RESORCYLIC ACID LACTONE L-783,277 INHIBITS THE GROWTH OF THE HUMAN ADRENAL CANCER CELL LINE H295R IN VITRO
H. LAWNICKA, M. KOWALEWICZ-KULBAT’, P. SICINSKA2, K.H. ALTMANN3, T. HOFMANN3 and H. STEPIEN
Department of Immunoendocrinology Ist Chair of Endocrinology, Medical University of Lodz, Lodz, Poland; 1Department of Immunology and Infectious Biology, University of Lodz, Lodz, Poland;
2 Department of Molecular Biophysics, University of Lodz, Lodz, Poland; 3Institute of Pharmaceutical Sciences Swiss Federal Institute of Technology (ETH) Zürich, Zürich, Switzerland
Received May 8, 2009 - Accepted October 13, 2009
The resorcylic acid lactone L-783,277, isolated from a Phoma sp. (ATCC 74403), is a potent and specific inhibitor of MEK (Map kinase kinase) that exerts very interesting pharmacological activities including anti-neoplastic properties. However, the role of this compound in the regulation of endocrine-related cancer cell growth and tumor progression remains unknown. In the present study we have evaluated the effect of L-783,277 on the viability, proliferation and cell cycle of the human adrenocortical carcinoma cell line H295R. L-783,277 inhibited viability (IC50 of 22 uM) and cell proliferation (IC50 of 21 uM) of H295R. At concentrations of 106-10-8M this effect was associated with the accumulation of H295R cells in S-phase, whereas at concentrations of 10-9-10-10M a prolonged G1-phase and reduced transition into S-phase were observed. Our findings demonstrate for the first time the anti-proliferative action of L- 783,277 on the human adrenocortical H295R cell line.
Adrenocortical carcinomas (ACCs) are rare, highly malignant tumors with an estimated prevalence of 4-12 per million population (1). This neoplasm is characterized by a poor prognosis, with only 16 to 38% of patients surviving for more than 5 years after diagnosis. Approximately 60% of patients present cortisol and/or androgen overproduction responsible for rapid development of specific clinical syndrome (e.g. virilization, Cushing’s syndrome). The remaining 40% are hormonally silent and identified only when they suffer from local abdominal mass symptoms or metastases (2). Complete tumor resection should be considered as first choice medical therapy. Surgery also plays a
role in local tumor recurrence and metastatic disease treatment. In advanced disease, the most promising therapeutic options (etoposide, doxorubicin, cisplatin plus mitotane, and streptozotocin plus mitotane) are currently being compared in an international phase III trial (3). Although a majority of patients have resectable disease at presentation, 75 to 85% have a relapse after radical resection and cytotostatic therapy (4). Thus, the necessity to search for new, alternative methods of pharmacological therapy in this group of tumors is of utmost importance.
Considerable progress has been made in understanding adrenocortical carcinoma pathogenesis from the study of genetics at the germinal level
Key words: resorcylic acid lactones, L-783,277, adrenocortical cancer, H295R, cell viability, cell proliferation, cell cycle
Mailing address: Prof. Henryk Stepien, MD, PhD
Medical University of Lodz, Dr Sterling 3 Street,
91-425 Lodz, Poland
Tel/fax: ++4842 6324854
e-mail: hstep@csk.umed.lodz.pl
0394-6320 (2009)
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in familial carcinomas, as well as at the somatic mutation level by analyzing molecular alterations in sporadic tumors. Some data support the presence of changes in the p53 tumor suppressor gene and its 53- kDa protein product (5-6). Also other genetic markers responsible for the regulation of cell proliferation, exiting from the cell cycle and differentiation of adrenal cortex included the H19, insulin-like growth factor II and p57kip2 gene products were studied in human adrenocortical cancer (7). With the increasing knowledge of the molecular basis of adrenocortical cancer development, the identification of new therapeutic drugs that could target the disease at the molecular level has been initiated. Recently, it has been shown that mitogen-activated protein kinases (MAPK) act as a common target of hormones, growth factors and oncogenes involved in the control of adrenal cortex cell growth, proliferation and survival (8). However, the significance of the MAPK system in the pathogenesis of adrenal cortical carcinomas has not been elucidated. In this context the assessment of drugs capable of inhibiting multiple kinases and affecting intracellular signaling as potential inhibitors for adrenal carcinomas is of particular interest. Thus, the aim of this study is to evaluate the influence of the natural resorcylic acid lactone L-783,277, which is a potent MEK inhibitor (9), on the proliferation and cell cycle of the human adrenocortical carcinoma cell line (H295R).
