Changes in the Hormone Receptors of Human Breast Carcinoma Xenografis in Nude Mice by Treatment with Cytotoxic Agents Jun-ichi KoH,* Eiichi SHIINA,*Yoichiro HOSODA,*Mitsumasa HASHIMOTO,* Osami YAMAMOTO,*Shoji SAKAI,* Tetsuro KtmOTA,** Koji ENOMOTO** and Osahiko ABE** ABSTRACT: We examined the effect of chemotherapeutic agents on the estrogen receptors (Ell) o f breast carcinomas in vivo using human breast carcinoma strains (Br-10, T-61) serially transplanted into nude mice. When the tumor size reached approximately 1 X 1 X 1 cm, mitomycin C (MMC) at doses o f 1, 2 and 4.5 m g / k g and cyclophosphamide (CPA) at a dose o f 120 mg/kg, were administered once intraperitoneally, and the ERs o f the rumors were measured sequentially by the dextran-coated charcoal method. Four days after the MMC administration at above doses, the binding sites o f ER in Br-10 were not reduced and binding affinity was not affected. When the changes in ER content with time after the treatment with 4.5 m g / k g MMC and 120 m g / k g CPA were investigated, the ER content was f o u n d to be stable until 4 days after the treatment with both drugs, although the growth ofT-61 had been significantly inhibited by the drugs. From these findings, it seems reasonable to initiate chemotherapy before endocrine therapy, since the chemotherapeutic agents did not reduce the ER content o f the breast cancer strains. KEY WORDS: therapy
estrogen receptor, breast cancer, nude mouse, c h e m o -
INTRODUCTION I n the past decade, significant progress has been achieved in the treatment o f breast carcinoma. Since Cooper* first reported a high rate of response to combined chemotherapy, many combined chemotherapy regimens have been developed. ~,3 With regard
*The Department of Surgery, Social Insurance Saitama Chuo Hospital, Saitama, Japan and ~*the Department of Surgery, School of Medicine, Keio University, Tokyo,Japan Reprint requests to:Jun-ichi Koh, MD, The Department of Surgery, Social Insurance Saitama Chuo Hospital, 4-9-3 Kitaurawa, Urawa City, Saitama 336,Japan
to endocrine therapy, estrogen receptor (ER) has been recognized as a biological indicator of endocrine sensitivity,* and agents such as tamoxifen 5 and medroxyprogesterone acerate 6 have been newly synthesized. Since it has been suggested that breast cancers are heterogeneous in terms o f estrogen receptors, consisting of ER-positive and -negative cells, 7,8 combined chemoendocrine therapy has been applied for more effective breast cancer treatment. T h e rationale for combined chemoendocrine therapy is based on the hypothesis that ER-positive cells will be controlled by endocrine therapy, while ER-negative cells will be killed by chemotherapeutic agents. Yang & Samaan ~ have reported, however,
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that vincristine, methotrexate and 5-fluorouracil reduce the ER content of MCF-7 cells in vitro. Clarke et al? ~ also reported that exposure of MCF-7 cells to chemotherapeutic agents resulted in a reduction of ER expression, and that the chemotherapeutic agent-induced abolition of ER expression might protect cells from the antiproliferative effect of tamoxifen. Although these investigations were conducted using the MCF-7 cell line in vitro, no information was obtained for an in vivo system with regard to the effect of chemotherapeutic agents on ER expression. Thus, while the system involving transplantation of h u m a n t u m o r xenografts into nude mice has b e e n widely applied for the screening of chemotherapeutic agents, ~ strains of h u m a n breast carcinoma xenografts that maintain their h o r m o n e dependency and ER expression have b e e n rarely reported. *=,~3In the present paper, we report the effect of chemotherapeutic agents on ER content in vivo using h u m a n breast carcinoma strains with ER serially transplanted into nude mice. MATERIALS AND METHODS
Mice Female nude mice with a BALB/c n u / n u genetic b a c k g r o u n d were o b t a i n e d f r o m CLEA Japan, Tokyo, and maintained u n d e r specific pathogen-free conditions using vinyl isolators at our institute. Six to seven week old mice each weighing 20-22 g were used for the experiments. Tumors Two h u m a n breast carcinoma strains, Br10 a n d T-61, serially transplanted into nude mice were used. Br-10 was established from the cancerous pleural effusion of a 43 year old w o m a n with c o m m o n ductal carcinoma in 1974 by Hirohashi et al., 12 and kindly supplied to our institute. T-61 was derived from the cancerous tissue of a 54 year old w o m a n with breast cancer by Bastert, ~4 and supplied by Dr. Brfinner of the University of Copenhagen. Both strains possessed ER, and their growth was d e p e n d e n t on estrogen.
