Potent Inhibitory Effects of Type I Interferons on Human Adrenocortical Carcinoma Cell Growth
Peter M. van Koetsveld, Giovanni Vitale, Wouter W. de Herder, Richard A. Feelders, Katy van der Wansem, Marlijn Waaijers, Casper H. J. van Eijck, Ernst-Jan M. Speel, Ed Croze, Aart-Jan van der Lely, Steven W. J. Lamberts, and Leo J. Hofland
Departments of Internal Medicine (P.M.v.K., G.V., W.W.d.H., R.A.F., K.v.d.W., M.W., A .- J.v.d.L., S.W.J.L., L.J.H.) and Surgery (C.H.J.v.E), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands; Department of Molecular Cell Biology (E .- J.M.S.), Research Institute for Growth and Development, University of Maastricht, NL-6200 MD Maastricht, The Netherlands; and Department of Immunology (E.C.), Berlex Bioscience Inc., Richmond, California 94006
Context: Adrenocortical carcinoma (ACC) is a rare tumor with a poor prognosis. Despite efforts to develop new therapeutic regimens for metastatic ACC, surgery remains the mainstay of treatment. Inter- ferons are known to exert tumor-suppressive effects in several types of human cancer.
Design: We evaluated the tumor-suppressive effects of type I inter- ferons (IFN)-a2b and IFNß on the H295 and SW13 human ACC cell lines.
Results: As determined by quantitative RT-PCR analysis and im- munocytochemistry, H295 and SW13 cells expressed the active type I IFN receptor (IFNAR) mRNA and protein (IFNAR-1 and IFNAR-2c subunits). Both IFNa2b and IFNß1a significantly inhibited ACC cell growth in a dose-dependent manner, but the effect of IFNß1a (IC50 5 IU/ml, maximal inhibition 96% in H295; IC50 18 IU/ml, maximal
inhibition 85% in SW13) was significantly more potent, compared with that of IFNa2b (IC50 57 IU/ml, maximal inhibition 35% in H295; IC50 221 IU/ml, maximal inhibition 60% in SW13). Whereas in H295 cells both IFNs induced apoptosis and accumulation of the cells in S phase, the antitumor mechanism in SW13 cells involved cell cycle arrest only. Inhibitors of caspase-3, caspase-8, and caspase-9 coun- teracted the apoptosis-inducing effect by IFNßla in H295 cells. In H295 cells, IFNßla, but not IFNa2b, also strongly suppressed the IGF-II mRNA expression, an important growth factor and hallmark in ACC.
Conclusions: IFNß1a is much more potent than IFNa2b to suppress ACC cell proliferation in vitro by induction of apoptosis and cell cycle arrest. Further studies are required to evaluate the potency of IFNß1a to inhibit tumor growth in vivo. (J Clin Endocrinol Metab 91: 4537-4543, 2006)
A DRENOCORTICAL CARCINOMA (ACC) is a rare tu- mor with a dismal prognosis (1, 2). Complete surgical resection is currently the only curative therapy for localized ACC (3). When complete resection is not possible, or in metastatic disease, the treatment of choice is chemotherapy with mitotane (2). Mitotane, being an adrenolytic compound (4), has been used in the treatment of patients with ACC as a single agent as well as in combination with other therapies. Overall, a response rate between 20 and 33% has been re- ported (5). However, treatment with mitotane is associated with several side effects, and long-term therapy is indicated only in case of a clinical response (2). In addition, there are no conclusive in vivo data showing favorable effects on sur- vival and quality of life in metastatic ACC after the treatment with mitotane, alone or in combination with chemotherapy. Therefore, novel treatment strategies are clearly required for this carcinoma (6)
In vitro and in vivo studies have shown the efficacy of type I interferons (IFNs) in the treatment of several tumors, alone or in combination with chemotherapy (7, 8). Type I IFNs, such as IFNa, IFNß, and IFN@, interact with the same re-
ceptor complex [type I IFN receptor (IFNAR)], composed by two subunits, i.e. IFNAR-1 and IFNAR-2 (9, 10). The type I IFNs modulate tumor-suppressive activity through different mechanisms, including induction of cell cycle arrest and apoptosis (11). It has been described that type I IFNs are able to down-regulate the expression of IGF-II at the mRNA and protein level in some cancers (12, 13). Considering that IGF-II is highly expressed in more than 90% of the ACC and that this growth factor is involved in adrenal growth and tumor- igenesis of ACC (14), type I IFNs may be of potential interest in the treatment of ACC.
