Breast Cancer Research and Treatment

, Volume 80, Issue 1, pp 49–62 | Cite as

Vitamin D Growth Inhibition of Breast Cancer Cells: Gene Expression Patterns Assessed by cDNA Microarray

  • Srilatha Swami
  • Nalini Raghavachari
  • Uwe R. Muller
  • Yijia P. Bao
  • David Feldman


1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth. Although it is evident that 1,25(OH)2D3 inhibits growth of both estrogen receptor alpha-positive [ERα(+)] and -negative [ERα(−)] breast cancer cells, the cellular pathways contributing to these effects remain unclear. We studied the gene expression patterns in ERα(+) MCF-7 and ERα(−) MDA MB 231 human breast cancer cells following 1,25(OH)2D3 treatment, using cDNA expression arrays. Both cell lines showed a significant induction of the 1,25(OH)2D3-dependent 24-hydroxylase gene, a marker for the actions of 1,25(OH)2D3. In MCF-7 cells, 51 genes were up-regulated and 19 genes were down-regulated. The up-regulated genes encoded cell adhesion molecules, growth factors/modulators, steroid receptors/co-activators, cytokines, kinases and transcription factors. Of the up-regulated genes, 40% were implicated in cell cycle regulation and apoptosis and included cyclin G1 and cyclin I, p21-activated kinase-1 (PAK-1), p53, retinoblastoma like-2 [Rb2 (p130)], insulin-like growth factor binding protein-5 (IGFBP5) and caspases. Among the down-regulated genes were ERα, growth factors, cytokines and several kinases. Some of these results were confirmed by real-time PCR. In MDA MB 231 cells, 20 genes were up-regulated and 13 genes were down-regulated. Very few genes directly implicated in cell cycle regulation were up-regulated. The matrix metalloproteinases formed a major class of genes that were down-regulated in the MDA MB 231 cells. Seven genes were commonly up-regulated in both cell lines and these included transforming growth factor (TGFβ2) and Rb2 (p130). In conclusion, the gene expression profiles of the two cell lines studied were different with a few overlapping genes suggesting that different cellular pathways might be regulated by 1,25(OH)2D3 to exert its growth inhibitory effects in ERα(+) and ERα(−) cells.

cDNA microarray estrogen receptor gene expression MCF-7 MDA MB 231 1,25(OH)2D3 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Mangelsdorf D, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans R: The nuclear receptor superfamily: the second decade. Cell 83: 835-839, 1995Google Scholar
  2. 2.
    Tsai MJ, O'Malley BW: Molecular mechanisms of action of steroid/thyroid receptor super family members. Annu Rev Biochem 63: 451-486, 1994Google Scholar
  3. 3.
    Osborne CK: Steroid hormone receptors in breast cancer management. Breast Cancer Res Treat 51: 227-238, 1998Google Scholar
  4. 4.
    Feldman D, Malloy PJ, Gross C: Vitamin D: Biology, Action and clinical implications. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, New York, 2001, pp 257-303Google Scholar
  5. 5.
    Colston KW, Hansen C: Mechanisms implicated in the growth regulatory effects of vitamin D in breast cancer. Endocr Relat Cancer 9: 45-59, 2002Google Scholar
  6. 6.
    Abe J, Nakano T, Nishii Y, Matsumoto T, Ogata E, Ikeda K: A novel vitamin D3 analog, 22-oxa-1,25-dihydroxyvitamin D3, inhibits the growth of human breast cancer in vitro and in vivo without causing hypercalcemia. Endocrinology 129: 832-837, 1991Google Scholar
  7. 7.
    Love-Schimenti CD, Gibson DFC, Ratnam AV, Bikle DD: Antiestrogens potentiation of antiproliferative effects of vitamin D3 analogs in breast cancer cells. Cancer Res 56: 2789-2794, 1996Google Scholar
  8. 8.
    Colston KW, Perks CM, Xie SP, Holly JM: Growth inhibition of both MCF-7 and Hs578T human breast cancer cell lines by vitamin D analogues is associated with increased expression of insulin-like growth factor binding protein-3. J Mol Endocrinol 20: 157-162, 1998Google Scholar
  9. 9.
    Xie SP, Pirianov G, Colston KW: Vitamin D analogues suppress IGF-I signalling and promote apoptosis in breast cancer cells. Eur J Cancer 35: 1717-1723, 1999Google Scholar
  10. 10.
