Antiinvasive Activity of Estramustine on Malignant MO4 Mouse Cells and on DU-145 Human Prostate Carcinoma Cells in Vitro1

(CANCER RESEARCH 48, 1842-1849, April 1, 1988] Antiinvasive Activity of Estramustine on Malignant MO4 Mouse Cells and on DU-145 Human Prostate Carcinoma Cells in Vitro1

Marc M. Marcel,2 Guy A. Storme, Christian H. Dragonetti, Georges K. De Bruyne, Beryl Hartley-Asp, Jeanine L. Segers, and Marcel L. Rabaey Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine [M. M. M., C. H. D., G. K. D. B.J, Department of Clinical Chemistry [J. L. S.J and Department of Ophthalmology [M. L. R.], University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium; Department of Radiotherapy and Oncology, University Hospital, Free University Brussels, Laarbeeklaan 103, B-1090 Brussels, Belgium [G. A. S.]; and Department of Pharmacology, AB Leo, Box 941, S-251 09 Helsingborg, Sweden [B. H-A.J

ABSTRACT finding suggested that the mechanism of action of EM on MTCs was different from that of the majority of microtubule Estramustine (EM) is a conjugate of estradiol and nor-nitrogen mus tard (nor-HN2), which is effective in the treatment of prostate cancer. inhibitors that act through binding to tubulin. We have compared the effect of EM with that of the known microtubule We have found that microtubule inhibitors stop tumor inva inhibitor vinblastine (VLB) on the following functions of malignant MO4 sion and that this antiinvasive activity is not dependent upon mouse cells and of DU-145 human prostate cancer cells in vitro: direc the way these agents interact with the assembly-disassembly tional migration, invasion; and the organization and the assembly/disas equilibrium of microtubules. For example, an antiinvasive ac sembly equilibrium of microtubule complexes. The circular area covered tivity was found for colchicine-like agents that lead to micro by cells migrating from an aggregate explanted on a solid substrate was tubule disassembly as well as for taxol that leads to overassem- taken as an index of directional migration. Invasion was studied through bly of microtubules (reviewed in Ref. 10). It was our aim to find confrontation of MO4 or DU-14S cells with fragments of embryonic chick out whether EM would share antiinvasive activity with other heart in organ culture. Microtubules were investigated immunocytochem- microtubule inhibitors tested so far (listed in Ref. 11). There ically and through immunodetection on protein blots. VLB and EM inhibited directional migration and invasion of MO4 and DU-145 cells in fore, we have examined (a) the effect of EM on invasion of a dose-dependent manner; equimolar combinations of estradiol plus nor- malignant mouse cells (MO4) in organ culture, a process that nitrogen mustard did not mimic these effects. At antiinvasive concentra is sensitive to microtubule inhibitors (12), (b) the effect of EM tions VLB led to partial disassembly of microtubule complexes, whereas and of the well-documented microtubule inhibitor VLB on the EM resulted in an abnormal pattern of microtubule complexes without invasion of human prostate carcinoma cells (DU-145) in organ alteration of the overall assembly/disassembly equilibrium. Combined culture not tested for sensitivity to antiinvasive agents so far, treatment with VLB and EM resulted in an enhanced VLB effect, namely and (c) the effect of EM and VLB and of combinations of both complete disassembly. In all tests DU-145 cells were more sensitive to on the organization of MTCs and on the microtubule assembly- both VLB and EM than were MO4 cells, and the effects were less disassembly equilibrium in MO4 and DU-145 cells by immu- reversible. The present experiments showed that EM shares an antiin nocytochemistry and by immunodetection on protein blots us vasive activity with other microtubule inhibitors. ing an antiserum against tubulin.

