Anti-neoplastic effect of protein kinase CK2 inhibitor, 2-dimethylamino-4,5,6,7-tetrabromobenzimidazole (DMAT), on growth and hormonal activity of human adrenocortical carcinoma cell line (H295R) in vitro
Hanna Lawnicka . Magdalena Kowalewicz-Kulbat ·
Paulina Sicinska · Zygmunt Kazimierczuk ·
Pawel Grieb . Henryk Stepien
Received: 12 January 2010 / Accepted: 26 February 2010 /Published online: 6 April 2010 C Springer-Verlag 2010
Abstract Several studies indicate the involvement of protein kinases in the progression of various malignancies.
This work was supported by European Cooperation in the field of Scientific and Technical Research (COST ACTION, grant no. CM0602) and the Polish Ministry of Science and Higher Education (grant no. 505- 04-001). The synthesis of DMAT was supported by the Foundation for the Development of Diagnostics and Therapy, Warsaw, Poland.
Conflict of interest statement All the above authors state that they have no financial or personal relationships with other people or organizations that could inappropriately influence (bias) this work.
H. Lawnicka . H. Stepien Department of Immunoendocrinology, 1st Chair of Endocrinology, Medical University of Lodz,
Dr. Sterling 3 Street, 91-425 Lodz, Poland e-mail: hstep@csk.umed.lodz.pl
M. Kowalewicz-Kulbat Department of Immunology and Infectious Biology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland
P. Sicinska Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland
Z. Kazimierczuk · P. Grieb
Department of Experimental Pharmacology, Mossakowski Medical Research Center, Polish Academy of Sciences, Pawinskiego 5, 02-124 Warsaw, Poland
Kinase inhibitors are therefore becoming important anti- cancer drugs. CK2 kinase (casein kinase-2) has been suggested to be a constituent of a neoplastic milleu, and its inhibition might represent a new approach to cancer therapy. Adrenocortical carcinomas (ACCs) are highly malignant neoplasms with poor overall prognosis. We have examined the effects of 2-dimethylamino-4,5,6,7-tetrabro- mobenzimidazole (DMAT), a potent CK2 inhibitor, on the H295R human adrenocortical cancer cell line. Treatment with DMAT decreases the secretion of aldosterone, dehy- droepiandrosterone sulfate, and androstendione and results in an accumulation of 17-OH-progesterone. Cell growth as measured by the MTT and 5-bromo-2’-deoxyuridine incor- poration assays is inhibited, and cell cycle analysis has revealed a slight induction of apoptosis. Thus, CK2 kinase activity is probably involved in human ACC endocrine activity and growth.
Keywords Caseine kinase 2 . 2-Dimethylamino-4,5,6, 7-tetrabromobenzimidazole (DMAT) . Adrenocortical cancer . H295R cell line (Human)
Introduction
Adrenocortical carcinomas (ACCs) are rare and extremely malignant tumors with an estimated incidence rate of 1-2 cases per million of the adult population and extremely poor overall prognosis (Fassnacht and Allolio 2009). ACC- induced mortality is related to its rapid spread and clinical
complications connected with excessive hormonal secre- tion. Approximately 60% of ACCs in adults and 90% in children occur with the uncontrolled production of high quantities of adrenal cortex steroids and/or their precursors. Oversecretion of cortisol and androgens is the most common effect and is responsible for severe Cushing syndrome and virilization (Kirschner 2006). Estrogen-secreting tumors are found in 10% of the ACCs, and aldosterone hypersecretion is detected in 2% (Kirschner 2006). Other steroid precursors such as 17-x-hydroxyprogesterone and deoxycorticosterone are frequently detected (Abiven et al. 2006; Libe et al. 2007; Patalano et al. 2009) in apparently non-functioning ACC cases.
Tumor surgical resection is considered the first choice therapy for ACC but is impossible or not efficient in most cases (Crucitti et al. 1996; Libe et al. 2007; Patalano et al. 2009). The application of DDT-derivative o,p’-DDD (mito- tane), given either alone or in combination with cytotoxic agents such as etoposide, doxorubicin, cisplatin, and streptozotocin, is another approach (Khan et al. 2000; Berruti et al. 2005; Kirschner 2006; Patalano et al. 2009), but the response rate remains poor (Khan et al. 2000; Kirschner 2006).
