[Rich66]

When employing estrogen against resistant ER+ BC, Glutathione inhibition is highly synergistic:


Medscape
Buthionine Sulfoximine Sensitizes Antihormone-Resistant Human Breast Cancer Cells to Estrogen-Induced Apoptosis


Joan S. Lewis-Wambi; Helen R. Kim; Chris Wambi; Roshani Patel; Jennifer R. Pyle; Andres J. Klein-Szanto; V. Craig Jordan Authors and Disclosures
Posted: 03/09/2009; Breast Cancer Research. 2008;10(6) © 2008 BioMed Central, Ltd.


FULL multi-section online article

Free PDF of original study

Abstract

Introduction: Estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis.
Methods Estrogen deprived MCF-7:2A cells were treated with 1 nM 17β-estradiol (E₂), 100 µM BSO, or 1 nM E₂ + 100 µM BSO combination in vitro, and the effects of these agents on cell growth and apoptosis were evaluated by DNA quantitation assay and annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. The in vitro results of the MCF-7:2A cell line were further confirmed in vivo in a mouse xenograft model.
Results: Exposure of MCF-7:2A cells to 1 nM E₂ plus 100 µM BSO combination for 48 to 96 h produced a sevenfold increase in apoptosis whereas the individual treatments had no significant effect on growth. Induction of apoptosis by the combination treatment of E₂ plus BSO was evidenced by changes in Bcl-2 and Bax expression. The combination treatment also markedly increased phosphorylated c-Jun N-terminal kinase (JNK) levels in MCF-7:2A cells and blockade of the JNK pathway attenuated the apoptotic effect of E₂ plus BSO. Our in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of BSO either as a single agent or in combination with E₂ significantly reduced tumor growth of MCF-7:2A cells.
Conclusions: Our data indicates that GSH participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to E₂-induced apoptotic cell death. We suggest that these data may form the basis of improving therapeutic strategies for the treatment of antihormone resistant ER-positive breast cancer.





Bisphosphonates suppress insulin-like growth factor 1-induced angiogenesis via the HIF-1α/VEGF signaling pathways in human breast cancer cells
International Journal of Cancer, 08/12/09
Tang X et al. - In a trial to investigate potential molecular mechanisms underlying the antiangiogenic effect of non-nitrogen-containing and nitrogen-containing bisphosphonates, clodronate and pamidronate, respectively, in insulin-like growth factor (IGF)-1 responsive human breast cancer cells, it was demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF-1-stimulated MCF-7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in inhibition of tumor angiogenesis in breast cancer cells.
Methods

• It was tested whether bisphosphonates had any effects on hypoxia-inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis.

Results

• Both pamidronate and clodronate significantly suppressed IGF-1-induced HIF-1α protein accumulation and VEGF expression in MCF-7 cells.
• Mechanistically, either pamidronate or clodronate did not affect mRNA expression of HIF-1α, but they apparently promoted the degradation of IGF-1-induced HIF-1α protein.
• The presence of pamidronate and clodronate led to a dose-dependent decease in the newly-synthesized HIF-1α protein induced by IGF-1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting inhibition of protein synthesis.
• The inhibitory effects of bisphosphonates on the HIF-1α/VEGF axis are associated with inhibition of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathways.

Stopping Treatment Can Reverse Acquired Resistance to Letrozole

Gauri J. Sabnis¹, Luciana F. Macedo¹, Olga Goloubeva², Adam Schayowitz¹ and Angela M.H. Brodie^1,2
1 Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine and ² University of Maryland Greenebaum Cancer Center, Baltimore, Maryland
Requests for reprints: Angela M.H. Brodie, Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Health Science Facility I, Room 580G, 685 West Baltimore Street, Baltimore, MD 21201. Phone: 410-706-3137; Fax: 410-706-0032; E-mail: abrodie@umaryland.edu .