To explore new possibilities of ACC treatment, we inves- tigated the in vitro effects and mechanism of action of IFNa2b and IFNßla on the growth of two established human ACC cell lines, e.g. H295 and SW13.
Materials and Methods
Cell lines and culture conditions
The human ACC cell line, NCI-H295R, was obtained from the Amer- ican Type Culture Collection (Manassas, VA). The SW13 ACC cell line was obtained from ECACC (Salisbury, Wiltshire, UK) .
The cells were cultured in 75-cm2 culture flasks (Corning Costar, Amsterdam, The Netherlands) at 37 C in a humidified incubator con- taining 5% CO2. The culture medium consisted of a 1:1 mixture of DMEM and F12K medium, supplemented with 5% fetal calf serum, penicillin (1 × 105 U/liter), Fungizone (0.5 mg/liter), and L-glutamine (2 mmol/liter). Cells were harvested with trypsin (0.05%)-EDTA (0.53 mM) and resuspended in culture medium. Cell viability always ex-
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ceeded 95%. Media and supplements were obtained from Gibco Bio-cult Europe (Invitrogen, Breda, The Netherlands).
Drugs and reagents
Human recombinant IFN&2b (Roferon-A) was purchased from Roche (Almere, The Netherlands). Human recombinant IFNßla was pur- chased from Serono Inc. (Rebif; Rockland, MA). Human recombinant IFNß1b was obtained from Schering (Mijdrecht, The Netherlands).
Caspase inhibitors Devd-cho, Lehd-cho, and Ietd-cho were pur- chased from Biosource (Brussels, Belgium).
Quantitative RT-PCR
For the detection of IFN receptors (IFNAR-1, IFNAR-2b, IFNAR-2c) and IGF-II, total RNA was isolated using a commercially available kit (high pure RNA isolation kit; Roche). cDNA synthesis and quantitative PCR using the TaqMan Gold nuclease assay was performed as described in detail previously (15). The primer and probe sequences were pur- chased from Biosource. The sequence of the primers IFNAR-1, IFNAR-2 total, IFNAR-2b, and IFNAR-2c as well as the concentrations of primers and probes used in the assay have been described by Vitale et al. (16). The sequences of the IGF-II primers were: IGF-II forward, 5’- CCAAGTCCGAGAGGGACGT-3’; IGF-II reverse, 5’-TTGGAAGAACT- TGCCCACG-3’; and IGF-II probe, 5’-FAM-ACCGTGCTTCCGGA- CAACTTCCC-TAMRA-3’. Dilution curves were constructed for calculating the PCR efficiency for every primer set. PCR efficiencies were: IFNAR-1, 1.90 ± 0.02; IFNAR-2,1.89 ±0.03; IFNAR-2b, 1.68 ± 0.04; IFNAR-2c, 1.86 ± 0.06; and IGF-II, 2.01 ± 0.07. The estimated copy numbers were obtained according to the method described by Swillens et al. (17). The amount of mRNA was normalized to the total amount of RNA.
To exclude genomic DNA contamination in the RNA, the cDNA reactions were also performed without reverse transcriptase and am- plified with each primer pair. To exclude contamination of the PCR mixtures, the reactions were also performed in the absence of cDNA template in parallel with cDNA samples. As a positive control for the PCR of type I IFN receptors and IGF-II, human DNA was amplified in parallel with the cDNA samples.
Immunocytochemistry
H295 and SW13 cells, cultured on coverslips (Invitrogen), were fixed with acetone (10 min at room temperature) and incubated for 30 min at room temperature with antibodies to human IFNAR-1 (rabbit polyclonal antibody; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and IFNAR- 2c. Finally, a standard streptavidin-biotinylated alkaline phosphatase detection system (IL Immunologic, Duiven, The Netherlands) was used according to the manufacturer’s recommendations to visualize the bound antibodies.
The IFNAR-2c antibody (monoclonal antibody 27D11, provided by E.C., Berlex Biosciences) was generated against the purified IFNAR-2c ectodomain expressed in baculovirus and purified using a FLAG affinity column. This method was previously described for the generation of a monoclonal antibody against the IFNAR-1 ectodomain (18). The use and specificity of the IFNAR-2c antibody has been described previously (16, 19). Negative controls for the immunohistochemistry included omission of the primary antibody.
