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Inhibition of hepatoma cell growth by a steroid anti-hormone
Cancer Letters, 4lf1988) 333- 343 Elsevier Scientific Publishers Ireland
333 Ltd.
INHIBITION OF HEPATOMA ANTI-HORMONE
SYLVETTE
CHASSEROT-GOLAZ
Institut de Biologic Molkculaire
CELL
and GISKLE et Cellulaire
GROWTH
BY A STEROID
BECK
du CNRS 15, Rue Descartes,
F-67000, Strasbourg
ilhncel (Received 31 March 1988) (Revised version received 13 June 1988) (Accepted 16 June 1988)
SUMMARY
The antiproliferative effect of the potent antiglucocorticoid and antiprogestin RU38486, an 11/Ssubstituted nor-steroid, was investigated on the HTC cell line. Its action clearly visualized in absence of serum, is reversible. Dexamethasone does whereas progesterone does not protect the cells against the cytostatic effect. Thus, the latter rather depends on the antiglucocorticoidand not on the antiprogestin-function of RU38486. Taking into account the antiproliferative action on HTC cells together with the antiglucocorticoid behaviour in hepatocytes and hepatomas, our results suggest that RU38486 might represent a potential drug for liver tumour therapy. Key
words:
Antiglucocorticoid
steroid; HTC cells; Cytostatic
effect.
INTRODUCTION
In the endocrine therapy of breast cancer, various hormonal agents have been used such as estrogens and anti-estrogens, androgens and anti-androgens, glucocorticoids. A steroid molecule with progestins or potent antiprogestational and antiglucocorticoid activities [22], initially indicated by its code name RU38486 and currently named Mifepristone, is the first antiprogestin or antiglucocorticoid available for therapy [16]. A preliminary clinical trial with RU38486 in progestin receptor positive breast cancer cells suggests a good long-term tolerance in the metastatic disease [ll]. In the case of Cushing’s syndrome the hyperproduction of glucocorticoids could also be antagonized by administration of RU38486 [15]. Correspondence
to: G. Beck.
03043835/88/$03.50 Published and Printed
0 1988 Elsevier in Ireland
Scientific
Publishers
Ireland Ltd.
334
The mechanism by which antihormones inhibit cell proliferation is very complex, as shown in breast cancer cells for the two sex steroid antagonists, tamoxifen and RU38486. For tamoxifen, which has frequently been used to successfully treat with estrogen dependent cancers, three series of effects have been reviewed by Bardon et al. [l]: (il
a cytostatic effect by which estrogen-receptor positive cells are blocked by the drug and which might be due to the decreased production of growth factor(s) induced by estrogen; (ii) a direct receptor mediated inhibition by a hitherto unknown mechanism; (iii) a rapid cytotoxic effect triggered by much higher concentrations (> 4 PM tamoxifenl in MCF 7 breast cancer cells. Whether RU 38486 acts through complementary mechanisms remains to be determined. The antiprogestin may act as an anti-hormone via the progesterone receptor and also as an antimitogenic drug by a mechanism presently unknown; an action related to certain growth factors as described for breast cancer cells may also be hypothesized [8]. In this paper the antiproliferative effect of RU38486 was investigated on a hepatoma cell line. HTC cells were chosen for two reasons: their growth is not hormone-dependent but these tumour cells contain a high level of glucocorticoid receptors and remain glucocorticoid-sensitive for the synthesis of a limited amount of proteins and especially for tyrosine aminotransferase [21]; moreover, we observed that RU38486 was rapidly metabolized in hepatocytes whereas this steroid undergoes no or only a minute degradation in HTC cells [4]. We next investigated if high concentrations of the steroid would selectively inhibit the growth of the tumour cells. This ability points to the possibility of using the antiglucocorticoid as a potent drug in the therapy of hepatocarcinoma in order to avoid subsequent partial hepatectomy. We successfully asked two questions. (i) Does RU38486 affect HTC cell growth? (ii) Is there a mediation by glucocorticoid receptors or by other cellular structures? MATERIALS
AND METHODS
Steroids and anti-estrogen Dexamethasone, RU38486 [17/3-hydroxy-11/I-(4-dimethylaminophenyl)-17a(lpropynyll-estra-4,9-dien-&one] and tamoxifen were kindly provided by RousselUclaf (Romainville, France). Steroids and the anti-estrogen were added to the culture medium in ethanolic solution. The final concentration of ethanol was 0.1% and an equivalent volume of solvent was added to the control. Cell culture HTC cells are derived from a Morris hepatoma 7288C [20]; they were grown in suspension culture with magnetic stirring (100 rev./mini in Swim’s S-77 medium (Gibco) supplemented with 10% newborn calf serum (Gibco) or 2% serum substitute. Ultroser SF (IBF).
