Estrogen-responsive Breast Cancer Cell Research Articles

Estrogen-induced reactive oxygen species, through epigenetic reprogramming, causes increased growth in breast cancer cells

Despite the progress made in cancer diagnosis and treatment, breast cancer remains the second leading cause of cancer-related death among the women. Exposure to elevated levels of endogenous estrogen or environmental estrogenic chemicals is an important risk factor for breast cancer. Estrogen metabolites and ROS generated during estrogen metabolism are known to play a critical role in estrogen carcinogenesis. However, the molecular mechanisms through which estrogen-induced ROS regulate gene expression is not clear. Epigenetic changes of DNA methylation and histone modifications are known to regulate genes expression. Therefore, the objective of this study was to evaluate whether estrogen-induced ROS, through aberrant expression of epigenetic regulatory genes and epigenetic reprogramming, causes growth of breast cancer cells. Estrogen responsive MCF-7 and T47D human breast cancer cells were exposed to natural estrogen 17 beta-estradiol (E2) and synthetic estrogen Diethylstilbestrol (DES) both alone and in combination with antioxidant N-acetyl cysteine. Effects of NAC-mediated scavenging of estrogen-induced ROS on cell growth, gene expression, and histone modifications were measured. The result of MTT and cell cycle analysis revealed significant abrogation of E2 and DES-induced growth by scavenging ROS through NAC. E2 and DES caused significant changes in expression of epigenetic regulatory genes for DNA methylation and histone modifications as well as changes in both gene activating and repressive marks in the Histone H3. NAC restored the expression of epigenetic regulatory genes and changes in histone marks. Novel findings of this study suggest that estrogen can induce growth of breast cancer cells through ROS-dependent regulation of epigenetic regulatory genes and epigenetic reprogramming of histone marks.

Chemical constituents from Neopestalotiopsis clavispora culture medium with estrogenic effects in MCF-7 cells

Estrogen is a vital hormone responsible for the development of the female reproductive system. Hence, estrogen deficiency in menopause increases various symptoms. This study aimed to discover biologically active compounds from an EtOAc-extract of Neopestalotiopsis clavispora culture medium which has previously shown estrogenic activity. A new α-pyrone (1) and a new tetramic acid derivative (2), together with hymenosetin (3) and pestalotiollide B (4), were isolated from the extract. The chemical structures of the new compounds were elucidated using MS and NMR spectroscopy, and the absolute configurations were established by quantum mechanical calculations of electronic circular dichroism and gauge-including atomic orbital NMR chemical shift, followed by DP4 + analysis. The isolated compounds were initially tested for their estrogenic activities using MCF-7 estrogen responsive human breast cancer cells. Hymenosetin (3) showed estrogenic activity by increasing the proliferation of MCF-7 cells at the concentration of 2.5 μM via the phosphorylation of estrogen receptor-α.

A new α-pyrone from Arthrinium pseudosinense culture medium and its estrogenic activity in MCF-7 cells.

A new α-pyrone analog, arthrifuranone A (1) was isolated from an EtOAc-extract of Arthrinium pseudosinense culture medium. The isolation workflow was guided by a Molecular Networking-based dereplication strategy. The chemical structure of the new compound was elucidated using MS and NMR spectroscopic techniques, and the absolute configuration was established by the Mosher's method and gauge-including atomic orbital NMR chemical shift calculations, followed by DP4 + analysis. The isolated compound was evaluated for its estrogenic activity using the MCF-7 estrogen responsive human breast cancer cells. Compound 1 showed estrogenic activity by increasing the proliferation of MCF-7 cells at the concentration of 3.125 μM via phosphorylation of estrogen receptor-α.

Anti-Cancer Effect of Melatonin via Downregulation of Delta-like Ligand 4 in Estrogen-Responsive Breast Cancer Cells.