Cell proliferation assay
Cells were plated in 1 ml of medium in 24-well plates at a density of 105 cells/well for H295 and 5 × 103 cells/ well for SW13. The plates were placed in a 37 C, 5% CO2 incubator. Two days later the cell culture medium was replaced with 1 ml/well medium containing the indicated concentrations of interferon in quadruplicate. After 3 and 6 d of treat- ment, the cells were harvested for DNA measurement. Plates for 6 d were refreshed after 3 d, and compounds were added again. Measure- ment of total DNA contents was performed as previously described (20).
Apoptosis assay
Apoptosis was evaluated by the analysis of the DNA fragmentation. After plating of 105 cells/well for H295 and 2 × 104 cells/well for SW13
on 24-well plates, cells were incubated at 37 C. After 2 d the cell culture medium was replaced with 1 ml/well medium containing various con- centrations of drugs (IFNa2b: 0-1000 IU/ml; IFNß1a: 0-100 IU/ml; IFNß1b: 0-100 IU/ml) in quadruplicate. After 1 d of incubation, apo- ptosis was assessed using a commercially available ELISA kit (Cell Death Detection ELISAPlus, Roche Diagnostic GmbH, Penzberg, Ger- many). The standard protocol supplied by the manufacturer was used. Apoptosis was expressed as percentage of control, untreated cells.
To evaluate the role of caspases involved in the induction of apoptosis after IFNß1a treatment, H295 cells (105 cells/well) were plated in 24-well plates and after 1 d of incubation; medium was changed by 1 ml of the medium containing 10 µM of different caspase inhibitors: Devd-cho (blocks caspase-3), Ietd-cho (blocks caspase-8), and Lehd-cho (blocks caspase-9). After 1 d of incubation, IFNß1a (5 IU/ml), in the absence or presence of the caspase inhibitors, was added, and the cells were incu- bated for an additional 24 h. Plates were then collected for detection of DNA fragmentation by ELISA, as described above.
Cell cycle analysis
Cells (2 × 105 for H295 and 5 × 105 for SW13) were plated on 12-well plates. After 1 d the medium was changed with fresh medium (control) or fresh medium plus IFNa2b (500 and 1000 IU/ml) or IFNß1a (50 and 100 IU/ml). After 3 d of incubation (confluency of about 60-70%), cells were harvested by gentle trypsinization, washed with ice-cold phos- phate-buffered calcium and magnesium-free saline (PBS) and collected by centrifugation. Cells were resuspended in 200 ul PBS and fixed in 70% ice-cold ethanol, followed by an overnight incubation at -20 C. After centrifugation, the cells were washed once with PBS and incubated for 30 min at 37 C in PBS containing 40 µg/ml propidium iodide (Sigma Aldrich, Zwijndrecht, The Netherlands) and 10 µg/ml of DNase-free RNase (Sigma Aldrich, Zwijndrecht, The Netherlands). For each tube, 20,000 cells were immediately measured on a FACScalibur flow cytom- eter (Becton Dickinson, Erembodegem, Belgium) and analyzed using CellQuest Pro Software.
Statistical analysis
All experiments were carried out at least three times and gave com- parable results. For statistical analysis GraphPad Prism 3.0 (GraphPad Software, San Diego, CA) was used. The comparative statistical evalu- ation among groups was performed by ANOVA. When significant dif- ferences were found, a comparison between groups was made using the Newman-Keuls test. The unpaired Student t test was used to analyze the differences in concentration effect curves and effects in cell cycle mod- ulation between different types of IFN preparations.
Results
Expression of type I IFN receptor mRNAs and proteins in H295 and SW13 cells
The effect of type I IFNs on cells is modulated by a com- mon receptor. We generated specific primers and probes to determine the expression of IFNAR-1 and IFNAR-2 (total, short and long form) mRNA in H295 and SW13 cells by quantitative RT-PCR. In both cell lines, the presence of IFNAR-1, IFNAR-2 total, IFNAR-2b, and IFNAR-2c (Fig. 1, upper panel) was detected. IFNAR-1 mRNA expression, was approximately three times higher than IFNAR-2 total in both cell lines. Among the IFNAR-2 subunits, IFNAR-2c was the form expressed at the relatively lowest level.