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Two tumor tissue fragments were inoculated into the subcutaneous tissue of the bilateral backs of ether anesthetized nude mice using a trocar needle. Agents Agents used for the experiments were mitomycin C (MMC; Kyowa Hakko, Tokyo) a n d c y c l o p h o s p h a m i d e (CPA; Shionogi, Osaka). MMC at doses of 1, 2 and 4.5 m g / k g and CPA at a dose of 120 m g / k g dissolved in 0.2 ml of saline were administered intraperitoneally w h e n the transplanted tumors had started exponential growth. Evaluation T u m o r s were m e a s u r e d with sliding calipers twice a week and the tumor weight (w) in mg was estimated by the m e t h o d o f Geran et al. 15 from the linear m e a s u r e m e n t using the formula: w=(width in mm) ~ X length in m m / 2 . T h e relative m e a n tumor weight was calculated, using the formula RW=Wi/Wo, where Wi was the m e a n tumor weight at any given time and Wo was the initial m e a n t u m o r weight. T h e growth curve was drawn by plotting RW against time, in days, after treatment. T / C ratios were calculated, where T was the RW of the treated group and C was the RW of the control group. Each group consisted of three to four mice. Hormone receptor assay Cytosol ER a n d p r o g e s t e r o n e r e c e p t o r (PgR) were m e a s u r e d by the dextran-coated charcoal m e t h o d as previously described26 Briefly, after the tumor tissues h a d been minced and h o m o g e n i z e d in four volumes of TED buffer, the h o m o g e n a t e was centrifuged for 60 rain at 2~ Following centrifugation, aliquots of supernatant were incubated with varying concentrations of [3H]estradiol in the presence or absence of 1,000 fold cold estradiol. After incubation for 16 h, 50 #1 of charcoal-coated dextran was added to each tube and vortexed 3 times for 15 rain. Each tube was then centrifuged for 10 rain, after which 0.1 ml aliquots of the supernatants were added to 10 ml of scintillation cocktail, then counted in a scintillation counter. To determine the dissociation constant (Kd),
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Scatchard's ~ analysis was performed. PgR was also measured with pH]R5020, corresponding to the ER assay? 8
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binding sites of ER were not altered remarkably at each dose of MMC in comparison with those of controls, and no significant difference was observed by Student's t test between the control group and the group treated with 4.5 mg MMC per kg. While the binding sites o f PgR following treatment with MMC at doses of 1 and 2 m g / k g were not reduced, the PgR of Br-10 could not be detected following treatment with MMC at a dose of 4.5 m g / k g in 2 of 3 tumors. Temporal changes in the ER of Br-10 treated with 4.5 m g / k g MMC and 120 m g / k g CPA are shown in Fig. 1. T h e data represent the mean and standard deviations of three materials consisting of two tumors for each ER assay. No significant reduction in the ER
RESULTS
The antitumor effects o f MMC and CPA on Br-10 in terms of the lowest T / C ratio are shown in Table 1. The antitumor effect of MMC was dose-dependent and the tumor growth significantly suppressed at 4.5 m g / k g MMC. The effect of CPA at a dose of 120 m g / k g was almost equivalent to that of MMC at a dose of 2 rng/kg. Changes in the ER and PgR of Br-10 four days after MMC administration at doses o f 1, 2 and 4.5 m g / k g are shown in Table 2. The
TaMe 1. The Antitumor Effect of Chemotherapeutic Agents on Br-10 Agents
Dose (mg/kg)
T/C Ratio (%)~
No. of Miceb)
1 2 4.5 120
69.3 55.1 40.8 58.6
4 4 4 4
Mitomycin C
Cyclophosphamide
a) The lowest T/C value of relative mean tumor weight during the experiment. b) Number of mice. Table 2. The Effect of MMC against the Hormone Receptors of Br-10 ERat Binding Site (fmol/mg protein)
Kd~ (10-~~
PgRb~ Binding Site (fmol/mg protein)
Control
123d) 127.3 128
0.26 0.07 0.07
118.7 111.8 111.9
2.43 2.43 2.7
Mitomycin C 1 mg/kg
90.7 107.3
0.95 0.98
70.2 85.6
0.05 0.71
Mitomycin C 2 mg/kg
115.1 112.8
0.29 0.29
98.4 115.5
1.9 0.05
Mitomycin C 4,5 mg/kg
122.7 113.6 120
1.0 0.03 0.03
94.6 UDr UD
1.0
a) b) c) d) e)
Cytosol estrogen receptor Cytosol progesterone receptor Dissociation constant Data shown are those obtained 4 days after ip treatment Undetectable; 0 fmol/mg protein
K d c/
(10-~~
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0 Control 9 CPA 120mg/kgX1 ip
fmol/mg protein 500-
0 MMC ~2.0
400: 300-
r
~
5v~ 200-
1.0
100
6 1
i2
2'4
48
9'6h
Hours after Treatment Days after Initial Treatment
MMC 4 . 5 m g / k g ip or C P A 120mg/kg ip
Fig. 1. Changes in cytosol ER with time after chemotherapy. Each point represents the mean of three materials with standard deviations. Estrogen receptors were not abolished at any time following the chemotherapy of MMC and CPA. o Control 9 MMC 4.5mg/kgX1 ip
Fig. 3. The effect of CPA on T-61. Each group consisted of six tumors. The antitumor activity of CPA was also evaluated as positive, although the effect of this drug was inferior to that of MMC. fmol/mg protein 56 40-
~ 20~
1.0-
6 1'0 1'3 Days after Initial Treatment
1'7
Fig. 2. The effect of MMC on T-61. Each group consisted of six tumors. The antitumor activity of MMC was evaluated as positive because the lowest T / C ratio of the estimated tumor weight was 18.8 per cent on day 17 with tumor regression. level was o b s e r v e d statistically at a n y time following these treatments. T h e a n t i t u m o r activity o f 4.5 m g / k g M M C a g a i n s t T-61 is illustrated in Fig. 2. T-61 t u m o r s r e g r e s s e d f o l l o w i n g MMC a d m i n i s tration, r e s u l t i n g in a T / C ratio f o r t u m o r w e i g h t o f 18.8 p e r cent, 17 days after t h e t r e a t m e n t . T h e effect o f 120 m g / k g CPA a g a i n s t T-61 is s h o w n in Fig. 3. T h e growth o f
Fig. 4. Time course of the ER of T-61 after mitomycin C treatment. Data are shown as the mean of two determinations consisting of four to six tumors. The ER expression of T-61 tumor was not abolished by MMC until 17 days after the MMC treatment. T-61 was also s u p p r e s s e d b y CPA, a n d the T / C ratio at 17 days was 49.0 p e r cent. T h e t i m e c o u r s e o f t h e ER level o f T-61 after M M C t r e a t m e n t is s h o w n in Fig. 4. Data are s h o w n with t h e m e a n v a l u e o f two m a t e r i a l s c o n s i s t i n g o f f o u r to six r e d u c e d tumors. It s e e m e d t h a t t h e b i n d i n g sites o f ER w e r e u n c h a n g e d o n Days 1 a n d 2. A l t h o u g h t h e ER levels w e r e slightly r e d u c e d o n Day 17, n o c o m p l e t e d i s a p p e a r a n c e o f ER was o b s e r v e d with r e g r e s s i o n o f t h e tumors.
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fmol/mg protein 60 50 ~ _ ~ 40 =~30.
