Tectoridin,a poor ligand of estrogen receptor α, exerts its estrogenic effects via an ERK-dependent pathway

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17β-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER α as compared to 17β-estradiol and genistein. Despite poor binding to ER α, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER α at Ser¹¹⁸. It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.     

Experimental study on the estrogen-like effect of mercuric chloride

Although mercuric chloride has toxicity on reproductive system, it is uncertain if such toxicity is induced by estrogen-like effect. To study whether mercuric chloride has the estrogen-like effect and its relevant mechanism, proliferation assay of MCF-7 human breast cancer cells, uterotrophic assay, peroxidase activity assay and estrogen receptor competitive binding assay were conducted to screen the estrogen-like effect of mercuric chloride. The MCF-7 cells proliferated in the stimulation of mercuric chloride and got to the peak at 10⁻⁷ mol/l concentration. And this proliferation could be completely blocked by estrogenic antagonist ICI182.780. In addition, mercuric chloride could increase the weight of uterus of ovariectomized SD rats and the peroxidase activity of uterus complying with dose-effect relationship. However, mercuric chloride could not affect the binding of estradiol (E₂) to estrogen receptor (ER). So mercuric chloride exhibits the estrogen-like effect through binding and activating ER rather than bind to ER by competing with E₂.     

Synthesis,computational studies and antiproliferative activities of coumarin-tagged 1,3,4-oxadiazole conjugates against MDA-MB-231 and MCF-7 human breast cancer cells

A novel library of coumarin tagged 1,3,4 oxadiazole conjugates was synthesized and evaluated for their antiproliferative activities against MDA-MB-231 and MCF-7 breast cancer cell lines. The evaluation studies revealed that compound 9d was the most potent molecule with an IC₅₀ value of <5?µM against the MCF-7 cell line. Interestingly, compounds 10b and 11a showed a similar trend with lower inhibitory concentration (IC₅₀?=?7.07?µM), in Estrogen Negative (ER?) cells than Estrogen Positive (ER+) cells. Structure–activity relationship (SAR) studies revealed that conjugates bearing benzyl moieties (9b, 9c and 9d) had superior activities compared to their alkyl analogues. The most potent compound 9d showed ～1.4?times more potent activity than tamoxifen against MCF-7 cell line; while the introduction of sulfone unit in compounds 11a, 11b and 11c resulted in significant cytotoxicity against both MCF-7 and MDA-MB-231 cell lines. These results were further supported by docking studies, which revealed that the stronger binding affinity of the synthesized conjugates is due to the presence of sulfone unit attached to the substituted benzyl moiety in their pharmacophores.     

Caspase-3 status is a determinant of the differential responses to genistein between MDA-MB-231 and MCF-7 breast cancer cells

Genistein, a soy isoflavone with anti-tumor properties, has both estrogenic and non-estrogenic activities. Genistein sensitive/estrogen receptor negative (ER-) MDA-MB-231 cells and genistein resistant/ER+MCF-7 cells are frequently cited as examples of differential responses to genistein due to different ER status. Other factors that may affect genistein response, however, are largely unknown. Based on our finding that MCF-7 is caspase-3 deficient, we examined whether caspase-3 status plays a role in the differential responses between the two cell lines. We demonstrate that reconstitution of caspase-3 significantly sensitizes MCF-7 cells to genistein. Specific knockdown of caspase-3 in MDA-MB-231 cells renders the cells resistant to genistein. We also found that caspases-4 and -10 were downregulated in MCF-7 cells. Reconstitution of caspase-10 in MCF-7 cells, however, resulted in little sensitization. Moreover, we show that caspase-3 downregulation is very common in breast cancer cell lines and tumor tissues. Taken together, our data indicate that caspase-3 is a critical determinant of cellular response to genistein, which may have important implications in studying soy/genistein-mediated anti-tumor activities.     

Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and apoptosis inducing agents

A new series of 1,2,4-triazole-linked urea and thiourea conjugates have been synthesized and evaluated for their in vitro cytotoxicity against selected human cancer cell lines namely, breast (MCF-7, MDA-MB-231), lung (A549) prostate (DU145) and one mouse melanoma (B16-F10) cell line and compared with reference drug. The compound 5t showed significant cytotoxicity on MCF-7 breast cancer cell line with a IC₅₀ value of 7.22?±?0.47?µM among all the tested compounds. Notably, induction of apoptosis by compound 5t on MCF-7 cells was evaluated using different staining techniques such as acridine orange/ethidium bromide (AO/EB), annexin V-FITC/PI, and DAPI. Further, clonogenic assay indicates the inhibition of colony formation on MCF-7 cells by compound 5t. Moreover, the flow-cytometric analysis also revealed that compound 5t caused the arrest of cells at G0/G1 phase of cell cycle. In addition, the compounds when tested on normal human cells (L-132) were found to be safer with low cytotoxicity profile.     

Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.     

Benzo[b]furan derivatives induces apoptosis by targeting the PI3K/Akt/mTOR signaling pathway in human breast cancer cells

The PI3K/Akt/mTOR signaling pathway plays a key regulatory function in cell survival, proliferation, migration, metabolism and apoptosis. Aberrant activation of the PI3K/Akt/mTOR pathway is found in many types of cancer and thus plays a major role in breast cancer cell proliferation. In our previous studies, benzo[b]furan derivatives were evaluated for their anticancer activity and the lead compounds identified were 26 and 36. These observations prompted us to investigate the molecular mechanism and apoptotic pathway of these lead molecules against breast cancer cells. Benzo[b]furan derivatives (26 and 36) were evaluated for their antiproliferative activity against human breast cancer cell lines MCF-7 and MDA MB-231. These compounds (26 and 36) have shown potent efficiency against breast cancer cells (MCF-7) with IC₅₀ values 0.057 and 0.051 μM respectively. Cell cycle analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7 cells. Western blot analysis revealed that these compounds inhibit the PI3K/Akt/mTOR signaling pathway and induced mitochondrial mediated apoptosis in human breast cancer cells (MCF-7).     

Estrogen receptor alpha mediates epithelial to mesenchymal transition,expression of specific matrix effectors and functional properties of breast cancer cells

The 17β-estradiol (E2)/estrogen receptor alpha (ERα) signaling pathway is one of the most important pathways in hormone-dependent breast cancer. E2 plays pivotal roles in cancer cell growth, survival, and architecture as well as in gene expression regulatory mechanisms. In this study, we established stably transfected MCF-7 cells by knocking down the ERα gene (designated as MCF-7/SP10 + cells), using specific shRNA lentiviral particles, and compared them with the control cells (MCF-7/c). Interestingly, ERα silencing in MCF-7 cells strongly induced cellular phenotypic changes accompanied by significant changes in gene and protein expression of several markers typical of epithelial to mesenchymal transition (EMT). Notably, these cells exhibited enhanced cell proliferation, migration and invasion. Moreover, ERα suppression strongly affected the gene and protein expression of EGFR and HER2 receptor tyrosine kinases, and various extracellular matrix (ECM) effectors, including matrix metalloproteinases and their endogenous inhibitors (MMPs/TIMPs) and components of the plasminogen activation system. The action caused by E2 in MCF-7/c cells in the expression of HER2, MT1-MMP, MMP1, MMP9, uPA, tPA, and PAI-1 was abolished in MCF-7/SP10 + cells lacking ERα. These data suggested a regulatory role for the E2/ERα pathway in respect to the composition and activity of the extracellular proteolytic molecular network. Notably, loss of ERα promoted breast cancer cell migration and invasion by inducing changes in the expression levels of certain matrix macromolecules (especially uPA, tPA, PAI-1) through the EGFR–ERK signaling pathway.In conclusion, loss of ERα in breast cancer cells results in a potent EMT characterized by striking changes in the expression profile of specific matrix macromolecules highlighting the potential nodal role of matrix effectors in breast cancer endocrine resistance.     

2-Phenyl-1-[4-(2-piperidine-1-yl-ethoxy)benzyl]-1H-benzimidazoles as ligands for the estrogen receptor: Synthesis and pharmacological evaluation

2-Phenyl-1H-benzimidazoles 7a–e were synthesized and tested for gene activation on ERα-positive MCF-7 breast cancer cells, stably transfected with the reporter plasmid ERE_wtcluc (MCF-7-2a cells). None of the compounds showed agonistic properties, but they antagonized dependent on hydroxyl groups at the benzimidazole core (5- or 6-OH) and at the aromatic ring in the 2-position (4-OH) in high concentrations the gene activation induced by estradiol (E2, 1 nM). All compounds exhibited significant antiproliferative properties on MCF-7 cells but they were inactive against hormone independent, ER negative MDA-MB-231 cells.     

A novel cytotoxic terpenoid from the flowers of Kaunia lasiophthalma Griseb

A phytochemical study of the flowers of Kaunia lasiophthalma G. (Asteraceae) yielded a novel triterpene (1) together with several known sesquiterpenoids. The structure of the new compound was elucidated by analysis of the spectroscopic data. The biosynthetic origin of 1 is proposed to be a dimerization of an oxidized derivative (3) of the germacrane sesquiterpene costunolide (2), also present in the flowers. The anticancer activity of 1 in the five breast cancer cell lines HCC1937, JIMT-1, L56Br-C1, MCF-7 and SK-BR-3 was compared with the cytotoxicity in the normal-like breast epithelial cell line MCF-10A. 1 exhibited high cytotoxicity in all investigated cancer cell lines with IC₅₀ values ranging from 0.67 to 7.0 μM, although it is lacking selectivity as the MCF-10A cells were almost as sensitive.     