MATERIALS AND METHODS
L-783,277 was obtained by total synthesis (10). The chemical structure of L-783,277 is given in Fig. 1.
Cell line and culture condition
The human pluripotent adrenocortical cell line H295R (CRL-2128) obtained from American Type Culture Collection (LGC Promochem, Poland) was used in the experiments. The cells were grown as monolayers in 25cm2 culture flasks (Nunc Eas Y Flask 25 cm2, NUNC) in ATCC complete growth medium: 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; 0.00535 mg/ml linoleic acid, 2.5% Nu-Serum I and 100 U/ml penicillin and 100 µg/ml streptomycin. The additives (insulin, transferrin, selenium, BSA and linoleic acid) as well as Nu-Serum I and antibiotics were obtained as ITS+Premix form (BD Biosciences). Cultures were incubated in a humidified atmosphere of 95% of air and
5% of CO2 at 37℃ and harvested weekly after 3-min incubation at 37°C in the presence of trypsin-EDTA (0.05 or 0.02%, respectively) in Hanks balanced solution (Sigma, USA). The cells were washed twice in complete medium and after the last centrifugation seeded at 2x105 cells in 5 ml of fresh medium.
Cell viability/cytotoxicity study
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) colorimetric assay was used as an indicator of cell viability/cytotoxicity (11). The method is based on the finding that functional mitochondria of living cells are capable of slightly reducing uncoloured tetrazolium salts into intensely coloured formazan derivates and provides global information on the number of viable/active cells.
H295R cells were seeded at density 2x105 cells/ml into 96-well microplates (NUNC, Denmark) in 180 ul/well of complete culture medium and cultured for 84 h (5% CO2 37°℃, 95% humidity). After 12 h of preincubation, 20 ul of L-783,277, previously dissolved in 96% ethanol and serum-free culture medium (30% of 96% ethanol in stock solution), was added to the appropriate wells at the final concentrations as indicated in the figure. The appropriate volume of serum-free culture medium and 96% ethanol in the final concentration identical to the solvent concentration in 10-4M groups was added to the control wells. After 72 h of incubation, cell viability was assessed using EZ4Y assay kit (The 4th Generation Non- Radioactive Cell proliferation & Cytotoxity Assay, Biomedica Gesellschaft mbH, Biomedica Gruppe Austria) according to the manufacturer’s instructions.
The optical density (OD) of each sample was measured by a microplate reader at 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 (12). H295R cells were seeded at density of 1.5x105 cells/ml into 96-well microplates (NUNC, Denmark) in 90 ul/well of complete culture medium and cultured for 84 h (5% CO2 37°℃, 95% humidity). After 12 h of preincubation, 10 ul of L-783,277, previously dissolved in 96% ethanol and serum free culture medium (30% of 96% ethanol in stock solution), was added to the appropriate wells at the final concentrations as indicated in the figure. The appropriate volume of serum-free culture medium and 96% ethanol in the final concentration identical with the solvent concentration in 10-4M groups was added to the control wells. After 72 h of incubation, cell proliferation was assessed using BrdU assay (Cell proliferation ELISA, BrdU, Roche) following
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O
O
O
O
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L-783277
the producer’s instructions. The optical density (OD) of each sample was measured by a microplate reader at 450.
