Surg Today
`Jpn J Surg (1999) 29:149—156
`
`
`
`
`A SURGERYTODAY
`8/ © Springer-Verlag 1999
`
`Effects of Experimental Chemoendocrine Therapy With a
`Combination of a Pure Antiestrogen and 5-Fluorouracil on
`Human Breast Cancer Cells Implanted in Nude Mice
`
`YUTAKA OGASAWARA, HIROYOSHI DOIHARA, KOUJI SHIROMA, YOSHIAKI KANAYA, and NOBUYOSHI SHIMIZU
`
`Department of Surgery II, Okayama University Medical School, 2—5—1 Shikata—cho, Okayama 700, Japan
`
`Abstract: The antitumor effects of an experimental chemo-
`endocrine therapy combining a new pure antiestrogen ICI
`182 780 and 5-fluorouracil (5-FU) were studied on MCF-7
`human breast cancer cells implanted in nude mice. ICI 182 780
`had a dose-dependent antitumor activity, which was potenti-
`ated by the concomitant use of S-FU. When compared with
`the control group, the estrogen receptor (ER) level in the ICI
`182780 group was lower and that in the combination group
`was markedly lower. Cell cycle analysis by flow cytometry
`(FCM) resulted in a lower percentage of S-phase cells (%S) in
`the treated mice. No significant difference was observed in the
`5-FU concentrations in tumor cells, while the 5-FU content in
`RNA was significantly higher in the combination group. The
`changes in free thymidylate synthetase (TS) concentration
`indicated TS synthesis after the administration of 5-FU to be
`more greatly suppressed in the combination group than in the
`5-FU group. These results suggest that ICI 182 780 and 5-FU
`exert their combination effect mainly on ER-positive cells,
`and that the suppression of TS synthesis in tumor cells and the
`potentiation of the S-FU-induced metabolic dysfunction of
`RNA are thus involved in the mode of action of this combina-
`
`tion therapy.
`
`Key Words: chemoendocrine therapy, pure antiestrogen,
`S-fluorouracil, nude mouse, breast cancer
`
`receptor (ER)-positive breast cancer is sensitive to
`endocrine therapy while ER-negative cancer is not very
`sensitive.1 Meanwhile, even apparently ER-positive
`breast cancer is intermingled with ER-negative cells, so
`that a combination of endocrine therapy with chemo-
`therapy which has a different mode of action is required
`to improve the response rate. In addition, a possible
`reduction of adverse reactions of chemotherapy by con-
`comitant endocrine therapy is of clinical significance.2
`Among the various forms of endocrine therapy
`for breast cancer, antiestrogens, especially tamo-
`xifen (TAM), have been the most widely used. TAM,
`although an antiestrogen, also has estrogenic activity}5
`and has recently been reported to be associated with the
`occurrence of endometrial cancer."”7 Among the newly
`developed antiestrogens,
`ICI 182780, a pure anti-
`estrogen free from estrogenic activity, is expected to be
`particularly useful in the treatment of breast cancerfi9
`In this experiment, the antitumor effects of a com-
`bination of ICI 182780 and a widely used fluorinated
`pyrimidine, 5-fluorouracil (5-FU) were studied, and
`the mode of action of this chemoendocrine therapy
`on human breast cancer implanted in nude mice was
`evaluated.
`
`Introduction
`
`Materials and Methods
`
`Breast cancer is a hormone-dependent cancer which
`is generally known to respond to chemoendocrine
`therapy. Evidence has also been obtained that estrogen
`
`Reprint requests to: Y. Ogasawara
`A summary of this paper was presented at the Fourth General
`Conference of the Japan Breast Cancer Society and the 34th
`General Meeting of the Japan Society for Cancer Therapy.
`(Received for publication on May 19, 1997; accepted on
`May 15, 1998)
`
`The animals used were BABL/c nu/nu female nude
`
`mice aged 7—8 weeks and weighing 19—22 g, obtained
`from Nippon Crea, Tokyo, Japan. The cell line used was
`MCF-7, a nude mouse transplantable human breast can-
`cer. It was a hormone-dependent, human breast cancer
`cell line originally established by Soule et al.10 in 1970
`from cancerous pleural effusion of a postmenopausal
`breast cancer patient who was 69 years old, donated
`3 years ago by Dr T. Kubota, Department of Surgery,
`Keio University Medical School, and since then has
`been serially cultivated by the anthors.
