TAMOXIFEN AS AN ANTI-TUMOUR AGENT: OESTROGEN BINDING AS A PREDICTIVE TEST FOR TUMOUR RESPONSE V. C. JORDAN AND T. JASPAN Department of Pharmacology, School of Medicine, Leeds, LS2 9NL
(Received 19 June 1975) SUMMARY
Rats with growing 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinomata were biopsied and oestrogen-binding capacity was measured using a Sephadex LH-20 chromatography method. Tumours were measured with calipers and animals were treated for 3 weeks with tamoxifen (50 \g=m\g/day, s.c.). Tumour response was determined by the size (cm2) before and after therapy. An increase in tumour regression (ten tumours) was seen with increasing oestrogen-binding sites determined by Scatchard analysis (P < 0\m=.\01).Thirty tumours were used to determine oestrogen binding with a single dose of [3H]\x=req-\ oestradiol. The percentage tumour regression was linearly correlated with oestrogen\x=req-\ binding capacity (P < 0\m=.\01),although some tumours with high oestrogen-binding capacities only partially regressed in response to tamoxifen therapy. The time of the oestrous cycle when biopsy occurred was not a critical factor in determining oestrogen binding for prediction of response. Oestrogen binding was reduced during tamoxifen therapy. INTRODUCTION
Tamoxifen (ICI 46,474; trans l-(/>-/?-dimethylaminoethoxyphenyl)-l,2-diphenyl but-1-ene) inhibits the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinomata and during therapy the binding of [3H]oestradiol-17/#, in vivo and in vitro, is significantly reduced (Jordan & Dowse, 1976). Although oestrogen-stimulated prolactin levels can be reduced by tamoxifen (Jordan, Koerner & Robison, 1975), prolactin levels are not correlated with anti-tumour activity (Jordan & Koerner, 1976). If the fundamental mechanism of action of tamoxifen is related to its ability to inhibit oestrogen binding, then a determination of oestrogen-binding capacities in biopsy samples may be useful in pre¬ dicting the future response of the tumour. In the present investigation, a Sephadex LH-20 method has been used to determine oestrogen binding in biopsy samples of DMBA-induced rat mammary carcinomata. The tumour responses have been assessed after 3 weeks of tamoxifen therapy (50 /¿g/day, s.c). METHODS
Mammary carcinomata were induced in 50-day-old female Sprague-Dawley rats by the administration (by gavage) of 20 mg DMBA (Sigma) in 2 ml peanut oil. Tumours were detected by palpation 50-100 days after DMBA administration and the increase in tumour area was determined at weekly intervals as previously described (Jordan & Dowse, 1976). Suitably sized tumours were selected randomly and biopsied at known times during the oestrous cycle (determined by vaginal smear) and tissue samples were weighed, immediately frozen in liquid nitrogen and processed as described in (b) below.
(a) Preparation of solutions Tamoxifen was prepared in a stock alcoholic solution. Aliquots were added to the required volume of peanut oil and the alcohol was evaporated, under a stream of nitrogen, on a warm (60 °C) water bath. [2,4,6,7-3H]Oestradiol-17/? (110 Ci/mmol) was obtained from the New England Nuclear Corporation, dissolved in benzene : ethanol (9:l,v/v). Purity was 98 % and the material was used without further purification. A known aliquot was evapo¬ rated to dryness under nitrogen and the [3H]oestradiol was then redissolved in a known volume of absolute ethanol to give a stock solution of 210 pmol/50 µ\. A 1:100 dilution was made with phosphate buffer (see below) to give an approximate 2 pmol/50 µ\ buffer solution and then the solutions for Scatchard analysis were made by serial dilutions (0-2, 0-1, 0-075, 0-05, 00375, 0-025, 0-015 pmol/50 µ phosphate buffer). Solutions were stored at 4 °C and fresh solutions were made every 2 weeks. Diethylstilboestrol (British Drug Houses Ltd) was prepared in a stock alcoholic solution. Aliquots were evaporated in a stream of nitrogen and then made up to the required volume with phosphate buffer (see below). The final solution contained 25 pmol/50 µ\ buffer. (b) Binding of[2,4,6,7-3H]oestradiol-17ß in vitro Tumour samples, frozen in liquid nitrogen, were powdered using a Thermovac tissue pulver¬ izer. Powdered tumours were homogenized (1:4, w/v) in phosphate buffer (pH 7-3, 001 Mdisodium phosphate, 0-25 M-sucrose, 0-5 mM-dithiothreitol [Sigma]) using 4 2 s bursts of an Ultraturax tissue homogenizer at 4 °C. Cooling periods of 15 s were used between bursts. Homogenates were centrifuged at 100000 g (4 °C) in a Christ Omega II 70000 