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High dose estrogen response of the hormone independent R3327-At Copenhagen rat prostatic tumor
57
Cancer Letters, 11 (1960) 57-61 0 ElsevierINorth-Holland Scientific Publishers Ltd.
HIGH DOSE ESTROGEN RESPONSE OF THE HORMONE R3327-At COPENHAGEN RAT PROSTATIC TUMOR*
INDEPENDENT
W.D.W. HESTON and D.W. LAZAN The Division of Urology, Washington University School of Medicine, St. Louis, Missouri 631 IO (U.S.A.). (Received 30 October 1979) (Accepted 22 July 1980) SUMMARY
In studies with the hormone independent R3327-At prostatic tumor, relatively high doses of estrogen were inhibitory to the growth of the R3327At rat prostatic tumor were observed. It was further observed that although tumor growth was inhibited, an increase in the [ 3H] thymidine labelling of the DNA of the tumor cells of the estrogen treated group relative to the castrate control was apparent.
INTRODUCTION
One of the few animal tumor models of prostatic carcinoma is the Copenhagen rat prostatic tumor R3327 and its derivative lines [3,5,8]. During initial investigations on the slow growing and androgen-dependent well differentiated R3327-H tumor, there arose a rapidly growing anaplastic tumor which grew nearly equally well in intact or castrated males. This rapidly growing hormone independent derivative was designated R3327-At [ 61. While both androgen and estrogen receptors were present in the R3327-H tumor, none have been found in the R3327-At tumor, correlating to its lack of hormone responsiveness [ 5,6]. The hormone responsive tumor indicated reduction in its growth potential when treated with daily doses of estrogen of 0.1 mg/kg body wt. No effect of estrogen was found on the growth of the anaplastic R3327-At tumor at this dose level; however, when the daily dosage of diethylstilbestrol was increased 200-fold to 20 mg/kg body wt, the growth of this hormone insensitive tumor was inhibited. Our preliminary findings on this high dose estrogen effect on the hormone insensitive R3327At prostatic tumor are presented. *Supported in part by Public Health Service grant CA23665 from the National Cancer Institute, National Institutes of Health, Education and Welfare.
58 MATERIALS
AND METHODS
Chemicals Estradiol-17P was obtained from Steraloids. Tamoxifen was supplied through the generosity of I.C.I. Americas, Inc. Diethylstilbestrol and all other non-radioactive chemicals were purchased from Sigma Chemical Co. [ 3H] Thymidine 5 mCi/mM was purchased from Amersham. Animals and tumors All animals were inbred Copenhagen rats and were supplied by the Mammalian Genetics Branch of the National Cancer Institute. The R3327-At tumor used was transplanted and maintained by techniques described previously [ 61. Tumor volume measurements were obtained by caliper measurements. Tumor volumes and apparent doubling times were calculated as previously described [ 61. To conserve animals, we inoculated two tumors per animal, one tumor per flank. We found the tumor growth rate to be identical for the R3327-At tumor, whether the animal was bearing 1 or 2 tumors. Two hours prior to sacrifice 0.5 pC!i of [3H]thymidine/g body weight was injected intraperitoneally. The tumors were surgically excised and weighed to the nearest hundredth of a gram. Aliquots of the tumors were removed and homogenized in ice-cold 25 mM phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 0.5 mM EDTA with the aid of a polytron. The resulting 25% homogenates were centrifuged at 50,000 X g for 2 h at 4°C. The supernatants were saved and the pellets examined for DNA content and [ 3H] thymidine incorporation by methods previously described.