~z 20" 10. bi~
DaysafterCyelophosphamide Treatment
~7
Fig. 5. Time course of the ER of T-61 after cyclophosphamide treatment. Data are shown as the mean of two determinations consisting of four to five tumors. The ER expression was stable until 17 days after the CPA treatment.
The effect of CPA on the ER content ofT-61 is shown in Fig. 5 as the m e a n value o f two materials consisting of four to five tumors. The binding sites o f ER remained positive during the experiments, as in the MMC treatment. These results thus revealed no evidence of reduction in ER levels in h u m a n breast carcinoma xenografts following exposure to the c h e m o t h e r a p e u t i c agents, MMC a n d CPA.
DISCUSSION Because the efficacy of endocrine therapy alone on ER-positive breast cancers is limited to 50-60 per cent, 4 c h e m o t h e r a p y is needed in order to control the remaining tumor cells through a different m o d e of action. In the clinical field, however, the results of chemoendocrine therapy are still controversial. CocconP 9 and Mouridsen 2~reported that the response rate for c h e m o e n d o c r i n e therapy' was significantly superior to that of chemotherapy alone in a r a n d o m i z e d prospective study of patients with advanced breast cancer, some trials have proven that adjuvant chemoendocrine therapy for primary breast
93
cancer reduced the recurrence rate in comparison with c h e m o t h e r a p y alone, ul#~ O n the other hand, Kardinal 2a and Bezwoda 24 noted that there was no advantage to be gained by the addition of c h e m o t h e r a p y to endocrine therapy, suggesting the possibility that tamoxifen serves to antagonize the effect of cyclespecific agents by accumulating tumor cells at the G1 phase. 23This indicated the n e e d for further studies to confirm the efficacy o f c h e m o e n d o c r i n e therapy. T h e present study was conducted to observe the stability o f ER expression in breast cancer cells in vivo following the exposure to chemotherapeutic agents. Although it has b e e n reported that chemotherapeutic agents reduce the ER content of MCF-7 cells in vitro, 9,l~ we observed no significant changes in the ER binding sites or binding affinity of h u m a n breast carcinoma xenografts following MMC and CPA treatment until 4 days after the administration. While the Kd values varied from 0.03 to 1.0 X 10 -1~ M, all of the values were less than 1 X 10 -9 M, which is generally accepted to be the specific binding affinity, 16 and each coefficient of correlation in regression equations using Scatchard's analysis was m o r e than 0.6, suggesting that these results indicate the specific binding affinity of ER to estrogen. T h e deviations of Kd values in h u m a n breast carcinoma xenografts might be concerned with the necrosis of the tumor caused by c h e m o t h e r a p y or tumor growth. Although MMC and CPA were eliminated from the serum a n d tumors of the nude mice on Day 4, the present doses were thought to be high enough to affect the cell kinetics o f the tumor cells because tumor growth was significantly i n h i b i t e d by these drugs in terms o f the T / C ratio and growth curve. I f MMC a n d CPA directly suppressed the ER protein synthesis o f the t u m o r cells, then the ER content would have b e e n reduced by these agents during the first 4 days. However, our results showed no remarkable change in the ER content of the breast carcinoma xenografts.
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Koh et al.
Moreover, to clarify the correlation between ER expression and tumor cell death, we investigated the alteration in the ER content of tumor cells caused by chemotherapy until tumor regression on Day 17. W h e n 4.5 m g / k g MMC was administered to T-61, the tumor growth was markedly suppressed with a T / C of 18.8 per cent, 17 days after treatment, but the content of ER was not abolished without alteration of the binding affinity. Similarly to MMC, CPA at a dose of 120 m g / k g did not reduce the content of ER 17 days after administration. Yang and Samaan 9 reported a reduction of ER expression in MCF-7 cells following exposure to vincristine, methotrexate and 5fluorouracil, but the reduction occurred only 4 h after exposure and no alteration in ER content was observed within 3 h. Although their report did not show how m a n y cells were killed by the exposure to cytotoxic agents for 4 h, the reduction of ER expression might have b e e n due to the death of MCF-7 cells. T h e y also noted that the cellkilling activities of these drugs, as determ i n e d by the colony formation technique, were proportional to the degree of ER reduction. Clarke et a12 ~ reported that exposure to cytotoxic agents resulted in a reduction of the ER content of MCF-7 cells and that a combination of 1 nM vincristine followed by 2 /.tM of tamoxifen produced no additive effect, although 0.5 nM vincristine, which reduced the ER content by approximately 70 per cent, did not influence the antiproliferative effect of tamoxifen? 5 In contrast with these in vitro results, the present study clearly demonstrated that the expression of ER in h u m a n breast carcinoma xenografts was stable after the treatment of MMC and CPA. This discrepancy of in vitro and in vivo results might have b e e n caused by the different cytotoxic agents a n d tumor cell lines used in these experiments. Adriamycin, vincristine, methotrexate and 5-fluorouracil which were reported in the in vitro systems 9,a~were not used in the present study. Adriamycin a n d 5-fluorouracil were not
Jpn. J. Surg. January 1990
adopted for the experiments because Br-10 and T-61 are insensitive to these drugs, which are thought to have little influence on the ER systems of these strains. Vincristine and methotrexate were also omitted from the study since these drugs are rarely used for breast cancer in our country. Although it might be more favorable to investigate the stability of the ER expression of Br-10 and T-61 in an in vitro system to compare it with the in vivo results, repeated trials failed to establish in vitro cell lines of these strains because of the contamination of stromal cells. However, if the various changes in ERs were caused by in vitro a n d in vivo discrepancies, h u m a n tumor xenografts could have the advantage of b e i n g able to present the clinical features, since cultured cell lines in vitro lack the cell to cell interaction, as well as the endocrine and immunological circumstances of the host. In a previous report, we stated that prior MMC treatment followed by tamoxifen is more effective than the reversed sequence and estimated that this discrepancy was due to the different m o d e of action of MMC and tamoxifen o n the t u m o r cell kinetics determined by a labeling index? ~ Furthermore, the c o m b i n e d therapy of MMC followed by tamoxifen was more effective against Br-10 and T-61 in vivo t h a n using each drug singly, without any additive side effectsY A clinical study also revealed that adjuvant chemo- and endocrine therapy used in operable breast cancer patients is m o r e effective for prolonging the overall a n d disease free survivals of ER positive and postmenopausal patients with Stage II and I I I a breast cancer when c o m p a r e d with c h e m o t h e r a p y alone. ~ These data suggested that tamoxifen could have an antiproliferative action in vivo after prior c h e m o t h e r a p y using MMC. T h e pre'sent p a p e r which focused on the changes in ER systems caused by c h e m o t h e r a p y indicated that ERs were not abolished by the prior c h e m o t h e r a p y of MMC and CPA. These data were compatible with the experimental and clinical results mentioned
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Changes in hormone receptor by chemotherapy
above, and the sequential chemo- and endoc r i n e t h e r a p y w a s t h o u g h t to b e r a t i o n a l from the viewpoint of the changes of ER e x p r e s s i o n i n b r e a s t c a r c i n o m a cells c a u s e d by c h e m o t h e r a p y .
