Responsive Human Breast Cancer Cells Research Articles

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.

Pomolic acid suppresses HIF1α/VEGF-mediated angiogenesis by targeting p38-MAPK and mTOR signaling cascades

BackgroundPomolic acid (PA), an active triterpenoid from Euscaphis japonica, inhibits the proliferation of a variety of cancer cells, but the molecular mechanisms of the anti-angiogenic potential of PA have not been fully elucidated in breast cancer cells. Hypothesis/PurposeWe investigated the molecular mechanisms underlying the anti-angiogenic effect of PA in epidermal growth factor (EGF)-responsive human breast cancer cells, MCF-7 and MDA-MB-231, and human umbilical vascular endothelial cells (HUVEC). Study design/MethodsEffects of PA on EGF-induced HIF1α/VEGF expression in MCF-7, MDA-MB-231 and HUVEC were assayed. As to the mechanisms, EGF-mediated MAPKs, PI3K/Akt, and mTOR signaling pathway were performed. Wound healing and invasion assay, tube formation assay, immunoblot assay, real-time PCR, luciferase gene assay, electrophoretic mobility shift assay and immunofluorescence staining were used for assessment. ResultsPA significantly and selectively suppressed EGF-induced HIF1α/VEGF expression, whereas it did not affect the expression of HIF1β in MCF-7 and MDA-MB-231. Furthermore, PA inhibited EGF-induced angiogenesis in vitro and downregulated HIF1α/VEGF expression in HUVEC. Mechanistically, we found that the inhibitory effects of PA on HIF1α/VEGF expression are associated with inhibition of HIF1α/VEGF expression through an EGF-dependent mechanism. In addition, PA suppressed the EGF-induced phosphorylation of p38-MAPK and mTOR. ConclusionPA suppresses EGF-induced HIF1α protein translation by inhibiting the p38-MAPK and mTOR kinase signaling pathways and plays a novel anti-angiogenic role.

Estrogenic activity of aromatic compounds present in mineral oil used in printing inks

Mineral oils (MO) used as ingredients for many printing inks are complex mixtures of hydrocarbons. Although migration of MO from paperboard food packaging made of recycled paper into food has been shown, the toxicological relevance of these findings is elusive. Therefore we investigated the estrogenic potential of mineral oil using different in vitro assays. Proliferative effects of MO in the estrogen responsive human breast cancer cell line MCF7 were assessed in the E-screen, and endogenous estrogen responsive gene expression was quantified by real-time PCR. Transactivation assays were performed in HeLa9903 containing the human estrogen receptor α (ERα) and an estrogen responsive element (ERE)-driven luciferase. MO primarily consist of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). These two fractions were separated by preparative HPLC and also assessed in the bioassays. MO in a thousand-fold dilution stimulated MCF7 proliferation but did not induce reporter gene expression in HeLa9903 cells. Furthermore, expression of the estrogen responsive genes PGR, TFF1 and GREB1 in MCF7 cells was induced after 24 h exposure to total MO. The estrogenic effects were derived from the MOAH fraction, as exposure of MCF7 cells to this fraction induced gene expression and increased proliferation. Estrogenic activity of 14 more tested MO correlated with the percentage of the MOAH fraction. In conclusion estrogenic action of aromatic substances derived from MO could be shown in vitro.

Abstract P5-09-02: Cell culture models to study mechanisms for endocrine resistant breast cancer, new treatment options and new biomarkers