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`150
`
`Y. Ogasawara et al.: Pure Antiestrogen and 5-FU in Nude Mice
`
`The tumor was cut into fragments measuring about
`8mm3 which were implanted into the dorsal subcu-
`taneous tissue of the mice by a trocar. For the tumor to
`grow in the exponential growth phase, immediately af-
`ter the implantation 5mg/kg estradiol dipropionate and
`250 mg/kg progesterone caproate were intramuscularly
`injected into the mice.11 The nude mice bearing tumors
`weighing 40—90 mg were then assigned, 14—16 days after
`implantation, to receive either one of the trial medica-
`tions. The tumor weight was estimated by the formula
`[long axis X (short axis)2 X 1/2], which has been re-
`ported to correlate with the actual tumor weight.12
`The drugs used were pure antiestrogen ICI 182780
`and 5-FU. ICI 182780 was formulated as a 50mg/ml
`solution in castor oil and 5-FU was dissolved in distilled
`
`water by an ultrasonic cleaner to make a 12 mg/ml con-
`centration. The nude mice in the ICI 182780 group were
`administered a single dose of 0.5, 1, 3, or 5 mg subcuta-
`neously on the first day of treatment. Those in the 5-FU
`group were administered intraperitoneally a 60mg/kg
`dose of 5-FU three times at 4-day intervals on and after
`the first day of treatment. The combination group re-
`ceived 1mg of ICI 182780 and 60mg/kg of 5-FU in the
`same manner as that for the individual monotherapy
`groups.
`
`Assessment of the Antitumor Effects
`The long and short axes of each tumor were measured
`by a sliding caliper twice a week over the period from
`day 1 to day 21 to estimate the tumor weight and the
`mean tumor weight for each group. The mean tumor
`weight on Day X (W) was divided by the initial mean
`weight (W0) and the obtained values (the relative mean
`tumor weight ratio: W/WO) were plotted against the
`time (days) after the start of the treatment so as to
`produce a tumor growth curve for each group. The ratio
`of W/WO of each treated group to W/WO of the control
`group was also calculated (T/C). For the ER measure-
`ments and cell cycle analysis by flow cytometry (FCM),
`the tumors were resected on day 21, cut in half to re-
`move any necrotized tissue, and then frozen until use.
`
`ER Measurements
`
`ERs in the frozen tissues were determined by the
`dextran-coated charcoal
`(DCC) method and the
`results were analyzed by Scatchard plots to determine
`the number of binding sites (BM).
`
`Cell Cycle Analysis by FCM
`The frozen tissues were minced, mixed with 0.1% ribo-
`nuclease and 0.1% polyoxyethylene(10)octylphenyl
`ether, and filtered with a 40-um nylon mesh. The filtrate
`was mixed with the same volume of propidium iodine
`solution (to make the final concentration of 50 ug/ml). A
`DNA histogram was prepared using a flow cytometer
`
`(FACScan, Becton Dickinson, San Jose, CA, USA) and
`the percentage of cells in S phase (%S) and G phases
`(%GOGl) were determined by the software Sum of
`Broaded Rectangles (SOBR). The tumor 5-FU and
`thymidylate synthetase (TS) concentrations, and 5-FU
`contents in tumor cell RNA were determined in the
`
`5-FU monotherapy and the combination groups. Nude
`mice bearing MCF-7 intramuscularly received 5mg/kg
`estradiol
`dipropionate
`and
`250mg
`progesterone
`caproate immediately and 14 days after implantation.
`At 28 days after implantation, treatment with 5-FU
`alone or in combination with ICI 182780 was started
`in the same manner as mentioned before. The tumors
`were resected at either 1 to 12h after the last adminis-
`
`tration of 5-FU, cut in half and frozen within 5 min after
`the necrotized tissue was removed. The tumors resected
`at 1 h after the last administration of 5-FU were used for
`
`the tissue 5-FU assay, those taken at 1 and 12h after the
`last administration of 5-FU for the TS assay, and those
`taken at 12h after the last administration of 5-FU for
`5-FU contents in RNA.
`
`Tumor 5-FU Assay
`The tumor tissue specimens were mixed with silica gel
`and cold acetonitorile, homogenized, and centrifuged.
`The obtained supernatant was concentrated to dryness,
`reconstituted with ethanol, passed through a silica gel
`column to be adsorbed by silica gel, eluted by acetone,
`then concentrated to dryness. The resulting solid was
`reconstituted with a solvent and subjected to high-
`performance liquid chromatography (HPLC).13
`
`Tumor TS Assay
`In accordance with the method reported by Spears
`et
`al.14 the tumor
`tissues were homogenized and
`centrifuged.
`Sufficient
`amounts
`of
`3H-labeled
`fiuorodeoxyuridine monophosphate ([3H]FdUMP) and
`methylenetetrahydrofolate (CHZFH4) were added to
`the resulting cytosol fraction to form a ternary complex.