ultracentrifuge for 1 h using a 6 5 ml titanium swing-out bucket rotor. A sample of the supernatant (cytosol) was used for protein determination by the method of Lowry, Rosebrough, Farr & Randall (1951). Oestrogen binding was determined using the methods described by Ginsberg, Greenstein, Maclusky, Morris & Thomas (1974). Incubations, in duplicate, were undertaken for 30 min at 30 °C using 150 µ\ cytosol, 50 µ\ buffer and 50 µ\ [3H]oestradiol. Each cytosol was incubated in parallel with 50 µ\ buffer containing excess diethylstilboestrol (DES) (instead of buffer alone) to determine non-specific binding. Samples were allowed to stand for 10 min at 23 °C with the DES before the addition of [3H]oestradiol. Nineteen tumour cytosols were incubated with a single concentration of [3H]oestradiol (0-2 pmol). Eleven tumour cytosols were incubated with seven [3H]oestradiol concentrations between 0-2-0 15 pmol. Sephadex LH-20 (Pharmacia Fine Chemicals Ltd) columns (6 0-4 cm) were prepared surrounded by a 4 °C ice bath and 200 µ\ samples of each incubate were layered on top. After 30 min on the columns, the protein-bound [3H]oestradiol was eluted with 800 µ phosphate buffer into scintillation vials. Samples were counted as previously described (Jordan & Dowse, 1976). Where applicable, the dissociation constant (Kd) and oestrogen-binding capacities (fmol/mg cytosol protein) were determined using methods described by Scatchard (1949). Lines of best fit were calculated by regression analysis. The oestrogen-binding capacities in other cases were calculated from the c.p.m./mg cytosol protein and expressed as fmol/mg cytosol protein. A similar calculation was performed upon the results for 0-2 pmol [3H]oestradiol concentration from the dilutions used to obtain results for Scatchard plots. For cytosols from tumours after tamoxifen therapy, a single concentration of 0-2 pmol [3H]oestradiol was used to determine oestrogen binding and the results were converted from c.p.m./mg cytosol protein to fmol/mg cytosol protein.
RESULTS
In response to 50/¿g tamoxifen/day for 3 weeks, the DMBA-induced mammary tumours produced a mixed response, with some tumours continuing to grow whereas others under¬ went rapid regression. The data presented in Fig. 1 are the weekly growth of DMBAinduced tumours, in a single rat, before and during tamoxifen therapy. Oestrous cycles stopped during tamoxifen administration and animals presented with dioestrous smears. About 1-2 weeks after the end of treatment, oestrous cycles restarted. Tamoxifen (50
/ig/day)
5 r
4
"
-
31*
U 27-57
fmol/mg protein
3 -
1
-
46-6
y/-
70
80
j_ 90
100
110 after DMBA
120
•l-B 130
fmol/mg protein 140
Days Fig. 1. Plot of dimethylbenz(e)anthracene (DMBA)-induced carcinomata area (cm2) on a single rat, before and during tamoxifen treatment. Tamoxifen (50 µ$) was administered daily (s.c.) for 3 weeks. Oestrogen-binding sites (fmol/mg cytosol protein) were measured in biopsy samples before therapy. U, ulcération of the tumour. The dissociation constants (Kd) and oestradiol-binding capacities were determined by Scatchard analysis and ranged from 5-2 10_10to l-56x 10~10mol/l and 5-67 fmol/mg cytosol protein respectively. Each of the 11 tumour biopsies was taken during dioestrus. With the exception of II/2, S^ which had an oestrogen-binding capacity of 48 fmol/mg cytosol protein (Ka 1-75 10~10 mol/1), there was an increased percentage decrease in tumour area (y) with tamoxifen therapy as the concentration of oestrogen-binding sites (x) in the tumour -0-735 (P < 0-01)) (Fig. 2). increased (y 92-61 -l-2x; r (correlation coefficient) Tumour II/2, Si was excluded from calculations since at autopsy the tumour was found to be necrotic and fluid filled thus leading to inaccuracies in determining viable tumour tissue. In a second experiment, tumour biopsies were taken at random stages of the oestrous cycle and oestrogen-binding capacities were determined using a single concentration of [3H]oestradiol. The results using the highest concentration of [3H]oestradiol in the Scatchard analysis were recalculated and the results are included on the graph (Fig. 3). Again with the exception of tumour II/2, S1; the percentage decrease in tumour area in response to tamoxifen (y) increased with increasing oestrogen-binding capacity (x)(y 85-64- l-18x; r= -0-515, < 001). Tumours which had oestrogen-binding sites < 11 fmol/mg cytosol protein, =
=
=
Growth 100
SO
Binding sites (fmol/mg protein) 10
c
20
30
40
50
60
70 -I
o
50
100 L