Abstract Introduction: Estrogen is the main driver of growth of the majority of breast cancers and many patients benefit from endocrine therapy. However, both innate and acquired resistance is a major problem. In order to study resistance mechanisms and to explore targeted treatment options for endocrine resistant breast cancer, we have developed in vitro cell culture models based on the estrogen receptor (ER) positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D. Methods: Panels of MCF-7 and T47D sublines with acquired resistant to tamoxifen, fulvestrant and aromatase inhibitors were established by selection of colonies surviving long term treatment. Long-term estrogen deprived (LTED) MCF-7/S9 cells were developed by step-wise serum reduction until transfer to serum-free medium. The endocrine resistant sublines were analysed with respect to expression and function of the ER and the HER system, with respect to response to 195 kinase inhibitors and to second-line endocrine therapy. Results: ER expression was reduced in tamoxifen and fulvestrant resistant MCF-7 sublines, unchanged in tamoxifen resistant T47D sublines, whereas fulvestrant resistant T47D sublines were ER negative. In general, ER was functional in the resistant MCF-7 cells. Both tamoxifen and fulvestrant resistant MCF-7 sublines expressed increased level of HER1, the activated form of HER3 and activated ERK1/2, whereas HER4 was severely decreased. In fulvestrant resistant T47D sublines, HER1 and HER4 expression were decreased whereas HER2 expression was highly upregulated. The kinase inhibitor screen disclosed HER receptors as common hits in the tamoxifen and fulvestrant resistant MCF-7 sublines. Regarding T47D, c-Src and Aurora were common hits for tamoxifen and fulvestrant resistant sublines. Tamoxifen resistant MCF-7 and T47D sublines were severely growth inhibited by ER knockdown with either siRNA or fulvestrant, but complete growth arrest required combined treatment with e.g ERK1/2 inhibitor. LTED MCF-7 cells were resistant to aromatase inhibitor treatment and sensitive to both tamoxifen and fulvestrant. Conclusions: In our large panels of MCF-7 and T47D derived endocrine resistant sublines, it was a general finding that ER is the main driver of growth of tamoxifen resistant tumor cells, and also of growth of LTED MCF-7 cells. The ER down modulator fulvestrant inhibited growth of tamoxifen resistant sublines but complete growth arrest required combined treatment with e.g. a kinase inhibitor targeting the activated growth promoting pathways in the resistant cells. In fulvestrant resistant sublines, ER played only a minor role in MCF-7 sublines and no role in T47D sublines. The HER system played a major role in fulvestrant resistant MCF-7 cells, whereas Aurora B and c-Src were new candidate targets for fulvestrant resistant T47D cells. These cell culture models reflect clinical findings and will be available for collaborative studies, e.g. to discover new predictive markers to select targeted therapy for resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-02.

Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α through an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action.

Abstract 1315: Regulation of Nodal expression by estrogen in hormone responsive human breast cancer cell lines.

Abstract Breast cancer is one of the most common cancers among Canadian women. Estrogen plays a key role in the in the normal growth and development of the breast and is also a major contributor to breast cancer growth. Recent studies have demonstrated that embryonic morphogen, Nodal is expressed in metastatic breast cancers and that its expression is correlated with breast cancer progression. In silico analysis of the Nodal promoter has revealed putative estrogen receptor binding sites (ERE) in proximity to the transcription start site. Our goal in this study is to investigate the role of estrogen on Nodal expression in human breast cancer cell-lines. Stimulation of MCF-7 and T47D breast cancer cell-lines that are estrogen receptor positive and express low levels of Nodal with 10nM of estradiol (E2) for varying amounts of time revealed that Nodal protein expression increases at about 48 hours after E2 exposure and persists up to 120 hours. Treatment with the E2 antagonist, ICI-182,780 (ICI) abrogates the E2-enhanced Nodal protein expression. Conversely, treating the MCF- 7 and T47D cells with recombinant Nodal resulted in a decreased expression of estrogen receptor in these cells both at the transcript and protein levels. These preliminary results suggest that there is a bidirectional interaction between estrogen receptor and Nodal in these cell-lines. Future experiments are aimed at understanding in depth this bidirectional phenomenon to provide better insight into hormonal regulation in metastatic breast cancer. Citation Format: Padmalaya Das, Daniela Quail, Lynne-Marie Postovit. Regulation of Nodal expression by estrogen in hormone responsive human breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1315. doi:10.1158/1538-7445.AM2013-1315