`Free [3H]FdUMP was removed by DDC for TS and
`radioactivity was determined by a liquid scintillation
`counter. Buffer, pH 8.0, was added to the cytosol frac-
`tion to cut the TS-FdUMP bond and, as in the free TS
`measurement, sufficient amounts of [3H]FdUMP and
`CHZFH4 were added, and radioactivity was counted by
`scintillation counter to determine the total TS. The TS
`
`inhibition rate (TSIR) was calculated by the equation
`[(total TS — free TS)/(total TS) X 100%].
`
`5-FU Assay in Tumor Cell RNA
`Gas chromatography—mass spectrometry (GC-MS) was
`used.15 The tumor tissue specimens were homogenized
`in distilled water.
`Ice-cold trichloroacetic acid was
`
`added, mixed, and centrifuged to remove the superna-
`tant. The precipitate was rinsed, hydrolyzed by potas-
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`Y. Ogasawara et al.: Pure Antiestrogen and 5-FU in Nude Mice
`
`151
`
`sium hydroxide, and then perchloric acid was added.
`After centrifugation, the supernatant was neutralized
`by potassium hydroxide, and newly appearing precipi-
`tate was removed to obtain a mononucleotide solution.
`This solution was used to determine the concentrations
`
`of RNA and 5-FU by GC-MS, and the 5-FU content in
`RNA was also calculated.
`
`regard to the antitumor activity, the difference between
`the combination and the control was significant (Fig. 2).
`The combination, however, failed to show a synergetic
`effect with a minimum T/C of 0.42 marginally exceeding
`the product of T/C of the two forms of monotherapy
`(0.61 X 0.68 : 0.41) (Table 1).
`
`Changes in the Body Weights 0f Nude Mice
`
`The body weights of the nude mice decreased slightly in
`those treated with both 5mg of ICI 182780 alone and
`with the combination. There was a significant difference
`between 1 mg and 5mg of ICI 182780, but not between
`the control and any of the treatment groups (Table 2).
`
`ER Values at the Time of Tumor Resection
`
`The mean ER value at the time of tumor resection
`
`(BM) was 605.5 fmol/mg protein in the control group,
`
`
`Table 1. Tumor response to trial medication
`
`Treatment Minimum T/C
`
`1.00 (on day 21)
`(n : 14)
`Control
`0.75 (on day 21)
`ICI182780 0.5 mg (n : 7)
`0.61 (on day 21)
`ICI182780 1mg
`(n : 11)
`0.51 (on day 21)
`ICI182780 3mg
`(n : 10)
`0.35 (on day 21)
`1C1182780 5mg
`(n :14)
`0.68 (on day 21)
`5-FU
`(n : 10)
`
`(n : 10)Combination 0.42 (on day 21)
`
`
`T/C, mean ratio of the estimated tumor weight to the baseline in the
`treated group/that in the control group
`5—FU, 5—fiuorouracil
`
`6 5 4
`
`Ratio of
`mean
`estimated
`tumort
`weight
`
`_\
`
` O
`NIlllillllilll
`
`14
`7
`Days after start of treatment
`
`2 1( days)
`
` O
` 0
`
`Criteria for Antitumor Effects
`The treatment was considered to be effective when the
`
`minimum T/C during the treatment was not higher than
`0.42.12 The significance of the intergroup difference in
`the mean tumor weight was tested by two-way repeated
`measures ANOVA (analysis of variance). The combi-
`nation effect was considered to be synergetic when T/C
`in the combination group was smaller than the product
`of T/Cs in the two forms of monotherapy. The two-way
`repeated measures ANOVA was used to test the signifi-
`cance of intergroup differences in the nude mouse body
`weight change, and Student’s t-test and the Mann-
`Whitney U—test were used for ER, %S, %GOGl, tumor
`5-FU concentrations, TS concentrations, and 5-FU con-
`tent in RNA. A difference with P-value less than 0.05
`
`was considered to be significant.
`
`Results
`
`Antiturnor Effects on MCF-7
`
`ICI 182780 exhibited a dose-dependent antitumor ac-
`tivity which was statistically greater at 3 and 5mg as
`compared with the control (Fig. 1). As the minimum
`T/C was as low as 0.35 ($0.42), 5 mg was assessed to be
`effective (Table 1).
`Although neither 1 mg of ICI 182 780 alone nor 5-FU
`alone was significantly different from the control with
`
`6 5
`
`Ratio of
`mean
`estimated
`IUIIlUI
`
`weight 3
`
`14
`7
`Days after Start of treatment
`
`21 (days)
`
`Fig. 1. Tumor response of MCF-7 to ICI 182 780. Vertical axis,
`mean estimated tumor weight on a given day/that on day 0.