INTRODUCTION EM3 is a steroid-alkylating agent, consisting of estradiol MATERIALS AND METHODS conjugated to nor-HN2 through a carbamate ester linkage. EM Cell Lines. The MO4 cell line was derived from an immortalized is a major metabolite of Estracyt (AB Leo, Helsingborg, Swe C3H/He fetal mouse cell line after transformation by Kirsten murine den), a drug effective in the treatment of advanced prostate sarcoma virus (13). MO4 cells are invasive in vitro (14) and metastatic cancer (1, 2). Originally, the anticancer activity of EM was in vivo (15). ascribed to its constituents estradiol and nor-HN2 (for review, The DU-145 cell line was isolated from a brain metastasis of a see Ref. 3). Our interest in EM comes from three recent aspects human prostate carcinoma (16). We obtained this cell line from Dr. Don Mickey (Duke University Medical Center, Durham, NC) at pas of its mechanisms of action: (a) EM per se was found to be sage 65 in 1979. DU-145 cells were used in the present experiments more potent than estradiol plus nor-HN2 in inhibition of pro between passages 99 and 110. The cell lines were maintained on 25- liferation of cultured cells (4, 5); (b) there exists a naturally cnr tissue culture substrate with minimum essential medium Eagle's occurring estramustine-binding protein (6); (c) the following (modified) with Earle's salts and nonessential amino acids (Flow Lab observations indicated that EM acted as a microtubule inhibi oratories, Irvine, Scotland) for MO4, and RPMI 1640 (Gibco Limited, tor: (a) increase of the mitotic index through metaphase arrest Paisley, Scotland) for DU-145, both supplemented with 10% (v/v) fetal in cultured cells (5); (/>)microtubule disassembly in squirrel fish bovine serum, 0.05% (w/v) L-glutamine, 250 lU/ml penicillin, and 100 erythrophores and in DU-145 cells (7); (c) interference with Â¿ig/mlstreptomycin (hereafter called culture medium). fast axonal transport in isolated nerve (8); (d) inhibition of Drugs. EM Â¡17/3-estradiol,3-[AyV-bis(2-chloroethyl)carbamate]|; M, 440.41 ; synthesized at AB Leo and 17/3-estradiol; M, 272.37; purchased microtubule assembly and induction of disassembly of isolated from Diosynth B.V., Oss, The Netherlands were dissolved in absolute brain microtubules (8); (e) binding to MAPs (9). The latter ethanol at 1 and 5 mg/ml, respectively. Nor-HN2 (synthesized at AB Received 8/11/87; revised 12/14/87; accepted 12/31/87. Leo; M, 141.8) was dissolved in distilled water at 0.5 mg/ml. VLB The costs of publication of this article were defrayed in part by the payment (Vinca leukoblastine; M, 811.00; obtained from Eli Lilly Benelux, of page charges. This article must therefore be hereby marked advertisement in Brussels, Belgium) was dissolved in Ringer's balanced salt solution at accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 mg/ml. Stock solutions were stored at -25Â°Cand diluted in culture 1Supported by grants from the Kankerfonds van de Algemene Spaar- en Lijfrentekas, the Fonds voor Geneeskundig Wetenschappelijk Onderzoek medium immediately before use. Control cultures contained equivalent (FGWO, 3.0095.87) and Eli Lilly, Brussels, Belgium. amounts of solvent. 2To whom requests for reprints should be addressed. 3The abbreviations used are; EM, estramustine; nor-HN2, nor-nitrogen mus Assay for Directional Migration. Multicellular spheroids of MO4 or DU-145 cells were prepared by incubation of a single cell suspension tard; MAP, microtubule-associated protein; MTC, microtubule complex; VLB, vinblastine; PHF, precultured embryonic chick heart fragment; SDS, sodium on a Gyrotory shaker (New Brunswick Scientific Company Inc., New dodecyl sulfate; CMTC, cytoplasmic MTC; SMTC, spindle MTC. Brunswick, NJ) as described earlier (14). Spheroids with a diameter of 1842

