Cancer Chemotherapy and Pharmacology

, Volume 59, Issue 4, pp 515–525 | Cite as

Additive antitumor effect of concurrent treatment of 4-hydroxy tamoxifen with 5-fluorouracil but not with doxorubicin in estrogen receptor-positive breast cancer cells

  • Junichi Kurebayashi
  • Mamoru Nukatsuka
  • Hideki Nagase
  • Tsunehisa Nomura
  • Mai Hirono
  • Yutaka Yamamoto
  • Yoshikazu Sugimoto
  • Toshinori Oka
  • Hiroshi Sonoo
Original Article



The sequential addition of tamoxifen (TAM) to chemotherapy seems superior to its concurrent addition in patients with breast cancer. This study was conducted to clarify the hypothesis that there are differential interactions among TAM and chemotherapeutic agents.


Estrogen receptor (ER)-α-positive or -negative breast cancer cells were treated with 4-hydroxy TAM (4OHT), 5-fluorouracil (FU) and/or doxorubicin (Dox). Changes in the expression levels of genes related to sensitivity and resistance to TAM, 5-FU or Dox were tested.


Concurrent treatment of 4OHT with 5-FU but not with Dox additively inhibited the growth of ER-α-positive cells. 5-FU did not change the expression levels of any tested genes related to either sensitivity or resistance to TAM. Although Dox did not change the expression levels of any genes related to the sensitivity to TAM, Dox significantly increased the expression levels of some genes related to TAM resistance, Eph A-2, ER-β, Fos and vascular endothelial growth factor. 4OHT significantly decreased thymidilate synthase (TS) activity.


Although the antitumor effect of concurrent 4OHT and 5-FU was additive, that of concurrent 4OHT and Dox was less than additive in ER-α-positive cells. The increased expression of genes related to TAM resistance by Dox might be responsible for the interaction. Decreased TS activity by 4OHT might increase the antitumor activity of 5-FU. These findings may provide a preclinical rationale for concurrent use with 5-FU and TAM.


Breast Cancer Concurrent Doxorubicin 5-Fluorouracil Tamoxifen 



Analysis of variance


Cycle threshold


Dextran-coated charcoal-stripped




Dihydropyrimidine dehydrogenase




Estrogen receptor


Fetal bovine serum




Fibroblast growth factor




Human epidermal growth factor receptor


Hypoxia inducible factor


50% inhibitory concentration




Orotate phosphoribosyl transferase


Phosphate-buffered saline


Progesterone receptor


Trefoil factor 1


Reverse transcriptional polymerase chain reaction


Stromal cell-derived factor






Thymidylate synthase


Vascular endothelial factor



This work was supported by Research Project Grants (16-501 S and 17-113 S) from Kawasaki Medical School and by a grant from the Japanese Breast Cancer Society.