In H295 and SW13 cells, specific expression of the two active IFN receptor subunits (IFNAR-1 and IFNAR-2c) was found by immunocytochemistry (Fig. 1). For IFNAR-1 as well as IFNAR-2c, staining was observed both in the cyto- plasm and at the cell membrane (Fig. 1, middle panels). No staining was observed in the negative controls (Fig. 1, lower panels).
Relative copy number of type | IFN receptors (mRNA / µg total RNA)
H295
SW13
400
400
300
300
200
200
100
100
0
AR-1
AR-2
AR-2a AR
R-2b
AR-2c
0
AR-1
AR-2
AR-2a
AR-2b
AR-2c
tot
tot
IFNAR-1
1
IFNAR-2c
Negative control
Effects of two different types of IFNß on cell growth and apoptosis
We first evaluated the effect of two commercially available IFNß preparations on cell proliferation and proapoptotic activity in H295 cells, e.g. IFNß1a (Serono, amino acid se- quence and glycosylation similar to native IFNß) and IFNß1b (Schering, one amino acid change and absence of glycosyl- ation, compared with the native IFNß).
Inhibition of cell proliferation by IFNßla was significantly stronger than with IFNß1b after 6 d of treatment (IC50 9.7 ± 2.7 vs. 87.0 ± 3.8 IU/ml, P < 0.01; maximal inhibitory effect on cell proliferation 88 ± 7 vs. 51 + 10%, P < 0.01, respec- tively, for IFNß1a and IFNß1b). This is shown in Fig. 2A.
In H295 cells both types of IFNß preparations increased DNA fragmentation in a dose-dependent manner. In agree- ment with the effect on cell proliferation, IFNß1a was more powerful than IFNß1b (EC50 4.0 ± 1.1 IU/ml, vs. EC50 24.9 ± 2 IU/ml, respectively, P < 0.01) (Fig. 2B). In subsequent experiments, we therefore decided to use IFNßla.
Comparison between the effects of IFNa2b and IFNßla on cell proliferation
After 6 d of incubation, IFNa2b and IFNß1a significantly suppressed the growth of H295 and SW13 cells in a dose- dependent manner (Fig. 3, upper panels). IFNßla was signif-
icantly more potent than IFNa2b, as determined by both the IC50 (H295: 5.4 ± 1.3 vs. 157.0 ± 2.3 IU/ml, respectively, P < 0.01; and SW13: 18.1 ± 1.3 vs. 221.1 ± 1.3 IU/ml, respectively, P < 0.01) and the maximal inhibition of cell proliferation after 6 d of treatment (H295: 96 ± 7 vs. 35 ± 2%, respectively, P < 0.01; and SW13: 85 ± 7 vs. 60 ± 3%, respectively, P < 0.01). After 6 d of incubation, IFNßla induced in both cell lines a statistically significant inhibition of cell growth, already at very low concentrations (1 IU/ml, P < 0.01, vs. control). Moreover, the concentration required to obtain the maximal inhibition of proliferation was 100 IU/ml for IFNßla and 10 times higher for IFNa2b (1000 IU/ml). Comparable results were found by a [3H]thymidine incorporation assay (data not shown). There was no statistically significant difference be- tween the IC50 values of 3 and 6 d of treatment (DNA control cells on d 3 and 6: 4330 ± 204 and 7257 ± 246 ng/well for H295, and 1685 ± 36 and 6870 ± 326 ng/well for SW13, respectively). H295 cells were slightly more sensitive to type I IFNs than SW13 cells in terms of IC50, whereas maximal inhibition of cell growth by IFNa2b was highest in SW13 cells.
Effects of type I IFN on cortisol secretion
In H295 cells, we did not observe any difference (IC50 or maximal inhibition) between the inhibitory effects of type I
DNA content (% of control)
125
A
100
75
50
25
0
0
1
10
100
DNA fragmentation (% of control)
600
B
400
200
0
0
1
10
100
Log IFN dose (IU/ml)
IFNs on forskolin-induced cortisol secretion on the one hand and on DNA content on the other hand. Cortisol production, normalized for DNA content, was not significantly changed by IFN treatment (data not shown). No detectable cortisol secretion by SW13 was found.