Recently, the inhibition of protein kinases as specific molecular targets in anticancer therapy has been intensely investigated and has become one of the most exciting and promising therapeutic approaches in oncology. The block- ing of these proteins may retard the growth and prolifera- tion of various tumors. Protein kinase inhibitors have demonstrated significant anticancer activity, and some of them, especially tyrosine kinase inhibitors, have been successfully approved for clinical use (imatinib [Gleevec], gefitinib [Irressa], erlotinib [Tarceva]).
In mammalian cells, protein kinase CK2 (EC. 2.7.11.1), also named casein kinase II (CK2), has been identified as a ubiquitous heterotetrameric protein that phosphorylates serine and threonine residues (Gietz et al. 1995; Graham and Litchfield 2000). Cellular and growth-related effects of CK2 activity arise from the phosphorylation of numerous nuclear-associated substrates involved in gene expression and cell growth (Ahmad et al. 2008). Direct phosphoryla- tion of oncogenic targets by CK2 is associated with the regulation of proliferation, differentiation, and apoptosis (Duncan and Litchfield 2008).
CK2 is believed to create a specific cellular environment favorable for cancer development; it is an antiapoptotic and prosurvival agent, promotes the transforming potential of oncogenes, affects the chaperone machinery protecting the “onco-kinome”, enhances the multidrug resistance (MDR) phenotype, counteracts the efficacy of antitumor drugs, and supports angiogenesis. The role of CK2 in cancer develop- ment has recently been proposed as an “non-oncogene addiction” (Ruzzene and Pinna 2010). CK2 activity is
consistently elevated in various human cancers and experi- mental tumors, such as kidney, mammary gland, lung, prostate, head, and neck cancers (Guerra and Issinger 1999; Tawfic et al. 2001; Ahmed et al. 2002; Duncan and Litchfield 2008; Cozza et al. 2009). Overexpression of CK2 is also correlated with tumor aggression (Faust and Montenarh 2000; Tawfic et al. 2001) and is considered an unfavorable prognostic marker in such cancers as prostate and lung cancers and in acute myeloid leukemia (O- charoenrat et al. 2004; Kim et al. 2007; Laramas et al. 2007).
Targeted CK2 inhibition has been postulated for anti- cancer therapy. However, the effect of CK2 inhibitors on the ACC has not yet been elucidated. The aim of our study has been to examine the effect of a small molecule proteine kinase CK2 inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo- benzimidazole (DMAT), on the growth, cell cycle, and hormonal activity of the human ACC cell line H295R in vitro.
Materials and methods
Cell culture
Human pluripotent adrenocortical cell line H295R (CRL- 2128) was obtained from the American Type Culture Collection (LGC Promochem, Poland) and was cultured as a monolayer in 25-cm2 culture flasks (Nunc Eas Y Flask 25 cm2, NUNC) in ATCC complete growth medium, viz., DMEM:F12 (supplemented with 0.00625 mg/ml insulin, 0.00625 mg/ml transferrin, 6.25 ng/ml selenium, 1.25 mg/ ml bovine serum albumin [BSA], 0.00535 mg/ml linoleic acid), 2.5% Nu-Serum I, 100 U/ml penicillin, and 100 µg/ ml streptomycin. The additives (insulin, transferrin, seleni- um, BSA, linoleic acid), Nu-Serum I, and antibiotics were obtained as ITS+Premix (BD Biosciences). The cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C and harvested weekly after a 3-min incubation at 37°C in the presence of trypsin-EDTA (0.05% or 0.02%) in Hanks’ balanced solution (Sigma, USA). The cells were washed twice in complete medium and, after the last centrifugation, were seeded at a density of 2×105 cells in 5 ml fresh medium.
Substances
DMAT was synthesized according the procedure previously described (Pagano et al. 2004).
Cell viability/cytotoxicity study
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tet- razolium bromide) colorimetric assay was used as an
indicator of cell viability/cytotoxicity (Gerlier and Thomasset 1986).
