Mechanistic Progress of Estrogen-induced Apoptosis in Estrogen-de
Journal of Cell Science & Therapy

Journal of Cell Science & Therapy
Open Access

ISSN: 2157-7013

Research Article - (2015) Volume 6, Issue 4

Mechanistic Progress of Estrogen-induced Apoptosis in Estrogen-deprived Breast Cancer Cells

Shuqiao Chai1,2 and Ping Fan2*
1McLean High School, 1633 Davidson Road, McLean, VA 22102, USA
2Department of Breast Medical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 1354, Houston, TX 77030, USA
*Corresponding Author: Ping Fan, MD PhD, Department of Breast Medical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 1354, Houston, TX 77030, USA, Tel: 713-792-2121 Email:


The laboratory discovery of estrogen-induced apoptosis has been translated to treat antihormone resistant patients and to reduce the incidence of breast cancer in postmenopausal hysterectomized women with estrogen replacement therapy (ERT). The key step is the selection pressure exerted by long-term antiestrogen therapy or over 5 years of menopause to specific breast cancer cell populations that will be vulnerable to estrogen-induced apoptosis. However, the mechanisms underlying estrogen-induced apoptosis are currently unclear. At the cellular level, estrogen-induced apoptosis is dependent upon the estrogen receptor (ER), which can be completely blocked by antiestrogen ICI 182,780 or 4-hydroxytamoxifen (4-OHT). Knockdown of ER alpha, but not ER beta, through specific small interfering RNAs effectively blocks estrogen-induced apoptosis, indicating that the ER alpha subtype participates in apoptosis. Further examinations demonstrate that estrogen-induced apoptosis is due to accumulation of endoplasmic reticulum stress, inflammatory responses, and oxidative stress, which, in turn, activate the intrinsic mitochondrial pathway and the extrinsic death receptor pathway to complete the process. This contrasts with paclitaxel, which causes G2 arrest with immediate apoptosis. These stress responses are modulated by glucocorticoids and the c-Src inhibitor to block estrogen-induced apoptosis, but the mechanism for estrogen action is through a genomic pathway rather than a non-genomic pathway. In the nucleus, estrogen activates classic ERE-regulated endogenous genes, but the ERE transcriptional pathway does not directly participate in the estrogen-induced apoptosis in vitro or in vivo. Simultaneously, estrogen activates a non-classic transcriptional pathway involving the interaction of ER with transcription factors such as activator protein-1 (AP-1), which may regulate proliferation, stress responses, or apoptosis. Investigation of how AP-1 modulates the stress responses to trigger estrogen-induced apoptosis will ultimately uncover the mechanisms underlying estrogen-induced apoptosis.

Keywords: Estrogen; Estrogen receptor; Estrogen-induced apoptosis; Breast cancer


Estrogen (E2) plays a pivotal role in the development and progression of breast cancers [1]. As a result, blockade of E2 signals through either aromatase inhibitors (AIs) or selective estrogen receptor modulators (SERMs) is an important therapeutic strategy to treat or prevent estrogen receptor (ER) positive breast cancers [2]. These endocrine therapies have significantly improved breast cancer survival [3-6]. Paradoxically, long-term endocrine therapy generates selection pressure for cell populations that evolve from acquired resistance to eventually expose a vulnerability that is expressed as E2-induced apoptosis in vitro [7,8] and in vivo [9-11]. All of these laboratory data with MCF-7 cells provide the scientific rationale for the subsequent finding that high dose (30 mg daily) or low dose (6 mg daily) E2 produces a 30% clinical benefit rate in patients failing aromatase inhibitor therapy [12]. Further, E2-induced apoptosis has been used to explain lower incidences of breast cancer and mortality of postmenopausal women in their 60s undergoing E2 replacement therapy (ERT) [13]. A recent study addresses the hypothesis that women taking traditional hormone replacement therapy (HRT) comprising of E2 plus medroxyprogesterone acetate (MPA) may have an increased risk of breast cancer, as MPA may act as a glucocorticoid and block E2-induced apoptosis in E2-deprived breast cancer cells [14]. All of these observations indicate the clinical implications of E2- induced apoptosis. However, mechanisms of E2-induced apoptosis are currently unclear. This review will focus on the progress of mechanistic discoveries in E2-induced apoptosis in breast cancer cells.