Induction of apoptosis by IFNa2b and IFNßla
After 1 d of treatment with IFNß1a and IFNa2b, we mea- sured the DNA fragmentation to investigate the induction of apoptosis (Fig. 3, lower panels). In H295, a potent dose- dependent induction of apoptosis was observed after IFNß1a treatment (EC50 5.0 ± 1.4 IU/ml). The maximal increase of DNA fragmentation induced by 100 IU/ml IFNßla was about six times, compared with untreated control cells. IFNßla stimulated apoptosis in H295 cells already at very low concentrations (1 IU/ml, P < 0.01). A less effective dose- dependent induction of apoptosis was observed after IFNa2b treatment of H295 cells (EC50 249 ± 2 IU/ml). The maximal increase of DNA fragmentation by 1000 IU/ml IFNa2b was about 2.5 times, compared with the control. Interestingly, DNA fragmentation was not observed in SW13 after treatment with IFNa2b and IFNßla. To study the role of caspases-3, -8, and -9 in the induction of apoptosis by IFNß1a in H295 cells, we evaluated whether specific caspase inhibitors were able to blunt IFNß-induced DNA fragmen- tation. As shown in Fig. 4, the stimulation of apoptosis by IFNß1a is completely blocked by Devd-cho, a specific inhib- itor of caspase-3, whereas it is only partially counteracted by
DNA fragmentation (% of control) DNA content (% of control)
125
H295
125
SW13
100
100-
75
75-
50
50-
25
25
0
0
0
1
10
100
1000
0
1
10
100
1000
600
H295
600
SW13
400
400
200
200
0
0
0
1
10
100
1000
0
1
10
100
1000
Log IFN (IU/ml)
Log IFN (IU/ml)
the use of specific inhibitors for caspase-8 (Ietd-cho) and caspase-9 (Lehd-cho).
Effects of IFNa2b and IFNßla on cell cycle progression
We also evaluated the effect of treatment with IFN&2b (1000 and 500 IU/ml) and IFNß1a (100 and 10 IU/ml) on cell cycle distribution after 3 d of incubation (Table 1).
Both IFNs induced a significant and dose-dependent ac- cumulation of cells in the S phase (IFN&2b 1000 IU/ml and IFNß1a 100 IU/ml: P < 0.001 vs. control in both cell lines; IFNa2b 500 IU/ml and IFNß1a 50 IU/ml: P < 0.05 vs. control in H295 and P < 0.01 vs. control in SW13 cells), whereas there was no significant change in G1/G0 distribution (Table 1). The accumulation of cells in S phase after treatment with both IFNs was significantly higher in SW13 cells, compared with H295 cells. In addition, the proportion of cells in the G2/M phase decreased significantly, compared with control cells in both cell lines. These data suggest that ACC cells exhibit a prolonged stay in S phase and fail to transit into the G2/M phase after treatment with type I IFNs.
In H295 cells the cell cycle analysis revealed an increase in cells with subdiploid DNA content (sub-Go phase) after treat- ment with IFNß1a (100 IU/ml: P < 0.001; 50 IU /ml: P < 0.05) or high-dose IFNa2b (1000 IU/ml: P < 0.05), thereby con- firming the induction of apoptosis by IFNß1a and IFNa2b, as previously shown by the DNA fragmentation analyses.
200
*
T
DNA fragmentation (% of control)
150
*,#
#
T
#
100-
T
T
T
50
0
IFN-B1a (5 IU/ml)
+
Devd-cho (10 μM)
+
letd-cho (10 μM)
Lehd-cho (10 μM)
+
Effects of IFNa2b and IFNßla treatment on IGF-II expression in ACC cells
The expression of IGF-II mRNA was detectable in H295 cells (20 ± 3 × 10° copies mRNA IGF-II per microgram RNA) but was undetectable in SW-13.
In H295 cells, the transcription of IGF-II gene is modulated by incubation with IFNßla. We observed a potent and dose- dependent decrease in the number of copies of IGF-II mRNA after 3 d of treatment with IFNßla (Fig. 5). In contrast, IFNa2b (1000 IU/ml) was unable to modify the expression of IGF-II gene in H295 cells (data not shown). mRNA ex- pression of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase was not affected by treatment with IFNß1a.