H295R cells were plated at a density of 2×104 cells/well into 96-well microplates (NUNC, Denmark) in complete culture medium and preincubated for 12 h (5% CO2, 37°℃, 95% humidity). DMAT in 96% ethanol and Nu-Serum-free culture medium was added to the appropriate wells at final concentrations of 10 4-10-10 M (the highest concentration of ethanol was 1.8% [vol] in the 10 4 M wells). The same volume of Nu-Serum-free culture medium and 96% ethanol was added to the control wells at the same concentration as the solvent in the 10 4 M group. Incubation was performed for 72 h under standard conditions (5% CO2, 37°C, 95% humidity). Cell viability was measured with the EZ4Y assay kit (4th Generation Non-Radioactive Cell Prolifera- tion & Cytotoxicity Assay, Biomedica Gesellschaft, Bio- medica Gruppe, Austria) according to the manufacturer’s instructions. The absorbance (OD, optical density) of each sample was measured with an enzyme-linked immunosor- bent assay (ELISA) microplate reader at a wavelength of 450 nm.
Cell proliferation study
Colorimetric immunoassay based on the measurement of 5-bromo-2’-deoxyuridine (BrdU) incorporation during DNA synthesis was used for the quantification of H295R cell proliferation (Heil and Reifferscheid 1992). H295R cells were plated into 96-well microplates (NUNC, Denmark) at a density of 1×104 cells/well in complete culture medium and preincubated for 12 h (5% CO2, 37°C, 95% humidity). DMAT in 96% ethanol and Nu-Serum-free culture medium was added to the appro- priate wells at final concentrations of 10 4-10-10 M (the highest concentration of ethanol was 1.8% [vol] in the 10 4 M wells). The same volume of Nu-Serum-free culture medium and 96% ethanol was added to the control wells at the same concentration as the solvent in the 10 4 M group. The incubation was performed for 72 h under standard conditions (5% CO2, 37°C, 95% humidi- ty). Cell proliferation was assessed by using BrdU assay (Cell proliferation ELISA, BrdU, Roche) following the producer’s instructions. The absorbance (OD) of each sample was measured with an ELISA microplate reader at a wavelength of 450 nm.
Cell cycle analysis
H295R cells were seeded at a density of 106 cells/ml into 12-well microplates (NUNC, Denmark) in complete culture medium and preincubated for 12 h (5% CO2, 37°℃, 95% humidity). DMAT in 96% ethanol and Nu- Serum-free culture medium was added to the appropriate
wells at final concentrations of 10-5-10-8 M. The same volume of Nu-Serum-free culture medium and 96% ethanol was added to the control wells at the same concentration as the solvent in the 10-4 M group. After 72 h of incubation under standard conditions (5% CO2, 37°C, 95% humidi- ty), cells were harvested by gentle trypsinization, centri- fuged, resuspended in phosphate-buffered calcium- and magnesium-free saline (PBS), and fixed in 70% ice-cold ethanol. After centrifugation at 4℃, the cells were finally suspended in PBS containing 75 umol/dm3 propidium iodide and 50 IU Kunitz/ml DNase-free RNase (Sigma Aldrich). After a 30-min incubation, the cell tubes were measured with an LSR II Flow Cytometer (Becton Dickinson). The percentage of cells in the G1, S, and G2/ M phases of the cell cycle and the percentage of cells undergoing apoptosis were determined with FlowJo analytical software.
Analysis of secretory activity of H295R cells
In order to determine hormone secretion, the H295R cells were seeded in 24-well microplates (NUNC, Denmark) and allowed to grow for 36 h in complete medium in a final volume of 1 ml/well. Then, the full medium was changed to Nu-Serum-free medium, and DMAT in 96% ethanol and Nu-Serum-free culture medium was added to the appropriate wells at final concentrations of 10-4_ 10-9 M (the highest concentration of ethanol was 1.8% [vol] in the 10 4 M wells). The same volume of Nu- Serum-free culture medium and 96% ethanol was added to the control wells at the same concentration as the solvent in the 10 4 M group. The cells were incubated for 72 h under standard conditions (5% CO2, 37°C, 95% humidi- ty). Subsequently, the cell-free supernatants, after centri- fugation at 1400 rpm for 10 min at 4℃, were collected and immediately stored at -80℃ for hormone release studies. These were as follows:
Cortisol
Chemiluminescence (Ortho-Clinical Diagnostics, Johnson & Johnson (Vitros), UK) was used to measure cortisol concentrations in the cell culture medium. The sensitiv- ity was <0.11 ug/dl with an inter-assay precision of <5.9%.
Aldosterone
The radioimmunoassay (RIA) method (ZenTech, Bel- gium) was used to measure aldosterone concentrations in cell culture medium. The detection limit was 1.4 pg/ml, with an intra- and inter-assay precision of <13.7% and <18.6%, respectively.