Evolution of Estrogen-induced Apoptosis is Selected by Antiestrogen Pressure

Laboratory findings with the MCF-7 breast cancer cell line grown in athymic mice first described tamoxifen-stimulated growth as a new mechanism of drug resistance to a therapeutic intervention [15]. However, discovery that re-transplantation of tamoxifen-stimulated tumors into successive generations of athymic mice over 5 years results in the selection of a resistant tumor cell population that is killed by physiological levels of E2 [9-11], which results in the new biology of E2- induced apoptosis [16].

A similar story has also occurred over the past 20 years with the development of models to study antihormone resistance to aromatase inhibtors (AIs). The discovery that breast cancer cells, in particular MCF-7 cells, have been grown routinely in the medium with phenol red [17] that contains estrogenic activity revolutionized options to solve the question of what happens to hormone-responsive cells once starved of E2 i.e. AI therapy. Wild-type MCF-7 cells grow with radioimmunologically undetectable levels of E2 (Figure 1), whereas long- term E2-deprived MCF-7 cells, MCF-7:5C undergo apoptosis by E2 (Figure 1). Thus, both E2 deprivation [7,8] and SERMs produce the same selective pressure on MCF-7 cells [9-11] to create selective cellular populations vulnerable to E2-induced apoptosis [18]. A period of 5-10 years is required to accomplish the described selection process [19]. The apoptotic action of E2 provides a new clinical treatment strategy for breast cancer patients following exhaustive antihormonal therapy [12,20,21]


Figure 1: Different growth responses to E2 between wild-type MCF-7 and MCF-7:5C cells. Wild-type MCF-7 cells were first transferred to E2-free medium for 3 days. Then, MCF-7 and MCF-7:5C cells were loaded in 24-well plate respectively. After one day, cells were treated with different concentrations of E2 as indicated. Cells were harvested after 7 days treatment. Cell viability was quantitated by determination of total DNA.

Estrogen induces a delayed apoptosis in breast cancer cells

The estrogen receptor (ER) is the initial trigger for E2 to induce apoptosis since antiestrogens ICI 182,780 and 4-hydroxytamoxifen (4- OHT) completely block apoptosis triggered by E2 [22]. Contradictory to the traditional apoptosis mechanism caused by cytotoxic chemotherapy, E2-induced apoptotic cells simultaneously undergo proliferation with an increased S phase of the cell cycle, resulting in an increased cell number [22-25]. The possibility that apoptotic impairment can be rescued by antiestrogen is a unique feature of E2-induced apoptosis, which differs from the rapid (12 hour) chemotherapy-induced cytotoxic apoptosis [24]. E2 exerts a dual function on E2-deprived cells, with both initial proliferation and subsequent apoptosis [22]. In other words, there is not any detected apoptotic change in the first 24 hours after E2 treatment [22], which gives researchers a possibility to reverse E2-induced apoptosis by antiestrogen within 24 hours [24]. Activation of apoptotic genes appears after 48 hours treatment with E2, and reaches a peak after 72 hours [22]. Our observations also indicate that insulin-like growth factor-1 receptor (IGF-1R)/phosphoinositide 3-kinase (PI3K) is a dominant growth driver after E2 treatment in two E2-deprived breast cancer cells [23,26], which activates Akt to promote cell growth [23, 26]. These data suggest that the higher rate of proliferation by E2 might activate other pathways to trigger apoptosis.