[61. RESULTS
AND DISCUSSION
In Fig. 1, it can be seen that there is no difference in the growth rate between castrate males or intact females; however, in intact males the tumor was slightly but significantly larger than the castrate or female at all time points. This led to the conclusion that the tumor is hormone independent in its growth characteristics. Indeed, if diethylstilbestrol is administered in daily doses of 0.1 mg/kg, no effects are seen on the tumor growth (data not shown). This dose is 10 times greater than the ED,, dose required for maximal reduction of the size of the prostate of normal male rats [ 11. At 0.1 mg/kg, activity of diethylstilbestrol is considered to be indirect in that it inhibits the release of pituitary luteinizing hormone, which in turn results in decreased production of testosterone by the testes. The resulting lower serum levels of testosterone are responsible for the reduced size of the prostate. Therefore, doses of diethylstilbestrol at 0.1 mg/kg being ineffectual in inhibiting the growth of the R3327-At tumor were consistent with the hormone independent nature of the R3327-At tumor. However, as we
59
-3 t
I o
6
2
4
6
8
0
I2
14
I6
16
20
22
DAYS
Fig. 1. Tumor volume change of the R3327-At tumor following inoculation of 2 X lo6 viable tumor cells into intact males (a), castrate males ((I), females (A), or diethylstilbestrol, 20 mg/kg, treated castrate males (0). Volumes were calculated from caliper measurements as 1 x w x h x 0.5236 and plotted as the natural log of the calculated volume. Each point represents the mean of the natural log of the volume + S.E.M. These are 10 tumors/group.
often administer doses of androgen in the 20 mg/kg range, we decided to demonstrate that even at this higher dose of estrogen that there was no effect on tumor growth; Figure 1 illustrates that in contrast to our expectations, there was an inhibitory effect on tumor growth at this high dose of diethylstilbestrol. As calculation of apparent tumor volume doubling times are about 2 days for the diethylstilbestrol group, as well as the other groups, it appeared that this high dose of diethylstilbestrol was affecting primarily the initial phase following the inoculation of tumor. Table I lists the results of an experiment in which the antiestrogen tamoxifen and the estrogens diethylstilbestrol and 17/l-estradiol were administered at daily doses of 20 mg/kg. In all cases and for all the parameters reported except DNA, the treated groups were significantly different from the controls at P < 0.05 level. However, tamoxifen, a weak estrogen agonist used primarily for its estrogen antagonistic activity, was the least active [4]. It reduced tumor wet weight by 46%, but only reduced DNA content, and hence tumor cellularity, by 23%; however, this decrease in DNA content was not significant at P < 0.05 by the Student’s t-test. 17/3-Estradiol reduced tumor weight by 67% and also reduced DNA content by 68%. Diethylstilbestrol was the most effective of the 3 compounds and reduced tumor weight by 75% and tumor DNA by 85%. When 17&estradiol and diethylstilbestrol only are compared, the final tumor wet weight did not differ significantly between 17/l-estradiol and diethylstilbestrol treatments, however, the DNA
60 TABLE I RESPONSE OF THE R3327-At TUMOR TO THE ADMINISTRATION OF THE ANTIESTROGEN TAMOXIFEN (TAM) AND THE ESTROGENS 17p-ESTRADIOL (E, ) AND DIETHYLSTILBESTROL (DES) Treatmenta
Tumor wetb wt
Total mg DNA
[ ‘H] ThymidineC incorporation
Castrate TAM E: DES
2.50 1.34 0.83 0.63
4.71 3.64 1.52 0.71
5500 8700 11,100 15,933
f 0.37 + 0.13d t O.lld t 0.12d
+ 0.62 + 0.52 ? 0.21d * 0.20d
+ 490 i 650d f 1070d * 1530d
aAll animals were castrated 1 week prior to inoculation of tumor. The castrate controls received sesame oil only 1.0 ml/kg S.C. daily. Tamoxifen, 17p-Estradiol and Diethylstilbestrol were dissolved in sesame oil and administered at 20 mg/kg S.C. bMean gram wet wt of 10 tumors t S.E.M. ‘Two hours prior to sacrifice the animals were given 0.5 PCi [ ‘Hlthymidine (5 mCi/mM)/g body wt. The values represent mean cpm of [‘Hlthymidine incorporated/mg DNA dDiffers significantly from control (P < 0.05) by Student’s f-test.