Influence of Anthocyanins, Derivative Pigments and Other Catechol and Pyrogallol-Type Phenolics on Breast Cancer Cell Proliferation

Anthocyanins (cyanidin-3-glucoside (Cy-3-gluc) and delphinidin-3-glucoside (Dp-3-gluc)) and their respective vinylpyranoanthocyanin-catechins (portisins) were studied in order to evaluate the cytotoxicity effect on the estrogen responsive human breast cancer cell line (ER+) MCF-7 and their effect on estrogen receptor (ER-alpha and ER-beta) expression. Other flavonoid classes and phenolic molecules were also tested, aiming to study possible structural features related with these effects. Also, the antiproliferative effect of Cy-3-gluc and Dp-3-gluc was studied by an immunofluorescence assay. Generally, all the anthocyanin pigments studied inhibited, in a dose-dependent manner, the growth of the (ER+) MCF-7. The cytotoxicity effect was higher when cells were treated with Dp-3-gluc and its respective portisin. Altogether, the results point to the ortho trihydroxylated moiety in the phenolic ring as an important structural feature for more potent cytotoxicity effect on MCF-7 cells comparatively to the effect observed with the similar dihydroxylated compounds. In order to elucidate the molecular mechanism involved, expression of estrogen receptor was assayed by RT-PCR and real time RT-PCR. The higher antiproliferative effect observed after cell treatment with Dp-3-gluc was not followed by modification on ER expression. However, the anthocyanin Cy-3-gluc was able to induce a downregulation of ER levels although with no significant effect on MCF-7 proliferation.

Antioxidant and Biological Properties of Bioactive Phenolic Compounds fromQuercus suberL.

Phenolic compounds, namely, hydrolyzable tannins and low molecular weight phenolic compounds, were isolated and purified from Portuguese cork from Quercus suber L. Some of these compounds were studied to evaluate their antioxidant activity, including free-radical scavenging capacity (DPPH method) and reducing capacity (FRAP method). All compounds tested showed significant antioxidant activity, namely, antiradical and reducing properties. The antiradical capacity seemed to increase with the presence of galloyl groups. Regarding the reducing capacity, this structure-activity relationship was not so clear. These compounds were also studied to evaluate the growth inhibitory effect on the estrogen responsive human breast cancer cell line (ER+) MCF-7 and two other colon cancer cell lines (Caco-2 and HT-29). Generally, all the compounds tested exhibited, after a continuous exposure during a 48 h period, a dose-dependent growth inhibitory effect. Relative inhibitory activity was primarily related to the number of phenolic hydroxyl groups (galloyl and HHDP moieties) found in the active structures, with more groups generally conferring increased effects, except for HHDP-di-galloyl-glucose. Mongolicain B showed a greater potential to inhibit the growth of the three cell lines tested, identical to the effect observed with castalagin. Since these compounds are structurally related with each other, this activity might be based within the C-glycosidic ellagitannin moiety.

Bisphosphonates suppress insulin‐like growth factor 1‐induced angiogenesis via the HIF‐1α/VEGF signaling pathways in human breast cancer cells

Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its antiresorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms, including antiangiogenesis. In this study, we investigated the 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. We tested whether bisphosphonates had any effects on hypoxia-inducible factor (HIF)-1alpha/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF-1-induced HIF-1alpha protein accumulation and VEGF expression in MCF-7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF-1alpha, but they apparently promoted the degradation of IGF-1-induced HIF-1alpha protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose-dependent decease in the newly-synthesized HIF-1alpha protein induced by IGF-1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF-1alpha/VEGF axis are associated with the inhibition of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathways. Consistently, we 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 the inhibition of tumor angiogenesis in breast cancer cells.