0.2 mm were selected under a Macroscope (Wild, Heerbrugg, Switzer Â¿Â¿I/lane.AfterSDS-polyacrylamide gel electrophoresis the proteins were land) and explanted on glass (for MO4 spheroids) or on tissue culture blotted onto nitrocellulose sheets (Hybond-C; Amersham, International plastic substrate (for DU-145 spheroids) as individual cultures. DU- pic, Buckinghamshire, United Kingdom). The electrophoretic transfer 145 cells attached poorly to glass. After attachment of the spheroid to was performed overnight at 200 mA and 50 V (27). Blocking of the the substrate, cells started to migrate radially from the spheroid and remaining free sites was done in phosphate buffered saline containing the diameter of the circular area covered by the cells was taken as an 0.5% (v/v) Tween 20. The primary antibody was antitubulin, as used index of directional migration (17). The direction of migration is for immunocytochemistry, diluted 1/250, supplemented with 1% (v/v) mediated through contact inhibition of ruffling and loss of contact normal goat serum and 5% (w/v) fat-free dry milk. Immunodetection inhibition results in random migration with reduced increase in the was done in accordance with Blake et al. (28) using anti-rabbit IgG- area covered by the cells (18). Drugs were added after the onset of alkaline phosphatase conjugate (Sigma) diluted 1/750. According to directional migration. We know from previous experiments that inhi the latter authors this method provides quantitative data over a wide bition of cell proliferation does not affect the index of directional range of antigen concentrations. Quantitation of blots was done by migration during the first 4 days of culture (17, 19). So far, a good image analysis (Vicom Inc., San Jose, CA). correlation was found between drug-induced inhibition of directional migration and inhibition of invasion (10). Assay for Invasion. Spheroids of MO4 cells and of DU-145 cells RESULTS (diameter, 0.2 mm) were confronted with precultered fragments of 9- day old embryonic chick heart (PHF; diameter, 0.4 mm) as described Assay for Directional Migration. Directional migration of earlier (14). Briefly, a spheroid was put into contact with a PHF on top MOi cells from an explanted aggregate was inhibited after of a semisolid agar medium. After attachment of both partners to each addition of 5.9 MgEM/ml (Fig. 1). The inhibition was clearly other the confronting pair was transferred into liquid medium and seen 1 day after addition of the drug and was found to be further incubated on a Gyrotory shaker for 4 days. Then, the confront ing pair was fixed in Bouin-Hollande's solution and processed for reversible within the first day after washing of the cultures with drug-free medium. A similar curve was obtained with VLB and embedding in paraffin. The whole pair was sectioned into 8-fim thick serial sections. Consecutive sections were stained with hematoxylin- with other Vinca alkaloids (29). A slight inhibitory effect was also noted for the combined treatment with estradici plus nor- eosin or with an antiserum against chick heart (20). Invasion was scored by two independent observers on the basis of occupation and replace HN2 at concentrations equimolar to 5.9 fig,EM/ml. ment of the heart tissue following a subjective grading (21). To determine concentrations of VLB useful for invasion The relevance of this assay in vitro for the present investigation is experiments we used the same assay and found that the direc supported by a good correlation between invasion in the assay and tional migration of DU-145 cells was sensitive to VLB at invasion in vivo (22) and by the finding that inhibitors of invasion in concentrations between 0.001 and 0.01 Mg/ml (Fig. 2). The vitro were antiinvasive also in vivo (23). effect of EM at 5.0 Mg/ml was similar to that of VLB at 0.01 Immunocytochemical Assay of Tubulin. Cell suspensions freshly tryp Mg/ml or higher, whereas cultures treated with combinations of sinized from stock cultures were seeded at a concentration of 1 x IO4 estradiol (up to 42 Mg/ml) P'us nor-HN2 (up to 7.4 Mg/ml) cells/ml (MO4) on round glass coverslips (diameter, 15 mm) or at 3.5 hardly differed from control cultures (data not shown). A dose- x 10" cells/ml (DU-145) on glass coverslips coated with collagen type response curve of the sensitivity of DU-145 cell directional IV (1 mg/ml; Sigma, St. Louis, MO). Cells on coverslips were incubated at 37Â°Cin a 24-well multidish (Nunc, Roskilde, Denmark) containing migration to EM is shown in Fig. 3. The data indicate that EM inhibits directional migration of DU-145 cells at concentrations 1 ml culture medium/well for 3 days prior to drug administration. Drugs were added at concentrations from noneffective to toxic and in between 1 (no effect) and 5 Mg/ml (maximal effect). Recovery different combinations for 3 h. Reversibility after drug treatment was from the inhibitory effect of EM on the directional migration evaluated as follows: cultures were washed three times with 1 ml drug- of DU-145 cells appeared only 4 to 6 days after removal of the free medium at room temperature and reincubated at 37Â°Cfor 3 or 20 drug (Fig. 4). This was explained by phase contrast microscopy h. Cultures incubated for 20 h were washed once more after 3 h. For of living cultures that showed necrosis of a number of DU-145 immunocytochemistry, cells were fixed immediately (without washing) in 1% glutaraldehyde (in 0.1 M cacodylate buffer) for 10 min at room temperature, treated with 0.1 % (w/v) NaBH4 in ethanol:distilled water, 1:1, and stained with an ant Â¡scrumagainst tubulin (kindly provided by Marc De Brabander and Jan De Mey, Janssen Pharmaceutica, Beerse, Belgium) according to the peroxidase-antiperoxidase procedure de scribed by De Mey et al. (24). Immunocytochemical preparations were scored by two independent observers on coded slides. Immunodetection of Tubulin on Protein Blots. We seeded 5 x 10s g 3 MO4 cells on 25-cm2 tissue culture flasks. After 4 days of incubation at 37Â°Ccultures were treated with VLB, or with EM, or with a combina tion of both for 3 h. Then, cultures were washed twice in Dulbecco's phosphate buffered saline and treated in a microtubule stabilizing buffer for the preparation of extractable and cytoskeletal proteins in accord ance with the method of Solomon et al. (25). As demonstrated by these authors, the extractable fraction contains the unassembled tubulin, whereas the cytoskeletal fraction contains tubulin from assembled microtubules. Monodimensional SDS-poIyacrylamide gel electrophoresis (26) was carried out in a 10.5% (w/v) separating gel. Sample 0 1234567 preparation was in 1.3% (w/v) SDS, 3.5% (v/v) 2-mercaptoethanol, â€¢* -Â» and 50 HIM2-(Ar-cyclohexylamino)-ethanesulfonic acid (CHES) buffer. time of culture [days] After heating at 95Â°Cfor 5 min, insoluble material was removed by Fig. 1. Directional migration of MO., cells from a spheroid explanted on glass, centrifugation. Prior to electrophoresis the samples were diluted 1:2 to in presence of 5.9 Mg/ml estramustine (â€¢)and 2.2 /Â¿g/mlnor-nitrogen mustard 1:6 in 0.062 M Tris-HCl buffer (pH 6.8), 2% (w/v) SDS, and 1% (v/v) plus 3.7 fig/ml estradiol (â€¢).as compared to untreated cells (O); drugs were washed out (Â») in some cultures ( ) 3 days after addition (Â»). Ordinate, 2-mercaptoethanol, in accordance with the results obtained with im diameter of circular area covered by MOÂ»cells, mean Â±SD (bars) from six munocytochemistry. The applied volume of the sample solution was 20 cultures; abscissa, time after explantation of MO4 cell spheroid. 1843

Table 1 Effects of estradici plus nor-HNl and EM on invasion ofMO4 cells in organ culture Grading" after