content did. DES treatment resulted in less than half (0.7 vs. 1.5) the tumor DNA content of that determined for the 17fl-estradiol group. This further illustrates that DES was the most effective of these agents in reducing total cellularity of the tumor. Even though estrogens at these doses tend to produce a weight loss, we found a significant decrease in tumor wet weight for all treated groups when the data is corrected for body weight. We find the 1 tumor wet wt/lOO g body wt to be 1.35, 0.77, 0.53 and 0.43 for the castrate control, tamoxifen, 17/3-estradiol and diethylstilbestrol groups, respectively. Further, we administered these high doses of estrogens for much longer periods of time and have not found them to be lethal. Unexpectedly, the use of these compounds resulted in an increased incorporation of [3H]thymidine into the DNA of the tumor. Tamoxifen increased the incorporation by 58%, 17P-estradiol by 202% and diethylstilbestrol by 290%. Previously, Sufrin et al. described a close correlation between in vitro [ 3H]thymidine uptake and the calculated and actual tumor doubling time of the R3327-At tumor [ 71. This suggested that there was no substantial ‘cell loss’ factor affecting the growth of the R3327-At tumor. Sufrin et al. also found substantial agreement between the in vitro and in vivo labelling techniques using [ 3H] thymidine. Because increased [ 3H] thymidine incorporation into the high dose estrogen treatment groups was found, it may mean that estrogen treatment has increased the rate of cell loss from the tumor. The higher apparent rate of DNA synthesis in the estrogen treated tumor may also mean that high dose estrogen treatment may make this tumor more susceptible to treatment with cell cycle specific chemotherapeutic agents.
61
It appears that estrogen in high doses has growth inhibitory properties on a tumor that would not be expected to be inhibited by estrogens. This may be due to a direct inhibitory effect or may be related to inhibitory substances produced in response to high dose not low dose estrogen administration. ACKNOWLEDGEMENT
The authors M. Baumann.
wish to acknowledge
the excellent
technical
assistance
of
REFERENCES 1 He&on, W.D.W. and Coffey, D.S. (1974) New concept in the control of abnormal prostatic growth: combination hormone therapy. The Pharmacologist, 12, 275 (abst.). 2 Heston, W.D.W., Menon, M., Tananis, C. and Walsh, P.C. (1979) Androgen, estrogen and progesterone receptors of the R-3327-H Copenhagen rat prostatic tumor. Cancer Lett., 6, 45-40. 3 Issacs, J.T., Heston, W.D.W., Weissman, R.M. and Coffey, D.S. (1978) Animal models of hormone sensitive and insensitive prostatic adenocarcinomas; Dunning R-3327~HIF, and R3327-AT. Cancer Res., 38,4353-4359. 4 Jordan, V.C. (1976) Antiestrogenic and antitumor properties of tamoxifen in laboratory animals. Cancer Treat. Rep., 60, 1409-1419. 5 Markland, F.J., Chopp, R.T., Coxgrove, M.D. and Howard, E.B. (1978) Characterization of steroid hormone receptors in the Dunning R-3327 rat prostatic adenocarcinoma. Cancer Res., 38, 2828-2925. 6 Smolev, J.K., Heston, W.D.W., Scott, W.W. and Coffey, D.S. (1977) Charact.erization of the Dunning R-3327-H prostatic carcinoma: an appropriate animal model for prostatic cancer. Cancer Treat. Rep. 16, 273-287. 7 Sufrin, G., Heston, W.D.W., Fair, W.R. and Meyer, J. (1979) Kinetic studies of the R3327 Dunning murine prostatic carcinoma. Proc. Am. Urol. Assoc., 74, 46 (Abst.). 8 Voight, W., Feldman, M. and Dunning, W.F. (1975) 5a-Dihydrotestosterone-binding proteins and androgen sensitivity in prostatic cancers of Copenhagen rats. Cancer Res., 35, 1840-1846.