335 Measurement
of the antiproliferative
effect
HTC cells were maintained in suspension in Swim’s S-77 medium containing 2% serum substitute Ultroser SF at least 5 h before starting the experiment. Cells were centrifuged at 600 x g for 5 min; suspension cultures of 50 ml (1.5 x lo5 cells/ml) were grown in each flask. The cell growth was evaluated every 24 h during 3 or 5 days by counting the cells with a Neubauer microcytometer; the number of living cells was determined by the Trypan Blue exclusion test. Experiments were run 3 times in duplicate. Extraction
of the lipid fraction
from newborn
calf serum
Serum (100 ml) was centrifuged for 48 h in 30 ml polycarbonate tubes at 105,000 x g. The upper fraction (l/3 of the content of the tube) was collected and the lipids were extracted by chloroform/methanol (2:1, v/v) during 40 min by sonication. The organic solvent was evaporated; the lipids were dissolved in 3 ml ethanol (50 mglml); the composition of the lipid fraction was: cholesterol 68 mM; triglycerides 14 mM; phospholipids 32 mM. RESULTS Antipro&ferative
effect of RI738486
on HTC cells
The influence of different concentrations of RU38486 on the growth of HTC cells was studied either in medium complemented with calf serum or in a defined medium containing 2% serum substitute Ultroser SF (Fig. 11. The concentrations tested were below 10s4M since above that, synthetic steroids become toxic [6]. In the presence of serum, RU38486 (40 PM) exerts no significant inhibition of cell growth whereas in defined medium a complete inhibition of cell growth was observed at a 20 FM concentration. The addition of twice or four times more RU38486 does not bring much change in the effect observed. No cell lysis occurs, as the Trypan Blue exclusion test shows only a small amount of coloured dead cells and no cell debris. Therefore we assume that the antagonist exerts a cytostatic effect rather than a cytotoxic action. Thereafter, we studied the reversibility of the effect of 2 concentrations of RU38486 (Fig. 21.When RU38486 (20 FM) has been in contact with the cells for a 24-h period, we observed a total recovery of cell growth without any latency as soon as the steroid has been washed out (Fig. 2Al. After a 48-h contact with the steroid, the recovery of cell growth still occurred, but with a 24-h latency. By adding a higher dose of steroid (40 PM), there is still a reversibility which happens after a slight decrease in cell number and an increase in the lag time (Fig. 2Bl. It can be assumed, that after contact with a high dose of steroid, the cells concentrate the drug and some damage is brought about; the viability drops slightly but most of the cells will just need more time to recover their regular growth rate. These results show that the cytostatic effect exerted by RU38486 on HTC cells can be reversed even after exposure to a relatively high dose (40 PM) and during quite a long period (48 hl.
1o--70
1
2
3
4
Time (days) Fig. 1. Antiproliferative effect of RU38486. HTC cells were grown in absence 6) or in presence of serum (ii). 6) HTC cells were cultured in defined medium and dispatched in several subcultures in order to measure, as a function of the incubation time, the effect of different concentrations of RU38486: 20 pM (A), 40 pM (0) or 80 PM (0); control culture ( x ). (ii) HTC cells were grown in medium complemented with loo/o newborn calf serum and treated with a 40 pM concentration of RU38486 @); control culture (m). The cell growth was evaluated every 24 h during 3 days. The data presented correspond to a typical experiment repeated at least 3 times.
Interaction of specific binding sites In order to understand the mechanism of the antiproliferative effect exerted by RU38486, we investigated whether specific hormonal receptors were involved. RU38486 exerts its glucocorticoid receptor blocking activity without any agonist effect as proven by both in vitro and in vivo experiments [9,13]. Thus, we investigated if an agonist, either a glucocorticoid (dexamethasonel or a progestin (progesterone), was able to protect cells against the cytostatic action of the antihormone by exerting an occupancy of the binding sites. We followed cell growth during 3 days after having treated the cells with RU38486 and dexamethasone or RU38486 and progesterone (Fig. 31. We observed that dexamethasone alone did not affect cell growth; therefore the antiproliferative effect of RU38486 is specific to this molecule and is not a general feature of synthetic steroids. Moreover, the presence of dexamethasone protects cells from the cytostatic effect exerted by RU38486; the protection was enhanced by increasing the concentration of dexamethasone from 20
VI-
P-
w-
N-
t
w
Cell concentration
(~10~ cells/ml) G
Cell concentration 8
(~10~ cells/ml) E
1
3
2
4
Time (days)
,
I
100 1
10
I
I
I
0
1
2
I 3
4
Time (days) Fig. 3. Influence of dexamethasone and progesterone on the inhibitory effect of RU38486. (Al HTC cells were grown in defined medium and treated either with dexamethasone (40 PM, 01 or simultaneously with RU38486 (20 FM) and two concentrations of dexamethasone: 20 PM (01.40 PM (Al. (Bl HTC cells were treated either with progesterone (20 PM, +l or simultaneously (Al with RU38486 (20 PM) and progesterone (20 PM). Control, cells untreated ( x ), or treated (Dl by RU38486 alone (20 PM). The data presented correspond to a typical experiment as described above.