The Notch signaling pathway has a key role in angiogenesis and Delta - Like Ligand 4 (DLL4) is one of the main ligands of Notch involved in cell proliferation in sprouting vessels. In this study, we aimed to evaluate the expression of DLL4 in primary breast tumors and to examine the effect of melatonin on DLL4 expression in vitro. Eighty-five breast tumor and paired adjacent non-tumor tissue samples were collected. Apoptosis assay was performed on breast cancer cells to evaluate melatonin effects. Western blot and quantitative RT-PCR were used to measure DLL4 expression. Then, we investigated the effect of melatonin on the expression of DLL4 in four breast cancer cell lines at RNA and protein levels. We also performed a probabilistic neural network analysis to study genes closely associated with DLL4 expression. Our results showed a significantly higher expression of DLL4 in tumor tissues compared to non-tumor tissues (P = 0.027). Melatonin treatment substantially attenuated DLL4 expression in BT474 and MCF-7 cells, but not in SK-BR-3 and MDA-MB-231 cells. Also, melatonin induced apoptosis in all four cell lines. Network analysis revealed a set of 15 genes that had close association and interaction with DLL4. DLL4 was overexpressed in breast cancer tissues as compared to the non-tumor tissues. It can be concluded that melatonin treatment attenuated DLL4 expression only in estrogen- responsive breast cancer cells and is able to induce apoptosis in breast cancer cells.

Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance

Estrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy.

Characterization of basal and estrogen-regulated antisense transcription in breast cancer cells: Role in regulating sense transcription

Estrogen-responsive breast cancer cells exhibit both basal and estrogen-regulated transcriptional programs, which lead to the transcription of many different transcription units (i.e., genes), including those that produce coding and non-coding sense (e.g., mRNA, lncRNA) and antisense (i.e., asRNA) transcripts. We have previously characterized the global basal and estrogen-regulated transcriptomes in estrogen receptor alpha (ERα)-positive MCF-7 breast cancer cells. Herein, we have mined genomic data to define three classes of antisense transcription in MCF-7 cells based on where their antisense transcription termination sites reside relative to their cognate sense mRNA and lncRNA genes. These three classes differ in their response to estrogen treatment, the enrichment of a number of genomic features associated with active promoters (H3K4me3, RNA polymerase II, open chromatin architecture), and the biological functions of their cognate sense genes as analyzed by DAVID gene ontology. We further characterized two estrogen-regulated antisense transcripts arising from the MYC gene in MCF-7 cells, showing that these antisense transcripts are 5′-capped, 3′-polyadenylated, and localized to different compartments of the cell. Together, our analyses have revealed distinct classes of antisense transcription correlated to different biological processes and response to estrogen stimulation, uncovering another layer of hormone-regulated gene regulation.

Design, synthesis, and anti-proliferative evaluation of 1H-1,2,3-triazole grafted tetrahydro-β-carboline-chalcone/ferrocenylchalcone conjugates in estrogen responsive and triple negative breast cancer cells

A series of 1H-1,2,3 triazole grafted tetrahydro-β-carboline-chalcone/ferrocenylchalcone conjugates were synthesized and in vitro evaluated against estrogen responsive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells.

Cycloartane and Stigmastane Type Triterpenoids from Pothos scandens Inhibit Estradiol (E2) Induced Proliferations in Breast Cancer Cells

Four cycloartane type triterpenoids and three stigmastane type steroids were isolated from the methanolic extract of stem and root part of Pothos scandens L. (Araceae), a Bangladeshi medicinal plant by high performance liquid chromatographic technique. The compounds were characterized as 24-methylenecycloartanol (1), 24-methylenecycloartenone (2), 24-en-cycloartenone (3), 24-methylenecycloartanyl ferulate (4), stigmast-4-en-3-one (5), stigmast-4,22-diene-3-one (6) and β-sitosterol glucoside (7) through extensive 1D and 2D NMR spectroscopic studies. All the isolates were evaluated for their estrogenic/antiestrogenic activity using the estrogen-responsive breast cancer cell lines, MCF-7 and T47D. The results showed that all the compounds possess mild to strong antiestrogenic activity in both cell lines which was compared to positive control tamoxifen. 24- Methylenecycloartanol (1), which contains a hydroxyl group in its C-3 position, inhibited 90% of estradiol (E2)- induced cell proliferation in MCF-7 and T47D cell lines at a concentration of 0.01 μM only. 24- Methylenecycloartanyl ferulate (4) and stigmast-4,22-diene-3-one (6) showed 90% of estradiol (E2)-induced cell proliferation in T47D cell only at a concentration of 0.01 μM whereas 10.0 μM was required for 24- methylenecycloartanyl ferulate (4) for the same activity in MCF-7 cells. This is the first report of isolation of these compounds from the plant along with their antiestrogenic property.
 Dhaka Univ. J. Pharm. Sci. 18(1): 93-102, 2019 (June)