4 days 8 days 4 + 4 days* 1.2 Treatment None IV (5)' NO* ND g 1.0 CH3CH2OH (0.2%) IV (5) ND ND Estradici (3.7 Mg/ml) + nor-HN2 (2.2 ^g/ml) IV (5) ND ND |0.8 EM (5.9 Mg/ml) 11(5) II (5) III (5) -50.6 " Grades I or II, confronting cells were at the periphery of the heart tissue (no invasion); Grades III and IV, confronting cells had occupied and/or replaced less than or more than one-half of the PHF, respectively (invasion) (21). I0'4 * Four days with drug, followed by washing and 4 days without drug. ^0.2 c Numbers in parentheses, number of cultures. **ND, not done. 0.0 0001 0.01 0.03 0.06

Fig. 2. Effect of VLB on directional migration of DU-145 cells from a spheroid cells after treatment with 3 or 4 /Â¿gEM/ml, followed by focal explanted on tissue culture plastic substrate. Ordinate, diameter of area covered proliferation of surviving cells after removal of EM. Increase by DU 145 cells 8 days after explantation, mean Â±SD (bars) from six cultures; in diameter of the area covered by DU-145 cells, used as the , extreme values from four untreated cultures; abscissa, concentration of VLB (log-scale). VLB was added 2 days after explantation of DU-145 cell index of directional migration, resumed after sufficient prolif spheroids. eration explaining the shape of the curves in Fig. 4. Recovery from inhibition of directional migration could not be shown after treatment with 5 ng EM/ml since focal proliferation 2.8H occurred in only 1 of 4 cultures. It is unlikely that this focus consisted of a subpopulation of EM-resistant DU-145 cells, since subsequent treatment with 5 ng EM/ml killed the whole culture. Assay for Invasion. Table 1 shows that addition of 5.9 fig EM/ml to the culture medium inhibited the invasion of MO4 cells into PHF, in contrast to the equimolar amount of estradici plus nor-HN2. Instead of occupying and replacing the heart tissue MO4 cells remained at the periphery of the PHF. The antiinvasive effect of EM on MO4 cells was reversible (Table 1). DU-145 cells extensively invaded the heart tissue within 4 days after confrontation with PHF (Fig. 5), and were, therefore, appropriate for antiinvasive drug testing. At 0.005 jig/ml VLB inhibited the invasion of DU-145 cells, and this inhibition was reversible. Higher concentrations of VLB were so toxic for DU- Fig. 3. Effect of EM on directional migration of DU-145 cells from a spheroid. Ordinate, diameter of area covered by DU-145 cells after 5 days over diameter of 145 cells as to kill the cells, allowing no recovery studies. area covered after 2 days (when EM was added), mean Â±SD (bars) from six Inhibition of invasion was demonstrated with EM at concentra cultures; abscissa, concentration of EM. tions between 1.0 and 5.9 Â¿ig/ml(Table 2). In some of these cultures invasion apparently occurred despite mitotic arrest since no postmetaphase figures were found on serial sections and blocked DU-145 metaphase figures were present inside the heart tissue (Fig. 5, E and F). At concentrations between 1.0 and 4.0 ng/m\ recovery from inhibition was obvious 4 days after washing. At a concentration of 5.9 ng EM/ml invasion

DC was absent in all cultures and relatively few DU-145 cells E 8 remained at the periphery of the PHF (Fig. 5, G and H); recovery of DU-145 cells from EM effects could not be dem onstrated. Combined treatment with estradiol plus nor-HN2 had no effect on invasion by DU-145 cells (Table 2 and Fig. 5, C and D). Immunocytochemical Assay for Tubulin. Untreated MO4 cells showed a normal CMTC with microtubules irradiating in a regular array from the perinuclear cytocenter towards the pe riphery of the cell (Fig. 6A); mitotic cells had a normal SMTC as described previously (30). Treatment with VLB at 0.1 tig/ml 4 6 8 10 12 for 3 h reduced the number of microtubules (Fig. 6B) and led time of treatment [days] to complete disappearance of microtubules at 0.3 tig/ml. Disas Fig. 4. Recovery from inhibition of directional migration of DU-145 cells sembly of microtubules by VLB was also demonstrated by after treatment with EM at concentrations of 1 (O), 3 (D), and 4 (â€¢)pg/ml. EM immunodetection of extractable (unassembled) and cytoskeletal was added 2 days after explantation of DU-145 cell spheroids and control cultures (assembled) tubulin on protein blots (Fig. 7). CMTCs (Fig. 6C) were treated with 0.2% CH3CH2OH (â€¢).Ordinate, diameter of area covered by DU-145 cells over diameter covered after 2 days, mean Â±SD (bars) from six and SMTC were restored to normal after washing and further cultures; abscissa, number of days after addition of EM. Â»,removal of drug. incubation without drug. After treatment with EM at 5.9 fig/ 1844

D H * <&Â»â€¢ A B .*-â€¢ -â€¢,

\ H' ' D H

c D Ã® .**-.*- : D . V* 3?, H ^ H 4

D H H

G Fig. 5. Photomicrograph of paraffin sections from confrontations between DU-145 cell (D) spheroids and embryonic chick heart (H) fragments in organ culture in presence of 0.2% (v/v) ethanol (A and A), 3.7 jig estradiol/ml plus 2.2 Â¿ignor-nitrogen mustard/ml (C and D), 2 (E and F) and 5.9 fig estramustine/ml (C and //). All cultures were fixed after 4 days and sections were stained with hematoxylin-eosin (A. C, E, and G) or with an antiserum against chick heart (B, D, F, H). Arrowheads, blocked metaphase figures inside the heart tissue; scale bars, 50 /jm.