339
to 40 PM, nevertheless we did not observe a total reversion of the inhibitory effect (Fig. 3Al. In contrast progesterone does not hinder the effect of RU38486 but rather increases the inhibition of growth. This result is not surprising since progesterone already slightly affects cell growth (Fig. 3BL Thus the progestin binding sites present in HTC cells do not seem to be involved in the cytostatic effect of RU38486. Prevention
of the cytostatic
effect ofRU38486
by the lipidfraction
of serum
Considering that a high dose of RU38486 (over 10 PM) is necessary to observe the antiproliferative effect and that this phenomenon is only observed in defined culture medium containing no calf serum (Fig. 11, it is reasonable to assume the existence of an additive mechanism which does not involve the glucocorticoid receptor. A variety of potentially toxic compounds exert a much higher inhibitory effect on growth in serum-deprived conditions. It has already been shown that the lipids and lipoproteins of the serum play an essential role in the expression of the cytotoxic activity of hydroxysterols in HTC cells [17]. The lipid fraction of the serum was extracted as described in Materials and Methods. When cells were incubated with RU38486 and in the presence of increasing concentrations of the lipid fraction, we observed a regular decrease of the antiproliferative effect. The addition of the lipid fraction to the control did not change the cell growth rate. A strong antagonism was observed in the presence of 100 d of lipid extract, i.e. the amount of lipids corresponding to a medium complemented with loo/o serum (Fig. 41. The effect of RU38486 on cell growth was compared to the one exerted by tamoxifen. Although tamoxifen does not belong to the steroid family, there is a similarity in structure between tamoxifen and RU38486 and a correlation between these two molecules has been shown for their antiproliferative action in breast cancer cells [16]. We investigated if the lipid fraction was also able to reverse the effect of tamoxifen on HTC cells (Fig. 41.A 20 PM concentration of tamoxifen exerted a stronger antiproliferative effect than RU38486. Taking into account the high amount used [l] and the absence of estrogen receptors in HTC cells but the presence of anti-estrogen binding sites (AEBS) in rat liver cells [2], tamoxifen may exert its effect by interacting with the AEBS [18]. The addition of serum lipid fraction to the cells treated with tamoxifen partially abolishes the cytotoxic effect of tamoxifen but the reversion observed is less important than for RU38486. Thus, for RU38486 as for tamoxifen, at least 2 mechanisms must be put forward in order to explain the antiproliferative effect; one may involve specific hormone receptors whereas the other involves different cellular structures. DISCUSSION
The antihormone RU38486 exerts an antiproliferative effect on hepatoma cells in vitro. This effect can be reversed; it is rather cytostatic than cytotoxic.
400
-
300
200
100
0 5
-
Control
RU38486
Tamoxifen
Fig. 4. Influence of the lipid fraction of serum (LF) on the inhibition of cellular gro h exerted by RU38486 or tamoxifen. The cells were grown in defined medium and treated by RU38486 (40 FM) or by tamoxifen (40 FM) alone (El1 or simultaneously with 50 4 (EN or 100 4 (Hl of the lipid fraction of the serum. After 3 days of contact, the cell concentration was determined. In the control, the cells were not treated (01 or received only 100 d of lipid fraction (ml. Results are the mean of triplicate determinations.
