Skip to main content
NCBI home page
F1000Research logoLink to F1000Research
. 2014 Jul 11;3:161. [Version 1] doi: 10.12688/f1000research.4753.1

Estrogen as Jekyll and Hyde: regulation of cell death

Wen Zhou 1,a, Xiaoxia Zhu 2
PMCID: PMC4243742  PMID: 25485093

Abstract

Estrogen has profound effects on growth, differentiation and function in male and female reproductive systems, and it is important for bone density, brain function and cholesterol mobilization. Despite beneficial estrogen functions, sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. This opinion article touches upon the dual role estrogen played in cancer and asks whether the use of estrogen in combination with other targeted therapy would be possible, considering the newly identified crosstalk pathway which can switch the effects of estrogen.

Introduction

Our research projects currently focus on understanding of the interplay between the different signaling cascades and estrogen receptor (ER) dependent transcription activation in breast cancer. For many years, those of us working in the field were used to looking at estrogen as a mitogen through its genomic function mediated by the ER-dependent transcription program. Now we realize that estrogen through ER, in addition to regulate gene expression, crosstalks with many non-genomic signaling pathways involved in cell growth, differentiation and apoptosis. These newly identified interplays may give a different flavor to long conceived mitogenic role of estrogen. In this opinion article, we generally reviewed the changing attitude toward estrogen in clinical use, with a focus on a new discovery that estrogen, in combination with IKKα, can induce breast cancer cell apoptosis effectively. We also discuss the possibility of estrogen and IKKα inhibitor dual-therapy strategy in cancer treatment.

Historical perspective and changing attitude to estrogen in clinical use

Sir George Thomas Beatson (1896) used oophorectomy to reduce the estrogen level in premenopausal women in order to prevent breast cancer occurrence, and he was the first to reveal the relationship between estrogen levels and breast cancer 1. Half a century later, Haddow et al. (1944) first used a high-dose synthetic estrogen (stillbestrol) to induce tumor regression in hormone-dependent breast cancer in postmenopausal women 2. Huggins et al. (1952) pioneered adrenalectomy to reduce estrogen level for treating mammary cancer 3. Huggins’ work is internationally recognized by the prestigious Nobel Prize (1966) for the contribution to the development of endocrine therapy in hormone-regulated cancer. Jensen E (1958) characterized the first receptor for estrogen - estrogen receptor alpha (ERα). Soon after these discoveries, extensive mechanistic studies have gained large information about estrogen’s physiological functions and carcinogenic roles. The Women's Health Initiative (WHI) research program (1991) initiated a 15-year study enrolled 161,808 generally healthy women aged 50–79 to evaluate the beneficial effects of postmenopausal hormone replacement therapy (HRT) on heart diseases, bone fractures, and cancers 4. Due to the increased incidence of breast cancer, stroke, and cardiovascular complications in women treated with estrogen alone or with a combination of estrogen and progesterone, the study was terminated prematurely in 2002. Though extensively studied, the definite understanding of the mechanism of estrogen action always challenges our mind.

The challenge: paradoxical role of estrogen in cell death

Estrogen regulates the proliferation and development of tissues expressing estrogen receptors and ERα is mainly expressed in breast epithelium, ovary and endometrium. Thus, estrogen is mitogenic for cultured ER positive breast cancer lines. The mitogenic effects of estrogen at the G1-to-S transition are mediated by the key effectors of estrogen action, c-Myc, cyclin D1 and E2F-1 5– 7. c-Myc expression occurs within 15 min of estrogen stimulation, among the earliest responses to estrogen. Estrogen also rapidly induces cyclin D1 expression. In the G1 phase, estrogen drives E2F-1 expression. Estrogen-triggered all these coherent genetic changes to guarantee the cell cycle progression. Nongenomically, Estrogen binding to the ER stimulates rapid activation of Src and signaling pathways MAPK and PI3K/Akt pathways that affect cell survival 8, 9. Based on these understandings of estrogen action, ER protein is assayed in newly diagnosed breast cancers because it is a clinically useful prognostic factor and predicts responsiveness to ER blocking drugs such as tamoxifen.