12.
13. ( R e c e i v e d f o r p u b l i c a t i o n o n M a r . 2, 1989) REFERENCES 1. Cooper R. Combination chemotherapy in human resistant breast cancer. Proc Am Assoc Cancer Res 1969; 10: 15. 2. Bonadonna G, Valagnssa P, Rossi A, Tancini G, Brambila C, Zambetti M, Veronesi U. Ten year experience with CMF-based adjuvant chemotherapy in resectable breast cancer. Breast Cancer Res 1985; 5: 95-115. 3. Abe O, Izuo M, Enomoto K, Kubo K, Koyama H, Sakai K, Terasawa T, Tominaga I~ Nomura Y. Combination chemotherapy of cydophosphamide, adriamycin and 5-ttuorouracil (CAF) in advanced and recurrent breast cancer. Gan to Kagaku Ryohou (Jpn J Cancer Chemother) 1982; 9: 866-873. (in Japanese) 4. Nomura Y, Takatani O, 8ugano H, Matsumoto K. Oestrogen and progesterone receptors and response to endocrine therapy in Japanese breast cancer. J Steroid Biochem 1980; 13: 565-566. 5. Mouridsen HT, Palshof T, Patterson J, Battersby L. Tamoxifen in advanced breast cancer. Cancer Treat Rev 1978; 5: 131-141. 6. Pannuti F, DiMarco A, Martoni A Medroxyprogesterone acetate in treatment of metastatic breast cancer. In: Role of medroxyprogesterone in endocrine-related tumors. New York: Raven Press 1982; 73-92. 7. Allegra JC, Barlock A, Huff KK, Lippman ME. Changes in multiple or sequential ER determinations in breast cancer. Cancer 1980; 45: 792-794. 8. Raam S, Nemeth E, Obrian S, Tamura H, CohenJL. Immunohistochemical localization of estrogen receptors in human breast carcinoma using antireceptors antibodies. Proc Am Assoc Cancer Res 1981; 22: 10. 9. Yang KP, Samaan NA. Reduction of estrogen receptor concentration in MCF-7 human breast carcinoma cells following exposure to chemotherapeutic drags. Cancer 1983; 43: 3534-3538. 10. Clarke R, Morwood J, van den Berg H, Nelson J, Murphy R. Effect of cytotoxic dnJgs on estrogen receptor expression and response to tamoxifen in MCF-7 cells. Cancer Res 1986; 46:6116-6119. 11. Kubota T, Shimosato Y, Nagai K. Experimental chemotherapy of carcinoma of the stomach and
14.
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17. 18.
19.
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colon serially transplanted in nude mice. Gann 1977; 69: 299-309. Hirohashi S, Shimosato Y, Kameya T, Nagai I~ Tsunematsu R. Horraone dependency of a serially transplantable human breast cancer (Br-10) in nude mice. Cancer Res 1977; 37: 3184-3189. Kubota T, Kubouchi K, KohJ, Enomoto K, Ishibiki K, Abe O. Human breast carcinoma (MCF-7) serially transplanted into nude mice. Jpn J Surg 1983; 13: 381-384. Briinner N, Spang-Thomsen M, Skovgaard SA, Engelholm A, Nielsen A, Vindelov L. Endocrine sensitivity of receptor positive T-61 human breast carcinoma serially grown in nude mice. Int J Cancer 1985; 35: 59-64. Geran RI, Greenberg NH, Macdonald MM, Shumacher AM, Abort BJ. Protocols for screening chemical and natural products against animal tumors and other biological systems. Cancer Chemother Rep 1972; 3: 51-61. Yamasaki T. Estrogen receptor in malignant and benign breast tumors and normal tissues of Japanese patients.JpnJ Clin Oncol 1978; 8: 37-47. Scatchard G. The attractions of proteins for small molecules and ions. Ann Sci 1949; 51: 660-672. Ikeda T. Studies on progesterone receptor in malignant and benign breast tumors and normal breast tissues - - i n relation to estrogen receptor--. Keio Igaku (Journal of the Keio Medical Society) 1983; 60: 489-501. (in Japanese) Clarke R, Morwood J, Nelson J, van den Berg H, Murphy R. The influence of vincristine on the oestrogen-binding capacity of two human breast cancer cell lines in vitro. Biochem Soc Trans 1986; 14: 449-450. Cocconi G, De Lisi V, Boni C, Mori P, Malacarne P, Amadori D, Giovanelli E. Chemotherapy versus combination of chemotherapy and endocrine therapy in advanced breast cancer, a prospective randomized study. Cancer 1983; 51: 581-588. Mouridsen HT, PalshofT, Engelman E, Sylvester R. CMF versus CMF plus tamoxifen in advanced breast cancer in postmenopausal woman. An EORTC trial. In: Mouridsen HT, Palshof T, eds. Breast cancer-experimental and clinical aspects. Oxford: Pergamon Press 1980; 119-123. Fisher B, Redmond C, Brown A, Wolmark N, Wittliff J, Fisher E, Plotkin D, Bowman D, Sachs S, WolterJ, Frelik R, Desser R, Licalzi N, Geggie P, Campbell T, Elias GE, Prager D, Koontz P, Volk H, Dimitrov N, Gardner B, Lerner H, Shibata H. Treatment of primary breast cancer with chemotherapy and tamoxifen. N EnglJ Med 1981; 305: 1-6. Hubay CA, Pearson OH, Marshall JS, Stellato TA, Rhodes RS, DeBanne SM, Rosenblatt J, Mansour EG, Hermann RE, Jones JC, Flynn WJ, Eckert C, McGuire WL. Adjuvant therapy of stage II breast