The activity of RU38486 depends on its antiglucocorticoid function since only dexamethasone and not progesterone counteracts the inhibition of cell growth. It is well demonstrated that RU38486 has progesterone- and glucocorticoidreceptor blocking abilities which may be involved in the inhibition of cell growth, depending on the origin of the tumour cell. For instance, in pituitary tumours which contain glucocorticoid- and progestin-receptors, the activity of RU38486 is mediated by both [lo] whereas in breast cancer cells the antiproliferative effect of RU38486 is only mediated by progestin receptors despite the presence of glucocorticoid receptors [l]. It can be assumed that, once bound to a specific hormone receptor, RU38486 may trigger the antiproliferative effect either by an induction of growth factor inhibitors or by decreasing the production of mitogens through other mechanisms. [19]. A complementary mechanism must be considered in order to explain that the degree of growth inhibition depends on the presence or the absence of serum. In the model used, cholesterol or serum lipoproteins seem to prevent the activity of RU38486. A similar observation has yet been described for other substances like estrogens [14] or hydroxysterols [17]. The lipids do not trap the
341
steroid since other biological activities of RU38486, like the inhibition of the synthesis of tyrosine aminotransferase, are not hindered by the presence of lipids [3]. For hydroxysterols it was hypothesized that the presence of serum lipids induces some change in the fluidity of the cellular membrane [7]. An action of RU38486 on the membrane could be imagined especially since it has been claimed that tamoxifen could act via the AEBS which may be membrane associated [12]. Moreover, we observed that lipids partially prevent the inhibitory effect of tamoxifen. It must be emphasized that RU38486 and tamoxifen have a similar side chain added to the structure of the agonist; this substitution might exert a common role for the expression of the antiproliferative activity [16]. For tamoxifen, the integrity of the side chain has been shown to be essential for the binding to AEBS [18]. It would be interesting to verify if RU38486 is able to interact with those sites and find out about the role of the 11/3-side chain in the expression of the antiproliferative effect of RU38486. We observed that. this part of the molecule undergoes a rapid metabolization by the cytochrome P-450 enzyme complex [5]. Thus, the action of RU38486 in HTC cells would appear to be dependent on the presence of glucocorticoid receptors and the absence of cytochrome P-450 isoenzymes. These two points may serve as a guide in the development of the therapeutical use of this molecule. These parameters will also be important to further our study on other hepatomas, either ascitic rat tumours in vivo or human hepatoma tumour cells in primary cultures. The treatment of advanced breast cancer by RU38486 has been attempted and this molecule is well tolerated in long term therapy; moreover, it also has a positive influence on pulmonary and cutaneous metastasis [ll]. The antiproliferative action on HTC cells is an interesting finding which must be considered with the other properties of RU38486: the antiglucocorticoid potency and the behaviour in different hepatoma cells or hepatocytes related to the cytochrome P-450 isoenzyme pattern [5]. Taken together, our results favour the idea that RU38486 might represent a possible drug for the treatment of hepatocarcinomas. ACKNOWLEDGEMENTS
This work has been supported by a grant from the Association pour la Recherche Contre le Cancer (no. 64961. S. C.-G. was a recipient from the Fondation pour la Recherche Medicale. We are grateful to D. Philibert, Roussel UCLAF Comp., for his help and interest and to Dr. J.P. Beck, U.L.P. Strasbourg, for many stimulating discussions. REFERENCES 1
2
Bardon, S., Vignon, F., Montccurrier, P. and Rochefort, H. (1987) Steroid receptor-mediated cytotoxicity of an antiestrogen and antiprogestin in breast cancer cells. Cancer Res., 47,14411448. Blankenstein, M.A., Besselink, C.M.L.T. and Thijssen, J.H.H. (198’7) Anti-estrogen binding
342
3 4
5
6 7
8
9
10
11
12
13
14
15
16 17
18 19
sites (AEBSl in human and rat breast cancer and liver tissues. J. Steroid Bioehem., 28 Supplement, Abstract no. D.024. Chasserot-Golaz, S. and Beck, G. (19841 An approach to the mechanism of the potent antisteroid RU38486. J. Steroid Biochem., 21,585- 591. Chasserot-Golaz, S., Flaig, C. and Beck, G. (1985) Steroid metabolism in normal and in transformed liver cells. Relation with glucocorticoid antagonism. Cancer Biochem. Biophys., 8,95101. Chasserot-Golaz, S. and Beck, G. (19861 Metabolism and antiproliferative effect of the glucoeorticoid antagonist RU38486 in cultured liver and hepatoma cells. J. Steroid Biochem., 24,423426. Dao-Pei Huang, Schwartz, C.E., Jen-Fu Chiu and Gok, J.R. (19841 Dexamethasone inhibition of rat hepatoma growth and o-fetal proteins synthesis. Cancer Res., 44,2976-2980. Hietter, H., Trifilieff, E., Riehert, L., Beck, J.P., Luu, B. and Ourisson, G. (1984) Antagonistic action of cholesterol towards the toxicity of hydroxysterols on cultured hepatoma cells. Biochem. Biophys. Res. Commun., 120,657- 