Cancer Letters, 11 (1960) 57-61 0 ElsevierINorth-Holland Scientific Publishers Ltd.
HIGH DOSE ESTROGEN RESPONSE OF THE HORMONE R3327-At COPENHAGEN RAT PROSTATIC TUMOR*
INDEPENDENT
W.D.W. HESTON and D.W. LAZAN The Division of Urology, Washington University School of Medicine, St. Louis, Missouri 631 IO (U.S.A.). (Received 30 October 1979) (Accepted 22 July 1980) SUMMARY
In studies with the hormone independent R3327-At prostatic tumor, relatively high doses of estrogen were inhibitory to the growth of the R3327At rat prostatic tumor were observed. It was further observed that although tumor growth was inhibited, an increase in the [ 3H] thymidine labelling of the DNA of the tumor cells of the estrogen treated group relative to the castrate control was apparent.
INTRODUCTION
One of the few animal tumor models of prostatic carcinoma is the Copenhagen rat prostatic tumor R3327 and its derivative lines [3,5,8]. During initial investigations on the slow growing and androgen-dependent well differentiated R3327-H tumor, there arose a rapidly growing anaplastic tumor which grew nearly equally well in intact or castrated males. This rapidly growing hormone independent derivative was designated R3327-At [ 61. While both androgen and estrogen receptors were present in the R3327-H tumor, none have been found in the R3327-At tumor, correlating to its lack of hormone responsiveness [ 5,6]. The hormone responsive tumor indicated reduction in its growth potential when treated with daily doses of estrogen of 0.1 mg/kg body wt. No effect of estrogen was found on the growth of the anaplastic R3327-At tumor at this dose level; however, when the daily dosage of diethylstilbestrol was increased 200-fold to 20 mg/kg body wt, the growth of this hormone insensitive tumor was inhibited. Our preliminary findings on this high dose estrogen effect on the hormone insensitive R3327At prostatic tumor are presented. *Supported in part by Public Health Service grant CA23665 from the National Cancer Institute, National Institutes of Health, Education and Welfare.
58 MATERIALS
AND METHODS
Chemicals Estradiol-17P was obtained from Steraloids. Tamoxifen was supplied through the generosity of I.C.I. Americas, Inc. Diethylstilbestrol and all other non-radioactive chemicals were purchased from Sigma Chemical Co. [ 3H] Thymidine 5 mCi/mM was purchased from Amersham. Animals and tumors All animals were inbred Copenhagen rats and were supplied by the Mammalian Genetics Branch of the National Cancer Institute. The R3327-At tumor used was transplanted and maintained by techniques described previously [ 61. Tumor volume measurements were obtained by caliper measurements. Tumor volumes and apparent doubling times were calculated as previously described [ 61. To conserve animals, we inoculated two tumors per animal, one tumor per flank. We found the tumor growth rate to be identical for the R3327-At tumor, whether the animal was bearing 1 or 2 tumors. Two hours prior to sacrifice 0.5 pC!i of [3H]thymidine/g body weight was injected intraperitoneally. The tumors were surgically excised and weighed to the nearest hundredth of a gram. Aliquots of the tumors were removed and homogenized in ice-cold 25 mM phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 0.5 mM EDTA with the aid of a polytron. The resulting 25% homogenates were centrifuged at 50,000 X g for 2 h at 4°C. The supernatants were saved and the pellets examined for DNA content and [ 3H] thymidine incorporation by methods previously described.