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cancer 48-month follow-up of a prospective randomized clinical trial. Breast Cancer Res Treat 1981; 1: 77-82. 24. Kardinal CG, Perry MC, Weinberg V, Wood W, Ginsberg S, Raju RN. Chemoendocrine therapy vs chemotherapy alone for advanced breast cancer in postmonopausal woman. Breast Cancer Res Treat 1983; 3: 365-372. 25. Bezwoda WR, Derman D, DeMoor NG, Lange M, Levin J. Treatment of metastatic breast cancer in estrogen receptor positive patients, a randomized trial comparing tamoxifen alone versus tamoxifen plus CMF. Cancer 1982; 50: 2747-2750. 26. Kubota T, Kubouchi K, KohJ, Asanuma F, Enomoto
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K, Ishibiki I~ Abe Ot Combined cytotoxic and endocrine therapy for human breast carcinoma (Br-10) serially transplanted into nude mice. Jpn J Surg 1984; 14: 78-84. 27. Koh J. Experimental chemo and chemoendocrine therapy of human breast carcinomas serially transplanted into nude mice. Nippon Geka Gakkai Zasshi (J Jpn Surg Soc) 1988; 89: 91-102. (in Japanese with English Abst.) 28. Enomoto K, Abe O. The evaluation of adjuvant c h e m o e n d o c r i n e therapy for breast cancers. --Comparison of tegafur and tegafur § tamoxif e n - - Gan no Rinsho (]pnJ Cancer Clinic) 1987; suppl 13: 165-181. (in Japanese)
estrogen receptor, breast cancer, nude mouse, c h e m o -
INTRODUCTION I n the past decade, significant progress has been achieved in the treatment o f breast carcinoma. Since Cooper* first reported a high rate of response to combined chemotherapy, many combined chemotherapy regimens have been developed. ~,3 With regard
*The Department of Surgery, Social Insurance Saitama Chuo Hospital, Saitama, Japan and ~*the Department of Surgery, School of Medicine, Keio University, Tokyo,Japan Reprint requests to:Jun-ichi Koh, MD, The Department of Surgery, Social Insurance Saitama Chuo Hospital, 4-9-3 Kitaurawa, Urawa City, Saitama 336,Japan
to endocrine therapy, estrogen receptor (ER) has been recognized as a biological indicator of endocrine sensitivity,* and agents such as tamoxifen 5 and medroxyprogesterone acerate 6 have been newly synthesized. Since it has been suggested that breast cancers are heterogeneous in terms o f estrogen receptors, consisting of ER-positive and -negative cells, 7,8 combined chemoendocrine therapy has been applied for more effective breast cancer treatment. T h e rationale for combined chemoendocrine therapy is based on the hypothesis that ER-positive cells will be controlled by endocrine therapy, while ER-negative cells will be killed by chemotherapeutic agents. Yang & Samaan ~ have reported, however,
JAPANESEJOU~'~ALOF SURGERY,VOU 20, NO. 1 pp. 89-96, 1990
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that vincristine, methotrexate and 5-fluorouracil reduce the ER content of MCF-7 cells in vitro. Clarke et al? ~ also reported that exposure of MCF-7 cells to chemotherapeutic agents resulted in a reduction of ER expression, and that the chemotherapeutic agent-induced abolition of ER expression might protect cells from the antiproliferative effect of tamoxifen. Although these investigations were conducted using the MCF-7 cell line in vitro, no information was obtained for an in vivo system with regard to the effect of chemotherapeutic agents on ER expression. Thus, while the system involving transplantation of h u m a n t u m o r xenografts into nude mice has b e e n widely applied for the screening of chemotherapeutic agents, ~ strains of h u m a n breast carcinoma xenografts that maintain their h o r m o n e dependency and ER expression have b e e n rarely reported. *=,~3In the present paper, we report the effect of chemotherapeutic agents on ER content in vivo using h u m a n breast carcinoma strains with ER serially transplanted into nude mice. MATERIALS AND METHODS
Mice Female nude mice with a BALB/c n u / n u genetic b a c k g r o u n d were o b t a i n e d f r o m CLEA Japan, Tokyo, and maintained u n d e r specific pathogen-free conditions using vinyl isolators at our institute. Six to seven week old mice each weighing 20-22 g were used for the experiments. Tumors Two h u m a n breast carcinoma strains, Br10 a n d T-61, serially transplanted into nude mice were used. Br-10 was established from the cancerous pleural effusion of a 43 year old w o m a n with c o m m o n ductal carcinoma in 1974 by Hirohashi et al., 12 and kindly supplied to our institute. T-61 was derived from the cancerous tissue of a 54 year old w o m a n with breast cancer by Bastert, ~4 and supplied by Dr. Brfinner of the University of Copenhagen. Both strains possessed ER, and their growth was d e p e n d e n t on estrogen.