Using the intratumoral aromatase xenograft model, we have observed that despite long-lasting growth inhibition, tumors eventually begin to grow during continued letrozole treatment. In cells isolated from these long-term letrozole-treated tumors (LTLT-Ca), estrogen receptor- (ER) levels were decreased, whereas signaling proteins in the mitogen-activated protein kinase cascade were up-regulated along with human epidermal growth factor receptor 2 (Her-2). In the current study, we evaluated the effect of discontinuing letrozole treatment on the growth of letrozole-resistant cells and tumors. The cells formed tumors equally well in the absence or presence of letrozole and had similar growth rates. After treatment was discontinued for 6 weeks, letrozole was administered again. Marked tumor regression was observed with this second course of letrozole treatment. Similarly, in MCF-7Ca xenografts, a 6-week break in letrozole treatment prolonged the responsiveness of the tumors to letrozole. To understand the mechanisms of this effect, LTLT-Ca cells were cultured in the absence of letrozole for 16 weeks. The resulting cell line (RLT-Ca) exhibited properties similar to MCF-7Ca cells. The cell growth was inhibited by letrozole and stimulated by estradiol. The expression of phosphorylated mitogen-activated protein kinase (MAPK) was reduced and ER and aromatase levels increased compared with LTLT-Ca cells and were similar to levels in MCF-7Ca cells. These results indicate that discontinuing treatment can reverse letrozole resistance. This could be a beneficial strategy to prolong responsiveness to aromatase inhibitors for patients with breast cancer. [Cancer Res 2008;68(12):4518–24]



FASEB J. 2010 Feb 12. [Epub ahead of print]
BCL2 and CASP8 regulation by NF-{kappa}B differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells.

Nehra R, Riggins RB, Shajahan AN, Zwart A, Crawford AC, Clarke R.
*Department of Oncology,Lombardi Comprehensive Cancer Center, andDepartment of Physiology and Biophysics, Georgetown University School of Medicine, Washington, DC, USA.
Resistance to endocrine therapies remains a major problem in the management of estrogen receptor-alpha (ER)-positive breast cancer. We show that inhibition of NF-kappaB (p65/RELA), either by overexpression of a mutant IkappaB (IkappaBSR) or a small-molecule inhibitor of NF-kappaB (parthenolide; IC50=500 nM in tamoxifen-resistant cells), synergistically restores sensitivity to 4-hydroxytamoxifen (4HT) in resistant MCF7/RR and MCF7/LCC9 cells and further sensitizes MCF-7 and MCF7/LCC1 control cells to 4HT. These effects are independent of changes in either cell cycle distribution or in the level of autophagy measured by inhibition of p62/SQSTM1 expression and cleavage of LC3. NF-kappaB inhibition restores the ability of 4HT to decrease BCL2 expression, increase mitochondrial membrane permeability, and induce a caspase-dependent apoptotic cell death in resistant cells. Each of these effects is reversed by a caspase 8 (CASP8)-specific inhibitor that blocks enzyme-substrate binding. Thus, increased activation of NF-kappaB can alter sensitivity to tamoxifen by modulating CASP8 activity, with consequent effects on BCL2 expression, mitochondrial function, and apoptosis. These data provide significant new insights into how molecular signaling affects antiestrogen responsiveness and strongly suggest that a combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER-positive breast cancers.-Nehra, R., Riggins, R. B., Shajahan, A. N., Zwart, A., Crawford, A. C., Clarke, R. BCL2 and CASP8 regulation by NF-kappaB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells.

PMID: 20154269 [PubMed - as supplied by publisher]





Mol Cancer Ther. 2008 Jul;7(7):2096-108.
Apigenin inhibits antiestrogen-resistant breast cancer cell growth through estrogen receptor-alpha-dependent and estrogen receptor-alpha-independent mechanisms.

Long X, Fan M, Bigsby RM, Nephew KP.
Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, IN 47405-4401, USA.
Breast cancer resistance to the antiestrogens tamoxifen (OHT) and fulvestrant is accompanied by alterations in both estrogen-dependent and estrogen-independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either OHT or fulvestrant. We found that apigenin can function as both an estrogen and an antiestrogen in a dose-dependent manner. At low concentrations (1 mumol/L), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (>10 mumol/L), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either OHT or fulvestrant showed synergistic, growth-inhibitory effects on both antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or an antiestrogen, effects of the drug on estrogen receptor-alpha (ERalpha); transactivation activity, mobility, stability, and ERalpha-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERalpha and its coactivator amplified in breast cancer-1. However, higher doses (>10 mumol/L) of apigenin inhibited ERalpha mobility (as determined by fluorescence recovery after photobleaching assays), down-regulated ERalpha and amplified in breast cancer-1 expression levels, and inhibited multiple protein kinases, including p38, protein kinase A, mitogen-activated protein kinase, and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and a protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERalpha-dependent and ERalpha-independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer.