Cell cycle analysis
H295R cells were seeded at a density of 106 cells/ml into 12-well microplates (NUNC, Denmark) in 900 ul/well of complete culture medium and cultured for 84 h (5% CO2 37°C, 95% humidity). After 12 h of preincubation, 100 ul of L-783,277, previously dissolved in 96% ethanol and serum-free culture medium (30% of 96% ethanol in stock solution), was added to the appropriate wells at the final concentrations as indicated in the figure. The same volume of serum free culture medium and 96% ethanol in the appropriate concentrations identical with the solvent concentration in 10-5M-10-10M groups was added to the control wells. After 72 h of incubation, cells were harvested by gentle trypsynization, centrifugated, resuspended in 100 ul of 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 (PI) and 50 IU Kunitz/ml of DNase-free RNase (Sigma Aldrich). After 30 min of incubation the cell tubes were measured with LSR II Flow Cytometer (Becton Dickinson). The percentage of cells in G1, S and G2/M phases of the cell cycle and the percentage of cells undergoing apoptosis were determined with FlowJo analytical software.
Statistical analysis
The results of cell viability and cell proliferation are expressed as the average percentage of the optical density (OD) of the control groups (not shown) and represent mean values of 2-3 separate experiments with n=10 in each group. The normality of distribution of the results was examined by the Kolmogorow-Smirnow test. Comparisons of individual groups were evaluated by analysis of variance (one-way ANOVA) following LSD test (Least Significant Difference). IC50 (50% inhibitory
concentration) was calculated using the regression analysis. The results of cell cycle are expressed as mean values + SD. Data were compared with Mann-Whitney rank sum test. Differences were considered significant if p<0.05.
RESULTS
In the present study we demonstrate for the first time that L-783,277 at the concentration range between 10-100 uM strongly inhibits cell viability as assessed by a MTT based test (Fig. 2) with an IC50 of 22 uM. This compound also significantly reduces BrdU incorporation (Fig. 3) with an IC50 of 21 µM in the human adrenocortical carcinoma cell line H295R after 72 h incubation in vitro.
A study of cell cycle phase distribution showed that at the concentration range between 10-6-10-8M this effect was associated with the accumulation of H295R cells in S-phase, whereas concentrations of 10-9-10-10M led to a prolongation of the G1-phase and to a reduced transition into S-phase (Table I, Fig. 4). Moreover, a significant increase of the percentage of apoptotic cells (vs Control) was observed predominantly at a concentration of 10-8M and was accompanied by cell cycle arrest in G1, S and G2/M (Fig. 4).
DISCUSSION
Resorcylic acid lactones (RALs) are a particularly interesting class of fungal mycotoxins which are emerging as an alternative chemotype of kinase inhibitors (13-14). RALs are unique among kinase inhibitors as they target mitogen-activated protein (MAP) kinase pathways including MAP kinase kinase (MEK)1/2, ERK1/2, and certain downstream ERK substrates (9). These groups of RALs contain the zearalenones, hypothemycin and Ro-09-2210, which have been reported to inhibit VEGF-mediated cell proliferation, and to have antitumor properties (15, 16, 17). Also of interest is their ability to inhibit JNK/p38 signaling pathway in cells, the autophosphorylation of the platelet-derived growth factor (PDGF) receptor or TAK1 (a MEKK) in vitro with low nanomolar concentration (18). However, despite their interesting pharmacological activities, no resorcylic acid lactone has been tested as an anti- angiogenic or anti-cancer agent in humans, or been
% of Control
H295R cell viability
**
120
T
*
100
**
80
T
T
60
**
**
**
**
**
**
**
40
20
0
0.8 ×10-5
10-5
0.2x10-4
0.25×10-4
0.4 ×10-4
0.5 ×10-4
0.6 ×10-4
0.75×10-4
0.8 ×10-4
10-4
M
% of Control
H295R cell proliferation
120
*
100
*
**
80
T
T
60
**
**
**
**
**
**
**
40
20
0
0.8 x10-5
10-5
0.2×10-4
0.25×10-4
0.4 ×10-4
0.5 x10-4
0.6 ×10-4
0.75×10-4
0.8 ×10-4
10-4
M
approved as a drug.