Structure and estrogenic activity of new lignans from Iryanthera lancifolia

Five new dibenzylbutane type lignans (1-5) were isolated from the stem bark of Iryanthera lancifolia. Their structures were determined by extensive 1D and 2D NMR spectroscopic studies and chemical evidence. Seventeen of the isolated compounds were tested for their estrogenic activities in the estrogen responsive human breast cancer cell line MCF-7 BUS using the E-Screen proliferation assay. Cell proliferation was evaluated by the SRB assay to calculate the estrogenic parameters. The majority of the compounds induced a mitogenic response. This effect, given as Relative Proliferative Effect (RPE) to reference estrogen 17beta-estradiol (E(2)), ranged between 14% and 84%.

An inactive pool of GSK-3 at the leading edge of growth cones is implicated in Semaphorin 3A signaling.

Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase that has been implicated in several aspects in embryonic development and several growth factor signaling cascades. We now report that an inactive phosphorylated pool of the enzyme colocalizes with F-actin in both neuronal and nonneuronal cells. Semaphorin 3A (Sema 3A), a molecule that inhibits axonal growth, activates GSK-3 at the leading edge of neuronal growth cones and in Sema 3A–responsive human breast cancer cells, suggesting that GSK-3 activity might play a role in coupling Sema 3A signaling to changes in cell motility. We show that three different GSK-3 antagonists (LiCl, SB-216763, and SB-415286) can inhibit the growth cone collapse response induced by Sema 3A. These studies reveal a novel compartmentalization of inactive GSK-3 in cells and demonstrate for the first time a requirement for GSK-3 activity in the Sema 3A signal transduction pathway.

Tocotrienols inhibit growth of ZR-75–1 breast cancer cells

The vitamin E component of palm oil provides a rich source of tocotrienols which have been shown previously to be growth inhibitory to two human breast cancer cell lines: responsive MCF7 cells and unresponsive MDA-MB-231 cells. Data presented here shows that the tocotrienol-rich fraction (TRF) of palm oil and individual fractions (alpha, gamma and delta) can also inhibit the growth of another responsive human breast cancer cell line, ZR-75-1. At low concentrations in the absence of oestrogen tocotrienols stimulated growth of the ZR-75-1 cells, but at higher concentrations in the presence as well as in the absence of oestradiol, tocotrienols inhibited cell growth strongly. As for MCF7 cells, α -tocopherol had no effect on growth of the ZR-75-1 cells in either the absence or presence of oestradiol. In studying the effects of tocotrienols in combination with antioestrogens, it was found that TRF could further inhibit growth of ZR-75-1 cells in the presence of tamoxifen (10 -7 M and 10 -8 M). Individual tocotrienol fractions (alpha, gamma, delta) could inhibit growth of ZR-75-1 cells in the presence of 10 -8 M oestradiol and 10 -8 M pure antioestrogen ICI 164 384. The immature mouse uterine weight bioassay confirmed that TRF could not exert oestrogen antagonist action in vivo. These results provide evidence of wider growth-inhibitory effects of tocotrienols beyond MCF7 and MDA-MB-231 cells, and with an oestrogen-independent mechanism of action, suggest a possible clinical advantage in combining administration of tocotrienols with antioestrogen therapy.

Limitations of the MCF-7 Cell Proliferation Assay for Detecting Xenobiotic Oestrogens

The MCF-7 cell assay has recently been proposed as a primary screen for the detection of xenobiotic oestrogens. The assay is based on the ability of human breast cancer cells (MCF-7) to proliferate in response to oestrogens. The use of this assay has a number of known drawbacks, such as the differing responses of cells from different sources. This paper describes the sources of variability which we have encountered with this assay. There was significant variability between experiments in the response of MCF-7 cells to oestradiol, from twofold to sevenfold increases, sometimes with no evidence of a dose response. The background rate of cell proliferation (in the absence of oestradiol) also varied between experiments, from a low basal rate in some experiments to rates approaching that of oestradiol-treated cells in others. The variability in our experiments may be due to selection of sub-clones of cells at passage. We also determined the effects of oestrogens on the non-oestrogen responsive human breast cancer cell line MDA-MB-231. These cells did not proliferate in response to oestradiol, but nonylphenol promoted a two- to threefold increase in cell number. Our data illustrate the potentially unpredictable nature of breast cancer cell lines when used routinely as a screening test for xenobiotic oestrogens.