PMID: 18645020 [PubMed - indexed for MEDLINE]




Clin Ther. 2009;31P2:2371-2378.
High-dose estrogen as salvage hormonal therapy for highly refractory metastatic breast cancer: A retrospective chart review.

Mahtani RL, Stein A, Vogel CL.
Boca Raton Comprehensive Cancer Center, Boca Raton, Florida.
Background: High-dose estrogens (HDEs) are an efficacious but widely overlooked treatment option for patients with metastatic breast cancer (MBC). This is due in part to the introduction of tamoxifen in the 1970s, which was proven to be equivalent in efficacy and associated with fewer adverse events (AEs). Objective: The aim of this study was to report our experience with the use of HDE in postmenopausal women with advanced breast cancer. Methods: Local institutional review board approval was obtained to conduct a retrospective chart review of patients with MBC treated with HDEs at the Boca Raton Comprehensive Cancer Center, Boca Raton, Florida, from 2001 through March 2009. Demographic information, response rates, and tolerability profiles were collected. Results: Of the 426 patients with MBC identified, we found 26 patients with MBC who were prescribed HDEs as a treatment in any line of therapy for advanced breast cancer. The median age at the start of HDE therapy was 59 years (range, 42-92 years). Three of the 26 patients (11.5%) were human epidermal growth factor receptor 2-positive determined via fluorescent in situ hybridization analysis. With the exception of 1 patient who had received no prior systemic treatment for metastatic disease, all patients received multiple lines of treatment (both chemotherapy and hormonal treatments) in the advanced setting (median, 7 lines; range, 0-12) prior to the initiation of HDE. Five of 20 patients (25%) with measurable metastatic disease (visceral and/or soft tissue metastases) had objective antitumor responses defined as either a partial response (PR) or a complete response (CR). Four additional patients (20%) had prolonged stable disease (SD) for >/=6 months. Three of 6 patients (50%) with nonmeasurable metastatic disease (bone-only) had prolonged SD for >/=6 months. Clinical benefit rate (defined as CR + PR + SD >/=6 months) for all patients was 46% (12/26), with a median duration of 10 months. Overall median progression-free survival for the 26 subjects was 5 months. Median survival from the start of HDE was 17 months (range, 3-54 months). AEs included fluid retention (8 [31%]), vaginal bleeding (7 [27%]), and nausea (4 [15%]). Two patients discontinued therapy after 1 month. Three of the remaining 24 patients discontinued estrogen therapy due to AEs.

Conclusions: This retrospective chart review details our facility's experience with the use of HDE in patients with advanced breast cancer, most of whom had received multiple prior treatments. Our data suggest that this treatment is another option for heavilytreated patients in whom further endocrine manipulation might still be appropriate. Copyright © 2009 Excerpta Medica Inc. All rights reserved.

PMID: 20110046 [PubMed - as supplied by publisher]




JAMA. 2009 Aug 19;302(7):774-80.
Lower-dose vs high-dose oral estradiol therapy of hormone receptor-positive, aromatase inhibitor-resistant advanced breast cancer: a phase 2 randomized study.

Ellis MJ, Gao F, Dehdashti F, Jeffe DB, Marcom PK, Carey LA, Dickler MN, Silverman P, Fleming GF, Kommareddy A, Jamalabadi-Majidi S, Crowder R, Siegel BA.
Department of Medicine, Division of Oncology, Washington University School of Medicine, St Louis, MO 63110, USA. mellis@dom.wustl.edu
Comment in:

• JAMA. 2009 Aug 19;302(7):797-8.