Estrogen induces apoptosis through nuclear estrogen receptor alpha (ERα)

The original target site of E2-induced apoptosis is ER alpha (ERα) [22,27], which can be completely blocked by the knockdown of ERα, but not ERβ, through small interfering RNA (siRNA) [28]. Oncogene c-Src functions as an important downstream signal of ERα in MCF- 7:5C cells, which is activated by E2 and demonstrates multiple levels of association with ERα [22]. A well-known function of c-Src is that it mediates the non-genomic pathway of E2 in E2-deprived breast cancer cells [22]. However, E2-induced apoptosis is not through a nongenomic pathway [22]. This conclusion is supported by the evidence that synthetic macromolecules, estrogen-dendrimer conjugates (EDCs) that remain outside the nucleus [29] activate the non-genomic pathway while are unable to activate the genomic pathway, thereby not inducing apoptosis in MCF-7:5C cells [22]. All of these observations indicate that nuclear ERα plays an important role in E2-induced apoptosis [22]. In the nucleus, E2 activates classic ERE-regulated endogenous genes in MCF-7:5C cells [22,27], but the ERE transcriptional pathway does not directly participate in the E2-induced apoptosis in vitro [22] or in vivo [30]. Our global gene array [27] data suggest that E2 signaling can occur through a non-classic transcriptional pathway involving the interaction of ER with transcription factors such as activator protein-1 (AP-1), which may regulate proliferation, stress responses, or apoptosis [31].

Accumulation of stresses activates estrogen-induced apoptosis

The endoplasmic reticulum and mitochondria are two key organelles involved in E2-induced apoptosis [22]. The sensors of unfolded protein response (UPR) and the oxidative stress sensor HMOX1 are activated by E2 as initial adaptive responses in an attempt to sustain a balance in cell survival. A cross-talk exists between the endoplasmic reticulum and mitochondria to activate apoptosis cascades during E2- induced apoptosis (Figure 2). UPR initially occurs after a few hours of treatment with E2 [22]. Three sensors of endoplasmic reticulum stress, protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring protein 1 alpha (IRE1α), and activating transcription factor 6 (ATF6) are activated by E2 [22,27]. PERK attenuates protein translation, which has been confirmed as an important inducer for E2-induced apoptosis [22]; On the other hand, ATF-6 and IRE1 increase endoplasmic reticulum folding capacity by up-regulating the endoplasmic reticulum chaperones and the endoplasmic reticulumassociated protein degradation (ERAD) machinery [32,33]. The initial aim of UPR is to restore the normal function of the cell; however, if the damage is too severe to repair, the UPR ultimately initiates cell death through activation of the apoptotic pathway [34]. Compelling evidence suggests that E2 induces apoptosis through accumulation of stress responses, including endoplasmic reticulum stress, oxidative stress, and inflammatory stress [22,23,27]. Glucocorticoid and the c-Src inhibitor are able to modulate stress responses to block E2-induced apoptosis [14,22]. Currently, it is under investigation how E2 activates the nuclear AP-1 complex to modulate the stress and apoptosis in E2-deprived breast cancer cells (Figure 2).


Figure 2: schematic diagram of the mechanisms underlying E2-induced apoptosis. (1) E2 activates nuclear ER to modulate multiple transcriptional factors including AP-1 complex [27]; (2) unfolded protein responsesare activated to reduce protein translation or increase protein degradation to reduce the burden of unfolded protein in the endoplasmic reticulum [22]; (3) failure to relieve endoplasmic reticulum stress induces apoptosis via crosstalk with mitochondria to increase reactive oxygen species (ROS) production or activate the mitochondrial pathway [22]; (4) the endoplasmic reticulum stress subsequently activates inflammation response and the extrinsic pathway of apoptosis [22,27]; (5) apoptosis can occur independent of the intrinsic and extrinsic pathways through activation of caspase 4, 12 [27].

Conclusion and Future Direction

Despite the limitations of ER positive breast cancer cell lines [35], long-term estrogen-deprived MCF-7 cell models [7,8] are invaluable tools to uncover the mechanisms underlying E2-induced apoptosis (Figure 2). This scientific rationale has been utilized in clinical trials which have confirmed that E2 can reduce the incidence of breast cancer in postmenopausal women [12,13]. Laboratory evidence for modification of apoptosis by E2 through the glucocorticoid-like action of MPA provides an important rationale to change the traditional HRT strategy [14]. Principles have emerged for understanding and applying physiological E2 therapy appropriately by targeting the correct patient populations [36,37]. However, new findings reflect that the rapid plasticity of hormone resistance occurs as a response to selection pressure [14,22,26], indicating that there is still long way to translate such treatment into bedside practice.