Discussion
Type I IFNs (IFNa, -B, -w) are pleiotropic molecules that are able to modulate several processes, including immune response, cell differentiation, cell growth, and antitumor de-
Relative copy number of IGF-II mRNA (mRNA / total RNA (/106))
20-
15
*
10-
5
*
0
Control
IFN-ß1a (1 U/ml)
IFN- ß1a (10 U/ml)
IFN- ß1a (100 U/ml)
fense (11, 12). These cytokines activate a common receptor complex composed of two subunits: IFNAR-1 and IFNAR-2. IFNAR-1 is considered the signal subunit chain. There are three forms of IFNAR-2, which are differentially spliced products of the same gene. IFNAR-2a, the soluble form, acts as a regulator of the free IFNs (21, 22). IFNAR-2b, the short form, binds type I IFN but does not couple to signal trans- duction because it lacks the signal transduction tail (23); IFNAR-2c, the long form, constitutes together with the IFNAR-1 subunit the predominant active form of the type I receptor complex. In the present study, we evaluated tumor suppressive capability of type I IFNs (IFNa2b, IFNß1a) in the human ACC cell lines H295 and SW13. First of all, the ex- pression of mRNA and protein for the active subunits of type I IFN receptor (IFNAR-1 and IFNAR-2c) was demonstrated in H295 and SW13 cells by quantitative RT-PCR and immu- nocytochemistry, respectively. Both IFNs showed a dose- dependent inhibitory effect on cell proliferation. The antitu- mor activity of IFNßla was significantly higher, compared with that of IFNa2b in both cell lines. This appeared to be associated with an increased induction of apoptosis in H295
| Cell line | G0/G1 | S phase | G2/M phase | Sub-G0 | ||||
|---|---|---|---|---|---|---|---|---|
| H295 | SW13 | H295 | SW13 | H295 | SW13 | H295 | SW13 | |
| Treatment control | 61.9 ± 1.5 | 64.3 ± 0.1 | 13.1 ± 0.4 | 15.6 ± 1.4 | 23.8 ± 0.4 | 17.1 ±1.0 | 1.4 ± 0.1 | 0.3 ± 0.1 |
| IFNa2b (1000 IU/ml) | 58.1 ± 1.3 | 60.5 ± 1.8 | 20.0 ± 0.7ª | 27.3 ± 0.6ª | 19.1 ± 0.16 | 12.6 ± 0.36 | 3.5 ± 0.16 | 0.3 ± 0.1 |
| IFNa2b (500 IU/ml) | 62.5 ±1.2 | 60.2 ± 1.7 | 14.8 ± 0.1b | 22.8 ± 0.6€ | 19.7 ± 1.2b | 14.4 ± 1.8 | 2.3 ± 0.6 | 0.4 ± 0.1 |
| IFNß1a (100 IU/ml) | 59.4 ± 4.0 | 59.1 ± 2.4 | 23.9 ± 0.05α | 27.7 ± 1.1ª | 15.7 ± 0.5€ | 12.6 ± 0.86 | 6.0 ± 0.1ª | 0.3 ± 0.1 |
| IFNß1a (50 IU/ml) | 58.8 ± 2.9 | 60.4 ± 0.4 | 15.0 ± 0.16 | 22.3 ± 0.6€ | 21.6 ± 0.4b | 13.6 ± 0.1b | 3.0 ± 0.16 | 0.2 ± 0.1 |
Cell cycle distribution of G0/G1, S, G2/M, and sub-G0 phases in SW13 and H295 cells. Cell cycle distribution was measured after 72 h of incubation with IFNa2b (1000 and 500 IU/ml) or IFNß1a (100 and 10 IU/ml). Each phase is expressed as the percentage of the total cell distribution after treatment.
ª P < 0.001.
b P < 0.05.
c P < 0.01.
cells only and accumulation of cells in S phase in both cell lines. In addition, the maximal inhibition of SW13 cell growth by IFNa2b appeared to be higher than that in H295 cells. This may be related to the absence of an effect of type I IFN on apoptosis and the more prominent role of cell cycle inhibition induced by type I IFNs in SW13 cells, compared with H295 cells, as was observed in the present study. The higher sen- sitivity of both ACC cell lines to IFNßla may be explained by differences in the structure of IFNa and IFNß. There is only 35% sequence identity between both IFNs (24, 25), and IFNß has a higher affinity for the type I IFN receptor, com- pared with IFNa (26).