    Simboli-Campbell M, Narvaez CJ, van Weelden K, Tenniswood M, Welsh JE: Comparative effects of 1,25(OH)2D3 and EB1089 on cell cycle kinetics and apoptosis in MCF-7 breast cancer cells. Breast Cancer Res Treat 42: 31-41, 1997Google Scholar
  11. 11.
    James SY, Mackay AG, Colston KW: Effects of 1,25 dihydroxyvitamin D3 and its analogues on induction of apoptosis in breast cancer cells. J Steroid Biochem Mol Biol 58: 395-401, 1996Google Scholar
  12. 12.
    Hansen C, Rohde L, Madsen MW, Hansen D, Colston KW, Pirianov G, Holm PK, Binderup L: MCF-7/VD(R): a new vitamin D resistant cell line. J Cell Biochem 82: 422-436, 2001Google Scholar
  13. 13.
    Narvaez CJ, Welsh J: Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells. J Biol Chem 276: 9101-9107, 2001Google Scholar
  14. 14.
    James SY, Mackey AG, Binderup L, Colston KW: Effects of a new synthetic vitamin D analogue, EB1089, on the oestrogenresponsive growth of human breast cancer cells. J Endocrinol 141: 555-563, 1994Google Scholar
  15. 15.
    Stoica A, Saceda M, Fakhro A, Solomon HB, Fenster BD, Martin MB: Regulation of estrogen receptor-alpha gene expression by 1,25-dihydroxyvitamin D in MCF-7 cells. J Cell Biochem 75: 640-651, 1999Google Scholar
  16. 16.
    Swami S, Krishnan AV, Feldman D: 1alpha,25-Dihydroxyvitamin D3 down-regulates estrogen receptor abundance and suppresses estrogen actions in MCF-7 human breast cancer cells. Clin Cancer Res 6: 3371-3379, 2000Google Scholar
  17. 17.
    Elstner E, Linker-Israeli M, Said J, Umiel T, de Vos S, Shintaku IP, Heber D, Binderup L, Uskokovic M, Koeffler HP: 20-epi-vitamin D3 analogues: a novel class of potent inhibitors of proliferation and inducers of differentiation of human breast cancer cell lines. Cancer Res 55: 2822-2830, 1995Google Scholar
  18. 18.
    Wang Q, Lee D, Sysounthone V, Chandraratna RAS, Christakos S, Korah R, Wieder R: 1,25-dihydroxyvitamin D3 and retonic acid analogues induce differentiation in breast cancer cells with function-and cell-specific additive effects. Breast Cancer Res Treat 67: 157-168, 2001Google Scholar
  19. 19.
    Raghavachari N, Bao YP, Li G, Xie X, Muller UR: Reduction of auto-fluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride. Anal Biochem 312: 101-105, 2003Google Scholar
  20. 20.
    Bao P, Frutos AG, Greef C, Lahiri J, Muller U, Peterson TC, Warden L, Xie X: High-sensitivity detection of DNA hybridization on microarrays using resonance light scattering. Anal Chem 74: 1792-1797, 2002Google Scholar
  21. 21.
    Bieche I, Parfait B, Tozlu S, Lidereau R, Vidaud M: Quantitation of androgen receptor gene expression in sporadic breast tumors by real-time RT-PCR: evidence that MYC is an ARregulated gene. Carcinogenesis 22: 1521-1526, 2001Google Scholar
  22. 22.
    Narvaez CJ, Zinser G, Welsh J: Functions of 1alpha,25-dihydroxyvitamin D(3) in mammary gland: from normal development to breast cancer. Steroids 66: 301-308, 2001Google Scholar
  23. 23.
    Davoodi F, Brenner RV, Evans SRT, Schumaker LM, Shabahang M, Nauta RJ, Buras RR: Modulation of vitamin D receptor and estrogen receptor by 1,25(OH)2D2-vitamin D3 in cDNA microarray analysis in breast cancer cells after vitamin D treatment 61 T47D human breast cancer cells. J. Steroid Biochem Mol Biol 54: 147-153, 1995Google Scholar
  24. 24.
    Hall JM, McDonnell DP: The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140: 5566-5578, 1999Google Scholar
  25. 25.
    Krishnan A, Feldman D: Regulation of vitamin D receptor abundance. In: Feldman D, Glorieux FH, Pike JW (eds) Vitamin D. Academic Press, San Diego, CA, 1997, pp 179-200Google Scholar
  26. 26.
    Gross C, Krishnan AV, Malloy PJ, Eccleshall TR, Zhao XY, Feldman D: The vitamin D receptor gene start codon polymorphism: a functional analysis of FokI variants. J Bone Miner Res 13: 1691-1699, 1998Google Scholar
  27. 27.