  1. Jensen EV, Jordan VC (2003) The estrogen receptor: a model for molecular medicine.  Clin Cancer Res 9: 1980-1989.
  2. Jordan VC, Brodie AM (2007) Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer. Steroids 72: 7-25.
  3. Jordan VC (2014) Tamoxifen as the first targeted long-term adjuvant therapy for breast cancer. Endocr Relat Cancer 21: R235-246.
  4. Early Breast Cancer Trialists' Collaborative Group (EBCTCG), Davies C, Godwin J, Gray R, Clarke M, et al. (2011) Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet 378: 771-784.
  5. Davies C, Pan H, Godwin J, Gray R, Arriagada R, et al. (2013) Long-term effects of continuing adjuvant tamoxifen to 10 years versus stopping at 5 years after diagnosis of oestrogen receptor-positive breast cancer: ATLAS, a randomised trial. Lancet 381: 805-816.
  6. Dowsett M, Cuzick J, Ingle J, Coates A, Forbes J, et al. (2010) Meta-analysis of breast cancer outcomes in adjuvant trials of aromatase inhibitors versus tamoxifen. J Clin Oncol 28: 509-518.
  7. Song RX, Mor G, Naftolin F, McPherson RA, Song J, et al. (2001) Effect of long-term estrogen deprivation on apoptotic responses of breast cancer cells to 17beta-estradiol. J Natl Cancer Inst 93: 1714-1723.
  8. Lewis JS, Meeke K, Osipo C, Ross EA, Kidawi N, et al. (2005) Intrinsic mechanism of estradiol-induced apoptosis in breast cancer cells resistant to estrogen deprivation. J Natl Cancer Inst 97: 1746-1759.
  9. Yao K, Lee ES, Bentrem DJ, England G, Schafer JI, et al. (2000) Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice. Clin Cancer Res 6: 2028-2036.
  10. Osipo C, Gajdos C, Liu H, Chen B, Jordan VC (2003) Paradoxical action of fulvestrant in estradiol-induced regression of tamoxifen-stimulated breast cancer. J Natl Cancer Inst 95: 1597-1608.
  11. Liu H, Lee ES, Gajdos C, Pearce ST, Chen B, et al. (2003) Apoptotic action of 17beta-estradiol in raloxifene-resistant MCF-7 cells in vitro and in vivo. J Natl Cancer Inst 95: 1586-1597.
  12. Ellis MJ, Gao F, Dehdashti F, Jeffe DB, Marcom PK, et al. (2009) Lower-dose vs high-dose oral estradiol therapy of hormone receptor-positive, aromatase inhibitor-resistant advanced breast cancer: a phase 2 randomized study. JAMA 302: 774-780.
  13. Anderson GL, Chlebowski RT, Aragaki AK, Kuller LH, Manson JE, et al. (2012) Conjugated equine oestrogen and breast cancer incidence and mortality in postmenopausal women with hysterectomy: extended follow-up of the Women's Health Initiative randomised placebo-controlled trial. Lancet Oncol 13: 476-486.
  14. Sweeney EE, Fan P, Jordan VC (2014) Molecular modulation of estrogen-induced apoptosis by synthetic progestins in hormone replacement therapy: An insight into the Women's Health Initiative study. Cancer Res 74:7060-7068.
  15. Gottardis MM, Jordan VC (1988) Development of tamoxifen-stimulated growth of MCF-7 tumors in athymic mice after long-term antiestrogen administration. Cancer Res 48: 5183-5187.
  16. Jordan VC (2008) The 38th David A. Karnofsky lecture: the paradoxical actions of estrogen in breast cancer--survival or death? J Clin Oncol 26: 3073-3082.
  17. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS (1986) Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci U S A 83: 2496-2500.
  18. Fan P, Jordan VC (2014) Acquired resistance to selective estrogen receptor modulators (SERMs) in clinical practice (tamoxifen&raloxifene) by selection pressure in breast cancer cell populations. Steroids 90:44-52.
  19. Obiorah I, Jordan VC (2013) The scientific rationale for a delay after menopause in the use of conjugated equine estrogens in postmenopausal women that causes a reduction in breast cancer incidence and mortality. Menopause 20:372-382.