Jpn. J. Surg. January 1990
Two tumor tissue fragments were inoculated into the subcutaneous tissue of the bilateral backs of ether anesthetized nude mice using a trocar needle. Agents Agents used for the experiments were mitomycin C (MMC; Kyowa Hakko, Tokyo) a n d c y c l o p h o s p h a m i d e (CPA; Shionogi, Osaka). MMC at doses of 1, 2 and 4.5 m g / k g and CPA at a dose of 120 m g / k g dissolved in 0.2 ml of saline were administered intraperitoneally w h e n the transplanted tumors had started exponential growth. Evaluation T u m o r s were m e a s u r e d with sliding calipers twice a week and the tumor weight (w) in mg was estimated by the m e t h o d o f Geran et al. 15 from the linear m e a s u r e m e n t using the formula: w=(width in mm) ~ X length in m m / 2 . T h e relative m e a n tumor weight was calculated, using the formula RW=Wi/Wo, where Wi was the m e a n tumor weight at any given time and Wo was the initial m e a n t u m o r weight. T h e growth curve was drawn by plotting RW against time, in days, after treatment. T / C ratios were calculated, where T was the RW of the treated group and C was the RW of the control group. Each group consisted of three to four mice. Hormone receptor assay Cytosol ER a n d p r o g e s t e r o n e r e c e p t o r (PgR) were m e a s u r e d by the dextran-coated charcoal m e t h o d as previously described26 Briefly, after the tumor tissues h a d been minced and h o m o g e n i z e d in four volumes of TED buffer, the h o m o g e n a t e was centrifuged for 60 rain at 2~ Following centrifugation, aliquots of supernatant were incubated with varying concentrations of [3H]estradiol in the presence or absence of 1,000 fold cold estradiol. After incubation for 16 h, 50 #1 of charcoal-coated dextran was added to each tube and vortexed 3 times for 15 rain. Each tube was then centrifuged for 10 rain, after which 0.1 ml aliquots of the supernatants were added to 10 ml of scintillation cocktail, then counted in a scintillation counter. To determine the dissociation constant (Kd),
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Scatchard's ~ analysis was performed. PgR was also measured with pH]R5020, corresponding to the ER assay? 8
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binding sites of ER were not altered remarkably at each dose of MMC in comparison with those of controls, and no significant difference was observed by Student's t test between the control group and the group treated with 4.5 mg MMC per kg. While the binding sites o f PgR following treatment with MMC at doses of 1 and 2 m g / k g were not reduced, the PgR of Br-10 could not be detected following treatment with MMC at a dose of 4.5 m g / k g in 2 of 3 tumors. Temporal changes in the ER of Br-10 treated with 4.5 m g / k g MMC and 120 m g / k g CPA are shown in Fig. 1. T h e data represent the mean and standard deviations of three materials consisting of two tumors for each ER assay. No significant reduction in the ER
RESULTS
The antitumor effects o f MMC and CPA on Br-10 in terms of the lowest T / C ratio are shown in Table 1. The antitumor effect of MMC was dose-dependent and the tumor growth significantly suppressed at 4.5 m g / k g MMC. The effect of CPA at a dose of 120 m g / k g was almost equivalent to that of MMC at a dose of 2 rng/kg. Changes in the ER and PgR of Br-10 four days after MMC administration at doses o f 1, 2 and 4.5 m g / k g are shown in Table 2. The
TaMe 1. The Antitumor Effect of Chemotherapeutic Agents on Br-10 Agents
Dose (mg/kg)
T/C Ratio (%)~
No. of Miceb)
1 2 4.5 120
69.3 55.1 40.8 58.6
4 4 4 4
Mitomycin C
Cyclophosphamide
a) The lowest T/C value of relative mean tumor weight during the experiment. b) Number of mice. Table 2. The Effect of MMC against the Hormone Receptors of Br-10 ERat Binding Site (fmol/mg protein)
Kd~ (10-~~
PgRb~ Binding Site (fmol/mg protein)
Control
123d) 127.3 128
0.26 0.07 0.07
118.7 111.8 111.9
2.43 2.43 2.7
Mitomycin C 1 mg/kg
90.7 107.3
0.95 0.98
70.2 85.6
0.05 0.71
Mitomycin C 2 mg/kg
115.1 112.8
0.29 0.29
98.4 115.5
1.9 0.05
Mitomycin C 4,5 mg/kg
122.7 113.6 120
1.0 0.03 0.03
94.6 UDr UD
1.0
a) b) c) d) e)
Cytosol estrogen receptor Cytosol progesterone receptor Dissociation constant Data shown are those obtained 4 days after ip treatment Undetectable; 0 fmol/mg protein
K d c/
(10-~~
Jpn. J. Surg. January 1990
Koh et al.
92
0 Control 9 CPA 120mg/kgX1 ip
fmol/mg protein 500-
0 MMC ~2.0
400: 300-
r
~
5v~ 200-
1.0
100
6 1
i2
2'4
48
9'6h
Hours after Treatment Days after Initial Treatment
MMC 4 . 5 m g / k g ip or C P A 120mg/kg ip
Fig. 1. Changes in cytosol ER with time after chemotherapy. Each point represents the mean of three materials with standard deviations. Estrogen receptors were not abolished at any time following the chemotherapy of MMC and CPA. o Control 9 MMC 4.5mg/kgX1 ip
Fig. 3. The effect of CPA on T-61. Each group consisted of six tumors. The antitumor activity of CPA was also evaluated as positive, although the effect of this drug was inferior to that of MMC. fmol/mg protein 56 40-
~ 20~
1.0-
6 1'0 1'3 Days after Initial Treatment
1'7
Fig. 2. The effect of MMC on T-61. Each group consisted of six tumors. The antitumor activity of MMC was evaluated as positive because the lowest T / C ratio of the estimated tumor weight was 18.8 per cent on day 17 with tumor regression. level was o b s e r v e d statistically at a n y time following these treatments. T h e a n t i t u m o r activity o f 4.5 m g / k g M M C a g a i n s t T-61 is illustrated in Fig. 2. T-61 t u m o r s r e g r e s s e d f o l l o w i n g MMC a d m i n i s tration, r e s u l t i n g in a T / C ratio f o r t u m o r w e i g h t o f 18.8 p e r cent, 17 days after t h e t r e a t m e n t . T h e effect o f 120 m g / k g CPA a g a i n s t T-61 is s h o w n in Fig. 3. T h e growth o f
Fig. 4. Time course of the ER of T-61 after mitomycin C treatment. Data are shown as the mean of two determinations consisting of four to six tumors. The ER expression of T-61 tumor was not abolished by MMC until 17 days after the MMC treatment. T-61 was also s u p p r e s s e d b y CPA, a n d the T / C ratio at 17 days was 49.0 p e r cent. T h e t i m e c o u r s e o f t h e ER level o f T-61 after M M C t r e a t m e n t is s h o w n in Fig. 4. Data are s h o w n with t h e m e a n v a l u e o f two m a t e r i a l s c o n s i s t i n g o f f o u r to six r e d u c e d tumors. It s e e m e d t h a t t h e b i n d i n g sites o f ER w e r e u n c h a n g e d o n Days 1 a n d 2. A l t h o u g h t h e ER levels w e r e slightly r e d u c e d o n Day 17, n o c o m p l e t e d i s a p p e a r a n c e o f ER was o b s e r v e d with r e g r e s s i o n o f t h e tumors.