Determination and analysis of agonist and antagonist potential of naturally occurring flavonoids for estrogen receptor (ER\u03b1) by various parameters and molecular modelling approach

Most estrogen receptor α (ERα) ligands target the ligand binding domain (LBD). Agonist 17β-estradiol (E2) and tamoxifen (TM, known SERM), bind to the same site within the LBD. However, structures of ligand-bound complexes show that E2 and TM induce different conformations of helix 12 (H12). During the molecular modelling studies of some naturally occurring flavonoids such as quercetin, luteolin, myricetin, kaempferol, naringin, hesperidin, galangin, baicalein and epicatechin with human ERα (3ERT and 1GWR), we observed that most of the ligands bound to the active site pocket of both 3ERT and 1GWR. The docking scores, interaction analyses, and conformation of H12 provided the data to support for the estrogenic or antiestrogenic potential of these flavonoids to a limited degree. Explicit molecular dynamics for 50 ns was performed to identify the stability and compatibility pattern of protein-ligand complex and RMSD were obtained. Baicalein, epicatechin, and kaempferol with 1GWR complex showed similar RMSD trend with minor deviations in the protein backbone RMSD against 1GWR-E2 complex that provided clear indications that ligands were stable throughout the explicit molecular simulations in the protein and outcome of naringin-3ERT complex had an upward trend but stable throughout the simulations and all molecular dynamics showed stability with less than overall 1 Å deviation throughout the simulations. To examine their estrogenic or antiestrogenic potential, we studied the effect of the flavonoids on viability, progesterone receptor expression and 3xERE/3XERRE-driven reporter gene expression in ERα positive and estrogen responsive MCF-7 breast cancer cells. Epicatechin, myricetin, and kaempferol showed estrogenic potential at 5 µM concentration.

Abstract 907: Targeting glycolysis enzyme, PFKFB3, in endocrine therapy resistant breast cancers

Abstract Reprogrammed glucose metabolism is recognized as one of the hallmarks of cancer. High energy demand in cancer cells leads to increased glycolysis to maintain anabolic processes that may be driven by altered enzyme levels. The second committed step in the glycolysis pathway is catalyzed by phosphofructokinase1 (PFK1), which converts fructose-6 phosphate (F6P) to fructose 1,6-bisphosphate (F1,6BP). Another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), converts F6P to fructose 2,6-bisphosphate (F2,6BP) that then functions as an allosteric activator of PFK1 and drives glycolysis. PFKFB3 enzyme is upregulated in many cancers including breast cancers. PFKFB3 is regulated by estrogen in ER-positive breast cancer cells but its role in endocrine-therapy resistant breast cancer cells is largely unknown. We investigated the expression of PFKFB3 mRNA and protein in estrogen-responsive MCF7 cells and compared it with LCC9 cells that are estrogen-independent and resistant to 4-hydroxytamoxifen (4OHT) and fulvestrant (Fulv). Twofold higher PFKFB3 mRNA and protein levels were expressed in LCC9 cells compared to MCF7 cells. In addition, the estrogen-mediated stimulation of PFKFB3 that was evident in MCF7 cells was not observed in LCC9 cells. We further studied the effect of pharmacological inhibition of PFKFB3, on the growth of therapy-resistant breast cancer cells using a compound PFK158, a potent inhibitor of the PFKFB3 enzyme. Treatment with PFK158 inhibited the growth of LCC9 cells as well as another cell line, MCF7:5C that is also resistant to 4OHT and partially resistant to Fulv. PFK158 alone was effective in suppressing the growth of both the cell lines at a concentration of 5 µM or higher. Notably, combining PFK158 with either 4OHT or Fulv significantly potentiated the effect of PFK158 treatment in blocking the growth of the cells. In MCF7:5C cells the combination treatment of 2.5 µM PFK158 and Fulv (500nM) or 4OHT(500nM) inhibited cell growth by ~70% as compared to ~30% inhibition in the presence of PFK 158 alone. In LCC9 cells >70% of growth was inhibited in the combination treatment as compared to no inhibition when PFK158, 2.5 µM or 4OHT or Fulv alone was used. In publicly available datasets of ER+, node-negative breast cancer patients, high expression of PFKFB3 was associated with adverse recurrence-free survival (hazard ratio = 4.12 and p= 5.5x10-5). Our study shows an increased basal expression of PFKFB3 mRNA and protein in estrogen-independent, endocrine-therapy resistant breast cancer cells as compared to estrogen-responsive breast cancer cells. Targeting PFKFB3 in combination with anti-estrogens should be explored for potential therapeutic intervention in endocrine therapy-resistant breast cancers. Citation Format: Surojeet Sengupta, Catherine M. Sevigny, Xiaoyi Liu, Lu Jin, Paula R. Pohlmann, Robert Clarke. Targeting glycolysis enzyme, PFKFB3, in endocrine therapy resistant breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 907.