1845

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1988 American Association for Cancer Research. ANTIINVASIVE EFFECT OF EM Table 2 Effects of estradiol plus nor-HN2, EM, and VLB on invasion ofDU-145 and sometimes interconnecting chromosomes. The immuno- cells in organ culture cytochemical data did not indicate EM-mediated disassembly afterTreatmentNoneCHjCH2OH Grading" of microtubules as described with VLB (compare Fig. 6, B with + 4days*IV daysIV(3),CIII(11)"I Z>);this was confirmed by immunodetection on protein blots (4)Not (Fig. 7). A reduced number of microtubules was observed when (0.2%)Estradiol (5)III doneNot +nor-HN2(3.7 Mg/ml) (5)11(4)11(4)11(5)III doneIII the concentration of EM was increased to 11 jig/ml, but further Mg/ml)VLB (2.2 increase up to 47 fig/ml did not result in disappearance of all (0.005Mg/ml)VLB (2), IV(2)11(3)Not microtubules. All EM-induced alterations of MTCs in MO4 (0.01Mg/ml)VLB (0.1Mg/ml)EM doneIII cells returned to normal 3 h after washing. Combined treat (0.5Mg/ml)EM (4)II (1), IV(3)IV ments with VLB and EM at concentrations that in single (1.0Mg/ml)EM (2), III(1)II (1)IV treatment, respectively, led to reduced and abnormal MTCs, (2.0Mg/ml)EM (1), III(3)H(l), (2)III (3.0Mg/ml)EM III(3)II (1), IV(2)0 resulted in complete disassembly (Fig. 7) with diffuse staining (4.0Mg/ml)EM (1), HI(2)11(5)4 (2), IV(1)II of unassembled tubulin only (Fig. 6E). Reversibility of the (5.9 Mg/ml)4 (2), 0 (3) Â°Grade 0, confronting cells were absent; Grades I or II, confronting cells were combined treatment was somewhat slower than that of single at the periphery of the heart tissue (no invasion); Grades III and IV, confronting treatments. Recovery from VLB (0.3 Mg/ml) treatment, leading cells had occupied and/or replaced less than or more than one-half of the PHF, to complete microtubule disassembly, in presence of EM (5.9 respectively (invasion) (21). Mg/ml) produced abnormal MTCs as described for EM alone Four days with drug, followed by washing and 4 days without drug. c Numbers in parentheses, number of cultures. (compare Fig. 6, D with F). Results of immunocytochemical data from single and combined treatments of MO4 cells are summarized in Fig. 8. ml MTCs were abnormal: the CMTC consisted of short and Results obtained with DU-145 cells are illustrated in Fig. 9 long microtubules which lay crisscrossed through the cytoplasm and summarized in Fig. 10. DU-145 cells were sufficiently and many of which did not reach the cell periphery (Fig. 6D). spread to reveal normal MTCs (Fig. 9A) as described in MO4 SMTCs were irregular with microtubules at the kinetochores cells (see Fig. 6A). At 0.01 Mg/ml VLB reduced the number of

A i

D -E Fig. 6. Immunocytochemical staining with antiserum against tubulin of MO, cells cultured on glass. .I. untreated, normal CMTCs; //. treated with O.I i.g VLB/ ml, reduced number of microtubules; C, treated with 0.3 ng VLB/ml followed by washing and further incubation without drugs, normal CMTCs as in A; D, treated with 5.9 ng EM/ml, abnormal CMTCs; E, treated with O.I /jg VLB/ml plus 5.9 ng EM/ml, absence of microtubules and diffuse cytoplasmic staining; F, treated with 0.3 jig VLB/ml followed by washing and further incubation with 5.9 pg EM/ml, abnormal CMTCs as in D. All treatments were for 3 h. Scale bars, 20 /im. Symbols, status of microtubules as used in Fig. 8. 1846

VLB + EM EM VLB CONTROL Immunodetection of Tubulin on Protein Blots. Immunodetec- tion of extractable and cytoskeletal tubulin on protein blots (see Cyt. Ext. Cyt. Ext. Cyt. Ext. Cyt. Ext. Fig. 7) from MO4 cell cultures confirmed the main immunocytochemical data in a quantitative way (Table 3). EM, at a concentration that inhibited invasion and directional migration, did not alter the overall assembly-disassembly equilibrium, in contrast with VLB which caused disassembly. Combined treat ment of EM plus VLB enhanced VLB induced disassembly.

