Mechanisms of naringenin-induced apoptotic cascade in cancer cells: involvement of estrogen receptor alpha and beta signalling

The flavanone naringenin (Nar), especially abundant in the Mediterranean diet, is reported to have anti-proliferative effects in many cancer cell lines. Antioxidant activities, kinase and glucose uptake inhibition have been proposed as molecular mechanisms for these effects. In addition, an anti-estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity could play a role in the Nar anti-tumoral effects. Here, we tested the ability of Nar to activate a specific, rapid signal transduction pathway committed to the generation of an apoptotic cascade in the presence of one of the two estrogen receptor (ER) isoforms (i.e., ERalpha or ERbeta). Cancer cells containing transfected (human cervix epitheloid carcinoma HeLa cells) or endogenous ERalpha (human hepatoma HepG2 cells) or ERbeta (human colon adenocarcinoma DLD-1 cells) were used. Our results show that Nar exerts an anti-proliferative effect only in the presence of ERalpha or ERbeta. Moreover, Nar stimulation induces the activation of p38/MAPK leading to the pro-apoptotic caspase-3 activation and to the poly(ADP-ribose) polymerase cleavage in all cancer cell lines considered. Notably, Nar shows an anti-estrogenic effect only in ERalpha containing cells; whereas in ERbeta containing cells, Nar mimics the 17beta-estradiol effects. These findings indicate new steps in the mechanism underlying ER-dependent anti-proliferative effects of Nar suggesting new potential chemopreventive actions of flavonoids on cancer growth.     

Plasma membrane estrogen receptors exist and functions as dimers

A small pool of estrogen receptors (ERalpha and -beta) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17beta-estradiol (E2). Interestingly, in endothelial cells made from ERalpha /ERbeta homozygous double-knockout mice, membrane ERalpha or ERbeta are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ERalpha. Only wild-type ERalpha supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gsalpha and Gqalpha at the membrane in cells expressing the wild-type, but not the dimer mutant, ERalpha. Intact, but not dimer mutant, ERalpha also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ERalpha. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology.     

S-palmitoylation modulates human estrogen receptor-alpha functions

17beta-Estradiol (E2)-induced rapid functions (from seconds to minutes) can be attributed to a fraction of nuclear estrogen receptor-alpha (ERalpha) localized at the plasma membrane. As a potential mechanism, we postulated that S-palmitoylation of the Cys447 residue may explain the ability of ERalpha to associate to plasma membrane making possible E2-dependent rapid functions [e.g., extracellular regulated kinase (ERK) activation]. Here, we report direct evidence that the mutation of the Cys447 residue to Ala impairs human ERalpha palmitoylation and E2-induced rapid ERK phosphorylation when transfected in ER-devoid HeLa cells. Moreover, the Cys447Ala mutation significantly decreases the E2-induced transactivation of an estrogen responsive element construct probe. Similar effects were obtained treating HeLa cells transfected with wild type ERalpha with the palmitoyl-acyltransferase inhibitor 2-bromo-hexadecanoic acid. Moreover, the deletion of the A-D domains (containing the DNA binding region) of ERalpha had no consequences on [(3)H]palmitate incorporation, whereas no palmitoylation occurred in the ERalpha mutant devoid of the E domain (i.e., ligand binding domain). These results point to the pivotal role of the Cys447 residue in ERalpha palmitoylation and in the modulation of E2-induced non-genomic functions.     

Crosstalk between estrogen receptor alpha and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteasomes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that activates the aryl hydrocarbon receptor (AhR) and disrupts multiple endocrine signaling pathways. T47D human breast cancer cells express a functional estrogen receptor alpha (ERalpha) and AhR, and treatment of these cells with 17beta-estradiol (E2) or TCDD resulted in a rapid proteasome-dependent decrease in immunoreactive ERalpha and AhR proteins (>60-80%), respectively. E2 did not affect the AhR, whereas TCDD induced proteasome-dependent degradation of both the AhR and ERalpha in T47D and MCF-7 human breast cancer cells, and these responses were specifically blocked by proteasome inhibitors. Thus, TCDD-induced degradation of ERalpha may contribute to the antiestrogenic activity of AhR agonists and this pathway may be involved in AhR-mediated disruption of other endocrine responses.     

Nature of functional estrogen receptors at the plasma membrane

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.     

Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol

A fraction of the nuclear estrogen receptor alpha (ERalpha) is localized to the plasma membrane region of 17beta-estradiol (E2) target cells. We previously reported that ERalpha is a palmitoylated protein. To gain insight into the molecular mechanism of ERalpha residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ERalpha membrane localization. The cancer cell lines expressing transfected or endogenous human ERalpha (HeLa and HepG2, respectively) or the ERalpha nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ERalpha enacts ERalpha association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D1 promoter activity, DNA synthesis). Moreover, E2 reduces both ERalpha palmitoylation and its interaction with caveolin-1, in a time- and dose-dependent manner. These data point to the physiological role of ERalpha palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation.     

Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i)

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.     

Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling

The capability of 17beta-estradiol (E2) to induce the non-genomic activities of its receptors (ER alpha and ER beta) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ER alpha or ER beta. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2-ER alpha complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2-ER beta complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase-3 activation and cleavage of poly(ADP-ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2-ER beta complex did not activate any of the E2-ER alpha-activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ER beta isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ER alpha non-genomic signaling and cell death through ER beta non-genomic signaling.     

Estrogen receptor alpha signaling in inflammatory leukocytes is dispensable for 17beta-estradiol-mediated inhibition of experimental autoimmune encephalomyelitis

Estrogen treatment has been shown to exert a protective effect on experimental autoimmune encephalomyelitis (EAE), and is under clinical trial for multiple sclerosis. Although it is commonly assumed that estrogens exert their effect by modulating immune functions, we show in this study that 17beta-estradiol (E2) treatment can inhibit mouse EAE without affecting autoantigen-specific T cell responsiveness and type 1 cytokine production. Using mutant mice in which estrogen receptor alpha (ERalpha) has been unambiguously inactivated, we found that ERalpha was responsible for the E2-mediated inhibition of EAE. We next generated irradiation bone marrow chimeras in which ERalpha expression was selectively impaired in inflammatory T lymphocytes or was limited to the radiosensitive hemopoietic compartment. Our data show that the protective effect of E2 on clinical EAE and CNS inflammation was not dependent on ERalpha signaling in inflammatory T cells. Likewise, EAE development was not prevented by E2 treatment in chimeric mice that selectively expressed ERalpha in the systemic immune compartment. In conclusion, our data demonstrate that the beneficial effect of E2 on this autoimmune disease does not involve ERalpha signaling in blood-derived inflammatory cells, and indicate that ERalpha expressed in other tissues, such as CNS-resident microglia or endothelial cells, mediates this effect.     

The neuroprotective effects of estrogen in SK-N-SH neuroblastoma cell cultures

Estrogen has been considered to be a neuroprotectant and a neuromodulator in many neuronal cell lines and tissue preparations. The protective effects of estrogen may be mediated through classical estrogen receptors (ERs), or may be due to its anti-oxidant properties which are independent of receptors. The current studies show that 17beta-estradiol (E2) is neuroprotective against beta-amyloid protein 25-35 (Abeta)-, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-, high density culture condition-, and serum deprivation-induced neuronal death in SK-N-SH human neuroblastoma cells. SK-N-SH cells express ERbeta, but not ERalpha, as detected by Western blot analysis. Among all the insults, MPTP, high density culture and serum deprivation induce apoptotic cell death in this cell system as detected by ELISA determination of mono/oligonucleosomes and DNA laddering, while Abeta induces necrotic cell death. The protective effects of E2 are abolished by the addition of tamoxifen and ICI 182,780 in the MPTP treated cells, but not in the other models, suggesting that the effect of E2 in the MPTP model is probably associated with activation of ERbeta. The addition of ICI 182,780 shows a mitogenic effect in SK-N-SH cells in the presence of E2 in control culture or in the Abeta treated groups. Also, ICI 182,780 induced expression of ERalpha. Collectively, the current studies suggest that E2 is neuroprotective in apoptotic and necrotic death induced by multiple insults in SK-N-SH human neuroblastoma cells. Involvement of ER is insult type dependent. ICI 182,780 is able to influence the expression of ERs, probably through upregulation of ERalpha when ERbeta is totally antagonized.     

Imine Resveratrol Analogues: Molecular Design,Nrf2 Activation and SAR Analysis

Resveratrol is a natural phenol with protective effects against cancer and inflammation-related diseases. Its mechanism of action involves the activation of nuclear factor E2 p45-related factor 2 (Nrf2), which plays a key role in regulation of genes driven by antioxidant response element (ARE). Inspired by the effect of resveratrol, here we synthesized a series of imine resveratrol analogs (IRAs), evaluated their abilities to activate Nrf2 by using cell based ARE-reporter assay. After the first-round screening, preliminary and quantitative structure-activity relationship (SAR) was analyzed, and the structural features determining Nrf2 activation ability were proposed. Two novel IRAs were designed and subsequently synthesized, namely 2-methoxyl-3,6-dihydroxyl-IRA and 2,3,6-trihydroxyl-IRA. They were proved to be the most potent Nrf2 activators among the IRAs.