CONTEXT: Estrogen deprivation therapy with aromatase inhibitors has been hypothesized to paradoxically sensitize hormone-receptor-positive breast cancer tumor cells to low-dose estradiol therapy. OBJECTIVE: To determine whether 6 mg of estradiol (daily) is a viable therapy for postmenopausal women with advanced aromatase inhibitor-resistant hormone receptor-positive breast cancer. DESIGN, SETTING, AND PATIENTS: A phase 2 randomized trial of 6 mg vs 30 mg of oral estradiol used daily (April 2004-February 2008 [enrollment closed]). Eligible patients (66 randomized) had metastatic breast cancer treated with an aromatase inhibitor with progression-free survival (> or = 24 wk) or relapse (after > or = 2 y) of adjuvant aromatase inhibitor use. Patients at high risk of estradiol-related adverse events were excluded. Patients were examined after 1 and 2 weeks for clinical and laboratory toxicities and flare reactions and thereafter every 4 weeks. Tumor radiological assessment occurred every 12 weeks. At least 1 measurable lesion or 4 measurable lesions (bone-only disease) were evaluated for tumor response. INTERVENTION: Randomization to receive 1 oral 2-mg generic estradiol tablet 3 times daily or five 2-mg tablets 3 times daily. MAIN OUTCOME MEASURES: Primary end point: clinical benefit rate (response plus stable disease at 24 weeks). Secondary outcomes: toxicity, progression-free survival, time to treatment failure, quality of life, and the predictive properties of the metabolic flare reaction detected by positron emission tomography/computed tomography with fluorodeoxyglucose F 18. RESULTS: The adverse event rate (> or = grade 3) in the 30-mg group (11/32 [34%]; 95% confidence interval [CI], 23%-47%) was higher than in the 6-mg group (4/34 [18%]; 95% CI, 5%-22%; P = .03). Clinical benefit rates were 9 of 32 (28%; 95% CI, 18%-41%) in the 30-mg group and 10 of 34 (29%; 95% CI, 19%-42%) in the 6-mg group. An estradiol-stimulated increase in fluorodeoxyglucose F 18 uptake (> or = 12% prospectively defined) was predictive of response (positive predictive value, 80%; 95% CI, 61%-92%). Seven patients with estradiol-sensitive disease were re-treated with aromatase inhibitors at estradiol progression, among which 2 had partial response and 1 had stable disease, suggesting resensitization to estrogen deprivation. CONCLUSIONS: In women with advanced breast cancer and acquired resistance to aromatase inhibitors, a daily dose of 6 mg of estradiol provided a similar clinical benefit rate as 30 mg, with fewer serious adverse events. The efficacy of treatment with the lower dose should be further examined in phase 3 clinical trials. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00324259.

PMID: 19690310 [PubMed - indexed for MEDLINE]








J Mammary Gland Biol Neoplasia. 2010 Jan 27. [Epub ahead of print]
Epigenetic Regulation in Estrogen Receptor Positive Breast Cancer-Role in Treatment Response.

Pathiraja TN, Stearns V, Oesterreich S.
Translational Biology and Molecular Medicine Graduate Program, Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA.
Recent advances in breast cancer treatment have allowed increasing numbers of patients with estrogen receptor (ER) positive (+) breast cancer to receive various forms of endocrine therapy. Unfortunately, de novo and acquired resistance to endocrine therapy remains a major challenge in the clinic. A number of possible mechanisms for drug resistance have been described, which include activation of growth factor receptor pathways, overexpression of ER coactivators, and metabolic resistance due to polymorphisms in metabolizing enzymes. While many of these changes are caused by genetic alterations, there is also increasing evidence to implicate epigenetic gene regulatory mechanisms in the development of endocrine resistance. Since epigenetic modifications are easier to reverse than genetic mutations, they are appealing therapeutic targets, and thus future improvements in medical care for breast cancer patients will depend upon a better understanding of the roles epigenetic modifications play in endocrine resistance. In this review we will focus on recent advances made in the understanding of epigenetic gene regulation in estrogen response and endocrine resistance in breast cancer. We will also summarize current clinical-translational advances in epigenetic therapy, and discuss potential future clinical use of epigenetic changes as therapeutic targets, especially with respect to endocrine treatment.

PMID: 20101445 [PubMed - as supplied by publisher]



Int J Cancer. 2010 Jan 15;126(2):545-62.
Endocrine resistance associated with activated ErbB system in breast cancer cells is reversed by inhibiting MAPK or PI3K/Akt signaling pathways.