664. Knabbe, C., Lippmann, M.E., Wakefield, L.M., Flanders, K.C., Kasid, A., Derynck, R. and Dikson, R.B. (19871 Evidence that transforming growth factor /3 is a hormonally regulated negative growth factor in human breast cancer cells. Cell, 48,417- 428. Lamberts, S.W.J., Uitterlinden, P., Bons, E.G. and Verleun, T. (1985) Comparison of the actions of RU38486 and Megestrol acetate in the model of a transplantable adenocorticotropinand prolactin-secreting rat pituitary tumor. Cancer Res., 45,1015- 1019. Lamberts, S.W.J. (19871 RU38486, a progestin and glucocorticoid antagonist in the treatment of experimental pituitary tumors. In: Hormonal Manipulation of Cancer: Peptides, Growth Factors, and New (Anti) Steroidal Agents, pp. 31-38. Editor: J.G.M. Klijn. Raven Press, New York. Maudelonde, T., Romieu, G., Ulmann, A., Pujol, H., Grenier, J., Khalaf, S., Cavalie, G. and Rochefort, H. (19871 First clinical trial on the use of the antiprogestin RU38486 in advanced breast cancer. In: Hormonal Manipulation of Cancer: Peptides, Growth Factors, and New (Anti) Steroidal Agents, pp. 55-59. Editor: J.G.M. Klijn. Raven Press, New York. Miler, M.A. and Katzenellenbogen, B.S. (19831 Characterization and quantitation of antiestrogen binding sites in estrogen receptor-positive and -negative human breast cancer cell lines. Cancer Res., 43,3094-3100. Moguilewsky, M. and Philibert, D. (19841 RU38486: a potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. J. Steroid Biochem., 20,271- 276. Mummery, C.L., Van Den Brinks, R., Van Der Saag, P.T. and De Paat, SW. (1983) Screening for cytotoxicity in neuroblastoma cells. I Dependence of growth inhibition on the presence of serum. Toxicol. Lett., 18,201- 209. Nieman, L.K., Chrousos, G.P., Spitz, I., Nisula, B.C., Cutler, G.B., Merriam, G.R., Bardin, C.W. and Loriaux, D.L. (19841 Successful treatment of Cushing’s syndrome with glucocorticoid antagonist RU38486. In: The 7th International Congress of Endocrinology. Quebec City, Canada, Abstract no. 1718. Rochefort, H. (19871 Do antiestrogens and antiprogestins act as hormone antagonists or receptor-targeted drugs in breast cancer? Trends Pharmacol. Sci. 8,126- 128. Richert, L., Bergmann, C., Beck, J.P., Rong, S., Luu, B. and Ourisson, G. (1983) The importance of the serum lipoproteins in the cytolytic action of 7/I-hydroxycholesterol on cultured hepatoma cells. Biochem. Biophys. Res. Commun., 117.851-858. Sutherland, R.L., Murphy, L.C., Ming San Foo, Green, M.D. and Whybourne, A.M. (1980) Highaffinity anti-estrogen binding site distinct from estrogen receptor. Nature, 288,273 - 275. Vignon. F., Bardon, S., Chalbos. D., Derocq, D., Gill, P. and Rochefort, H. (1987) Antiproliferative effect of progestins and antiprogestins in human breast cancer cells. In: Hormonal Manipulation of Cancer: Peptides, Growth Factors, and New (Anti1 Steroidal Agents, pp. 47-54. Editor: J.G.M. Klijn. Raven Press, New York.
343 20
21
22
Thompson, E.B., Tomkins, G.M. and Curran, J.F. (19661 Induction of tyrosine aminotransferase by steroid hormones in a newly established tissue culture cell line. Proc. Natl. Acad. Sci. USA, 56.296 - 303. Tomkins, G.M., Gelehrter, T.D., Granner, D.K., Martin, D.W., Samuels, H.H. and Thompson, E.B. (19691 Expression of mammalian genes may be controlled by repressors acting on the translation of messenger RNA. Science, 166,14’76- 1480. Philibert, D., Dereadt, R. and Teutsch, G. (19811 RU38486: a potent antiglucocorticoid in viwo. In: The VIII International Congress of Pharmacology, Tokyo, Abstract no. 1463.
333 Ltd.
INHIBITION OF HEPATOMA ANTI-HORMONE
SYLVETTE
CHASSEROT-GOLAZ
Institut de Biologic Molkculaire
CELL
and GISKLE et Cellulaire
GROWTH
BY A STEROID
BECK
du CNRS 15, Rue Descartes,
F-67000, Strasbourg
ilhncel (Received 31 March 1988) (Revised version received 13 June 1988) (Accepted 16 June 1988)
SUMMARY
The antiproliferative effect of the potent antiglucocorticoid and antiprogestin RU38486, an 11/Ssubstituted nor-steroid, was investigated on the HTC cell line. Its action clearly visualized in absence of serum, is reversible. Dexamethasone does whereas progesterone does not protect the cells against the cytostatic effect. Thus, the latter rather depends on the antiglucocorticoidand not on the antiprogestin-function of RU38486. Taking into account the antiproliferative action on HTC cells together with the antiglucocorticoid behaviour in hepatocytes and hepatomas, our results suggest that RU38486 might represent a potential drug for liver tumour therapy. Key
words:
Antiglucocorticoid
steroid; HTC cells; Cytostatic
effect.
INTRODUCTION
In the endocrine therapy of breast cancer, various hormonal agents have been used such as estrogens and anti-estrogens, androgens and anti-androgens, glucocorticoids. A steroid molecule with progestins or potent antiprogestational and antiglucocorticoid activities [22], initially indicated by its code name RU38486 and currently named Mifepristone, is the first antiprogestin or antiglucocorticoid available for therapy [16]. A preliminary clinical trial with RU38486 in progestin receptor positive breast cancer cells suggests a good long-term tolerance in the metastatic disease [ll]. In the case of Cushing’s syndrome the hyperproduction of glucocorticoids could also be antagonized by administration of RU38486 [15]. Correspondence
to: G. Beck.