`Open circles, control (n : 14); closed triangles, 0.5 mg (n : 7);
`dotted circles, 1mg (n : 11); open triangles, 3mg (n : 10);
`closed circles, 5 mg (n : 14). *P < 0.05 compared with control
`
`Fig. 2. Tumor response of MCF-7 to the combination of ICI
`182780 and 5-fiuorouracil. Vertical axis, mean estimated tu-
`mor weight on a given day/that on day 0. Open circles, control
`(n : 14); dotted circles, 1mg ICI 182 780 (n : 11); closed
`circles, 5-fiuorouracil (n : 10); triangles, combination (n :
`10). *P < 0.05 compared with control
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`152
`
`Y. Ogasawara et al.: Pure Antiestrogen and 5-FU in Nude Mice
`
`
`Table 2. Changes in body weights (BW) of the nude mice
`
`Treatment
`BW at baseline (g)
`BW on day 21 of treatment (g)
`
`Control
`
`(n : 7)
`
`20.51 i 1.43
`
`21.40 i 2.17
`
`23.70 i 1.42 7 *
`22.00 i 1.20
`ICI 182780 1mg (n : 4)
`
`20.78 i 1.24 7
`20.85 i 1.13
`ICI 182780 5mg (n : 6)
`5-FU
`(n : 5)
`20.38 i 1.88
`21.02 i 3.16
`
`Combination
`(n : 6)
`23.03 i 1.28
`21.37 i 2.27
`
`*P < 0.05
`
`Table 3. Estrogen receptor (ER) count on day 21 of treat-
`
`ment (by dextran-coated charcoal method)
`
`Treatment
`ER content (fmol/mg protein)
`
`
`605.5 i 48.3 **
`(n : 4)
`Control
`198.5 : 83.2 :T *
`1C1182780 1mg (n : 5)
`5-FU
`(n : 4)
`418.1 : 164.7:* *
`
`Combination
`(n : )
`52.3 i 76.8 ;
`
`
`
`
`
`
`*P < 0.05
`
`while the mean ER decreased to 198.5, 418.1, and
`52.3 fmol/mg protein after the administration of 1 mg of
`ICI 182780, 5-FU, and the combination, respectively.
`The fall of ER was significantly greater with 1 mg of ICI
`182780 than with 5-FU, and with the combination than
`with either monotherapy (Table 3).
`
`Cell Cycle Analysis by Flow Cytometry
`
`The mean %S in the control group was 35.6%, while it
`was 14.2% for 1 mg of ICI 182780, 19.2% for 5-FU, and
`19.8% for the combination therapy. The mean %G0G1
`was 55.4% for the control, while the values in the
`treated groups were 82.8%, 76.4%, and 76.8%, respec-
`tively (Table 4). The treated groups showed a significant
`reduction in the %S and a significant increase in the
`%G0G1 as compared with the control group. ICI 182 7 80
`at 1mg resulted in a greatest reduction of the %S and
`the greatest elevation of the %G0G1, with a significant
`difference in the %S from that of the combination
`
`therapy, and a significant difference in the %G0G1 as
`compared with 5-FU alone and the combination
`therapy.
`
`5-FU and TS Concentrations in Tumors and 5-FU
`Contents in Tumor Cell RNA
`
`The mean 5-FU concentration in the tumors was
`
`302.0 ng/g in the 5-FU group, which was not significantly
`different from that of the combination group, 201.9 ng/g
`(Table 5). On the other hand, the mean 5-FU content in
`RNA was significantly higher in the combination group
`
`(102.6ng/mg RNA) than in the 5-FU group (65.2ng/mg
`RNA) (Table 6).
`The total TS at 1h after the last administration of
`
`5-FU was 30.8pmol/g tissue in the 5-FU group and
`30.5pmol/g tissue in the combination group, with no
`significant difference in TSIR (94.1% and 97.2%,
`respectively). At 12h after the last administration of
`5-FU, however, both the total and free TS (61.9 and
`24.1 pmol/g tissue) in the 5-FU group were significantly
`higher than those in the combination group (51.3 and
`17 .5 pmol/g tissue, respectively) (Table 7).
`
`Discussion
`
`Breast cancer is a hormone-dependent tumor for which
`endocrine therapy is widely accepted as being effective.
`TAM is one of the most commonly used antiestrogens.