Regression Fig. 2. Graph of the percentage change in dimethylbenz(e)anthracene-induced tumour area after 3 weeks of tamoxifen therapy (50 /
(Received 19 June 1975) SUMMARY
Rats with growing 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinomata were biopsied and oestrogen-binding capacity was measured using a Sephadex LH-20 chromatography method. Tumours were measured with calipers and animals were treated for 3 weeks with tamoxifen (50 \g=m\g/day, s.c.). Tumour response was determined by the size (cm2) before and after therapy. An increase in tumour regression (ten tumours) was seen with increasing oestrogen-binding sites determined by Scatchard analysis (P < 0\m=.\01).Thirty tumours were used to determine oestrogen binding with a single dose of [3H]\x=req-\ oestradiol. The percentage tumour regression was linearly correlated with oestrogen\x=req-\ binding capacity (P < 0\m=.\01),although some tumours with high oestrogen-binding capacities only partially regressed in response to tamoxifen therapy. The time of the oestrous cycle when biopsy occurred was not a critical factor in determining oestrogen binding for prediction of response. Oestrogen binding was reduced during tamoxifen therapy. INTRODUCTION
Tamoxifen (ICI 46,474; trans l-(/>-/?-dimethylaminoethoxyphenyl)-l,2-diphenyl but-1-ene) inhibits the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinomata and during therapy the binding of [3H]oestradiol-17/#, in vivo and in vitro, is significantly reduced (Jordan & Dowse, 1976). Although oestrogen-stimulated prolactin levels can be reduced by tamoxifen (Jordan, Koerner & Robison, 1975), prolactin levels are not correlated with anti-tumour activity (Jordan & Koerner, 1976). If the fundamental mechanism of action of tamoxifen is related to its ability to inhibit oestrogen binding, then a determination of oestrogen-binding capacities in biopsy samples may be useful in pre¬ dicting the future response of the tumour. In the present investigation, a Sephadex LH-20 method has been used to determine oestrogen binding in biopsy samples of DMBA-induced rat mammary carcinomata. The tumour responses have been assessed after 3 weeks of tamoxifen therapy (50 /¿g/day, s.c). METHODS
Mammary carcinomata were induced in 50-day-old female Sprague-Dawley rats by the administration (by gavage) of 20 mg DMBA (Sigma) in 2 ml peanut oil. Tumours were detected by palpation 50-100 days after DMBA administration and the increase in tumour area was determined at weekly intervals as previously described (Jordan & Dowse, 1976). Suitably sized tumours were selected randomly and biopsied at known times during the oestrous cycle (determined by vaginal smear) and tissue samples were weighed, immediately frozen in liquid nitrogen and processed as described in (b) below.
(a) Preparation of solutions Tamoxifen was prepared in a stock alcoholic solution. Aliquots were added to the required volume of peanut oil and the alcohol was evaporated, under a stream of nitrogen, on a warm (60 °C) water bath. [2,4,6,7-3H]Oestradiol-17/? (110 Ci/mmol) was obtained from the New England Nuclear Corporation, dissolved in benzene : ethanol (9:l,v/v). Purity was 98 % and the material was used without further purification. A known aliquot was evapo¬ rated to dryness under nitrogen and the [3H]oestradiol was then redissolved in a known volume of absolute ethanol to give a stock solution of 210 pmol/50 µ\. A 1:100 dilution was made with phosphate buffer (see below) to give an approximate 2 pmol/50 µ\ buffer solution and then the solutions for Scatchard analysis were made by serial dilutions (0-2, 0-1, 0-075, 0-05, 00375, 0-025, 0-015 pmol/50 µ phosphate buffer). Solutions were stored at 4 °C and fresh solutions were made every 2 weeks. Diethylstilboestrol (British Drug Houses Ltd) was prepared in a stock alcoholic solution. Aliquots were evaporated in a stream of nitrogen and then made up to the required volume with phosphate buffer (see below). The final solution contained 25 pmol/50 µ\ buffer. (b) Binding of[2,4,6,7-3H]oestradiol-17ß in vitro Tumour samples, frozen in liquid nitrogen, were powdered using a Thermovac tissue pulver¬ izer. Powdered tumours were homogenized (1:4, w/v) in phosphate buffer (pH 7-3, 001 Mdisodium phosphate, 0-25 M-sucrose, 0-5 mM-dithiothreitol [Sigma]) using 4 2 s bursts of an Ultraturax tissue homogenizer at 4 °C. Cooling periods of 15 s were used between bursts. Homogenates were centrifuged at 100000 g (4 °C) in a Christ Omega II 70000 ultracentrifuge for 1 h using a 6 5 ml titanium swing-out bucket rotor. A sample of the supernatant (cytosol) was used for protein determination by the method of Lowry, Rosebrough, Farr & Randall (1951). Oestrogen binding was determined using the methods described by Ginsberg, Greenstein, Maclusky, Morris & Thomas (1974). Incubations, in duplicate, were undertaken for 30 min at 30 °C using 150 µ\ cytosol, 50 µ\ buffer and 50 µ\ [3H]oestradiol. Each cytosol was incubated in parallel with 50 µ\ buffer containing excess diethylstilboestrol (DES) (instead of buffer alone) to determine non-specific binding. Samples were allowed to stand for 10 min at 23 °C with the DES before the addition of [3H]oestradiol. Nineteen tumour cytosols were incubated with a single concentration of [3H]oestradiol (0-2 pmol). Eleven tumour cytosols were incubated with seven [3H]oestradiol concentrations between 0-2-0 15 pmol. Sephadex LH-20 (Pharmacia Fine Chemicals Ltd) columns (6 0-4 cm) were prepared surrounded by a 4 °C ice bath and 200 µ\ samples of each incubate were layered on top. After 30 min on the columns, the protein-bound [3H]oestradiol was eluted with 800 µ phosphate buffer into scintillation vials. Samples were counted as previously described (Jordan & Dowse, 1976). Where applicable, the dissociation constant (Kd) and oestrogen-binding capacities (fmol/mg cytosol protein) were determined using methods described by Scatchard (1949). Lines of best fit were calculated by regression analysis. The oestrogen-binding capacities in other cases were calculated from the c.p.m./mg cytosol protein and expressed as fmol/mg cytosol protein. A similar calculation was performed upon the results for 0-2 pmol [3H]oestradiol concentration from the dilutions used to obtain results for Scatchard plots. For cytosols from tumours after tamoxifen therapy, a single concentration of 0-2 pmol [3H]oestradiol was used to determine oestrogen binding and the results were converted from c.p.m./mg cytosol protein to fmol/mg cytosol protein.
RESULTS
In response to 50/¿g tamoxifen/day for 3 weeks, the DMBA-induced mammary tumours produced a mixed response, with some tumours continuing to grow whereas others under¬ went rapid regression. The data presented in Fig. 1 are the weekly growth of DMBAinduced tumours, in a single rat, before and during tamoxifen therapy. Oestrous cycles stopped during tamoxifen administration and animals presented with dioestrous smears. About 1-2 weeks after the end of treatment, oestrous cycles restarted. Tamoxifen (50
/ig/day)
5 r
4
"
-
31*
U 27-57
fmol/mg protein
3 -
1
-
46-6
y/-
70
80
j_ 90
100
110 after DMBA
120
•l-B 130
fmol/mg protein 140
Days Fig. 1. Plot of dimethylbenz(e)anthracene (DMBA)-induced carcinomata area (cm2) on a single rat, before and during tamoxifen treatment. Tamoxifen (50 µ$) was administered daily (s.c.) for 3 weeks. Oestrogen-binding sites (fmol/mg cytosol protein) were measured in biopsy samples before therapy. U, ulcération of the tumour. The dissociation constants (Kd) and oestradiol-binding capacities were determined by Scatchard analysis and ranged from 5-2 10_10to l-56x 10~10mol/l and 5-67 fmol/mg cytosol protein respectively. Each of the 11 tumour biopsies was taken during dioestrus. With the exception of II/2, S^ which had an oestrogen-binding capacity of 48 fmol/mg cytosol protein (Ka 1-75 10~10 mol/1), there was an increased percentage decrease in tumour area (y) with tamoxifen therapy as the concentration of oestrogen-binding sites (x) in the tumour -0-735 (P < 0-01)) (Fig. 2). increased (y 92-61 -l-2x; r (correlation coefficient) Tumour II/2, Si was excluded from calculations since at autopsy the tumour was found to be necrotic and fluid filled thus leading to inaccuracies in determining viable tumour tissue. In a second experiment, tumour biopsies were taken at random stages of the oestrous cycle and oestrogen-binding capacities were determined using a single concentration of [3H]oestradiol. The results using the highest concentration of [3H]oestradiol in the Scatchard analysis were recalculated and the results are included on the graph (Fig. 3). Again with the exception of tumour II/2, S1; the percentage decrease in tumour area in response to tamoxifen (y) increased with increasing oestrogen-binding capacity (x)(y 85-64- l-18x; r= -0-515, < 001). Tumours which had oestrogen-binding sites < 11 fmol/mg cytosol protein, =
=
=
Growth 100
SO
Binding sites (fmol/mg protein) 10
c
20
30
40
50
60
70 -I
o
50
100 L
Regression Fig. 2. Graph of the percentage change in dimethylbenz(e)anthracene-induced tumour area after 3 weeks of tamoxifen therapy (50 /