Paradoxically, estrogen induces apoptosis under certain circumstances. As mentioned above, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in postmenopausal women before the introduction of tamoxifen 2. This regimen is of clinical interest, given that long-term treatment of breast cancer with anti-estrogen drug tamoxifen often leads to drug resistance and that sustained tamoxifen exposure may sensitize breast cancer cells to high-dose or even low-dose estrogen therapy 10. The field of the mysterious dual effects of estrogen on apoptosis have not much progressed until recently.

Recent research breakthrough

Recently, Perillo’s Group from Second University of Naples identified a key player, IKKα in the switch of estrogen action in apoptosis 11. They found that ER agonist 17β-estradiol (E2) and IKKα kinase specific inhibitor BAY11-7082 (BAY) in combination can induce apoptosis in an ERα-positive breast cancer cell line. Dual-therapy now receives more and more attention.

In the journal Cell Death & Differentiation, Perillo et al. recently reported that the inhibition of IKKα by BAY switched the effect of estrogens on breast cancer cells from anti- to pro-apoptotic, which leads the exploration of therapeutic benefits of estrogen into a new era 11. IKKα is the kinase responsible for histone H3 Ser 10 phosphorylation (H3pS10) 12. H3pS10 is known to inhibit H3 Lys 9 dimethylation (H3K9me2) in a space repulsion model 13. Thus, inhibiting H3pS10 by targeting IKKα facilitates estrogen-triggered ER-dependent recruitment of histone methyltransferase Suv39H1. Histone demethylase LSD1 demethylating the Suv39H1 target sites H3K9me2 was increased concomitantly. LSD1-mediated demethylation process is known to produce reactive oxygen species (ROS) and cause ROS-mediated DNA damaging effects 14. The net results after IKKα knowndown is causing more DNA damages to cancer cells through estrogen triggered ER-dependent Suv39H1 and LSD1 binding to ER target gene promoter ( Figure 1).

Figure 1. Involvement of IKKα in estrogen-triggered ER-dependent activation of the pS2 promoter.

Figure 1.

Open in a new tab

Upper panel: chromatin landscape and factors present at the pS2 (as known as TFF1) promoter in the presence of IKKα. The pS2 promoter is enriched with nucleosomes (blue and white cylinders) that dwell in positions proximal to the transcription start site (+1 position) and at ER binding sites. Only low levels of histone H3 lysine 9 dimethylation (H3K9me2) exist due to the space repulsion of histone methyltransferases binding to H3K9 from IKKα residing at the neighboring H3S10 site. RNA polymerase holoenzyme (Pol II) (yellow oval) is present at the proximal promoter region near the transcription start site (TSS, shown by black vertical line) of the pS2 gene. Lower panel: chromatin landscape and factors present at pS2 promoter following IKKα knockdown or IKKα inhibitor BAY11-7082 treatment. Once levels of IKKα have decreased, ER recruits the histone methyltransferase Suv39H1 or demethylase LSD1 proteins to bind within the pS2 promoter. Once the LSD1 is activated and demethylates its target H3K9me2, it generates reactive oxygen species (ROS) to cause DNA damage effects including base oxidation and nicks results from DNA damage itself and related DNA repair. In sum, the inhibition of IKKα results in the reversion of estrogen triggered anti-apoptotic effects to pro-apoptotic effects.

In short, Perillo's group identifies a novel crosstalk between IKKα and estrogen signaling and shows that inhibition of IKKα-mediated histone phosphorylation switch ER-mediated anti-apoptotic effects to ER-dependent ROS-mediated breast cell death, which implicates potential dual-therapy of ER agonist (E2) together with IKKα inhibitor (BAY) in a variety of hormone-regulated cancers.