Dehydroepiandrosterone sulfate (DHEAS)
RIA (Immunotech, France) was used to measure DHEAS concentrations in the cell culture medium. The
detection limit was 6 µg/dl, with an intra- and inter-assay precision of <10.6% and <7.4%, respectively.
Androstendione
RIA (Diagnostic Systems Laboratories, USA) was used to measure androstendione concentrations in the cell culture medium. The detection limit was 0.03 ng/ml, with an intra- and inter-assay precision of <5.6% and <9.8%, respectively. 17-Hydroxyprogesterone
RIA (Diagnostic Systems Laboratories, USA) was used to measure 17-hydroxyprogesterone (17-OH-progesterone) concentrations in the cell culture medium. The detection limit was 0.01 ng/ml, with an intra- and inter-assay precision of <19.3% and <10.8%, respectively.
Statistical analysis
The results of cell viability and proliferation are presented as means±SEM of the OD. Data concerning the cell cycle are expressed as means+SD. The results of the secretory activity of the cells are given as the mean of the concentrations of the hormones in the culture medium: cortisol and DHEAS [ug/ dl], aldosterone [ng/dl], and 17-OH-progesterone and androstendione [ng/ml].
Statistical significance was determined by one-way analysis of variance with a post-hoc least significant difference (LSD). The significance of the differences was then analyzed with Student’s t-test. Data concerning cell cycle analysis were compared with the Mann-Whitney rank sum test. Differences were considered significant when P<0.05.
Results
Effect of DMAT on viability and proliferation of H295R cells
Treatment with DMAT significantly inhibited (P<0.001) the viability of H295R cells in 72-h culture in the concentration range from 10-4 to 10-10 M (Fig. 1a).
The strong inhibitory effect of DMAT on H295R cell proliferation after 72 h of culture was observed only for concentrations of 10 4 and 10-5 M (P<0.001) as compared with the control (Fig. 1b).
Effect of DMAT on H295R cell cycle
A significant increase of apoptosis was observed in H295R cells treated with 10 8 M DMAT compared with the control cells (Table 1, Fig. 2). Moreover, the number of H295R cells in the G1 phase was increased in a 10-5 M concentration of DMAT. This effect was accompanied by a decrease in the
a
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number of cells in the S-phase, thereby indicating cell cycle arrest in the G1 phase (Table 1).
Effect of DMAT on cortisol secretion by H295R cells
None of the DMAT concentrations resulted in any signif- icant changes in cortisol concentration in the supernatants of H295R cells after 72 h of incubation. However, a slight inhibitory effect was found for the 10 4 and 10 6-10 9 M concentrations (data not shown).
Effect of DMAT on aldosterone secretion by H295R cells
DMAT at concentrations of 10 6-10 9 M significantly decreased (P<0.001 for 10-7-10 9 M and P<0.05 for 106 M) aldosterone release into supernatants of 72- h H295R cell cultures as compared with the control (Fig. 3a). A stimulatory effect was associated with 10-5 M DMAT (P<0.05).
| Cell cycle stage | Control | 10-5 | 10-6 | 10-7 | 10-8 |
|---|---|---|---|---|---|
| G1 | 51.5±8.5 | 59.2±3.7 | 46.3±10.4 | 51.6±13.2 | 54.7±8.3 |
| S | 31.7±5.4 | 25.6±6.5 | 30.8±4 | 31.6±4.9 | 30.3±4.1 |
| G2 | 11±2.8 | 12.6±3.6 | 10±4.6 | 10.3±3.8 | 7.3±8 |
| Apoptosis | 2.7±2.1 | 8±6.2 | 7.3±4.7 | 7.8±6.9 | 7.6±1.5* |
*P<0.05 vs control
Effect of DMAT on secretion of dehydroepiandrosterone sulfate by H295R cells
Compared with the control, the secretion of dehydroepian- drosterone sulfate by H295R cells was significantly inhibited by 10 4 M (P<0.001), 10-7 M (P<0.01), and 10 9 M (P<0.05) concentrations of DMAT (Fig. 3b) when cultured together for 72 h.