Estrogen increases intracellular p26Bcl-2 to p21Bax ratios and inhibits taxol-induced apoptosis of human breast cancer MCF-7 cells.

Recent studies have demonstrated that following estrogen ablation, estrogen responsive breast cancer cells undergo apoptosis. In addition, estrogen receptor (ER) expression has been strongly correlated with the expression of the bcl-2 gene product, p26Bcl-2 protein, which is known to inhibit apoptosis. In the present studies, we investigated whether estrogen affects the intracellular levels of p26Bcl-2 and thereby modulates taxol-induced apoptosis of estrogen responsive human breast cancer MCF-7 cells. Transfer of MCF-7 cells to a culture-medium without estrogens reduced their intracellular p26Bcl-2 levels by 50%. Inclusion of 0.1 microM estradiol in the medium produced approximately a four-fold increase in p26Bcl-2, but not p29Bcl-x1, or p21Bax levels; the expression of the c-myc and mdr-1 genes remained unchanged. Estradiol-induced four-fold increase in the ratio of the p26Bcl-2 to p21Bax levels caused a significant decline in the lethal, kilobase size DNA fragments of apoptosis, which had resulted when MCF-7 cells were cultured in a medium without estrogen. In addition, in MCF-7 cells, estradiol-induced increase in the intracellular p26Bcl-2 to p21Bax ratios was associated with a significant reduction in the large-sized DNA fragmentation induced by treatment with taxol. The increased ratios also protected MCF-7 cells against taxol-mediated cytotoxicity as assessed by the MTT assay. These results suggest that by modulating p26Bcl-2 levels, estrogens may affect the antitumor activity of taxol and potentially of other anti-breast cancer drugs against estrogen responsive human breast cancer cells.

Induction of Transforming Growth Factor β by the Antiestrogens Droloxifene, Tamoxifen, and Toremifene in MCF-7 Cells

We have previously shown that transforming growth factor beta (TGF beta) is a hormonally regulated negative growth factor in estrogen responsive MCF-7 human breast cancer cells. We have now compared the antiestrogens tamoxifen, droloxifene (3-hydroxytamoxifen), and toremifene in their ability to induce the secretion of autoinhibitory TGF beta by MCF-7 cells. The main results are as follows: induction of TGF beta secretion by droloxifene is about two to three times higher than by identical concentrations of tamoxifen or toremifene. A 5-10 times higher concentration of tamoxifen or toremifene than droloxifene is necessary to reach a similar induction of TGF beta secretion. In contrast to tamoxifen, intermittent application of droloxifene is as effective as continuous treatment in inducing TGF beta secretion. We conclude from these data that TGF beta proteins represent markers of antiestrogen action and might also play a pivotal role in their mechanism of action. Droloxifene is a more effective inducer of TGF beta and a more potent growth inhibitor for estrogen responsive human breast cancer cells than tamoxifen and toremifene in vitro. Therefore, droloxifene might also possess a higher antiestrogenic potential in treatment of human breast cancer.

Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells.

A cDNA library was constructed from the mRNA of the ZR-75 oestrogen responsive human breast cancer cell line and screened for oestrogen regulated mRNA sequences. Of the recombinants isolated, 30 contained cDNA that corresponded to a single mRNA species of 2.1 kb that was induced between 10 and 15 fold by oestradiol treatment. The sequence of the entire open reading frame and 3' non-coding region of the mRNA was determined and shown to encode the aspartyl protease cathepsin D. The induction of cathepsin D mRNA is specific for oestrogen and is maximal at 3 X 10(-10) M. Cathepsin D mRNA levels were increased by oestrogen in 3 oestrogen responsive breast cancer cell lines. Cathepsin D mRNA was expressed but not regulated in an oestrogen receptor negative breast tumour cell line, BT 20, and in 2 other malignant cell lines, Hela and A431.