Future perspectives

In the last few years, there have been significant shifts in the attitudes towards the use of estrogen in clinic. Estrogen exhibits a broad range of functions that regulates cell proliferation and homeostasis in many tissues. Despite beneficial estrogen functions, sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary 15. The International Agency for Research on Cancer (IARC) has listed estrogen as known human carcinogen 16. Now, the success of the combination of E2 and BAY will certainly become an “accelerator” to the alternative use of estrogen in treating cancers and we expect to see more positive pre-clinical and/or clinical results in the near future.

Acknowledgements

The authors thank laboratory members for helpful discussions.

Funding Statement

This work was partially supported by US Department of Defense pre-doctoral grant W81XWH-11-1-0097 (W.Z.).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

v1; ref status: approved with reservations 2

References

  • 1.Beatson GT: On treatment of inoperable cases of carcinoma of the mamma: suggestions for a new method of treatment with illustrative cases. Lancet. 1896;148(3802):104–7 10.1016/S0140-6736(01)72307-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Haddow A, Watkinson JM, Paterson E, et al. : Influence of Synthetic Oestrogens on Advanced Malignant Disease. Br Med J. 1944;2(4368):393–8. 10.1136/bmj.2.4368.393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Huggins C, Bergenstal DM: Effect of Bilateral Adrenalectomy on Certain Human Tumors. Proc Natl Acad Sci U S A. 1952;38(1):73–6. 10.1073/pnas.38.1.73 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Prentice RL, Anderson GL: The women’s health initiative: lessons learned. Annu Rev Public Health. 2008;29:131–50. 10.1146/annurev.publhealth.29.020907.090947 [DOI] [PubMed] [Google Scholar]
  • 5.Musgrove EA, Caldon CE, Barraclough J, et al. : Cyclin D as a therapeutic target in cancer. Nat Rev Cancer. 2011;11(8):558–72. 10.1038/nrc3090 [DOI] [PubMed] [Google Scholar]
  • 6.Caldon CE, Musgrove EA: Distinct and redundant functions of cyclin E1 and cyclin E2 in development and cancer. Cell Div. 2010;5:2. 10.1186/1747-1028-5-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zhou W, Srinivasan S, Nawaz Z, et al. : ERα, SKP2 and E2F-1 form a feed forward loop driving late ERα targets and G1 cell cycle progression. Oncogene. 2014;33(18):2341–53. 10.1038/onc.2013.197 [DOI] [PubMed] [Google Scholar]
  • 8.Sun J, Zhou W, Kaliappan K, et al. : ERα phosphorylation at Y537 by Src triggers E6-AP-ERα binding, ERα ubiquitylation, promoter occupancy, and target gene expression. Mol Endocrinol. 2012;26(9):1567–77. 10.1210/me.2012-1140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Kato S, Endoh H, Masuhiro Y, et al. : Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science. 1995;270(5241):1491–4. 10.1126/science.270.5241.1491 [DOI] [PubMed] [Google Scholar]
  • 10.Ariazi EA, Cunliffe HE, Lewis-Wambi JS, et al. : 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. 2011;108(47):18879–86. 10.1073/pnas.1115188108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Perillo B, Di Santi A, Cernera G, et al. : Phosphorylation of H3 serine 10 by IKKα governs cyclical production of ROS in estrogen-induced transcription and ensures DNA wholeness. Cell Death Differ. 2014. 10.1038/cdd.2014.91 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Park KJ, Krishnan V, O’Malley BW, et al. : Formation of an IKKalpha-dependent transcription complex is required for estrogen receptor-mediated gene activation. Mol Cell. 2005;18(1):71–82. 10.1016/j.molcel.2005.03.006 [DOI] [PubMed] [Google Scholar]
  • 13.Rea S, Eisenhaber F, O–Carroll D, et al. : Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature. 2000;406(6796):593–599. 10.1038/35020506 [DOI] [PubMed] [Google Scholar]
  • 14.Perillo B, Ombra MN, Bertoni A, et al. : DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression. Science. 2008;319(5860):202–206. 10.1126/science.1147674 [DOI] [PubMed] [Google Scholar]
  • 15.Zhou W, Slingerland JM: Links between oestrogen receptor activation and proteolysis: relevance to hormone-regulated cancer therapy. Nat Rev Cancer. 2014;14(1):26–38. 10.1038/nrc3622 [DOI] [PubMed] [Google Scholar]
  • 16.IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Combined estrogen-progestogen contraceptives and combined estrogen-progestogen menopausal therapy. IARC Monogr Eval Carcinog Risks Hum. 2007;91:1–528. [PMC free article] [PubMed] [Google Scholar]
F1000Res. 2014 Sep 22. doi: 10.5256/f1000research.5075.r6196