Ghayad SE, Vendrell JA, Larbi SB, Dumontet C, Bieche I, Cohen PA.
Université de Lyon, France.
Endocrine therapy resistance is one of the main challenges in the treatment of estrogen receptor positive (ER+) breast cancer patients. This study showed that two ER+ human breast carcinoma cell lines derived from MCF-7 (MVLN cells) that have acquired under OH-Tamoxifen selection two distinct phenotypes of endocrine resistance both displayed constitutive activation of the PI3K/Akt and MAPK pathways. Aberrant expression and activation of the ErbB system (phospho-EGFR, phospho-ErbB2, phospho-ErbB3, over-expression of ErbB4 and over-expression of several ErbB ligands) were also observed in the two resistant cell lines, suggesting the existence of an autocrine loop leading to constitutive activation of MAPK and PI3K/Akt survival pathways. The recent clinical use of specific signal transduction inhibitors is one of the most promising therapeutic approaches in breast cancers. The MEK inhibitor PD98059 and the PI3K inhibitor LY294002 were both able to enhance the cytostatic effect of OH-Tamoxifen or fulvestrant on MVLN sensitive cells. In the two resistant cell lines, inhibition of the MAPK or the PI3K/Akt pathways associated with endocrine therapy was sufficient to reverse OH-Tamoxifen or fulvestrant resistance. Investigating the effect of a combination of both inhibitors on the reversion of OH-Tamoxifen and fulvestrant resistance in the two resistant cell lines suggested that, in clinical practice, a strategy combining the two inhibitors would be the best approach to target the different endocrine resistance phenotypes possibly present in a tumor. In conclusion, the combination of MAPK and PI3K inhibitors represents a promising strategy to overcome endocrine therapy resistance in ER+ breast cancer patients.

PMID: 19609946 [PubMed - indexed for MEDLINE]

1: Eur J Gynaecol Oncol. 2001;22(5):347-9.Links

Herbal complex suppresses telomerase activity in chemo-endocrine resistant cancer cell lines.

Lian Z, Fujimoto J, Yokoyama Y, Niwa K, Tamaya T.
Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan.
A herbal complex consisting of Hoelen, Angelicae radix, Scutellariae radix and Glycyrrhizae radix suppressed cell viability and telomerase activity in hormone-refractory and chemo-resistant cancer cell lines, namely poorly differentiated uterine endometrial cancer cell line AN3 CA, adriamycin-resistant breast cancer cell line MCF7/ADR and cisplatin-resistant ovarian cancer cell line A2780. Furthermore, the herbal complex suppressed the expression of the full length of human telomerase reverse transcriptase (hTERT), which is related to telomerase activity. This indicates that the herbal complex can suppress the tumor growth of chemoendocrine resistant cancers, at least in part via suppression of telomerase activity associated with down-regulated hTERT.
PMID: 11766737

Surprise!

Herbal mix has patent: http://www.freepatentsonline.com/7527812.html

Int J Cancer. 2008 Apr 1;122(7):1506-11.
Induction of acquired resistance to antiestrogen by reversible mitochondrial DNA depletion in breast cancer cell line.

Naito A, Carcel-Trullols J, Xie CH, Evans TT, Mizumachi T, Higuchi M.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, USA.


FREE TEXT

Abstract

Although the net benefits of tamoxifen in adjuvant breast cancer therapy have been proven, the recurrence of the cancer in an aggressive and hormone independent form has been highly problematic. We previously demonstrated the important role mitochondrial DNA (mtDNA) plays in hormone-independence in prostate cancer. Here, the role of mtDNA in breast cancer progression was investigated. We established hydroxytamoxifen (4-OHT) resistant HTRMCF by growing MCF-7, human breast adenocarcinoma cells, in the presence of 4-OHT. HTRMCF was cross-resistant to 4-OHT and ICI182,780 concurrent with the depletion of mtDNA. To further investigate the role of mtDNA depletion, MCF-7 was depleted of mtDNA by treatment with ethidium bromide. MCF Rho 0 was resistant to both 4-OHT and ICI182,780. Furthermore, cybrid (MCFcyb) prepared by fusion MCF Rho 0 with platelet to transfer mtDNA showed susceptibility to antiestrogen. Surprisingly, after withdrawal of 4-OHT for 8 weeks, HTRMCF and their clones became susceptible to both drugs concurrent with a recovery of mtDNA. Herein, our results substantiated the first evidence that the depletion of mtDNA induced by hormone therapy triggers a shift to acquired resistance to hormone therapy in breast cancer. In addition, we showed that mtDNA depletion can be reversed, rendering the cancer cells susceptible to antiestrogen. The fact that the hormone independent phenotype can be reversed should be a step toward more effective treatments for estrogen-responsive breast cancer. (c) 2007 Wiley-Liss, Inc.

PMID: 17990320 [PubMed - indexed for MEDLINE]PMCID: PMC2293290Free PMC Article