  1. 1.
    Albain KS, Green SJ, Ravdin PM, Cobau CD, Levine EG, Ingle JN, Pritchard KI, Schneider DJ, Abeloff MD, Norton L, Henderson IC, Lew D, Livingston RB, Martino S, Osborne CK (2002) Adjuvant chemohormonal therapy for primary breast cancer should be sequential instead of concurrent: initial results from intergroup trial 0100 (SWOG-8814). Proc ASCO 21:37a (Abstract 143)Google Scholar
  2. 2.
    Pico C, Martin M, Jara C, Barnadas A, Pelegri A, Balil A, Camps C, Frau A, Rodriguez-Lescure A, Lopez-Vega JM, De La Haba J, Tres A, Alvarez I, Alba E, Arcusa A, Oltra A, Batista N, Checa T, Perez-Carrion R, Curto J (2004) Epirubicin–cyclophosphamide adjuvant chemotherapy plus tamoxifen administered concurrently versus sequentially: randomized phase III trial in postmenopausal node-positive breast cancer patients. A GEICAM 9401 study. Ann Oncol 15:79–87PubMedCrossRefGoogle Scholar
  3. 3.
    Goldhirsch A, Glick JH, Gelber RD, Coates AS, Thurlimann B, Senn HJ; Panel members (2005) Meeting highlights: international expert consensus on the primary therapy of early breast cancer 2005. Ann Oncol 16:1569–1583PubMedCrossRefGoogle Scholar
  4. 4.
    Noguchi S, Koyama H, Uchino J, Abe R, Miura S, Sugimachi K, Akazawa K, Abe O (2005) Postoperative adjuvant therapy with tamoxifen, tegafur plus uracil, or both in women with node-negative breast cancer: a pooled analysis of six randomized controlled trials. J Clin Oncol 23:2172–2184PubMedCrossRefGoogle Scholar
  5. 5.
    Osborne CK (1983) Combined chemo-hormonal therapy in breast cancer: a hypothesis. Breast Cancer Res Treat 1:121–123CrossRefGoogle Scholar
  6. 6.
    Benz C, Cadman E, Gwin J, Wu T, Amara J, Eisenfeld A, Dannies P (1983) Tamoxifen and 5-fluorouracil in breast cancer: cytotoxic synergism in vitro. Cancer Res 43:5298–5303PubMedGoogle Scholar
  7. 7.
    Leonessa F, Jacobson M, Boyle B, Lippman J, McGarvey M, Clarke R (1994) Effect of tamoxifen on the multidrug-resistant phenotype in human breast cancer cells: isobologram, drug accumulation, and M(r) 170,000 glycoprotein (gp170) binding studies. Cancer Res 54:441–447PubMedGoogle Scholar
  8. 8.
    Hug V, Hortobagyi GN, Drewinko B, Finders M (1985) Tamoxifen-citrate counteracts the antitumor effects of cytotoxic drugs in vitro. J Clin Oncol 3:1672–1677PubMedGoogle Scholar
  9. 9.
    Osborne CK, Kitten L, Arteaga CL (1989) Antagonism of chemotherapy-induced cytotoxicity for human breast cancer cells by antiestrogens. J Clin Oncol 7:710–717PubMedGoogle Scholar
  10. 10.
    Woods KE, Randolph JK, Gewirtz DA (1994) Antagonism between tamoxifen and doxorubicin in the MCF-7 human breast tumor cell line. Biochem Pharmacol 47:1449–1452PubMedCrossRefGoogle Scholar
  11. 11.
    Kurebayashi J, Kurosumi M, Sonoo H (1995) A new human breast cancer cell line, KPL-1 secretes tumour-associated antigens and grows rapidly in female athymic nude mice. Br J Cancer 71:845–853PubMedGoogle Scholar
  12. 12.
    Kurebayashi J, Otsuki T, Yamamoto S, Kurosumi M, Nakata T, Akinaga S, Sonoo H (1998) A pure antiestrogen, ICI 182,780, stimulates the growth of tamoxifen-resistant KPL-1 human breast cancer cells in vivo but not in vitro. Oncology 55:23–34PubMedCrossRefGoogle Scholar
  13. 13.
    Kunisue H, Kurebayashi J, Otsuki T, Kunisue H, Kurebayashi J, Otsuki T, Tang CK, Kurosumi M, Yamamoto S, Tanaka K, Doihara H, Shimizu N, Sonoo H (2000) Anti-HER2 antibody enhances the growth inhibitory effect of anti-oestrogen on breast cancer cells expressing both oestrogen receptors and HER2. Br J Cancer 82:46–51PubMedCrossRefGoogle Scholar
  14. 14.
    Saotome K, Morita H, Umeda M (1989) Cytotoxicity test with simplified crystal violet staining method using microtitre plates and its application to injection drugs. Toxicol In Vitro 3:317–321CrossRefGoogle Scholar
  15. 15.
    van der Flier S, Brinkman A, Look MP, Kok EM, Meijer-van Gelder ME, Klijn JG, Dorssers LC, Foekens JA (2000) Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment. J Natl Cancer Inst 92:120–127PubMedCrossRefGoogle Scholar
  16. 16.
    Wilcken NR, Prall OW, Musgrove EA, Sutherland RL (1997) Inducible overexpression of cyclin D1 in breast cancer cells reverses the growth-inhibitory effects of antiestrogens. Clin Cancer Res 3:849–854PubMedGoogle Scholar
  17. 17.
    Lu M, Miller KD, Gokmen-Polar Y, Jeng MH, Kinch MS (2003) EphA2 overexpression decreases estrogen dependence and tamoxifen sensitivity. Cancer Res 63:3425–3429PubMedGoogle Scholar
  18. 18.
    Speirs V, Malone C, Walton DS, Kerin MJ, Atkin SL (1999) Increased expression of estrogen receptor β mRNA in tamoxifen-resistant breast cancer patients. Cancer Res 59:5421–5424PubMedGoogle Scholar