`However, TAM is reported to have partial estrogenic
`activity, and it has also been reported to produce estro-
`genic effects on some targets and antiestrogenic effects
`on others?5
`
`ICI 182780, a new pure antiestrogen free from estro-
`genic activity, is a promising new therapy modality. One
`of the features of the drug is the lack of effect on gona-
`dotropins.8 This means that the drug does not block
`cerebral ERs and therefore does not interfere with the
`
`hypothalamus-pituitary-ovary axis in breast cancer pa-
`tients. In this sense, the drug is expected to be especially
`beneficial to premenopausal patients.8 Unlike TAM,
`in which the uterotropic activity has been recently
`reported to be associated with a risk of endometrial
`cancerfi7 ICI 182780 is devoid of uterotropic activity
`and therefore free from this concern.
`
`Meanwhile, even apparently ER-positive breast can-
`cer heterogeneously consists of both ER-positive and
`ER-negative tumor cells. Antiestrogens are not so ef-
`fective for ER-negative tumor cells.1 Therefore, a com-
`bination of hormone therapy with chemotherapy, which
`has a different mode of action, can be expected to im-
`prove the response rate. There are a number of clinical
`reports using randomized controlled trials to compare
`chemotherapy alone and chemoendocrine therapy in
`recurrent breast cancer”29 and some studies comparing
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`Y. Ogasawara et al.: Pure Antiestrogen and 5-FU in Nude Mice
`
`
`Table 4. Cell cycle analysis on day 21 of treatment (by flow cytometry)
`
`Treatment
`%S
`%G0G1
`
`
`
`35.6 i 2.4 7
`(n : 5)
`Control
`55.4 i 237*
`
`
`14.2 i 3.3 : * *
`ICI 182780 1mg (n : 4)
`82.8 i 2.9:: * *
`
`19.2 i 4.3
`*
`5-FU
`(n : 5)
`76.4 + 4.67 *
`
`Combination 76.8 i 2.6 i (n : 4) 19.8 i 2.4 *7
`
`
`
`
`
`
`%S, percentage of cells in S phase; % GOG“ percentage of cells in G phases
`*P < 0.05
`
`153
`
`
`Table 5. 5-FU concentrations in the tumor“ Table 6. 5-FU contents in tumor cell RNA“
`
`
` Treatment 5-FU concentration (ng/g)
`
`5-FU content in RNA
`
`Treatment
`(ng/mg RNA)
`
`302.0 : 154.61NS
`(n : 6)
`5-FU
`
`Combination (n : 8) 201.9 i 83.7
`
`NS, not significant
`“Tumors resected at 1 h after the last administration of 5—FU
`
`(n : 6)
`5-FU
`65.2 : 1807*
`
`
`Combination (n : 5) 102.6 i 34.67
`* P < 0.05
`“Tumors resected at 12h after the last administration of 5—FU
`
`
`Table 7. Concentrations of thymidylate synthetase (TS)
`TS total
`TS free
`
`Treatment
`(pmol/g tissue)
`(pmol/g tissue)
`TSIR (%)
`
`(n : 9)
`5-FU at 1h“
`Combination at 1h (n : 11)
`
`30.8 i 13.4
`30.5 i 7.1
`
`2.2 i 2.6
`0.9 i 0.7
`
`94.1 i 4.8
`97.2 i 2.2
`
`61.0 i 2.1
`61.9 i 691*
`(n : 6)
`5-FU at 12hb
`24.1 i 2.3T*
`
`
`
`51.3 i 8.1Combination at 12h (n : 5) 65.2 i 7.2 17.5 i 2.17
`
`
`TSIR, TS inhibition rate
`* P < 0.05
`“Tumors resected at 1 h after the last administration of 5—FU
`bTumors resected at 12h after the last administration of 5—FU
`
`chemoendocrine
`and
`alone
`therapy
`endocrine
`therapy.”23 Most of these reports show higher res-
`ponse rates to the combination therapy. However, the
`expected combination effect of the chemoendocrine
`therapy has yet to be clinically demonstrated.
`On the other hand, experimentally, Watanabe24 re-
`ported that
`the chemoendocrine therapy combining
`TAM, the most widely used drug in clinical practice, and
`the fluorinated pyrimidine 5-FU, which is relatively
`widely used, exerted a synergetic effect on R-27 in vivo,
`while Kubota et a1.25 failed to demonstrate a synergic
`effect of a combination of TAM and 1 M tegafur—4M
`uracil (UFT) on Br-10. In vitro, Benz et al.2‘”27 reported a
`synergetic effect of the combination therapy on MCF-7.