DISCUSSION We found that human prostate carcinoma cells DU-145 were invasive in vitro and that their invasiveness was sensitive to the microtubule inhibitor VLB, as was shown for other cell types (10). EM, like VLB, was antiinvasive for both MO4 and DU- 145 cells. Although the responses of both MO4 and DU-145 cells to the microtubule inhibitors VLB and EM were essentially the 1:2 1:6 1:3 1=5 1:2 1:6 1:3 1:5 same, differences in sensitivity between both cell types were Fig. 7. Immunodeleclion on protein blot of cytoskeletal (Cyt.) and extractable noted. Complete inhibition of invasion by VLB was achieved (Ext.) tubulin from MOÂ«cell cultures either untreated (control) or treated for 3 h at a lower concentration for DU-145 cells (present data) than with 0.1 *igvinblastine/ml, or with S.9 *tgestramustine/ml, or with O.I jig VLB/ ml plus 5.9 >igEM/ml. Numerical values, dilutions of samples. for MO4 cells (29). Recovery from inhibition of invasion was more obvious for MO4 than for DU-145 cells (compare Tables 1 and 2). It is unlikely that the differences in recovery between MO4 and DU-145 cells can be ascribed to the presence in DU- - .3 noÂ© â€¢^Em-. 145 cells of the estramustine-binding protein (about 20 ng/mg 2â€” protein)4 for two reasons: (a) differences hold true for both EM 1C> oâ€¢ 4:)â€¢ and VLB, whereas estramustin-binding protein is highly spe Â©Â®0 â€¢20

Fig. 9. Immunocytochemical staining with antiserum against tubulin of DU-145 cells cultured on collagen IV coated glass. .(. untreated, normal CMTCs; T. normal mitotic spindle. B, treated with 0.01 jig VLB/ml, reduced number of microtubules; <â€”.blockedmetaphase ligure with an abnormal mitotic spindle. C, treated with 0.03 Â»igVLB/ml, absence of microtubules. D, treated with 5.0 (ig EM/ml, abnormal CMTCs with empty ruffling lamellae. E, treated with 20.0 (ig EM/ml, reduced number of microtubules as in It. F, treated with 30.0 (ig EM/ml followed by washing and further incubation in drug-free medium, restored I'M 11 . All treatments were for 3 h. Scale bars, 20 fini. Symbols, status of microtubules as used in Fig. 10.

Table 3 Tubulin assembly/disassembly equilibria calculated from in accordance with other observations (7), but not to complete immunostained blots of proteins from MO, cells treated with VLB and EM We constructed graphs from measurements of average optical densities of 100 disassembly as observed with VLB. It is unlikely that the cross-sections through the blot taking into account the background. From these abnormal pattern of microtubules induced by EM represents graphs and from values of sample dilutions, percentages of extractable and incipient disassembly for two reasons: (a) immunoblots did not cytoskeletal tubulin were calculated. show a reduced amount of assembled tubulin; (b) when after Percentage of tubulin VLB-induced disassembly microtubules were allowed to reas TreatmentNone semble in the presence of EM the same abnormal pattern was observed, indicating that EM allows some kind of microtubule VLB (0.1 jig/ml) 87 13344 assembly. One explanation for both the disturbed microtubule EM (5.9 ng/ml) 66 VLB (0.1 ,ig/ml) + EM (5.9 Mg/ml)Extractable6196Cytoskeletal39 assembly in the presence of EM and the complete disassembly with combinations of EM plus VLB is offered by the concept of dynamic instability of individual microtubules (35). The Our immunocytochemical data on changes of the CMTC concept is based on real-time video recordings of isolated single induced by microtubule inhibitors suggest that EM affects the microtubules and states that growing and shortening microtu microtubule assembly-disassembly equilibrium in a way differ bules coexist under steady-state conditions in the absence of ent from that of VLB. Whereas VLB causes progressive disas MAPs. MAPs would stabilize microtubules in the growing sembly in a dose-dependent manner, antiinvasive concentra phase. It has been demonstrated quite convincingly that EM tions of EM cause an abnormal pattern of microtubules without binds to MAPs presumably at tubulin binding sites (9, 36), alteration of the overall equilibrium. This abnormal pattern making them inaccessible for binding to tubulin. This would could hardly be ascribed to shape changes of the cells as shown then permit the shortening phase of microtubules to occur, previously by optical diffractometry and Fourier analysis (34). causing length fluctuations that might explain the abnormal Higher concentrations of EM that are not relevant for their pattern. Since under these circumstances the microtubule length antiinvasive effect but could be helpful to understand its mech loss is balanced by elongation in the growing phase, the overall anism of action led to a reduction in the number of microtubules assembly-disassembly ratio remains constant, as shown by our 1848