    Ly LH, Zhao XY, Holloway L, Feldman D: Liarozole actssynergistically with 1alpha,25-dihydroxyvitamin D3 to inhibit growth of DU 145 human prostate cancer cells by blocking 24-hydroxylase activity. Endocrinology 140: 2071-2076, 1999Google Scholar
  28. 28.
    Verlinden L, Verstuyf A, Convents R, Marcelis S, Van Camp M, Bouillon R: Action of 1,25(OH)2D3 on the cell cycle genes, cyclin D1, p21 and p27 in MCF-7 cells. Mol Cell Endocrinol 142: 57-65, 1998Google Scholar
  29. 29.
    Kobayashi T, Hashimoto K, Yoshikawa K: Growth inhibition of human keratinocytes by 1,25-dihydroxyvitamin D3 is linked to dephosphorylation of retinoblastoma gene product. Biochem Biophys Res Commun 196: 487-493, 1993Google Scholar
  30. 30.
    Hansen CM, Binderup L, Hamberg KJ, Carlberg C: Vitamin D and cancer: effects of 1,25(OH)2D3 and its analogs on growth control and tumorigenesis. Front Biosci 6: D820-D848, 2001Google Scholar
  31. 31.
    Jensen SS, Madsen MW, Lukas J, Binderup L, Bartek J: Inhibitory effects of 1alpha,25-dihydroxyvitamin D(3) on the G(1)-S phase-controlling machinery. Mol Endocrinol 15: 1370-1380, 2001Google Scholar
  32. 32.
    Verlinden L, Verstuyf A, Van Camp M, Marcelis S, Sabbe K, Zhao XY, De Clercq P, Vandewalle M, Bouillon R: Two novel 14-Epi-analogues of 1,25-dihydroxyvitamin D3 inhibit the growth of human breast cancer cells in vitro and in vivo. Cancer Res 60: 2673-2679, 2000Google Scholar
  33. 33.
    Mathiasen IS, Lademann U, Jaattela M: Apoptosis induced by vitamin D compounds in breast cancer cells is inhibited by Bcl-2 but does not involve known caspases or p53. Cancer Res 59: 4848-4856, 1999Google Scholar
  34. 34.
    Thomas A, Giesler T, White E: p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway. Oncogene 19: 5259-5269, 2000Google Scholar
  35. 35.
    Bates S, Rowan S, Vousden KH: Characterisation of human cyclin G1 and G2: DNA damage inducible genes. Oncogene 13: 1103-1109, 1996Google Scholar
  36. 36.
    Okamoto K, Prives C: A role of cyclin G in the process of apoptosis. Oncogene 18: 4606-4615, 1999Google Scholar
  37. 37.
    Kimura SH, Ikawa M, Ito A, Okabe M, Nojima H: Cyclin G1 is involved in G2/M arrest in response to DNA damage and in growth control after damage recovery. Oncogene 20: 3290-3300, 2001Google Scholar
  38. 38.
    Chen L, Shinde U, Ortolan TG, Madura K: Ubiquitinassociated (UBA) domains in Rad23 bind ubiquitin and promote inhibition of multi-ubiquitin chain assembly. EMBO Rep 2: 933-938, 2001Google Scholar
  39. 39.
    Bertolaet BL, Clarke DJ, Wolff M, Watson MH, Henze M, Divita G, Reed SI: UBA domains of DNA damage-inducible proteins interact with ubiquitin. Nat Struct Biol 8: 417-422, 2001Google Scholar
  40. 40.
    Takahashi T, Caviness Jr VS: PCNA-binding to DNA at the G1/S transition in proliferating cells of the developing cerebral wall. J Neurocytol 22: 1096-1102, 1993Google Scholar
  41. 41.
    Paunesku T, Mittal S, Protic M, Oryhon J, Korolev SV, Joachimiak A, Woloschak GE: Proliferating cell nuclear antigen (PCNA): ringmaster of the genome. Int J Radiat Biol 77: 1007-1021, 2001Google Scholar
  42. 42.
    Zamparelli A, Masciullo V, Bovicelli A, Santini D, Ferrandina G, Minimo C, Terzano P, Costa S, Cinti C, Ceccarelli C, Mancuso S, Scambia G, Bovicelli L, Giordano A: Expression of cell-cycle-associated proteins pRB2/p130 and p27kip in vulvar squamous cell carcinomas. Hum Pathol 32: 4-9, 2001Google Scholar
  43. 43.