03043835/88/$03.50 Published and Printed
0 1988 Elsevier in Ireland
Scientific
Publishers
Ireland Ltd.
334
The mechanism by which antihormones inhibit cell proliferation is very complex, as shown in breast cancer cells for the two sex steroid antagonists, tamoxifen and RU38486. For tamoxifen, which has frequently been used to successfully treat with estrogen dependent cancers, three series of effects have been reviewed by Bardon et al. [l]: (il
a cytostatic effect by which estrogen-receptor positive cells are blocked by the drug and which might be due to the decreased production of growth factor(s) induced by estrogen; (ii) a direct receptor mediated inhibition by a hitherto unknown mechanism; (iii) a rapid cytotoxic effect triggered by much higher concentrations (> 4 PM tamoxifenl in MCF 7 breast cancer cells. Whether RU 38486 acts through complementary mechanisms remains to be determined. The antiprogestin may act as an anti-hormone via the progesterone receptor and also as an antimitogenic drug by a mechanism presently unknown; an action related to certain growth factors as described for breast cancer cells may also be hypothesized [8]. In this paper the antiproliferative effect of RU38486 was investigated on a hepatoma cell line. HTC cells were chosen for two reasons: their growth is not hormone-dependent but these tumour cells contain a high level of glucocorticoid receptors and remain glucocorticoid-sensitive for the synthesis of a limited amount of proteins and especially for tyrosine aminotransferase [21]; moreover, we observed that RU38486 was rapidly metabolized in hepatocytes whereas this steroid undergoes no or only a minute degradation in HTC cells [4]. We next investigated if high concentrations of the steroid would selectively inhibit the growth of the tumour cells. This ability points to the possibility of using the antiglucocorticoid as a potent drug in the therapy of hepatocarcinoma in order to avoid subsequent partial hepatectomy. We successfully asked two questions. (i) Does RU38486 affect HTC cell growth? (ii) Is there a mediation by glucocorticoid receptors or by other cellular structures? MATERIALS
AND METHODS
Steroids and anti-estrogen Dexamethasone, RU38486 [17/3-hydroxy-11/I-(4-dimethylaminophenyl)-17a(lpropynyll-estra-4,9-dien-&one] and tamoxifen were kindly provided by RousselUclaf (Romainville, France). Steroids and the anti-estrogen were added to the culture medium in ethanolic solution. The final concentration of ethanol was 0.1% and an equivalent volume of solvent was added to the control. Cell culture HTC cells are derived from a Morris hepatoma 7288C [20]; they were grown in suspension culture with magnetic stirring (100 rev./mini in Swim’s S-77 medium (Gibco) supplemented with 10% newborn calf serum (Gibco) or 2% serum substitute. Ultroser SF (IBF).
335 Measurement
of the antiproliferative
effect
HTC cells were maintained in suspension in Swim’s S-77 medium containing 2% serum substitute Ultroser SF at least 5 h before starting the experiment. Cells were centrifuged at 600 x g for 5 min; suspension cultures of 50 ml (1.5 x lo5 cells/ml) were grown in each flask. The cell growth was evaluated every 24 h during 3 or 5 days by counting the cells with a Neubauer microcytometer; the number of living cells was determined by the Trypan Blue exclusion test. Experiments were run 3 times in duplicate. Extraction
of the lipid fraction
from newborn
calf serum
Serum (100 ml) was centrifuged for 48 h in 30 ml polycarbonate tubes at 105,000 x g. The upper fraction (l/3 of the content of the tube) was collected and the lipids were extracted by chloroform/methanol (2:1, v/v) during 40 min by sonication. The organic solvent was evaporated; the lipids were dissolved in 3 ml ethanol (50 mglml); the composition of the lipid fraction was: cholesterol 68 mM; triglycerides 14 mM; phospholipids 32 mM. RESULTS Antipro&ferative
effect of RI738486
on HTC cells
The influence of different concentrations of RU38486 on the growth of HTC cells was studied either in medium complemented with calf serum or in a defined medium containing 2% serum substitute Ultroser SF (Fig. 11. The concentrations tested were below 10s4M since above that, synthetic steroids become toxic [6]. In the presence of serum, RU38486 (40 PM) exerts no significant inhibition of cell growth whereas in defined medium a complete inhibition of cell growth was observed at a 20 FM concentration. The addition of twice or four times more RU38486 does not bring much change in the effect observed. No cell lysis occurs, as the Trypan Blue exclusion test shows only a small amount of coloured dead cells and no cell debris. Therefore we assume that the antagonist exerts a cytostatic effect rather than a cytotoxic action. Thereafter, we studied the reversibility of the effect of 2 concentrations of RU38486 (Fig. 21.When RU38486 (20 FM) has been in contact with the cells for a 24-h period, we observed a total recovery of cell growth without any latency as soon as the steroid has been washed out (Fig. 2Al. After a 48-h contact with the steroid, the recovery of cell growth still occurred, but with a 24-h latency. By adding a higher dose of steroid (40 PM), there is still a reversibility which happens after a slight decrease in cell number and an increase in the lag time (Fig. 2Bl. It can be assumed, that after contact with a high dose of steroid, the cells concentrate the drug and some damage is brought about; the viability drops slightly but most of the cells will just need more time to recover their regular growth rate. These results show that the cytostatic effect exerted by RU38486 on HTC cells can be reversed even after exposure to a relatively high dose (40 PM) and during quite a long period (48 hl.