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Changes in hormone receptor by &emotherapy
fmol/mg protein 60 50 ~ _ ~ 40 =~30.
~z 20" 10. bi~
DaysafterCyelophosphamide Treatment
~7
Fig. 5. Time course of the ER of T-61 after cyclophosphamide treatment. Data are shown as the mean of two determinations consisting of four to five tumors. The ER expression was stable until 17 days after the CPA treatment.
The effect of CPA on the ER content ofT-61 is shown in Fig. 5 as the m e a n value o f two materials consisting of four to five tumors. The binding sites o f ER remained positive during the experiments, as in the MMC treatment. These results thus revealed no evidence of reduction in ER levels in h u m a n breast carcinoma xenografts following exposure to the c h e m o t h e r a p e u t i c agents, MMC a n d CPA.
DISCUSSION Because the efficacy of endocrine therapy alone on ER-positive breast cancers is limited to 50-60 per cent, 4 c h e m o t h e r a p y is needed in order to control the remaining tumor cells through a different m o d e of action. In the clinical field, however, the results of chemoendocrine therapy are still controversial. CocconP 9 and Mouridsen 2~reported that the response rate for c h e m o e n d o c r i n e therapy' was significantly superior to that of chemotherapy alone in a r a n d o m i z e d prospective study of patients with advanced breast cancer, some trials have proven that adjuvant chemoendocrine therapy for primary breast
93
cancer reduced the recurrence rate in comparison with c h e m o t h e r a p y alone, ul#~ O n the other hand, Kardinal 2a and Bezwoda 24 noted that there was no advantage to be gained by the addition of c h e m o t h e r a p y to endocrine therapy, suggesting the possibility that tamoxifen serves to antagonize the effect of cyclespecific agents by accumulating tumor cells at the G1 phase. 23This indicated the n e e d for further studies to confirm the efficacy o f c h e m o e n d o c r i n e therapy. T h e present study was conducted to observe the stability o f ER expression in breast cancer cells in vivo following the exposure to chemotherapeutic agents. Although it has b e e n reported that chemotherapeutic agents reduce the ER content of MCF-7 cells in vitro, 9,l~ we observed no significant changes in the ER binding sites or binding affinity of h u m a n breast carcinoma xenografts following MMC and CPA treatment until 4 days after the administration. While the Kd values varied from 0.03 to 1.0 X 10 -1~ M, all of the values were less than 1 X 10 -9 M, which is generally accepted to be the specific binding affinity, 16 and each coefficient of correlation in regression equations using Scatchard's analysis was m o r e than 0.6, suggesting that these results indicate the specific binding affinity of ER to estrogen. T h e deviations of Kd values in h u m a n breast carcinoma xenografts might be concerned with the necrosis of the tumor caused by c h e m o t h e r a p y or tumor growth. Although MMC and CPA were eliminated from the serum a n d tumors of the nude mice on Day 4, the present doses were thought to be high enough to affect the cell kinetics o f the tumor cells because tumor growth was significantly i n h i b i t e d by these drugs in terms o f the T / C ratio and growth curve. I f MMC a n d CPA directly suppressed the ER protein synthesis o f the t u m o r cells, then the ER content would have b e e n reduced by these agents during the first 4 days. However, our results showed no remarkable change in the ER content of the breast carcinoma xenografts.
94
Koh et al.
Moreover, to clarify the correlation between ER expression and tumor cell death, we investigated the alteration in the ER content of tumor cells caused by chemotherapy until tumor regression on Day 17. W h e n 4.5 m g / k g MMC was administered to T-61, the tumor growth was markedly suppressed with a T / C of 18.8 per cent, 17 days after treatment, but the content of ER was not abolished without alteration of the binding affinity. Similarly to MMC, CPA at a dose of 120 m g / k g did not reduce the content of ER 17 days after administration. Yang and Samaan 9 reported a reduction of ER expression in MCF-7 cells following exposure to vincristine, methotrexate and 5fluorouracil, but the reduction occurred only 4 h after exposure and no alteration in ER content was observed within 3 h. Although their report did not show how m a n y cells were killed by the exposure to cytotoxic agents for 4 h, the reduction of ER expression might have b e e n due to the death of MCF-7 cells. T h e y also noted that the cellkilling activities of these drugs, as determ i n e d by the colony formation technique, were proportional to the degree of ER reduction. Clarke et a12 ~ reported that exposure to cytotoxic agents resulted in a reduction of the ER content of MCF-7 cells and that a combination of 1 nM vincristine followed by 2 /.tM of tamoxifen produced no additive effect, although 0.5 nM vincristine, which reduced the ER content by approximately 70 per cent, did not influence the antiproliferative effect of tamoxifen? 5 In contrast with these in vitro results, the present study clearly demonstrated that the expression of ER in h u m a n breast carcinoma xenografts was stable after the treatment of MMC and CPA. This discrepancy of in vitro and in vivo results might have b e e n caused by the different cytotoxic agents a n d tumor cell lines used in these experiments. Adriamycin, vincristine, methotrexate and 5-fluorouracil which were reported in the in vitro systems 9,a~were not used in the present study. Adriamycin a n d 5-fluorouracil were not
Jpn. J. Surg. January 1990