Effect of DMAT on androstendione secretion by H295R cells
Similar to the results concerning the release of DHEAS, the secretion of androstendione by H295R was significantly decreased by a 72-hour treatment of 10 4 M (P<0.001),
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10-7 M (P<0.01), or 10-9 M (P<0.05) concentrations of DMAT as compared with the control (Fig. 3c).
Effect of DMAT on 17-OH-progesterone secretion by H295R cells
Interestingly, in contrast to other hormones, an accumula- tion of 17-OH-progesterone was seen in the culture medium of H295R cells incubated for 72 h with DMAT at concentrations of 10-5-10 9 M (P<0.001; Fig. 3d). Only a dose of 10 4 M DMAT inhibited the release (P<0.001) of 17-OH-progesterone into the cell culture supernatant as compared with the control.
Discussion
Until now, little success has been achieved by inhibiting kinases in endocrine tumors such as ACC (Fassnacht et al. 2009). However, two recent case reports have described clinical responses to multikinase inhibitors (sunitinib, sorafenib) in advanced ACCs (Butler et al. 2009; Lee et al. 2009). Moreover, resorcylic acid lactone L-783,277, a potent and specific inhibitor of MEK (Map kinase kinase) has been shown to have an inhibitory effect on the growth of the human adrenal cancer cell line H295R in vitro (Lawnicka et al. 2009).
Among the kinases, protein kinase CK2 (casein kinase- 2) is believed to represent a multipurpose target for cancer treatment (Ruzzene and Pinna 2010). DMAT is character- ized by its high affinity and selectivity toward CK2 with an IC50 value of approximately 0.14 uM and a Ki value of approximately 40 nM (Pagano et al. 2004; Cozza et al. 2009) and has been shown to inhibit the growth of various cancer cell lines. Studies of Jurkat cells derived from human T-cell leukemia have revealed a dose-dependent effect of DMAT on cell viability (Pagano et al. 2004). In the last-mentioned study, treatment of Jurkat cells with 5 AM and 10 AM DMAT results in the strong inhibition of endogenous CK2 activity (60% and 30% of control values, respectively). According to Pagano et al. (2004), the cell death induced by DMAT is in part caused by apoptosis. The
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viability of PLC1, B1, and B2 lymphoblastic leukemia cell lines, derived from various individual BCR/ABL transgenic mice has also been seen to be inhibited (Mishra et al. 2007). Interestingly, in an examination of DMAT toxicity, this compound has been found to inhibit the proliferation of normal primary mouse fibroblasts but does not cause significant cell death. Treatment with 1-10 AM DMAT has been shown to inhibit cell growth in a study of five anti-estrogen-resistant MCF-7 sublines of human breast cancer cells in 72-h culture as measured by colorimetric assay (Yde et al. 2007). This inhibitory effect is associated with DMAT-induced cell death mediated by caspases and with changes in CK2 enzymatic activity (reduced to 30%- 40% of control values). To our knowledge, prior to the present study, no data have previously been available with regard to the influence of protein kinase CK2 inhibitors on ACC cells.
The present study is the first to demonstrate the inhibitory effect of the CK2 inhibitor, DMAT, on the growth of human ACC H295R cells. Our results on these cells are similar with those mentioned above and indicate
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that the growth inhibition of this cell line by DMAT is linked to apoptosis.
The cell viability assay in our studies is based on the determination of functional mitochondria and provides global information on the number of viable/active cells but does not differentiate between the effects of changes attributable to proliferation, necrosis, or apoptosis. Com- parison of our results on H295R cell viability (inhibition by all tested DMAT concentrations) and proliferation (strong inhibition only for 10-4 and 10 5 M concentrations) indicates a major role of DMAT in inducing apoptosis, rather than being involved in suppressing H295R cell proliferation. Our observations have been confirmed by the results of the cell cycle analysis and are in agreement with the dual function of CK2: promotion of cell growth and concomitant inhibition of cell death (Ahmad et al. 2005). However, the potency and effectiveness of CK2 inhibitors in inducing apoptosis vary and depend on the cell type (Wang et al. 2005). Moreover, the pro-apoptotic effect of CK2 downregulation can be related to the activity of several downstream targets. These include, for example,
caspases 2, 3, 8, and 9 (Shin et al. 2005; Wang et al. 2006) and pro-caspase-2 (Shin et al. 2005). Apoptosis associated with CK2 downregulation can be mediated via caspase inhibitor protein (ARC), lamin A cleavage, components of the Bcl-2 family, Wnt, PI3K/Akt, NF-KB signaling, or some mitochondrial pathways (Ahmad et al. 2008; Ruzzene and Pinna 2010). Intracellular H2O2 production, survivin, IAPs (inhibitor of apoptosis proteins), and IGFBP-3 might also be involved. However, CK2 itself seems to have a global impact on these complex apoptotic mechanisms and numerous downstream signaling pathways, which eventu- ally cooperate to promote cell survival (Ahmad et al. 2008; Ruzzene and Pinna 2010).