Referee response for version 1

Philippa Saunders 1

New ideas about the way oestrogens might affect the growth, differentiation and functional development of cancers, including those of the breast, are always welcome. The authors' Opinion Article questions whether giving oestrogens (rather than the more common anti-oestrogens) may merit exploration as an alternative treatment for breast cancers when provided in combination with agents that alter inflammatory signalling pathways. A limitation of the commentary is that it has focused on data from studies using breast cancer cell lines such as the MCF-7 cells cited in the studies by the Perillo group 1 - 3. A limitation of all studies using tissue-culture adapted cell lines such as MCF-7 is that they do not reflect the genomic heterogeneity we now appreciate exists in different breast tumours 4, only a proportion of which are considered classically activated by oestrogens because they are ER alpha positive. In spite of these reservations the authors do raise some interesting points.

Firstly, they highlight the potential for inflammatory mediators such as those that activate the NFκB signalling pathway as targets for therapies in breast cancer treatment. One example provided is the data from the Perillo group, reporting that inhibition of IKKα kinase specific inhibitor BAY11-7082 might be beneficial in switching the effects of oestrogens from anti- to pro-apoptotic. These studies are in agreement with those from a number of other authors claiming that combination therapies are likely to be beneficial in treatment of this malignancy. Notably, there is an interesting paper by Biswas et al. published in 2004 5 which examines how NFκB activation in breast cancer specimens might have a role in cell proliferation and apoptosis. Notably, in the Biswas paper, they report results suggesting that activated NFκB is predominantly detected in ER negative rather than ER positive breast tumours. This paper, as well as the recent genomic proflling of breast cancers from 2000 patients 4 highlights the importance of considering the both the immediate microenvironment of the tumour as well as the adjacent non-tumour tissue. Notably, in considering how oestrogen therapy might be utilised, it is important to take into account the hormonal status of the woman (pre or post menopausal) as well as the compelling data that has demonstrated intra-tissue oestrogen biosynthesis in fat and other cells close to the malignant epithelial cells in situ 6. A paper that has just appeared in the Journal of Clinical Endocrinology and Metabolism by Savolainen-Peltonen and colleagues 7 is particularly relevant. In this paper, the authors compared the metabolic pathways producing active oestradiol in breast subcutaneous adipose tissue of postmenopausal women with and without cancer, and these showed there were differences depending on whether the adipose tissue was proximal or distal to a tumour. If the tumour is already bathed in high concentrations of oestrogen either from the periphery or local biosynthesis then addition of more ligand is unlikely to be effective.

The local inflammatory environment of the tumour is an important factor in tumour progression and it is influenced by activation of NFκB activity leading to production of inflammatory mediators such as TNFα, IL-1β and prostaglandin E2 from adipocyteassociated macrophages and COX-2 inhibitors have been cited as effective in reducing recurrence of cancer 8. Local increases in inflammatory mediators in turn stimulate adipocyte aromatase expression/activity and hence oestradiol production 9 that may be particularly relevant to obese women 9 , 10. Thus, when considering suppression of NFκB activity in combination therapy there may be many context-dependent impacts on the tumour microenvironment to consider.

The role of NFκB activation as a regulator of inflammatory signalling is particularly important to consider in the context of endocrine resistant breast cancer which cannot be addressed using MCF-7 cells. A new paper in Molecular Cellular Endocrinology by Litchfield et al.  11 notes that regulation of endocrine-resistant breast cancer cells may also be mediated by other transcription factors such as COUPTFII. So the keys questions to be addressed in considering the value of the future prospective advanced within this Opinion piece are the following:

  1. Are studies in isolated cell lines such as the one described really relevant when it comes to translating new therapies into practice?