GREB1 isoforms regulate proliferation independent of ERα co-regulator activities in breast cancer.

Activation of the transcription factor estrogen receptor α (ERα) and the subsequent regulation of estrogen-responsive genes play a crucial role in the development and progression of the majority of breast cancers. One gene target of ERα, growth regulation by estrogen in breast cancer 1 (GREB1), is associated with proliferation and regulation of ERα activity in estrogen-responsive breast cancer cells. The GREB1 gene encodes three distinct isoforms: GREB1a, GREB1b and GREB1c, whose molecular functions are largely unknown. Here, we investigate the role of these isoforms in regulation of ERα activity and proliferation. Interaction between GREB1 and ERα was mapped to the amino terminus shared by all GREB1 variants. Analysis of isoform-specific regulation of ERα activity suggests none of the GREB1 isoforms possess potent co-regulator activity. Exogenous expression of GREB1a resulted in elevated expression of some ER-target genes, independent of ERα activity. Despite this slight specificity of GREB1a for gene regulation, exogenous expression of either GREB1a or GREB1b resulted in decreased proliferation in both ER-positive and ER-negative breast carcinoma cell lines, demonstrating an ER-independent function of GREB1. Interestingly, we show an increase in the expression of GREB1b and GREB1c mRNA in malignant breast tissue compared to normal patient samples, suggesting a selective preference for these isoforms during malignant transformation. Together, these data suggest GREB1a has an isoform-specific function as a transcriptional regulator while all isoforms share an ER-independent activity that regulates proliferation.

Correlates of Breast Cancer and Indole-3-Carbinol: Medicinal Development and Strategies of Natural Products

Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor ER and ERß. Indole-3-carbinol (I3C) strongly down-regulated ER protein and transcript levels, without altering the level of ERß protein, in both cell lines. In cells transfected with the ER promoter linked to a luciferase gene reporter, I3C ablated ER promoter activity1-5. Dietary indole-3-carbinol prevents the development of estrogen-enhanced cancers including breast. Whereas estrogen increases the growth and survival of tumors, indole-3-carbinol causes growth arrest and increased apoptosis and ameliorates the effects of estrogen. In these findings best use indole-3-carbinol together with other nutrients (genistein) to achieve maximum benefits for cancer prevention. Investigator evaluated whether genistein, which is the major isoflavonoid in soy, would alter the ability of indole-3-carbinol/DIM to cause apoptosis and decrease expression driven by the estrogen receptor (ER)-alpha. Synergistic effect of indole-3-carbinol and genistein for induction of GADD expression, thus increasing apoptosis, and for decrease of expression driven by ER-alpha. Because of the synergistic effect of indole-3-carbinol and genistein, the potential exists for prophylactic or therapeutic efficacy of lower concentrations of each phytochemical when used in combination. I3C inhibits the enzyme elastase. High levels of elastase in breast cancer cells are suggestive of a poor prognosis because elastase shortens cyclin E, a cellular chemical involved in controlling the cell cycle, and the shortened version of cyclin E speeds up the cell cycle, therefore making cancer cells proliferate faster.I3C prevents the elastase shortening of cyclin E, thus halting the growth of breast cancer cells.