1o--70
1
2
3
4
Time (days) Fig. 1. Antiproliferative effect of RU38486. HTC cells were grown in absence 6) or in presence of serum (ii). 6) HTC cells were cultured in defined medium and dispatched in several subcultures in order to measure, as a function of the incubation time, the effect of different concentrations of RU38486: 20 pM (A), 40 pM (0) or 80 PM (0); control culture ( x ). (ii) HTC cells were grown in medium complemented with loo/o newborn calf serum and treated with a 40 pM concentration of RU38486 @); control culture (m). The cell growth was evaluated every 24 h during 3 days. The data presented correspond to a typical experiment repeated at least 3 times.
Interaction of specific binding sites In order to understand the mechanism of the antiproliferative effect exerted by RU38486, we investigated whether specific hormonal receptors were involved. RU38486 exerts its glucocorticoid receptor blocking activity without any agonist effect as proven by both in vitro and in vivo experiments [9,13]. Thus, we investigated if an agonist, either a glucocorticoid (dexamethasonel or a progestin (progesterone), was able to protect cells against the cytostatic action of the antihormone by exerting an occupancy of the binding sites. We followed cell growth during 3 days after having treated the cells with RU38486 and dexamethasone or RU38486 and progesterone (Fig. 31. We observed that dexamethasone alone did not affect cell growth; therefore the antiproliferative effect of RU38486 is specific to this molecule and is not a general feature of synthetic steroids. Moreover, the presence of dexamethasone protects cells from the cytostatic effect exerted by RU38486; the protection was enhanced by increasing the concentration of dexamethasone from 20
VI-
P-
w-
N-
t
w
Cell concentration
(~10~ cells/ml) G
Cell concentration 8
(~10~ cells/ml) E
1
3
2
4
Time (days)
,
I
100 1
10
I
I
I
0
1
2
I 3
4
Time (days) Fig. 3. Influence of dexamethasone and progesterone on the inhibitory effect of RU38486. (Al HTC cells were grown in defined medium and treated either with dexamethasone (40 PM, 01 or simultaneously with RU38486 (20 FM) and two concentrations of dexamethasone: 20 PM (01.40 PM (Al. (Bl HTC cells were treated either with progesterone (20 PM, +l or simultaneously (Al with RU38486 (20 PM) and progesterone (20 PM). Control, cells untreated ( x ), or treated (Dl by RU38486 alone (20 PM). The data presented correspond to a typical experiment as described above.
339
to 40 PM, nevertheless we did not observe a total reversion of the inhibitory effect (Fig. 3Al. In contrast progesterone does not hinder the effect of RU38486 but rather increases the inhibition of growth. This result is not surprising since progesterone already slightly affects cell growth (Fig. 3BL Thus the progestin binding sites present in HTC cells do not seem to be involved in the cytostatic effect of RU38486. Prevention
of the cytostatic
effect ofRU38486
by the lipidfraction
of serum
Considering that a high dose of RU38486 (over 10 PM) is necessary to observe the antiproliferative effect and that this phenomenon is only observed in defined culture medium containing no calf serum (Fig. 11, it is reasonable to assume the existence of an additive mechanism which does not involve the glucocorticoid receptor. A variety of potentially toxic compounds exert a much higher inhibitory effect on growth in serum-deprived conditions. It has already been shown that the lipids and lipoproteins of the serum play an essential role in the expression of the cytotoxic activity of hydroxysterols in HTC cells [17]. The lipid fraction of the serum was extracted as described in Materials and Methods. When cells were incubated with RU38486 and in the presence of increasing concentrations of the lipid fraction, we observed a regular decrease of the antiproliferative effect. The addition of the lipid fraction to the control did not change the cell growth rate. A strong antagonism was observed in the presence of 100 d of lipid extract, i.e. the amount of lipids corresponding to a medium complemented with loo/o serum (Fig. 41. The effect of RU38486 on cell growth was compared to the one exerted by tamoxifen. Although tamoxifen does not belong to the steroid family, there is a similarity in structure between tamoxifen and RU38486 and a correlation between these two molecules has been shown for their antiproliferative action in breast cancer cells [16]. We investigated if the lipid fraction was also able to reverse the effect of tamoxifen on HTC cells (Fig. 41.A 20 PM concentration of tamoxifen exerted a stronger antiproliferative effect than RU38486. Taking into account the high amount used [l] and the absence of estrogen receptors in HTC cells but the presence of anti-estrogen binding sites (AEBS) in rat liver cells [2], tamoxifen may exert its effect by interacting with the AEBS [18]. The addition of serum lipid fraction to the cells treated with tamoxifen partially abolishes the cytotoxic effect of tamoxifen but the reversion observed is less important than for RU38486. Thus, for RU38486 as for tamoxifen, at least 2 mechanisms must be put forward in order to explain the antiproliferative effect; one may involve specific hormone receptors whereas the other involves different cellular structures. DISCUSSION
The antihormone RU38486 exerts an antiproliferative effect on hepatoma cells in vitro. This effect can be reversed; it is rather cytostatic than cytotoxic.