L-783,277, a cis-enone RAL was found to be a potent and irreversible inhibitor of MEK (the cis-enone moiety was essential for its biological activity) and this compound also targets the ATP- binding pockets of MAP kinases (9). We have shown for the first time that L-783,277 inhibited viability (IC50 of 22 µM) and cell proliferation (IC50 of 21 uM) of H295R cells. In concentrations of 10-6-10-8M this effect was associated with the accumulation of
H295R cells in S-phase, whereas in concentrations of 10-9-10-10M a prolonged G1-phase and reduced transition into S-phase were observed.
It is known that also other cis-enone RALs (hypothemycin, 5Z-7-oxozeaenol) inhibit mammalian cell proliferation and tumor growth of a variety of experimental tumor cell lines in the in vitro/in vivo experimental conditions (19). The several possible molecular mechanisms by which the examined compound displays the anti-proliferative
300
Control
10-5
10-6
10-7
G1
200
S
Counts
G2/M
100
0
300
Control
10-8
10-9
10-10
G1
200
S
Counts
Apoptosis
G2/M
100
0
64K
128K
192K
256K
64K
128K
192K
256K
64K
128K
192K
256K
64K
128K
192K
256K
PE
| Control | 10-5 | 10-6 | 10-7 | 10-8 | 10-9 | 10-10 | |
|---|---|---|---|---|---|---|---|
| % G1 | 57.6±1.3 | 52.5±5.5 | 55.5±4.2 | 55.3±5.6 | 62±2.6* | 66.5±4.9* | 61.6±0.6* |
| % S | 26.8±3.2 | 31.3±5.8 | 33.5±3* | 32.5±3.2* | 18.7±1.5* | 22.3±5.5 | 25.3±2.3 |
| % G2/M | 9.4±3 | 8±1.7 | 4.2±1.5* | 3.3±1.2* | 3.3±0.6* | 5.7±2.1 | 9.7±1.5 |
| % apoptosis | 6.1±3 | 9.4±3.9 | 7±3.1 | 8.8±4.4 | 16±2* | 8.3±4 | 3.7±0.58 |
Values are means + SD. * p<0.05 vs Control
effect could be postulated. In fact, L-783,277 is a potent inhibitor of H297R cancer cell line dependent on ERK pathway-activating kinase mutants that are molecular targets of RALs. In addition, L-783,277 has been shown to be a potent in vitro inhibitor of MEK1, competitive with ATP (9). Furthermore, proliferation of several oncogenic cell lines carrying activating mutations in the mitogen receptors c- KIT, FLT3, or PDGFRa (all cis-enone RAL targets) were inhibited by hypothemycin and correlated
with phosphorylation of the downstream MEK1/2 and ERK1/2 (9, 20). Moreover, hypothemycin is selectively potent against all tested melanoma, colon and breast cancer cell lines having the ERK pathway-activating B-RAF V600E mutation (21- 22). Based on chemical structure similarity with hypothemycin, it can be speculated that inhibitory effect of L-783,277 on adrenocortical carcinoma cell line is related, at least in part, to its inhibitory action on MAP kinases intracellular signaling pathways.
In conclusion, our current findings are consistent with the data concerning the inhibitory effect of RALs on the growth of cancer cell lines in vitro. However, further experimental studies and preclinical trials could be useful to fully elucidate mechanism of anti-proliferative L-783,277 action and possible pharmacological strategy for the future treatment of patients with adrenocortical cancers.
ACKNOWLEDGEMENTS
This paper was supported by European Cooperation in the field of Scientific and Technical Research - COST ACTION, Grant No. CM0602 and Polish Ministry of Science and Higher Education, Grant No. 505-04-001.
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