H295 cells were significantly more sensitive to IFNß1a treatment than SW13 cells. This could be a consequence of the IFN-mediated proapoptotic activity in H295 but not in SW13 cells, as shown by the increase in DNA fragmentation and increase in cells in subGo phase after the treatment with IFNß1a or IFN&2b. Because caspases play an important role in the induction of apoptosis, we evaluated the role of caspases in the induction of DNA fragmentation after IFNß1a treatment in H295 cells. In the classical model, caspases are divided into initiator caspases (such as caspase-8, -9) and executioner caspases (caspase-3, -6, -7), according to their function and sequence of activation. There are at least two major apoptotic pathways. The first involves the death receptor or extrinsic pathway that is initiated by TNF receptor family members that recruit adaptor and sig- naling molecules to assemble the death-inducing signaling complex. This complex leads to activation of caspase-8 and/or -10. An alternative mitochondrial pathway involves activation of caspase-9 on recruitment to the mitochondria by cytochrome c and apoptosis protease activation factor-1. More downstream, the initiator caspases lead to the activa- tion of executioner caspase-3, -6, and -7, which in turn cleave specific proteins resulting in the DNA fragmentation (27, 28). In H295 cells the stimulation of DNA fragmentation by IFNßla is completely blocked by a specific inhibitor of caspase-3, whereas it is only partially counteracted by the use of specific inhibitors for caspase-8 and -9. Therefore, IFNßla seems to induce apoptosis through both the extrinsic and mitochondrial pathways in ACC.
At present, two recombinant IFNßs (IFNß1a and IFNß1b) are available as registered drugs. IFNßla is produced in mammalian cells, with an amino acid sequence and glyco- sylation identical with that of natural IFNß. In contrast, IFNß1b is produced in Escherichia coli bacteria and is not glycosylated. Furthermore, IFNß1b has one amino acid se- quence different from the native form of human IFNß. We observed in H295 cells that the effects of IFNßla on cell proliferation inhibition and stimulation of DNA fragmenta- tion were much more potent, compared with IFNß1b. This is in agreement with the observation that the absence of glycosylation in IFNß1b can reduce the biological activity (29). Because IFN is currently in clinical use for renal cell carcinoma and in trials for other malignancies, studies com- paring the effects of IFNß and IFNa in tumor cell models other than ACC will help determine whether the potent cytostatic and cytotoxic effects of IFNß are also present in other tumor models, rather than being restricted to selected tumor types.
IGF-II is considered to be an important growth factor in ACC. Therefore, we also evaluated the effects of IFNßla on IGF-II mRNA expression. H295, but not SW13, cells ex- pressed IGF-II. In H295 cells, IGF-II mRNA was inhibited in a dose-dependent manner by IFNß1a after 3 d of incubation. This effect was not observed after incubation with IFN&2b. It is well known that in ACC an up-regulation of the IGF-II system represents a main pathway involved in the patho- genesis through inducing proliferation and inhibiting apo- ptosis (30). This could partially explain why only in H295 cells is IFNßla able to induce apoptosis.
In conclusion, this is the first study showing that type I IFNs, particularly IFNß1a, are powerful inhibitors of prolif- eration of the H295 and SW13 human ACC cells. This effect is correlated with the induction of apoptosis and/or a cell cycle arrest in the S phase. Our findings support the clinical attractiveness to use IFNß in the treatment of ACC because of its ability to inhibit cell proliferation and stimulate apo- ptosis already at very low concentrations (1-10 IU/ml). In vivo, 12.3 IU/ml is the maximal IFNß serum concentration reported in healthy subjects after sc administration of this cytokine (31). In addition, recent studies report on the de- velopment of PEGylated IFNß with improved pharmacoki- netic properties, compared with the unmodified protein (32). Finally, an important finding of the present study is that IFNßla potently inhibits the expression of IGF-II at the tran- scriptional level. Further studies using primary ACC cul- tures as well as in vivo studies are required to evaluate these promising tumor suppressive effects, using IFNßla not only as a single compound but also in combination with the cur- rently used cytostatic drug mitotane.
Acknowledgments
Received March 21, 2006. Accepted August 7, 2006.
Address all correspondence and requests for reprints to: Leo J. Hof- land, Department of Internal Medicine, Erasmus Medical Center, Room Ee53ob, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: 1.hofland@erasmusmc.nl.
This study was supported by Grant 122 from the Vanderes Foundation. Disclosure statement: The authors have nothing to declare.
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