`Sato28 reported that dimethylbenzanthracene-induced
`rat mammary tumors responded to the combination
`therapy, while Yamamoto et a1.29 failed to demonstrate
`any synergetic effect. Although differences in the cell
`lines, dosages, and the types of administration tested
`
`prevent us from making a simple comparison across
`these studies, there is still no agreement on the efficacy of
`this combination therapy. Under these circumstances,
`the authors investigated the antitumor effects and mode
`of action of the experimental chemoendocrine therapy
`combining pure antiestrogen and 5-FU.
`A single administration of ICI 182780 at 0.5, 1, 3, and
`5 mg showed a dose-dependent antitumor activity with a
`significant difference between 3 or 5 mg and the control.
`At 5mg, the drug was assessed to be “effective” with a
`minimum T/C as low as 0.35. The toxic effect of the drug
`was considered to be minimal, as there were neither any
`deaths of the treated animals nor toxic symptoms other
`than a reduction of the mean body weight in the 5-mg
`group, which was not as significant as compared with the
`control.
`
`Referring to the report of Kondo et al.30 who reported
`the sensitivity of 5-FU on the tumor implanted in nude
`mice, the mice in the 5-FU group were administered
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`Y. Ogasawara et al.: Pure Antiestrogen and 5-FU in Nude Mice
`
`60 mg/kg of 5-FU three times at 4-day intervals on and
`after the first day of treatment. The 5-FU group failed to
`exert an antitumor effect with the minimum T/C of 0.68,
`and the effect was not significantly different from that
`of the control group. These findings agree with those
`of Koh31 who reported that MCF-7 did not respond to
`5-FU in vivo.
`
`In contrast to the minimum T/C of 0.61 for 1 mg ICI
`182780 alone and 0.68 for 60mg/kg 5-FU alone which
`were both assessed to be ineffective, the combination
`was assessed to be effective with a minimum T/C of 0.42,
`which was significantly different from that of the control
`but fell marginally short of the synergic effect. The com-
`bination therapy was associated with a reduction in the
`mean body weight, which was not significant as com-
`pared with the control. There were also no deaths seen
`in the treated mice. These results thus suggest the use-
`fulness of this combination therapy.
`A comparison of the ER values showed a significantly
`lower Bmax in the 1-mg ICI 182780 group than that of the
`control. This may be accounted for by the antitumor
`effect of ICI 182780 on ER-positive cells, as anti-
`estrogen is expected to work through its effect on ER.
`The ER value in the 5-FU group decreased slightly but
`not significantly from that of the control. Judging from
`the observed mild antitumor effect of 5-FU and its
`
`mode of action, 5-FU seems to exert its effect directly
`on tumor cells regardless of the ER status. In contrast,
`the authors observed a marked reduction of the ER
`
`value in the combination group, which closely agreed
`with the findings of Watanabe who reported a marked
`reduction in the ER value in R-27 treated with a com-
`
`bination of TAM and 5-FU.24 These findings as well as
`the increased antitumor response to the combination
`therapy thus suggest a potentiation of the effects on the
`ER-positive cells using this therapy.
`The cell cycle analysis by FCM resulted in a decrease
`in the %S and an increase in the %G0G1 by ICI 182780.
`TAM was reported to reduce the ratio of the cells in the
`S phase and accumulate cells in the GOG1 phase.32 Like
`TAM, ICI 182780 also seems to slow down the cycle
`progression. On the other hand, 5-FU at a low dose is
`known to prolong the S phase and at a high dose
`to accumulate cells as in the G1 phase.33 Our findings of
`the decreased %S and the increased %G0G1 support
`Watanabe24 who reported a reduction of the %S in R-27
`treated with 5-FU.
`
`An earlier report that DNA is synthesized in the S
`phase and the %S is inversely proportional to the tumor
`doubling time34 is consistent with our findings that the
`%S was significantly lower in the groups which re-
`sponded to the treatment than that of the control. The
`FCM used, however, failed to explain the combination
`effect from the results of the cell cycle analysis, as the
`greatest %S reduction was not related to the greatest
`
`tumor response, which was seen in the combination
`group.
`
`is said that 5-FU may have two major modes
`It
`of action. First, 5-FU is phosphorylated to FdUMP in
`the body, which formulates a ternary complex with
`methylenetetrahydrofolate and TS to inactivate TS and
`consequently inhibit the synthesis of DNA. Secondly, 5-
`FU is phosphorylated to fiuorouridine triphosphate
`(FUTP), which is taken up by RNA instead of UTP and
`consequently interferes with the metabolism of RNA.
`Although much controversy remains as to which makes
`a greater contribution to the antitumor effect of the
`drug,14’35’37 the authors tried to elucidate the mode of
`action of the combination therapy from both possible
`modes of action of 5-FU, that is, the inhibition of the
`DNA synthesis and the metabolic dysfunction of RNA,
`by determining the 5-FU and TS concentrations in
`tumor cells and 5-FU contents in RNA for the 5-FU
`
`and the combination groups.