In vitro and in vivo antiproliferative activity of organo-nickel SCS-pincer complexes on estrogen responsive MCF7 and MC4L2 breast cancer cells. Effects of amine fragment substitutions on BSA binding and cytotoxicity.

A family of organonickel complexes has been prepared, fully characterized, and tested for their antiproliferative activity against estrogen-responsive human breast cancer cells (MCF7). The three SCS-type pincer ligands HL1, HL2, and HL3 and their corresponding Ni(ii) complexes NiL1, NiL2, and NiL3 have been synthesized and fully characterized, including by single crystal diffraction studies for the complexes. The complexes possess square planar geometry with two symmetrical 5-membered nickellacycles. Fluorescence spectroscopy, circular dichroism measurements, molecular modeling, colorimetric based assay and tumor transplantation studies were used to evaluate the protein binding and antiproliferative activities of these organometallic complexes both in vitro and in vivo. Fluorescence quenching was used to investigate bovine serum albumin (BSA) interaction at different temperatures (293, 303 and 313 K), and the results were analyzed using the classical Stern-Volmer equation, allowing us to propose a dynamic quenching mechanism. Studies in vitro on the antiproliferative activity of the three organonickel complexes against estrogen-responsive human breast cancer cells (MCF7) showed promising antitumor activity for NiL1 containing pyrrolidine fragments. In vivo administration of this compound significantly inhibits tumor growth in estrogen-dependent MC4L2 cancer cells in female BALB/c mice.

Abstract 5758: Elevated level of estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells

Abstract Effectiveness of chemotherapy depends on the age of breast cancer patients. Breast tissues are estrogen sensitive and the levels of ovarian estrogen are age-dependent variable among the breast cancer patients. Whether this variation in estrogen levels influences the chemotherapeutic efficacy in breast cancer patients is unclear. Therefore, the objective of this study was to determine the effects of natural estrogen 17 beta-estradiol on the efficacy of chemotherapy in breast cancer cells. Estrogen responsive MCF-7 breast cancer cells were long-term exposed to 100 pg/ml estrogen for three months. Using these estrogen exposed MCF-7 cells, the efficacy of chemotherapeutic drugs doxorubicin and cisplatin were determined. The result of cell viability and cell cycle analysis revealed increased efficacy of doxorubicin and cisplatin in estrogen-exposed MCF-7 cells as compared to control MCF-7 cells. Gene expression analysis of marker genes for cell cycle, anti-apoptosis, DNA repair, and drug transporter genes further confirmed the increased efficacy of chemotherapeutic drugs in estrogen-exposed cells at molecular level. To further understand the role of epigenetic mechanism in enhanced chemotherapeutic efficacy by estrogen, cells were pre-treated with epigenetic drugs, 5-Aza-2-deoxycytidine and Trichostatin A before doxorubicin and cisplatin treatments. The enhanced efficacy of doxorubicin and cisplatin in estrogen-treated cells was significantly decreased by 5-aza-2 deoxycytidine pre-treatment, suggesting the role of estrogen-induced hypermethylation in enhanced sensitivity of these drugs in estrogen-exposed cells. In conclusion, results of this study demonstrate that sensitivity to chemotherapy depends on the levels of estrogen in breast cancer cells. Findings of this study will have clinical implications in selecting the type and dose of chemotherapy in breast cancer patients depending on the serum estrogen levels that varies in between pre- and post-menopausal age of the patients. Citation Format: Yu-Wei Chang, Kamaleshwar Singh. Elevated level of estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5758. doi:10.1158/1538-7445.AM2017-5758

Long-term exposure to estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells.