  19. 19.
    Reimer T, Koczan D, Muller H, Friese K, Thiesen HJ, Gerber B (2002) Tumour Fas ligand:Fas ratio greater than 1 is an independent marker of relative resistance to tamoxifen therapy in hormone receptor positive breast cancer. Breast Cancer Res 4:R9PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang L, Kharbanda S, Hanfelt J, Kern FG (1998) Both autocrine and paracrine effects of transfected acidic fibroblast growth factor are involved in the estrogen-independent and antiestrogen-resistant growth of MCF-7 breast cancer cells. Cancer Res 58:352–361PubMedGoogle Scholar
  21. 21.
    Vandermoere F, El Yazidi-Belkoura I, Adriaenssens E, Lemoine J, Hondermarck H (2005) The antiapoptotic effect of fibroblast growth factor-2 is mediated through nuclear factor-κB activation induced via interaction between Akt and IκB kinase-β in breast cancer cells. Oncogene 24:5482–5491PubMedCrossRefGoogle Scholar
  22. 22.
    McLeskey SW, Kurebayashi J, Honig SF, Zwiebel J, Lippman ME, Dickson RB, Kern FG (1993) Fibroblast growth factor 4 transfection of MCF-7 cells produces cell lines that are tumorigenic and metastatic in ovariectomized or tamoxifen-treated athymic nude mice. Cancer Res 53:2168–2177PubMedGoogle Scholar
  23. 23.
    Gee JM, Willsher PC, Kenny FS, Robertson JF, Pinder SE, Ellis IO, Nicholson RI (1999) Endocrine response and resistance in breast cancer: a role for the transcription factor Fos. Int J Cancer 84:54–61PubMedCrossRefGoogle Scholar
  24. 24.
    van Agthoven T, van Agthoven TL, Portengen H, Foekens JA, Dorssers LC (1992) Ectopic expression of epidermal growth factor receptors induces hormone independence in ZR-75-1 human breast cancer cells. Cancer Res 52:5082–5088PubMedGoogle Scholar
  25. 25.
    Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Shepard HM, Osborne CK (1993) Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Res Treat 24:85–95CrossRefGoogle Scholar
  26. 26.
    Kurebayashi J, Otsuki T, Moriya T, Sonoo H (2001) Hypoxia reduces hormone responsiveness of human breast cancer cells. Jpn J Cancer Res 92:1093–1101PubMedGoogle Scholar
  27. 27.
    Smith LM, Wise SC, Hendricks DT, Sabichi AL, Bos T, Reddy P, Brown PH, Birrer MJ (1999) cJun overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype. Oncogene 18:6063–6070PubMedCrossRefGoogle Scholar
  28. 28.
    Berns EM, Klijn JG, van Putten WL, de Witte HH, Look MP, Meijer-van Gelder ME, Willman K, Portengen H, Benraad TJ, Foekens JA (1998) p53 protein accumulation predicts poor response to tamoxifen therapy of patients with recurrent breast cancer. J Clin Oncol 16:121–127PubMedGoogle Scholar
  29. 29.
    Manders P, Beex LV, Tjan-Heijnen VC, Span PN, Sweep CG (2003) Vascular endothelial growth factor is associated with the efficacy of endocrine therapy in patients with advanced breast carcinoma. Cancer 98:2125–2132PubMedCrossRefGoogle Scholar
  30. 30.
    Spears CP, Shahinian AH, Moran RG, Heidelberger C, Corbett TH (1982) In vivo kinetics of thymidylate synthase inhibition in 5-fluorouracil-sensitive and resistant murine colon adenocarcinomas. Cancer Res 42:450–456PubMedGoogle Scholar
  31. 31.
    Takechi T, Okabe H, Fujioka A, Murakami Y, Fukushima M (1998) Relationship between protein levels and gene expression of dihydropyrimidine dehydrogenase in human tumor cells during growth in culture and in nude mice. Jpn J Cancer Res 89:1144–1153PubMedGoogle Scholar
  32. 32.
    Shirasaka T, Shimamoto Y, Fukushima M (1993) Inhibition by oxonic acid of gastrointestinal toxicity of 5-fluorouracil without loss of its antitumor activity in rats. Cancer Res 53:4004–4009PubMedGoogle Scholar
  33. 33.
    Beck A, Etienne MC, Cheradame S, Fischel JL, Formento P, Renee N, Milano G (1994) A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil. Eur J Cancer 30A:1517–1522PubMedCrossRefGoogle Scholar
  34. 34.
    Boumendjel A, Baubichon-Cortay H, Trompier D, Perrotton T, Di Pietro A (2005) Anticancer multidrug resistance mediated by MRP1: recent advances in the discovery of reversal agents. Med Res Rev 25:453–472PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Junichi Kurebayashi
    • 1
  • Mamoru Nukatsuka
    • 2
  • Hideki Nagase
    • 2
  • Tsunehisa Nomura
    • 1
  • Mai Hirono
    • 1
  • Yutaka Yamamoto
    • 1
  • Yoshikazu Sugimoto
    • 2
  • Toshinori Oka
    • 2
  • Hiroshi Sonoo
    • 1
  1. 1.Department of Breast and Thyroid SurgeryKawasaki Medical SchoolOkayamaJapan
  2. 2.Optimal Medication Research LaboratoryTaiho Pharmaceutical Co., LtdTokushimaJapan

Personalised recommendations