adopted for the experiments because Br-10 and T-61 are insensitive to these drugs, which are thought to have little influence on the ER systems of these strains. Vincristine and methotrexate were also omitted from the study since these drugs are rarely used for breast cancer in our country. Although it might be more favorable to investigate the stability of the ER expression of Br-10 and T-61 in an in vitro system to compare it with the in vivo results, repeated trials failed to establish in vitro cell lines of these strains because of the contamination of stromal cells. However, if the various changes in ERs were caused by in vitro a n d in vivo discrepancies, h u m a n tumor xenografts could have the advantage of b e i n g able to present the clinical features, since cultured cell lines in vitro lack the cell to cell interaction, as well as the endocrine and immunological circumstances of the host. In a previous report, we stated that prior MMC treatment followed by tamoxifen is more effective than the reversed sequence and estimated that this discrepancy was due to the different m o d e of action of MMC and tamoxifen o n the t u m o r cell kinetics determined by a labeling index? ~ Furthermore, the c o m b i n e d therapy of MMC followed by tamoxifen was more effective against Br-10 and T-61 in vivo t h a n using each drug singly, without any additive side effectsY A clinical study also revealed that adjuvant chemo- and endocrine therapy used in operable breast cancer patients is m o r e effective for prolonging the overall a n d disease free survivals of ER positive and postmenopausal patients with Stage II and I I I a breast cancer when c o m p a r e d with c h e m o t h e r a p y alone. ~ These data suggested that tamoxifen could have an antiproliferative action in vivo after prior c h e m o t h e r a p y using MMC. T h e pre'sent p a p e r which focused on the changes in ER systems caused by c h e m o t h e r a p y indicated that ERs were not abolished by the prior c h e m o t h e r a p y of MMC and CPA. These data were compatible with the experimental and clinical results mentioned
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Changes in hormone receptor by chemotherapy
above, and the sequential chemo- and endoc r i n e t h e r a p y w a s t h o u g h t to b e r a t i o n a l from the viewpoint of the changes of ER e x p r e s s i o n i n b r e a s t c a r c i n o m a cells c a u s e d by c h e m o t h e r a p y .
12.
13. ( R e c e i v e d f o r p u b l i c a t i o n o n M a r . 2, 1989) REFERENCES 1. Cooper R. Combination chemotherapy in human resistant breast cancer. Proc Am Assoc Cancer Res 1969; 10: 15. 2. Bonadonna G, Valagnssa P, Rossi A, Tancini G, Brambila C, Zambetti M, Veronesi U. Ten year experience with CMF-based adjuvant chemotherapy in resectable breast cancer. Breast Cancer Res 1985; 5: 95-115. 3. Abe O, Izuo M, Enomoto K, Kubo K, Koyama H, Sakai K, Terasawa T, Tominaga I~ Nomura Y. Combination chemotherapy of cydophosphamide, adriamycin and 5-ttuorouracil (CAF) in advanced and recurrent breast cancer. Gan to Kagaku Ryohou (Jpn J Cancer Chemother) 1982; 9: 866-873. (in Japanese) 4. Nomura Y, Takatani O, 8ugano H, Matsumoto K. Oestrogen and progesterone receptors and response to endocrine therapy in Japanese breast cancer. J Steroid Biochem 1980; 13: 565-566. 5. Mouridsen HT, Palshof T, Patterson J, Battersby L. Tamoxifen in advanced breast cancer. Cancer Treat Rev 1978; 5: 131-141. 6. Pannuti F, DiMarco A, Martoni A Medroxyprogesterone acetate in treatment of metastatic breast cancer. In: Role of medroxyprogesterone in endocrine-related tumors. New York: Raven Press 1982; 73-92. 7. Allegra JC, Barlock A, Huff KK, Lippman ME. Changes in multiple or sequential ER determinations in breast cancer. Cancer 1980; 45: 792-794. 8. Raam S, Nemeth E, Obrian S, Tamura H, CohenJL. Immunohistochemical localization of estrogen receptors in human breast carcinoma using antireceptors antibodies. Proc Am Assoc Cancer Res 1981; 22: 10. 9. Yang KP, Samaan NA. Reduction of estrogen receptor concentration in MCF-7 human breast carcinoma cells following exposure to chemotherapeutic drags. Cancer 1983; 43: 3534-3538. 10. Clarke R, Morwood J, van den Berg H, Nelson J, Murphy R. Effect of cytotoxic dnJgs on estrogen receptor expression and response to tamoxifen in MCF-7 cells. Cancer Res 1986; 46:6116-6119. 11. Kubota T, Shimosato Y, Nagai K. Experimental chemotherapy of carcinoma of the stomach and
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cancer 48-month follow-up of a prospective randomized clinical trial. Breast Cancer Res Treat 1981; 1: 77-82. 24. Kardinal CG, Perry MC, Weinberg V, Wood W, Ginsberg S, Raju RN. Chemoendocrine therapy vs chemotherapy alone for advanced breast cancer in postmonopausal woman. Breast Cancer Res Treat 1983; 3: 365-372. 25. Bezwoda WR, Derman D, DeMoor NG, Lange M, Levin J. Treatment of metastatic breast cancer in estrogen receptor positive patients, a randomized trial comparing tamoxifen alone versus tamoxifen plus CMF. Cancer 1982; 50: 2747-2750. 26. Kubota T, Kubouchi K, KohJ, Asanuma F, Enomoto
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K, Ishibiki I~ Abe Ot Combined cytotoxic and endocrine therapy for human breast carcinoma (Br-10) serially transplanted into nude mice. Jpn J Surg 1984; 14: 78-84. 27. Koh J. Experimental chemo and chemoendocrine therapy of human breast carcinomas serially transplanted into nude mice. Nippon Geka Gakkai Zasshi (J Jpn Surg Soc) 1988; 89: 91-102. (in Japanese with English Abst.) 28. Enomoto K, Abe O. The evaluation of adjuvant c h e m o e n d o c r i n e therapy for breast cancers. --Comparison of tegafur and tegafur § tamoxif e n - - Gan no Rinsho (]pnJ Cancer Clinic) 1987; suppl 13: 165-181. (in Japanese)