Chemotherapy is the most common clinical option for treatment of breast cancer. However, the efficacy of chemotherapy depends on the age of breast cancer patients. Breast tissues are estrogen responsive and the levels of ovarian estrogen vary among the breast cancer patients primarily between pre- and post-menopausal age. Whether this age-dependent variation in estrogen levels influences the chemotherapeutic efficacy in breast cancer patients is not known. Therefore, the objective of this study was to evaluate the effects of natural estrogen 17 beta-estradiol (E2) on the efficacy of chemotherapeutic drugs in breast cancer cells. Estrogen responsive MCF-7 and T47D breast cancer cells were long-term exposed to 100 pg/ml estrogen, and using these cells the efficacy of chemotherapeutic drugs doxorubicin and cisplatin were determined. The result of cell viability and cell cycle analysis revealed increased sensitivities of doxorubicin and cisplatin in estrogen-exposed MCF-7 and T47D cells as compared to their respective control cells. Gene expression analysis of cell cycle, anti-apoptosis, DNA repair, and drug transporter genes further confirmed the increased efficacy of chemotherapeutic drugs in estrogen-exposed cells at molecular level. To further understand the role of epigenetic mechanism in enhanced chemotherapeutic efficacy by estrogen, cells were pre-treated with epigenetic drugs, 5-aza-2-deoxycytidine and Trichostatin A prior to doxorubicin and cisplatin treatments. The 5-aza-2 deoxycytidine pre-treatment significantly decreased the estrogen-induced efficacy of doxorubicin and cisplatin, suggesting the role of estrogen-induced hypermethylation in enhanced sensitivity of these drugs in estrogen-exposed cells. In summary, the results of this study revealed that sensitivity to chemotherapy depends on the levels of estrogen in breast cancer cells. Findings of this study will have clinical implications in selecting the chemotherapy strategies for treatment of breast cancer patients depending on the serum estrogen levels that varies among pre- and post-menopausal age of the patients.

Secretion modification region-derived peptide blocks exosome release and mediates cell cycle arrest in breast cancer cells.

PurposeDiscovery and development of a novel anticancer PEG-SMR-Clu peptide to prevent breast cancer metastasis. How breast cancer cells and primary mammary epithelial cells interact and communicate with each other to promote tumorigenesis and how to prevent tumor metastasis has long been a concern of researchers. Cancer cells secrete exosomes containing proteins and RNA. These factors can influence tumor development by directly targeting cancer cells and tumor stroma. In this study, we determined the effects of a peptide as an inhibitor of exosome secretion on breast tumors. We developed a peptide derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein that was modified with PEG on the N-terminus and with a Clusterin (Clu)-binding peptide on the C-terminus. Attachment of PEG to the SMR peptide, termed PEGylation, offers improved water solubility and stability as well as reduced clearance through the kidneys, leading to a longer circulation time. The 12-mer Clu-binding peptide plays multiple roles in tumor development and metastasis. The Clu peptide can be detected by antibody in vivo, thus it has the potential to be used to monitor tumor status and treatment efficacy in animal studies and eventually in cancer patients.ResultsPEG-SMRwt-Clu and PEG-SMRwt peptides inhibited the growth of both of MCF-7 (estrogen responsive, ER+) and MDA-MD-231 (estrogen non-responsive, ER-) human breast cancer cells in a dose and time-dependent manner, without inducing cytotoxic effects. The SMRwt peptide, combined with paclitaxel, induced G2/M phase cell cycle arrest on MCF-7 and MDA-MB-231 cells but did not promote apoptosis. PEG-SMRwt-Clu peptide treatment blocked exosome release from both MCF-7 and MDA-MB-231 cells. This effect was blocked by knockdown of the chaperone protein mortalin by either antibody or siRNA.Materials and methodsMCF-7 and MDA-MB-231 breast tumor cells were treated with PEG-SMR-Clu peptide alone and in combination with paclitaxel and cisplatin. Cell proliferation and viabilty were determined via cell cycle analysis using Cellometer imaging cytometry, Annexin V and MTT assays. The effects of the PEG-SMR-Clu peptide on tumor exosome release were determined by testing isolated exosome fractions, for (i) expression of CD63 and Alix proteins by Western blotting, (ii) NanoSight nanoparticle tracking analysis (NTA 10) to measure exosomes size and concentration, and (iii) measurement of acetylcholinesterase (AchE) for exosome specific enzyme activity.ConclusionsPEG-SMRwt-CLU peptides inhibited the growth of human breast cancer cells and blocked tumor exosome release in vitro. The peptide alone did not cause increased cytotoxicity or apoptosis induction, but did cause cell cycle G2/M phase arrest in both estrogen responsive and non-responsive breast cancer cells. These data suggest a potential therapeutic value of SMR to prevent breast cancer metastasis and as an adjuvant for the chemotherapeutic treatment of human breast cancer.