400
-
300
200
100
0 5
-
Control
RU38486
Tamoxifen
Fig. 4. Influence of the lipid fraction of serum (LF) on the inhibition of cellular gro h exerted by RU38486 or tamoxifen. The cells were grown in defined medium and treated by RU38486 (40 FM) or by tamoxifen (40 FM) alone (El1 or simultaneously with 50 4 (EN or 100 4 (Hl of the lipid fraction of the serum. After 3 days of contact, the cell concentration was determined. In the control, the cells were not treated (01 or received only 100 d of lipid fraction (ml. Results are the mean of triplicate determinations.
The activity of RU38486 depends on its antiglucocorticoid function since only dexamethasone and not progesterone counteracts the inhibition of cell growth. It is well demonstrated that RU38486 has progesterone- and glucocorticoidreceptor blocking abilities which may be involved in the inhibition of cell growth, depending on the origin of the tumour cell. For instance, in pituitary tumours which contain glucocorticoid- and progestin-receptors, the activity of RU38486 is mediated by both [lo] whereas in breast cancer cells the antiproliferative effect of RU38486 is only mediated by progestin receptors despite the presence of glucocorticoid receptors [l]. It can be assumed that, once bound to a specific hormone receptor, RU38486 may trigger the antiproliferative effect either by an induction of growth factor inhibitors or by decreasing the production of mitogens through other mechanisms. [19]. A complementary mechanism must be considered in order to explain that the degree of growth inhibition depends on the presence or the absence of serum. In the model used, cholesterol or serum lipoproteins seem to prevent the activity of RU38486. A similar observation has yet been described for other substances like estrogens [14] or hydroxysterols [17]. The lipids do not trap the
341
steroid since other biological activities of RU38486, like the inhibition of the synthesis of tyrosine aminotransferase, are not hindered by the presence of lipids [3]. For hydroxysterols it was hypothesized that the presence of serum lipids induces some change in the fluidity of the cellular membrane [7]. An action of RU38486 on the membrane could be imagined especially since it has been claimed that tamoxifen could act via the AEBS which may be membrane associated [12]. Moreover, we observed that lipids partially prevent the inhibitory effect of tamoxifen. It must be emphasized that RU38486 and tamoxifen have a similar side chain added to the structure of the agonist; this substitution might exert a common role for the expression of the antiproliferative activity [16]. For tamoxifen, the integrity of the side chain has been shown to be essential for the binding to AEBS [18]. It would be interesting to verify if RU38486 is able to interact with those sites and find out about the role of the 11/3-side chain in the expression of the antiproliferative effect of RU38486. We observed that. this part of the molecule undergoes a rapid metabolization by the cytochrome P-450 enzyme complex [5]. Thus, the action of RU38486 in HTC cells would appear to be dependent on the presence of glucocorticoid receptors and the absence of cytochrome P-450 isoenzymes. These two points may serve as a guide in the development of the therapeutical use of this molecule. These parameters will also be important to further our study on other hepatomas, either ascitic rat tumours in vivo or human hepatoma tumour cells in primary cultures. The treatment of advanced breast cancer by RU38486 has been attempted and this molecule is well tolerated in long term therapy; moreover, it also has a positive influence on pulmonary and cutaneous metastasis [ll]. The antiproliferative action on HTC cells is an interesting finding which must be considered with the other properties of RU38486: the antiglucocorticoid potency and the behaviour in different hepatoma cells or hepatocytes related to the cytochrome P-450 isoenzyme pattern [5]. Taken together, our results favour the idea that RU38486 might represent a possible drug for the treatment of hepatocarcinomas. ACKNOWLEDGEMENTS
This work has been supported by a grant from the Association pour la Recherche Contre le Cancer (no. 64961. S. C.-G. was a recipient from the Fondation pour la Recherche Medicale. We are grateful to D. Philibert, Roussel UCLAF Comp., for his help and interest and to Dr. J.P. Beck, U.L.P. Strasbourg, for many stimulating discussions. REFERENCES 1
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