[61. RESULTS
AND DISCUSSION
In Fig. 1, it can be seen that there is no difference in the growth rate between castrate males or intact females; however, in intact males the tumor was slightly but significantly larger than the castrate or female at all time points. This led to the conclusion that the tumor is hormone independent in its growth characteristics. Indeed, if diethylstilbestrol is administered in daily doses of 0.1 mg/kg, no effects are seen on the tumor growth (data not shown). This dose is 10 times greater than the ED,, dose required for maximal reduction of the size of the prostate of normal male rats [ 11. At 0.1 mg/kg, activity of diethylstilbestrol is considered to be indirect in that it inhibits the release of pituitary luteinizing hormone, which in turn results in decreased production of testosterone by the testes. The resulting lower serum levels of testosterone are responsible for the reduced size of the prostate. Therefore, doses of diethylstilbestrol at 0.1 mg/kg being ineffectual in inhibiting the growth of the R3327-At tumor were consistent with the hormone independent nature of the R3327-At tumor. However, as we
59
-3 t
I o
6
2
4
6
8
0
I2
14
I6
16
20
22
DAYS
Fig. 1. Tumor volume change of the R3327-At tumor following inoculation of 2 X lo6 viable tumor cells into intact males (a), castrate males ((I), females (A), or diethylstilbestrol, 20 mg/kg, treated castrate males (0). Volumes were calculated from caliper measurements as 1 x w x h x 0.5236 and plotted as the natural log of the calculated volume. Each point represents the mean of the natural log of the volume + S.E.M. These are 10 tumors/group.
often administer doses of androgen in the 20 mg/kg range, we decided to demonstrate that even at this higher dose of estrogen that there was no effect on tumor growth; Figure 1 illustrates that in contrast to our expectations, there was an inhibitory effect on tumor growth at this high dose of diethylstilbestrol. As calculation of apparent tumor volume doubling times are about 2 days for the diethylstilbestrol group, as well as the other groups, it appeared that this high dose of diethylstilbestrol was affecting primarily the initial phase following the inoculation of tumor. Table I lists the results of an experiment in which the antiestrogen tamoxifen and the estrogens diethylstilbestrol and 17/l-estradiol were administered at daily doses of 20 mg/kg. In all cases and for all the parameters reported except DNA, the treated groups were significantly different from the controls at P < 0.05 level. However, tamoxifen, a weak estrogen agonist used primarily for its estrogen antagonistic activity, was the least active [4]. It reduced tumor wet weight by 46%, but only reduced DNA content, and hence tumor cellularity, by 23%; however, this decrease in DNA content was not significant at P < 0.05 by the Student’s t-test. 17/3-Estradiol reduced tumor weight by 67% and also reduced DNA content by 68%. Diethylstilbestrol was the most effective of the 3 compounds and reduced tumor weight by 75% and tumor DNA by 85%. When 17&estradiol and diethylstilbestrol only are compared, the final tumor wet weight did not differ significantly between 17/l-estradiol and diethylstilbestrol treatments, however, the DNA
60 TABLE I RESPONSE OF THE R3327-At TUMOR TO THE ADMINISTRATION OF THE ANTIESTROGEN TAMOXIFEN (TAM) AND THE ESTROGENS 17p-ESTRADIOL (E, ) AND DIETHYLSTILBESTROL (DES) Treatmenta
Tumor wetb wt
Total mg DNA
[ ‘H] ThymidineC incorporation
Castrate TAM E: DES
2.50 1.34 0.83 0.63
4.71 3.64 1.52 0.71
5500 8700 11,100 15,933
f 0.37 + 0.13d t O.lld t 0.12d
+ 0.62 + 0.52 ? 0.21d * 0.20d
+ 490 i 650d f 1070d * 1530d
aAll animals were castrated 1 week prior to inoculation of tumor. The castrate controls received sesame oil only 1.0 ml/kg S.C. daily. Tamoxifen, 17p-Estradiol and Diethylstilbestrol were dissolved in sesame oil and administered at 20 mg/kg S.C. bMean gram wet wt of 10 tumors t S.E.M. ‘Two hours prior to sacrifice the animals were given 0.5 PCi [ ‘Hlthymidine (5 mCi/mM)/g body wt. The values represent mean cpm of [‘Hlthymidine incorporated/mg DNA dDiffers significantly from control (P < 0.05) by Student’s f-test.