Physical interaction of estrogen receptor with MnSOD: implication in mitochondrial O2.- upregulation and mTORC2 potentiation in estrogen-responsive breast cancer cells.

Augmented reactive oxygen species levels consequential to functional alteration of key mitochondrial attributes contribute to carcinogenesis, either directly via oxidative DNA damage infliction or indirectly via activation of oncogenic signaling cascades. We previously reported activation of a key oncogenic signaling cascade via mammalian target of rapamycin (mTOR) signaling complex-2 (mTORC2) owing to estrogen receptor (ER-α)-dependent augmentation of O2.- within the mitochondria of 17-β-estradiol (E2)-stimulated breast cancer cells. Manganese superoxide dismutase (MnSOD) is the principal mitochondrial attribute governing mitochondrial O2.- homeostasis, raising the possibility that its functional alteration could be instrumental in augmenting mitochondrial O2.- levels in breast cancer cells. Here we show ER-dependent transient inhibition of MnSOD catalytic function in breast cancer cells. Catalytic function of MnSOD is tightly regulated at the post-translational level. Post-translational modifications such as phosphorylation, nitration and acetylation represent key regulatory means governing the catalytic function of MnSOD. Acetylation at lysine-68 (K68) inhibits MnSOD catalytic activity and thus represents an important post-translational regulatory mechanism in human cells. Using reciprocal immunoprecipitation and proximity ligation assay, we demonstrate the occurrence of direct physical interaction between ER-α and MnSOD in human breast cancer cells, which in turn was associated with potentiated acetylation of MnSOD at K68. In addition, we also observed diminished interaction of MnSOD with sirtuin-3, the key mitochondrial deacetylase that deacetylates MnSOD at critical K68 and thereby activates it for scavenging O2.-. Consequently, compromised deacetylation of MnSOD at K68 leading to its inhibition and a resultant buildup of O2.- within the mitochondria culminated in the activation of mTORC2. In agreement with this, human breast cancer tissue specimen exhibited a positive correlation between acetyl-MnSODK68 levels and phospho-Ser2481 mTOR levels. In addition to exposing the crosstalk of ER-α with MnSOD post-translational regulatory mechanisms, these data also unravel a regulatory role of ER/MnSOD interaction as an important control switch for redox regulation of ER-α-responsive oncogenic signaling cascades. Furthermore, our study provides a mechanistic link for ER-α-dependent O2.- potentiation and resultant mTORC2 activation in breast cancer cells.

The Impact of Soy Isoflavones on MCF-7 and MDA-MB-231 Breast Cancer Cells Using a Global Metabolomic Approach.

Despite substantial research, the understanding of the chemopreventive mechanisms of soy isoflavones remains challenging. Promising tools, such as metabolomics, can provide now a deeper insight into their biochemical mechanisms. The purpose of this study was to offer a comprehensive assessment of the metabolic alterations induced by genistein, daidzein and a soy seed extract on estrogen responsive (MCF-7) and estrogen non-responsive breast cancer cells (MDA-MB-231), using a global metabolomic approach. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that all test compounds induced a biphasic effect on MCF-7 cells and only a dose-dependent inhibitory effect on MDA-MB-231 cells. Proton nuclear magnetic resonance (1H-NMR) profiling of extracellular metabolites and gas chromatography-mass spectrometry (GC-MS) profiling of intracellular metabolites confirmed that all test compounds shared similar metabolic mechanisms. Exposing MCF-7 cells to stimulatory concentrations of isoflavones led to increased intracellular levels of 6-phosphogluconate and ribose 5-phosphate, suggesting a possible upregulation of the pentose phosphate pathway. After exposure to inhibitory doses of isoflavones, a significant decrease in glucose uptake was observed, especially for MCF-7 cells. In MDA-MB-231 cells, the glutamine uptake was significantly restricted, leading to alterations in protein biosynthesis. Understanding the metabolomic alterations of isoflavones represents a step forward in considering soy and soy derivates as functional foods in breast cancer chemoprevention.