  2. Is inhibition of inflammatory mediators such as that using the BAY compound really most effective in cells that are not actually estrogen responsive?This merits further investigation.

  3. Finally, how can we move forward with the field?

 

In our opinion, an excellent way to advance these studies would be to conduct further investigations using methods which better reflect the microenvironment of an intact tumour in situ, this should include studies on the interplay between malignant epithelial cells, fat and vascular and inflammatory cells as this likely to influence response to therapy and it cannot be under-estimated that the individual responses to oestrogens by these different cell types may vary considerably. Examples of such studies currently include 3 being explored in the context of breast cancer, including organoids, tissue slices or xenografts. These approaches are more likely than studies in MCF-7 cells to inform choice of new combination therapies for testing in clinical trials; oestrogen combined with NFκB inhibition should be tested in these systems 12 - 14.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References

  • 1.Perillo B, Di Santi A, Cernera G, Ombra MN, Castoria G, Migliaccio A: Phosphorylation of H3 serine 10 by IKKα governs cyclical production of ROS in estrogen-induced transcription and ensures DNA wholeness. Cell Death Differ.2014;21(9) : 1611503-1503 10.1038/cdd.2014.91Reference source [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Perillo B, Di Santi A, Cernera G, Ombra MN, Castoria G, Migliaccio A: Nuclear receptor-induced transcription is driven by spatially and timely restricted waves of ROS: The role of Akt, IKKα, and DNA damage repair enzymes. Nucleus.2014;5(5) : 161 10.4161/nucl.36274Reference source [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Perillo B, Ombra MN, Bertoni A, Cuozzo C, Sacchetti S, Sasso A, Chiariotti L, Malorni A, Abbondanza C, Avvedimento EV: DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression. Science.2008;319(5860) : 161202-206 10.1126/science.1147674Reference source [DOI] [PubMed] [Google Scholar]
  • 4.Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Graf S, Ha G, Haffari G, Bashashati A, Russell R, McKinney S, METABRIC Group: The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature.2012;486(7403) : 161346-352 10.1038/nature10983Reference source [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Biswas DK, Shi Q, Baily S, Strickland I, Ghosh S, Pardee AB, Iglehart JD: NF-kappa B activation in human breast cancer specimens and its role in cell proliferation and apoptosis. Proc Natl Acad Sci U S A.2004;101(27) : 16110137-10142 10.1073/pnas.0403621101Reference source [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Simpson HW, Candlish W, Pauson AW, McArdle CS, Griffiths K, Small RG: Genesis of breast cancer is in the premenopause. Lancet.1988;2(8602) :74-76Reference source [DOI] [PubMed] [Google Scholar]
  • 7.Savolainen-Peltonen H, Vihma V, Leidenius M, Wang F, Turpeinen U, Hamalainen E, Tikkanen MJ, Mikkola TS: Breast Adipose Tissue Estrogen Metabolism in Postmenopausal Women with or without Breast Cancer. J Clin Endocrinol Metab.2014; Reference source [DOI] [PubMed] [Google Scholar]
  • 8.Bowers LW, Maximo IX, Brenner AJ, Beeram M, Hursting SD, Price RS, Tekmal RR, Jolly CA, deGraffenried LA: NSAID Use Reduces Breast Cancer Recurrence in Overweight and Obese Women: Role of Prostaglandin-Aromatase Interactions. Cancer Res.2014;74(16) : 1614446-4457 10.1158/0008-5472.CAN-13-3603Reference source [DOI] [PubMed] [Google Scholar]
  • 9.Subbaramaiah K, Hudis C, Chang SH, Hla T, Dannenberg AJ: EP2 and EP4 receptors regulate aromatase expression in human adipocytes and breast cancer cells. Evidence of a BRCA1 and p300 exchange. J Biol Chem.2007;283(6) : 1613433-3444 10.1074/jbc.M705409200Reference source [DOI] [PubMed] [Google Scholar]
  • 10.Subbaramaiah K, Howe LR, Port ER, Brogi E, Fishman J, Liu CH, Hla T, Hudis C, Dannenberg AJ: HER-2/neu status is a determinant of mammary aromatase activity in vivo: evidence for a cyclooxygenase-2-dependent mechanism. Cancer Res.2006;66(10) : 1615504-5511 10.1158/0008-5472.CAN-05-4076Reference source [DOI] [PubMed] [Google Scholar]
  • 11.Litchfield LM, Appana SN, Datta S, Klinge CM: COUP-TFII inhibits NFkappaB activation in endocrine-resistant breast cancer cells. Mol Cell Endocrinol.2014;382(1) : 161358-367 10.1016/j.mce.2013.10.010Reference source [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Walsh AJ, Cook RS, Sanders ME, Aurisicchio L, Ciliberto G, Arteaga CL, Skala MC: Quantitative optical imaging of primary tumor organoid metabolism predicts drug response in breast cancer. Cancer Res.2014;74(18) : 1615184-5194 10.1158/0008-5472.CAN-14-0663Reference source [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Sun X, Mao Y, Wang J, Zu L, Hao M, Cheng G, Qu Q, Cui D, Keller ET, Chen X, Shen K, Wang J: IL-6 secreted by cancer-associated fibroblasts induces tamoxifen resistance in luminal breast cancer. Oncogene.2014;33(35) : 1614450. 10.1038/onc.2014.224Reference source [DOI] [PubMed] [Google Scholar]
  • 14.Holliday DL, Moss MA, Pollock S, Lane S, Shaaban AM, Millican-Slater R, Nash C, Hanby AM, Speirs V: The practicalities of using tissue slices as preclinical organotypic breast cancer models. J Clin Pathol.2013;66(3) :253-255Reference source [DOI] [PubMed] [Google Scholar]
F1000Res. 2014 Aug 11. doi: 10.5256/f1000research.5075.r5459