content did. DES treatment resulted in less than half (0.7 vs. 1.5) the tumor DNA content of that determined for the 17fl-estradiol group. This further illustrates that DES was the most effective of these agents in reducing total cellularity of the tumor. Even though estrogens at these doses tend to produce a weight loss, we found a significant decrease in tumor wet weight for all treated groups when the data is corrected for body weight. We find the 1 tumor wet wt/lOO g body wt to be 1.35, 0.77, 0.53 and 0.43 for the castrate control, tamoxifen, 17/3-estradiol and diethylstilbestrol groups, respectively. Further, we administered these high doses of estrogens for much longer periods of time and have not found them to be lethal. Unexpectedly, the use of these compounds resulted in an increased incorporation of [3H]thymidine into the DNA of the tumor. Tamoxifen increased the incorporation by 58%, 17P-estradiol by 202% and diethylstilbestrol by 290%. Previously, Sufrin et al. described a close correlation between in vitro [ 3H]thymidine uptake and the calculated and actual tumor doubling time of the R3327-At tumor [ 71. This suggested that there was no substantial ‘cell loss’ factor affecting the growth of the R3327-At tumor. Sufrin et al. also found substantial agreement between the in vitro and in vivo labelling techniques using [ 3H] thymidine. Because increased [ 3H] thymidine incorporation into the high dose estrogen treatment groups was found, it may mean that estrogen treatment has increased the rate of cell loss from the tumor. The higher apparent rate of DNA synthesis in the estrogen treated tumor may also mean that high dose estrogen treatment may make this tumor more susceptible to treatment with cell cycle specific chemotherapeutic agents.
61
It appears that estrogen in high doses has growth inhibitory properties on a tumor that would not be expected to be inhibited by estrogens. This may be due to a direct inhibitory effect or may be related to inhibitory substances produced in response to high dose not low dose estrogen administration. ACKNOWLEDGEMENT
The authors M. Baumann.
wish to acknowledge
the excellent
technical
assistance
of
REFERENCES 1 He&on, W.D.W. and Coffey, D.S. (1974) New concept in the control of abnormal prostatic growth: combination hormone therapy. The Pharmacologist, 12, 275 (abst.). 2 Heston, W.D.W., Menon, M., Tananis, C. and Walsh, P.C. (1979) Androgen, estrogen and progesterone receptors of the R-3327-H Copenhagen rat prostatic tumor. Cancer Lett., 6, 45-40. 3 Issacs, J.T., Heston, W.D.W., Weissman, R.M. and Coffey, D.S. (1978) Animal models of hormone sensitive and insensitive prostatic adenocarcinomas; Dunning R-3327~HIF, and R3327-AT. Cancer Res., 38,4353-4359. 4 Jordan, V.C. (1976) Antiestrogenic and antitumor properties of tamoxifen in laboratory animals. Cancer Treat. Rep., 60, 1409-1419. 5 Markland, F.J., Chopp, R.T., Coxgrove, M.D. and Howard, E.B. (1978) Characterization of steroid hormone receptors in the Dunning R-3327 rat prostatic adenocarcinoma. Cancer Res., 38, 2828-2925. 6 Smolev, J.K., Heston, W.D.W., Scott, W.W. and Coffey, D.S. (1977) Charact.erization of the Dunning R-3327-H prostatic carcinoma: an appropriate animal model for prostatic cancer. Cancer Treat. Rep. 16, 273-287. 7 Sufrin, G., Heston, W.D.W., Fair, W.R. and Meyer, J. (1979) Kinetic studies of the R3327 Dunning murine prostatic carcinoma. Proc. Am. Urol. Assoc., 74, 46 (Abst.). 8 Voight, W., Feldman, M. and Dunning, W.F. (1975) 5a-Dihydrotestosterone-binding proteins and androgen sensitivity in prostatic cancers of Copenhagen rats. Cancer Res., 35, 1840-1846.