9. Wallin, M., Deinum, J., and FridÃ©n,B.Interaction of estramustine phosphate SMTC with microtubule-associated proteins. FEBS Lett., 779: 289-293, 1985. .10 oo .10 10. Marcel, M. M., and De Mets, M. Effect of microtubule inhibitors on invasion and on related activities of tumour cells. Int. Rev. Cytol., 90:125-168, 1984. .08 OBi 11. Marcel, M. M., Bruyneel, E. A., De Bruyne, G. K., Dragonetti, C., and Van Cauwenberge, R. M-L. Growth and invasion: separate activities of malignant JÃŠ.06 00 - MO4 cell populations in vitro. In: T. Galeotti et al. (eds.). Membranes in Tumour Growth, pp. 223-232. Amsterdam: Elsevier/North-Holland > .04 cc Biomedicai Press, 1982. a. Â®oo 12. Mareel, M. M. K., and De Brabander, M. J. Effect of microtubule inhibitors .02 on malignant invasion in vitro. J. Nati. Cancer Inst., 61: 787-792, 1978. Â®<8>Â© Â© 13. Billiau, A., Sobis, II.. Eyssen, II.. and Van Den Berghe, H. Non-infectious intracisternal A-type particles in a sarcoma-positive, leukemia-negative 0 mouse cell line transformed by murine sarcoma virus (MSV). Arch. Gesamte 0 1 2 3 4 5 10 20 30 0 1 2 3 4 5 10 20 30 Virusforsch., 43: 345-351, 1973. 14. Mareel, M., Kint, J., and Meyvisch, C. Methods of study of the invasion of malignant C3H mouse fibroblasts into embryonic chick heart in vitro. V'ir CMTC chows Arch. B Cell Pathol., 30:95-111, 1979. .10 00 .10 15. Meyvisch, C., and Mareel, M. Site-induced differences in spontaneous me tastasis of MÃœ4mouse fibrosarcoma cells. Invasion Metastasis, 5:185-192, .08 .08 1985. 16. Stone, K. R., Mickey, D. D., Wunderli, H., Mickey, G. H., and Paulson, D. Â®o J .06- F. Isolation of a human prostate carcinoma cell line (DU 145). Int. J. Cancer, or 27:274-281, 1978. > DC.04 Â«>.04- 17. Storme, G., and Mareel, M. Effect of anticancer agents on directional migration of malignant OH mouse fibroblastic cells in vitro. Cancer Res., Â¿0:943-948, 1980. .02 .02 18. Mareel, M., Kieler, J., and Van Cauwenberge, R. Loss of directional migra ) Â© Â® Â© tion after round cell transformation of malignant ST/A mouse lung cells. 0 Cell Biol. Int. Rep., 5:921-928, 1981. 012345 o 30 0 1 2 3 74 5 10 20 30 19. Mareel, M. M., Dragonetti, C. H., De Bruyne, G. K., and Van Cauwenberge, R. M-L. Glycosylated podophyllotoxin congeners: loss of microtubule inhi Pg EM/ml bition and permission of invasion in vitro. J. Nati. Cancer Inst., 69: 1367- 1374, 1982. Fig. 10. Graphic representation of CMTC and SMTC in DU-145 cells treated 20. Mareel, M. M., De Bruyne, G. K., Vandesande, F., and Dragonetti, C. with VLB alone (ordinate), with EM alone (abscissa), or with combinations of Immunohistochemical study of embryonic chick heart invaded by malignant both. Cells were fixed immediately after treatment (left) or 3 h after washing and cells in three-dimensional culture. Invasion metastasis, 7: 195-204, 1981. further incubation in drug-free medium (right). â€¢(normal), O (abnormal), (â€¢) 21. Bracke, M. E., Van Cauwenberge, R. M-L., and Mareel, M. M. (+)-Catechin (reduced), O (absent), as described in the text and illustrated in Fig. 9. Combined inhibits the invasion of malignant fibrosarcoma cells into chick heart in vitro. symbols, cells with two types of MTCs were found in the same culture. Clin. Exp. Metastasis, 2:161-170, 1984. 22. Mareel, M M. K. The use of embryo organ cultures to study invasion in vitro. immunoblots. In combination with VLB the growing phase of In: L. A. Liotta and I. R. Hart (eds.), Tumor Invasion and Metastasis, pp. 207-230. The Hague: Martinus Nijhoff Publishers, 1982. microtubules would also be prevented due to binding of VLB 23. Distelmans, W., Van Gynckel, R., Vanherck, W., and De Brabander, M. The to tubulin, so that an unbalanced shortening phase would lead kidney invasion test: an assay allowing macroscopic quantification of malig to complete disassembly. nant invasion in vivo. Invasion Metastasis, 5: 170-184, 1985. 24. De Mey, J., Hoebeke, J., De Brabander, M., Geuens, G., and Joniau, M. We conclude that at least in vitro, EM has an antiinvasive Immunoperoxidase visualisation of microtubules and microtubular proteins. activity on malignant mouse MO., cells and on human DU-145 Nature (Lond.), 264: 273-275, 1976. prostate carcinoma cells which, at least for MO4 cells, can be 25. Solomon, F., Magendantz, M., and Salzman, A. Identification with cellular microtubules of one of the co-assembling microtubule-associated proteins. ascribed to structural and functional alterations of the CMTC. Cell, 18:431-438, 1979. 26. Laemmli, U. K. Cleavage of structural proteins during the assembly of the ACKNOWLEDGMENTS head of bacteriophage T4. Nature (Lond.), 227:680-685, 1970. 27. