK2 differing from other protein kinase agents (
As a selective CK2 kinase inhibitor, apigenin has been demonstrated to induce cell death in ovarian cancer cells and reduce the risk of ovarian cancer. The present study provided the first evidence, to the best of our knowledge, that apigenin inhibits the self-renewal capacity of SKOV3-derived SFCs through downregulation of the expression of Gli1 by inhibiting CK2α. Due to the emerging importance of Hh/Gli signaling in tumor initiation and progression, these results provide important evidence for the potential benefits of CK2 inhibitors, including apigenin.
The authors would like to thank Professor Jian-Guo Cao and Dr Xi-Yun Deng (Medical College, Hunan Normal University, Changsha, Hunan, China) for their critical input into the scientific content. The present study was supported by the Construct Program of the Key Discipline of Basic Medicine in Hunan Province, the Youth Fund of Hunan Normal University (grant no. 110637) and the Project of Hunan Provincal Natural Science Foundation (grant no. 13JJ3061).
Apigenin inhibits the self-renewal of SKOV3-derived SFCs. (A) Apigenin decreased the size of spheroids in suspension in a dose-dependent manner (magnification, ×400). (B) Apigenin inhibited the sphere formation efficiency of SKOV3-derived SFCs in a dose-dependent manner. *P<0.05, compared with the control group; #P<0.05, for the comparison between the groups treated with 20 or 40 μmol/l apigenin and the group treated with 10 μmol/l apigenin. SFC, sphere-forming cell; Medium, control group.
Apigenin downregulates the protein expression of CK2α and Gli1 in SKOV3-derived SFCs. (A) CK2α expression was higher in the SKOV3-derived SFCs than in the PCs. (B) Apigenin downregulated the expression of CK2α in SKOV3-derived SFCs in a dose-dependent manner. (C) Expression of Gli1 was higher in SKOV3-derived SFCs compared with PCs. (D) Apigenin downregulated the expression of Gli1 in SKOV3-derived SFCs in a dose-dependent manner. CK2α, casein kinase 2α; Gli1, glioma-associated oncogene 1; SFC, sphere forming cell; PC, parental cell; Medium, control group.
Inhibition of CK2α downregulates Gli1 and enhances the apigenin-inhibition of self-renewal of the SKOV3-derived SFCs. (A) CK2α siRNA inhibited the protein expression of CK2α and Gli1. (B) Inhibition of CK2α by siRNA further enhanced the inhibitory effects of apigenin (20.0 μmol/l) on the sphere-forming efficiency of SKOV3-derived SFCs. *P<0.05, compared with the control group (0 μmol/l apigenin with CK2α siRNA control); #P<0.05, compared with the groups treated with 20 μM/l apigenin or CK2α siRNA alone. CK2α, casein kinase 2α; Gli1, glioma-associated oncogene 1; SFC, sphere forming cell; Medium, control group.
Overexpression of CK2α leads to Gli1 upregulation and attenuates apigenin inhibiting self-renewal in SKOV3-derived SFCs. (A) Gli1 protein expression increased in the SKOV3 cells transfected with pcDNA3.1-CK2α. (B) Overexpression of CK2α protein increased the sphere formation of the SKOV3 cells. *P<0.05, compared with the pcDNA3.1 group and medium (control) group; #P<0.05, compared with the pcDNA3.1 group. (C) Overexpression of CK2α attenuated the apigenin-induced downregulation of CK2α and Gli1 protein expression. (D) Overexpression of CK2α reduced the inhibition of sphere formation by apigenin in the SKOV3-derived SFCs. *P<0.05, compared with the control group (0 μmol/l apigenin+pcDNA3.1); #P<0.05, compared with the pcDNA3.1 group. CK2α, casein kinase 2α; Gli1, glioma-associated oncogene 1; SFC, sphere forming cell; Medium, control group.