    Ravid A, Rocker D, Machlenkin A, Rotem C, Hochman A, Kessler-Icekson G, Liberman UA, Koren R: 1,25-Dihydroxyvitamin D3 enhances the susceptibility of breast cancer cells to doxorubicin-induced oxidative damage. Cancer Res 59: 862-867, 1999Google Scholar
  44. 44.
    Koren R, Hadari-Naor I, Zuck E, Rotem C, Liberman UA, Ravid A: Vitamin D is a prooxidant in breast cancer cells. Cancer Res 61: 1439-1444, 2001Google Scholar
  45. 45.
    Lindner DJ, Hofmann ER, Karra S, Kalvakolanu DV: The interferon-beta and tamoxifen combination induces apoptosis using thioredoxin reductase. Biochim Biophys Acta 1496: 196-206, 2000Google Scholar
  46. 46.
    Rozen F, Yang XF, Huynh H, Pollak M: Antiproliferative action of vitamin D-related compounds and insulin-like growth factor-binding protein 5 accumulation. J Natl Cancer Inst 89: 652-656, 1997Google Scholar
  47. 47.
    Boyle BJ, Zhao XY, Cohen P, Feldman D: Insulinlike growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin d(3) growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1. J Urol 165: 1319-1324, 2001Google Scholar
  48. 48.
    Zumkeller W: IGFs and IGFBPs: surrogate markers for diagnosis and surveillance of tumour growth? Mol Pathol 54: 285-288, 2001Google Scholar
  49. 49.
    Schmidt M, Lichtner RB: EGF receptor targeting in therapyresistant human tumors. Drug Resist Updat 5: 11-18, 2002Google Scholar
  50. 50.
    Zhang W, Wang H, Song SW, Fuller GN: Insulin-like growth factor binding protein 2: gene expression microarrays and the hypothesis-generation paradigm. Brain Pathol 12: 87-94, 2002Google Scholar
  51. 51.
    Wu G, Fan RS, Li W, Srinivas V, Brattain MG: Regulation of transforming growth factor-beta type II receptor expression in human breast cancer MCF-7 cells by vitamin D3 and its analogues. J Biol Chem 273: 7749-7756, 1998Google Scholar
  52. 52.
    Yang L, Yang J, Venkateswarlu S, Ko T, Brattain MG: Autocrine TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in breast cells. J Cell Physiol 188: 383-393, 2001Google Scholar
  53. 53.
    Hu X, Zuckerman KS: Transforming growth factor: signal transduction pathways, cell cycle mediation, and effects on hematopoiesis. J Hematother Stem Cell Res 10: 67-74, 2001Google Scholar
  54. 54.
    Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al.: A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266: 66-71, 1994Google Scholar
  55. 55.
    Wang SC, Lin SH, Su LK, Hung MC: Changes in BRCA2 expression during progression of the cell cycle. Biochem Biophys Res Commun 234: 247-251, 1997Google Scholar
  56. 56.
    MacDougall JR, Matrisian LM: Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis. Cancer Metastasis Rev 14: 351-362, 1995Google Scholar
  57. 57.
    Koli K, Keski-Oja J: 1alpha,25-dihydroxyvitamin D3 and its analogues down-regulate cell invasion-associated proteases in cultured malignant cells. Cell Growth Diff 11: 221-229, 2000Google Scholar
  58. 58.
    Akutsu N, Lin R, Bastien Y, Bestawros A, Enepekides DJ, Black MJ, White JH: Regulation of gene Expression by 1alpha,25-dihydroxyvitamin D3 and its analog EB1089 under growth-inhibitory conditions in squamous carcinoma Cells. Mol Endocrinol 15: 1127-1139, 2001Google Scholar
  59. 59.
    Lin R, Nagai Y, Sladek R, Bastien Y, Ho J, Petrecca K, Sotiropoulou G, Diamandis EP, Hudson TJ, White JH: Expression profiling in squamous carcinoma cells reveals pleiotropic effects of vitamin D3 analog EB1089 signaling on cell proliferation, differentiation, and immune system regulation. Mol Endocrinol 16: 1243-1256, 2002Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Srilatha Swami
    • 1
  • Nalini Raghavachari
    • 2
  • Uwe R. Muller
    • 2
  • Yijia P. Bao
    • 2
  • David Feldman
    • 1
  1. 1.Department of MedicineStanford University School of MedicineStanfordUSA
  2. 2.Biochemical Technologies, Corning Inc.Painted PostUSA

Personalised recommendations