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer of proteins We thank Lieve Baeke, Bea Buysse, and Ariette Verspeelt from the from polyacrylamide gels to nitrocellulose sheets: procedure and some appli Laboratory of Experimental Cancerology for technical assistance, A. cations. Proc. Nati. Acad. Sci. USA, 76:4350-4354, 1979. Claeys and L. Fortran from the Oncologie Center, Free University 28. Blake, M. S., Johnston, K. H., Russell-Jones, G. J., and Gotschlich, E. C. A Brussels, for quantitative analysis of protein blots, Jean Roels van rapid, sensitive method for detection of alkaline phosphatase-conjugaled antiantibody on Western blots. Anal. Biochem., 136: 175-179, 1984. Kerckvoorde from the Laboratory of Experimental Cancerology for 29. Mareel, M. M., Storme, G. A., De Bruyne, G. K., and Van Cauwenberge, R. preparing the illustrations, and Ghislaine Matthys-De Smet from the M. L. Vinblastine, vincristine and vindesine: antiinvasive effect on MO4 Department of Radiotherapy and Nuclear Medicine for typing the mouse fibrosarcoma cells in vitro. Eur. J. Cancer, 18: 199-210, 1982. manuscript. 30. De Brabander, M., Geuens, G., De Mey, J., and Joniau, M. Light microscopic and ultrastructural distribution of immunoreactive tubulin in mitotic mam malian cells. Biol. Cell., 34: 213-226, 1979. REFERENCES 31. Forsgren, B., Gustafsson, J-A., Pousette, A., and HÃ¶gberg, B. Binding 1. JÃ¶nsson,G., HÃ¶gberg,B.. and Nilsson, T. Treatment of advanced prostatic characteristics of a major protein rat ventral prostate cytosol that interacts carcinoma with estramustine phosphate (Estracyt). Scand. J. Urol. Nephrol., with estramustine, a nitrogen mustard derivative of 17/3-estradioI. Cancer 77:231-238, 1977. Res., 59:5155-5164, 1979. 2. Gunnarsson, P. O., Plym Forshell, G., Fritjofsson, A., and NorlÃ©n,B. J. 32. Hartley-Asp, B., and Gunnarsson, P. O. Growth and cell survival following Plasma concentrations of estramustine phosphate and its major metabolites treatment with estramustine, nor-nitrogen mustard, estrudiÂ»!and testoster in patients with prostatic carcinoma treated with different doses of estramus one of a human prostatic cancer cell line (DU 145). J. Urol., 727: 818-822, tine phosphate (Estracyt). Scand. J. Urol. Nephrol., 75: 201-206, 1981. 1982. 3. Sandberg, A. A. Metabolic aspects and actions unique to Estracyt. Semin. 33. Mareel, M., Storme, G., De Bruyne, G., and Van Cauwenberge, R. Anti- Oncol., 10 (Suppl. 3): 3-15,1983. invasive effect of microtubule inhibitors in vitro. In: M. De Brabander and J. 4. Tew, K. D., Erickson, L. C, White, G., Wang, A. L., Schein, P. S., and De Mey (eds.), Microtubules and Microtubule Inhibitors, pp. 535-544. Hartley-Asp, B. Cytotoxicity of estramustine, a steroid-nitrogen mustard Amsterdam: Elsevier/North-Holland BiomÃ©dicalPress, 1980. derivative, through non-DNA targets. Mpl. Pharmacol., 24: 324-328, 1983. 5. Hartley-Asp, B. Estramustine-induced mitotic arrest in two human prostatic 34. Mareel, M. M., De Bruyne, G. K., Ostrowski, K., Rozycka, M., Strojny, P., carcinoma cell lines DU 145 and PC-3. The Prostate, 5: 93-100, 1984. Dziedzic-Goclawska, A., Lenczowski, S., Grzesik, W., and Kieler, J. Numer 6. BjÃ¶rk,P., Forsgren, B., Gustafsson, J-A., Pousett, A., and HÃ¶gberg, B. ical evaluation of changes in the cytoplasmic microtubule complex of C3H Partial characterization and "quantitation" of a human prostatic estramus- mouse cells by optical diffractometry and of changes in cell shape by Fourier tine-binding protein. Cancer Res., 42: 1935-1942,1982. analysis. Cytometry, 7: 18-24, 1986. 7. Stearns, M. E., and Tew, K. D. Antimicrotubule effects of estramustine, an 35. I lorio. T., and Hotani, H. Visualization of the dynamic instability of individ antiprostatic tumor drug. Cancer Res., 45: 3891-3897, 1985. ual microtubules by dark-field microscopy. Nature (Lond.), 321: 605-607, 8. Kanje, M., Deinum, J., Wallin, M., EkstrÃ¶m,P., EdstrÃ¶m,A., and Hartley- 1986. Asp, B. Effect of estramustine phosphate on the assembly of isolated bovine 36. FridÃ©n,B.,Wallin, M., Deinum, J., Prasad, V., and Luduena, R. Interaction brain microtubules and fast axonal transport in the frog sciatic nerve. Cancer of estramustine phosphate with TAU.MAP2 and the tubulin binding frag Res., 45:2234-2239, 1985. ment of MAP2. Arch. Biochem. Biophys., in press, 1988. 1849

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1988 American Association for Cancer Research. Antiinvasive Activity of Estramustine on Malignant MO4 Mouse Cells and on DU-145 Human Prostate Carcinoma Cells in Vitro

Marc M. Mareel, Guy A. Storme, Christian H. Dragonetti, et al.

Cancer Res 1988;48:1842-1849.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/7/1842

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/48/7/1842. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.