`There was no significant difference in the 5-FU con-
`centrations in tumor cells, although they were slightly
`higher in the 5-FU group (mean of 302.0ng/g) than
`in the combination group (201.9 ng/g). Antiestrogen
`ICI 182780 is, therefore, unlikely to interfere with the
`uptake of 5-FU by tumor cells.
`In contrast, the 5-FU content in RNA was signifi-
`cantly higher in the combination group than in the 5-FU
`group. The metabolic dysfunction of RNA was thus
`suggested to contribute to the increased tumor response
`of the combination. In addition to ICI 182780, 5-FU
`also slowed down cell cycle progression and accumu-
`lated cells in the GOG1 phase, as indicated by FCM. Ueki
`reported a decreased %S and increased %G0G1 of SC-
`115, an androgen-responsive mouse mammary tumor,
`at 24h after administration of 5-FU, and a reincrease of
`%S at 1 week after administration.38 The action of ICI
`
`182780 to decrease %S and increase %G0G1 seems to
`last for some time, while that of 5-FU may disappear
`shortly after administration. 5-FU is said to work on
`RNA in the G1 phase.39 In our study, 5-FU was admin-
`istered in the combination group, when the %G0G1
`level was maintained at a high level by ICI 182780
`and, as a result, the uptake of 5-FU by RNA may be
`facilitated.
`
`There was no significant difference between 5-FU
`alone and the combination group with regard to the
`total TS, free TS, or TSIR at 1 h after the last adminis-
`tration of 5-FU. The TSIR approximated 100% in both
`groups. At 12h after the last administration of 5-FU, the
`TSIR was of the order of 60% in both groups without
`any significant difference. The TSIR in both groups was
`as high as near 100% shortly after the administration of
`5-FU and remained at a relatively high level of approxi-
`mately 60% even at 12h after the last administration of
`5-FU. These findings suggest the possible contribution
`
`lnnoPharma Exhibit 1103.0006
`
`

`

`Y. Ogasawara et al.: Pure Antiestrogen and 5-FU in Nude Mice
`
`155
`
`of the inhibition of DNA synthesis by 5-FU to the anti-
`tumor effect in both groups. On the other hand, no
`difference in the TSIR between the groups suggests that
`a potentiation of the inhibition of DNA synthesis by
`5-FU is not attributable to the increase in the antitumor
`
`response to the combination. The total TS and free TS
`in both groups at 12h after the last administration of 5-
`FU were higher than those at 1 h after the last adminis-
`tration of 5-FU. These parameters were not significantly
`different between the groups at 1 h after the last admin-
`istration of 5-FU, but were significantly lower in the
`combination group than in the 5-FU group at 12h after
`the last administration of 5-FU. These findings thus sug-
`gested that the TS synthesis observed in the tumor cells
`in order to produce 5-FU induced a rapid decrease in
`free TS to be suppressed by the combination therapy.
`As the antitumor response is reported to correlate with
`the free TS concentration,40 TS synthesis was more
`greatly suppressed by the combination therapy based
`on the higher antitumor response to the combination
`than to the 5-FU alone.
`
`These results suggest the antitumor effect of ICI
`182780 in ER-positive breast cancer cells, which was
`potentiated by the concomitant administration of 5-FU.
`
`Conclusion
`
`ICI 182780 showed a dose-dependent activity
`(1)
`against MCF-7, which was potentiated by the concomi-
`tant administration of 5-FU. (2) The observed marked
`reduction in the ER value in the combination therapy
`group of ICI 182780 and 5-FU suggests that the combi-
`nation therapy may mainly work on ER-positive tumor
`cells. (3) ICI 182780 seems to slow down the cycle pro-
`gression, as indicated by the decrease in the %S and the
`increase in the %G0G1. (4) In the combination group,
`the synthesis of TS after the administration of 5-FU was
`suppressed and the 5-FU-induced metabolic dysfunc-
`tion of RNA was potentiated.
`
`References
`
`1. Furr BJ, Patterson JS, Richardson DN, Slater SR, Wakeling AE
`(1979) Tamoxifen. Pharmacological and biochemical properties
`of drug substances 2. American pharmaceutica association acad—
`emy of pharmaceutical sciences, Washington, pp 355399
`2. Ashizawa T, Ishida H, Okabe M, Gomi K (1993) Effect of
`medroxyprogesterone acetate on antitumor efficacies and side
`effect on 5—fluorouracil
`(in Japanese with English abstract).