  20. Jordan VC, Lewis JS,Osipo C, Cheng D (2005) The apoptotic action of estrogen following exhaustive antihormonal therapy: a new clinical treatment strategy. Breast 14:624-630.
  21. Lønning PE, Taylor PD, Anker G, Iddon J, Wie L, et al. (2001) High-dose estrogen treatment in postmenopausal breast cancer patients heavily exposed to endocrine therapy. Breast Cancer Res Treat 67: 111-116.
  22. Fan P, Griffith OL, Agboke FA, Anur P, Zou X, et al. (2013) c-Src modulates estrogen-induced stress and apoptosis in estrogen-deprived breast cancer cells. Cancer Res 73: 4510-4520.
  23. Sweeney EE, Fan P, Jordan VC (2014) Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cells. Int J Oncol 44: 1529-1538.
  24. Obiorah I, Sengupta S, Fan P, Jordan VC (2014) Delayed triggering of oestrogen induced apoptosis that contrasts with rapid paclitaxel-induced breast cancer cell death. Br J Cancer 110: 1488-1496.
  25. Obiorah IE, Jordan VC (2014) Differences in the rate of oestrogen-induced apoptosis in breast cancer by oestradiol and the triphenylethylene bisphenol. Br J Pharmacol 171: 4062-4072.
  26. Fan P, Agboke FA, McDaniel RE, Sweeney EE, Zou X, et al. (2014) Inhibition of c-Src blocks oestrogen-induced apoptosis and restores oestrogen-stimulated growth in long-term oestrogen-deprived breast cancer cells. Eur J Cancer 50: 457-468.
  27. Ariazi EA, Cunliffe HE, Lewis-Wambi JS, Slifker MJ, Willis AL, et al. (2011) Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time. Proc Natl Acad Sci U S A 108: 18879-18886.
  28. Obiorah IE, Fan P, Jordan VC (2014) Breast cancer cell apoptosis with phytoestrogens is dependent on an estrogen-deprived state. Cancer Prev Res (Phila) 7: 939-949.
  29. Harrington WR, Kim SH, Funk CC, Madak-Erdogan Z, Schiff R, et al (2006) Estrogen dendrimer conjugates that preferentially activate extranuclear, nongenomic versus genomic pathways of estrogen action. Mol Endocrinol 20:491-502.
  30. Zhang Y, Zhao H, Asztalos S, Chisamore M, Sitabkhan Y, et al. (2009) Estradiol-induced regression in T47D:A18/PKCalpha tumors requires the estrogen receptor and interaction with the extracellular matrix. Mol Cancer Res 7: 498-510.
  31. Shaulian E, Karin M (2002) AP-1 as a regulator of cell life and death. Nat Cell Biol 4: E131-136.
  32. Fan P, Cunliffe HE, Maximov PY, Agboke FA, McDaniel RE, et al. (2015) Integration of Downstream Signals of Insulin-like Growth Factor-1 Receptor by Endoplasmic Reticulum Stress for Estrogen-induced Growth or Apoptosis in Breast Cancer Cells. Mol Cancer Res .
  33. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, et al. (2000) Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 287: 664-666.
  34. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, et al (2000) Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403:98-103.
  35. Sweeney EE, McDaniel RE, Maximov PY, Fan P, Jordan VC (2012) Models and Mechanisms of Acquired Antihormone Resistance in Breast Cancer: Significant Clinical Progress Despite Limitations. Horm Mol Biol Clin Investig 9: 143-163.
  36. Jordan VC (2015) The new biology of estrogen-induced apoptosis applied to treat and prevent breast cancer. Endocr Relat Cancer 22: R1-31.
  37. Jordan VC (2014) Linking estrogen-induced apoptosis with decreases in mortality following long-term adjuvant tamoxifen therapy. J Natl Cancer Inst 106.
Citation: Chai S, Fan P (2015) Mechanistic Progress of Estrogen-induced Apoptosis in Estrogen-deprived Breast Cancer Cells. J Cell Sci Ther 6:218.

Copyright: © 2015 Chai S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.