Novel estrogen receptor (ER) modulators containing various hydrophobic bent-core structures

We previously discovered m-carborane-containing estrogen receptor (ER) modulator 4, which exhibits weak ER-agonistic and antagonistic activities in transactivation assays. With the aim of developing novel ER partial agonists, we designed and synthesized various analogues of 4 with a bent-core structure, that is, pseudo cyclic structure (5), tetrahydropyrimidinone (6), m-benzene (7), adamantane (8), and 9,10-dimethyl-m-carborane (9), in place of the m-carborane moiety. Compound 9 showed greater binding affinity than 4 in ER-binding assay using [6,7-³H]-17β-estradiol and was a more effective partial agonist than 4 in MCF-7 cell proliferation assay. It appears to be a promising candidate as a selective ER modulator (SERM).     

Expression profiling of the genes responding to zearalenone and its analogues using estrogen-responsive genes

To compare gene expression profiles in response to estrogen or 17β-estradiol (E₂) and a mycotoxin, zearalenone (ZEA), and its analogues (collectively termed ZEA compounds), breast cancer MCF-7 cells were treated with 10 nM of E₂ or ZEA compounds including ZEA, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol. Expression profiles for 120 estrogen-responsive genes were subjected to cluster and statistical analyses using correlation coefficients or R-values. We found that all of the ZEA compounds stimulated the growth of MCF-7 cells, as much as E₂, and showed similar expression profiles to that of E₂ (R-values ranged from 0.82 to 0.96). The effect of ZEA compounds was likely mediated by estrogen-receptor-dependent Erk1/2-signaling. These results provide clues to understand the mechanism of their estrogen-like action.     

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines

The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by ¹H NMR and GC–MS to know the metabolites in each extract. In GC–MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC₅₀ values of 45.9 μg/mL and the dichloromethane extract of the leaves (DLE) showed IC₅₀ values of 47.3 μg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.     

Design,synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC₅₀ value of 4.52 ± 2.47 μM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure–activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.     

One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxicity in breast cancer cells

A series of cinnamylideneacetophenones were synthesized via a modified Claisen–Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer cells using the Alamar Blue™ assay. Derivatives 17 and 18 bearing a 2-nitro group on the B ring, exhibited sub-micromolar cytotoxicity in MCF-7 cells (IC₅₀ = 71 and 1.9 nM), respectively. Derivative 17 also displayed sub-micromolar (IC₅₀ = 780 nM) cytotoxicity in MDA-MB-468 cells. Additionally, 17 and 18 displayed significantly less cytotoxicity than the chemotherapeutic doxorubicin in non-tumorigenic MCF-10A cells. This study provides evidence supporting the continued development of nitro-substituted cinnamylideneacetophenones as small molecules to treat breast cancer.     

17beta-Estradiol inhibits osteoprotegerin production by the estrogen receptor-alpha-positive human breast cancer cell line MCF-7

Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-κB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-α, 17β-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p < 0.0001 by ANOVA). The inhibition of OPG production by 17β-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17β-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.     

Different anticancer effects of Saxifragifolin A on estrogen receptor-positive and estrogen receptor-negative breast cancer cells

BackgroundBreast cancer is the leading cause of cancer-related death among women worldwide. For treating breast cancer, numerous natural products have been considered as chemotherapeutic drugs.Hypothesis/purposeThe present study aims to investigate the apoptotic effect of Saxifragifolin A (Saxi A) isolated from Androsace umbellata in two different human breast cancer cells which are ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells, and examine the molecular basis for its anticancer actions.Study designThe inhibitory effects of Saxi A on cell survival were examined in MCF-7 cells and MDA-MB-231 cells in vitro.MethodsMTT assays, Annexin V/PI staining analysis, ROS production assay, Hoechst33342 staining and Western blot analysis were performed.ResultsOur results showed that MDA-MB-231 cells were more sensitive to Saxi A-induced apoptosis than MCF-7 cells. Saxi A induced apoptosis in MDA-MB-231 cells through ROS-mediated and caspase-dependent pathways, whereas treatment with Saxi A induced apoptosis in MCF-7 cells in a caspase-independent manner. In spite of Saxi A-induced activation of MAPKs in both breast cancer cell lines, only p38 MAPK and JNK mediated Saxi A-induced apoptosis. In addition, cell survival of shERα-transfected MCF-7 cells was decreased, while MDA-MB-231 cells that overexpress ERα remained viable.ConclusionSaxi A inhibits cell survival in MCF-7 cells and MDA-MB-231 cells through different regulatory pathway, and ERα status appears to be important for regulating Saxi A-induced apoptosis in breast cancer cells. Thus, Saxi A may have a potential therapeutic use for treating breast cancer.