Protein kinase CK2 has been identified as a positive regulator of Wnt signaling (Seldin et al. 2005). Recently, the role of incorrect Wnt signal activation connected with abnormal ß-catenin accumulation in adrenocortical adeno- ma and carcinoma pathogenesis has been confirmed (Tissier et al. 2005; Soon et al. 2008). Somatic activating mutations of the ß-catenin gene (CTNNB1) have also been found to be the most frequent genetic event in adrenocor- tical adenomas and ACCs (Tissier et al. 2005; Gaujoux et al. 2008). The H295R cells used in our experiments have been identified to harbor the CTNNB1 mutation, resulting in the constitutive overactivity of the ß-catenin signaling pathway (Tissier et al. 2005; Tadjine et al. 2008). Thus, the DMAT-induced inhibition of H295R cell growth might be achieved by targeting the Wnt pathway/ß-catenin signaling.
Pharmacological inhibition of tumor growth and hor- monal secretion is important, since hormonally active ACCs significantly affect patient mortality. In our studies, the treatment of H295R cells with DMAT results in an inhibition of aldosterone, DHEAS, and androstendione secretion. In contrast to other hormones, the concentration of 17-OH-progesterone (17-OH-P) is higher in samples from DMAT-treated cells compared with controls. In our opinion, this is not an effect of the stimulation of 17-OH-P production, but rather the consequence of a blockage in the steroidogenesis pathways in H295R cells as caused by DMAT. This results in the accumulation of 17-OH- progesterone and inhibition of sequential steroid synthesis, as a blockade in steroid and/or mineralocorticoid synthesis would cause an accumulation of 17-OH-P and decrease the concentration of hormones synthesized after the block. Moreover, the accumulation of 17-OH-progesterone indi- cates that the inhibition of aldosterone, DHEAS, and androstendione secretion by DMAT-treated H295R cells is attributable not only to the influence of this compound on proliferation or cell viability, but also to its influence on the secretory activity of these cells.
Our studies on the inhibition of CK2 in human ACC cells seem also to be noteworthy in the light of single-agent therapy not being recommended in ACC (Ahlman et al.
2001). This is connected with the high expression of the multidrug resistance protein, MDR1, in most ACCs (Flynn et al. 1992). Interestingly, the activity of CK2 appears to be connected with the multidrug resistance phenotype. CK2 overexpression has been shown to protect cells against drug-induced apoptosis (Guo et al. 2001), and CK2 blockade increases the accumulation of cytotoxic drugs in cells previously resistant to chemically mediated apoptosis (Di Maira et al. 2008). In vitro studies of human cancer cells have shown that the inhibition of CK2 sensitizes cancer cells against both receptor-mediated (Izeradjene et al. 2005) and intracellular (Yamane and Kinsella 2005) apoptosis. In studies of human MDR lines, the pharmaco- logical downregulation of CK2 activity induces cell death in both R-CEM (T lymphoblastoid cell line) and U2OS (osteosarcoma cell line) with CK2« overexpression or normal CK2x expression, respectively (Di Maira et al. 2007). Thus, a blockade of CK2 activity might be considered a promising tool to overcome the MDR phenotype. Moreover, combined treatment with tyrosine kinase inhibitor, Imatinib, and DMAT has been tried with good results in lymphoblastic leukemia cells, PLC1 exhibiting significantly decreased cell viability and in- creased apoptotic cell number in cultures treated with both drugs as compared with monotherapy (Mishra et al. 2007).
In conclusion, we have demonstrated, for the first time, the inhibitory effect of the CK2 inhibitor, DMAT, on the growth, cell cycle, and hormonal activity of the human ACC cell line H295R in vitro. Further studies are needed in order fully to elucidate the mechanisms of actions of CK2 inhibitors in adrenocortical malignancies.
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