`Gan To Kagaku Ryoho 20:9417947
`3. Harper MJK, Walpole AL (1966) Contrasting endocrine activities
`of cis and trans isomers in a series of substituted triphenyle—
`thylenes. Nature 212:87
`4. Boccardo F, Guarneri D, Rubagotti A, Casertelli GL, Bentivoglio
`G, Conte N, Campanella G, Gaggero G, Comelli G, Zanardi S,
`
`
`
`Nicolo G (1984) Endocrine effects of tamoxifen in postmeno—
`pausal breast cancer patients. Tumori 70:61768
`. Fornander T, Rutqvist LE, Wilking N, Carstrom K, Schoultz BV
`(1993) Oestrogenic effects of adjuvant tamoxifen in postmeno—
`pausal breast cancer. Eur J Cancer 29A:4977500
`Nayfield SG, Karp JE, Ford LG, Dorr FA, Kramer BS (1991)
`Potential role of tamoxifen in prevention of breast cancer. J Natl
`Cancer Inst 83:145071459
`
`. Jaiyesimi IA, Buzdar AU, Decker DA, Hortobagyi GN (1995)
`Use of tamoxifen for breast cancer:
`twenty—eight years later.
`J Clin Oncol 13:5137529
`. Wakeling AE (1993) The future of new pure antiestrogens in
`clinical breast cancer. Breast Cancer Res Treat 25:179
`
`10.
`
`11.
`
`. Parker MG (1993) Action of “pure” antiestrogens in inhibiting
`estrogen receptor action. Breast Cancer Res Treat 26:1317137
`Soule HD, Vazquez J, Long A, Albert S, Brennan M (1973) A
`human cell line from a pleural effusion derived from a breast
`carcinoma. J Natl Cancer Inst 51:140971413
`Takeuchi T (1995) Chemo— and endocrino—therapy of breast car—
`cinoma xenografts in the dormant or exponential growth phase
`(in Japanese with English abstract). Nippon Geka Gakkai Zasshi
`(J Jpn Surg Soc) 96:3797387
`Hanatani Y, Kubota T, Yamada Y, Tsuyuki K, Nakada M,
`Matsumoto S, Kumai K, Yoshino K, Ishibiki K, Abe O (1980)
`Experimental chemotherapy of human carcinomas
`serially
`transplanted to nude mice (in Japanese with English abstract).
`Nippon Gan Chiryo Gakkai Shi (J Jap Soc Cancer Ther) 15:11147
`1120
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`
`18.
`
`19.
`
`20.
`
`21.
`
`22.
`
`Masuike T, Watanabe I, Takemoto Y (1985) Quantitative method
`of 5—fluorouracil and its metabolites in biological samples using
`high performance liquid chromatography (in Japanese with
`English abstract). Yakugaku Zasshi 105:105&1064
`Spears CP, Shahinian AH, Moran RG, Heidelberger C, Corbett
`TH (1982) In vivo kinetics of thymidylate synthetase inhibition in
`5—fluorouracil—sensitive and— resistant murine colon adenocar—
`cinoma. Cancer Res 42:450 456
`
`Masuike T, Kikuchi K, Saito A, Hashimoto T, Takemoto Y (1995)
`Determination of 5—fluorouracil incorporated into RNA in tissue
`using gas chromatography—mass spectrometry (in Japanese with
`English abstract). Antibiot Chemother 11:1627167
`Cocconi G, De Lisi V, Boni C, Mori P, Malacarne P, Amadori D,
`Giovanelli E (1983) Chemotherapy versus combination of chemo—
`therapy and endocrine therapy in advanced breast cancer. Cancer
`51:5817588
`Kardinal CG, Perry MC, Weinberg V, Wood W, Ginsberg S, Raju
`RN (1983) Chemoendocrine therapy vs chemotherapy alone for
`advanced breast cancer in postmenopausal women: preliminary
`report of a randomized study. Breast Cancer Res Treat 3:3657371
`Mouridsen HT, Rose C, Engelsman E, Sylvester R, Rotmensz N
`(1985) Combined cytotoxic and endocrine therapy in postmeno—
`pausal patients with advanced breast cancer. A randomized study
`of CMF vs CMF plus tamoxifen. Eur J Cancer Clin Oncol 21:2917
`299
`
`Viladiu P, Alonso MC, Avella A, Beltran M, Borras J, Ojeda B,
`Bosch FX (1985) Chemotherapy versus chemotherapy plus
`hormonotherapy in postmenopausal advanced breast cancer
`patients. A randomized trial. Cancer 56:274572750
`Falkson G, Falkson HC, Glidewell O, Weinberg V, Leone L,
`Holland JF (1979) Improved remission rates and remission du