Estrogen Down-regulator Fulvestrant Potentiates Antitumor Activity of Fluoropyrimidine in Estrogen-responsive MCF-7 Human Breast Cancer Cells

Abstract

Background: Endocrine therapy is clinically administered in hormone-responsive breast cancer. Combinations of fluoropyrimidine S-1 and an aromatase inhibitor or anti-estrogen are considered beneficial in Japan. Herein we assessed new combinations of S-1 and fulvestrant.

Patients and methods: Cytotoxicity of fulvestrant and 5-fluorouracil (5-FU) was assessed in hormone-responsive (MCF-7) and non-responsive (MDA-MB-231) breast cancer cell cultures. Fulvestrant and S-1 were evaluated for antitumor activity in mice and their effects on estrogen receptor (ER)-α and progesterone receptor (PgR) levels in MCF-7 xenografts using immunohistochemical methods.

Results: Fulvestrant inhibited growth of MCF-7, but not of MDA-MB-231 xenografts. Combinations of 5-FU and fulvestrant were superior to monotherapy in vitro. In vivo antitumor activity of S-1/fulvestrant combination therapy was significantly (p<0.05) enhanced compared to that of both monotherapies. Fulvestrant partially down-regulated expression of ERα and PgR, but in combination with S-1, it almost completely blocked their expression.

Conclusion: Chemo-endocrine combination therapy using S-1 and fulvestrant is beneficial in estrogen-responsive breast cancer.

Keywords: Breast cancer; S-1; chemo-endocrine combination therapy; chemotherapy; endocrine therapy; estrogen receptor; fulvestrant; progesterone receptor.

Figure 1. Growth inhibition by single-drug treatment. 5-Fluorouracil (5-FU) (A) and fulvestrant (B) were tested for growth inhibition of estrogen receptor (ER)-positive human breast cancer cell line MCF-7 and ERnegative human breast cancer cell line MDA-MB-231 in the presence of 0.1 nM estradiol. The values are the means and SD of cell growth relative to that of the controI (T/C) from three independent experiments.

Figure 2. An isobologram curve for the combination treatment with 5-fluorouracil (5-FU) and fulvestrant. Da and Db indicate the ratio of the concentration inhibiting cell growth by 50% (IC50) for 5-FU and fulvestrant in combination therapy to monotherapy, respectively. An envelope of additivity is enclosed by mode I, IIa, and IIb. Data points within the envelope of interaction between 5-FU and fulvestrant indicate additivity, whereas data points below the envelope indicate synergy.

Figure 3. Tumor volume of MCF-7 xenograft in vivo. Nude mice bearing MCF-7 xenograft were randomized into four groups (n=7) on day 0, which was 7 days after the SE-121 pellet (containing 0.025 mg estradiol for 60 days release) had been subcutaneously implanted. Fulvestrant (5 mg/mouse) was administered subcutaneously on day one, S-1 (10 mg/kg) was administered orally once daily from day 1 to 18. Tumor volume was measured twice a week until day 19. The values are the means and SD of tumor volume (n=7). Significantly different on day 15 at ***p<0.001 vs. control by Dunnett’s test; #p<0.05 vs. either monotherapy by Aspin–Welch t-test.

Figure 4. Immunohistochemical staining of estrogen receptor-α (ERα) (upper panel) and progesterone receptor (PgR) (lower panel) in a MCF-7 xenograft subjected to monotherapy with either S-1 (10 mg/kg, days 1-18, orally) or fulvestrant (1 mg/animal, day 1, subcutaneously), or to combination therapy. Tumors were excised on day 19. The bar indicates 100 μm. The original magnification was 400×.