Five furofuranone lignan glucosides from Terminalia citrina inhibit in vitro E2-enhanced breast cancer cell proliferation

Five new polyalkoxylated furofuranone lignan glucosides, terminalosides L–P (1–5), were isolated from EtOAc extracts of the leaves of Terminalia citrina, a Bangladeshi medicinal plant. The structures of the isolates were deduced primarily by NMR spectroscopy, and four of the isolates were found to contain rare tetraoxygenated aryl groups in their structures. The absolute configurations and conformations of the furofuranone ring were confirmed by ECD spectroscopy. All of the isolates were evaluated for their estrogenic and/or antiestrogenic properties using two estrogen responsive breast cancer cell lines, T47D and MCF-7. At a concentration of 10nM, terminaloside L (1) suppressed E2-enhanced T47D cell proliferation by 90%, while terminaloside M (2) showed 90% antiestrogenic activity against MCF-7 cells. Compared to 2, the antiestrogenic activity of terminaloside O (4) and P (5) was weak, possibly due to the different attachment positions of the sugar moiety that they share in common. This is the first report of furofuranone lignans from any Terminalia species, and also of their antiestrogenic activity.

Insulin-like growth factors are essential to prevent anoikis in oestrogen-responsive breast cancer cells: importance of the type I IGF receptor and PI3-kinase/Akt pathway.

BackgroundDetachment of epithelial cells from the extracellular matrix initiates programmed cell death by a process termed anoikis. Malignant cells must acquire anoikis resistance to leave the primary tumour and metastasise. Multiple signal transduction pathways can activate anoikis and confer anoikis resistance, but these are not understood in breast cancer.MethodsModels for anoikis of oestrogen-responsive breast cancer cells were established and the protective effects of IGF-1 tested. Cleaved PARP was measured by western transfer and cleaved caspase 3 by flow cytometry. Pathways involved in anoikis and in anoikis resistance were investigated with PI3-kinase, Akt, and MEK1 and MEK2 inhibitors. The importance of the type I IGF receptor was investigated by IGF-concentration dependence, siRNA knockdown and pharmacological inhibition. Association between IGF-1R expression and relapse with distant metastasis was analysed in 1609 patients by log rank test.ResultsUnattached breast cancer cells required culture in serum-free medium to induce anoikis. Rapid loss of FAK, Akt and Bad phosphorylation was concurrent with anoiks induction, but ERK1 and ERK2 phosphorylation increased which suggested that anoikis resistance is mediated by the PI3-kinase/Akt rather than the Grb2/Ras/MAP-kinase pathway. IGF-1 conferred anoikis resistance in serum-free medium. IGF-1 activated the PI3-kinase/Akt and Grb2/Ras/MAP-kinase pathways but experiments with PI3-kinase, Akt and MEK1 and MEK2 inhibitors showed that IGF protection is via the PI3-kinase/Akt pathway. The concentration dependence of IGF protection, knockdown experiments with siRNA and pharmacological inhibition with figitumumab, showed that IGF-1 signals through the type I IGF receptor. The crucial role of the type I IGF receptor was demonstrated by induction of anoikis in full serum by figitumumab. High IGF-1R expression was associated with reduced time to relapse with distant metastases in oestrogen receptor-positive patients, especially those with aggressive disease which confirms its relevance in vivo.ConclusionsAnoikis resistance of oestrogen-responsive breast cancer cells depends upon IGF activation of the type I IGF receptor and PI3-kinase/Akt pathway. Because IGF-dependent evasion of anoikis will facilitate metastasis by malignant breast cancer cells, effective inhibition of IGF signal transduction should be included in combinations of targeted drugs designed to treat metastatic oestrogen receptor-positive breast cancers.