Referee response for version 1

Steven L Young 1

In this opinion manuscript, Zhou and Zhu highlight recent research that suggests estrogen can act on opposing signaling pathways to either promote proliferation or cell death in breast cancer cells and, further, that the signaling can be biased toward cell death by inhibition of IKKa action. The manuscript is thought provoking and timely and the argument is logical and generally well-constructed. The title is eye-catching and appropriate. The figure is attractive and highlights the key point of the manuscript. If the areas of concern (below) are addressed, the conclusions will be justified by the argument presented.

I have four areas of concern:

  1. The manuscript is marred by frequent grammatical and English language usage errors as well as occasional typographic errors. While readable, it is sometimes difficult to determine the precise meaning of the authors’ statements. 

  2. In the introduction, the authors state that estrogen, in combination with IKKa, can induce breast cancer apoptosis. However, it is IKKa inhibition that is used, suggesting that it is the absence of IKKa effect that is important - this should be clarified as currently the penultimate sentence of the introduction paragraph appears to be contradicted by the sentence following.

  3. The authors propose that IKKa inhibition has great promise for clinical use, but fail to remark on factors that would be important for feasibility of such therapy (see below):

     
    1. If IKKa were inhibited systemically, would apoptosis be largely limited to the cancer cells? What about other estrogen target tissues?
    2. Are there known toxicities of BAY or other IKKa inhibitors that would need to be considered? 
    3.  Are the concentrations of estradiol and IKKa inhibitor necessary to induce cell death achievable in humans?
    4. Are the concentrations of estradiol required for induction of cell death in the normal pre-menopausal range, or are these pharmacological levels: important implications for treatment of women with functioning ovaries and for the thrombogenic side effects of estrogen therapy, which are dose-related.

4. The abstract does not effectively summarize the manuscript.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Articles from F1000Research are provided here courtesy of F1000 Research Ltd

RESOURCES