Estrogen-Induced Apoptosis of Breast Epithelial Cells Is Blocked by NO/cGMP and Mediated by Extranuclear Estrogen Receptors

Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. The action of 17β-estradiol (E2) at ER is known to elicit NO signaling via activation of NO synthase (NOS) in many tissues. The MCF-10A nontumorigenic, mammary epithelial cell line is genetically stable and insensitive to estrogenic proliferation. In this cell line, estrogens or NOS inhibitors alone had no significant effect, whereas in combination, apoptosis was induced rapidly in the absence of serum; the presence of inducible NOS was confirmed by proteomic analysis. The application of pharmacological agents determined that apoptosis was dependent upon NO/cGMP signaling via cyclic GMP (cGMP)-dependent protein kinase and could be replicated by inhibition of the phosphatidylinositol 3 kinase/serine-threonine kinase pathway prior to addition of E2. Apoptosis was confirmed by nuclear staining and increased caspase-3 activity in E2 NOS inhibitor-treated cells. Apoptosis was partially inhibited by a pure ER antagonist and replicated by agonists selective for extranuclear ER. Cells were rescued from E2-induced apoptosis after NOS blockade, by NO-donors and cGMP pathway agonists; preincubation with NO donors was required. The NOS and ER status of breast cancer tissues is significant in etiology, prognosis, and therapy. In this study, apoptosis of preneoplastic mammary epithelial cells was triggered by estrogens via a rapid, extranuclear ER-mediated response, after removal of an antiapoptotic NO/cGMP/cGMP-dependent protein kinase signal. (Endocrinology 151: 5602–5616, 2010

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD+ biosynthesis via salvage of NAM formed from catabolism of NAD+ by proteins with NADase activity (e.g., PARPs, SIRTs, CD38). Depletion of NAD+ in aging, neurodegeneration, and metabolic disorders is addressed by NAD+ supplementation. Conversely, NAMPT inhibitors have been developed for cancer therapy: many discovered by phenotypic screening for cancer cell death have low nanomolar potency in cellular models. No NAMPT inhibitor is yet FDA-approved. The ability of inhibitors to act as NAMPT substrates may be associated with efficacy and toxicity. Some 3-pyridyl inhibitors become 4-pyridyl activators or “NAD+ boosters”. NAMPT positive allosteric modulators (N-PAMs) and boosters may increase enzyme activity by relieving substrate/product inhibition. Binding to a “rear channel” extending from the NAMPT active site is key for inhibitors, boosters, and N-PAMs. A deeper understanding may fulfill the potential of NAMPT ligands to regulate cellular life and death

Raloxifene and Desmethylarzoxifene Block Estrogen- Induced Malignant Transformation of Human Breast Epithelial Cells

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention

DataSheet_1_Estradiol improves behavior in FAD transgenic mice that express APOE3 but not APOE4 after ovariectomy.docx

Increasing evidence suggests that female individuals have a higher Alzheimer’s disease (AD) risk associated with post-menopausal loss of circulating estradiol (E2). However, clinical data are conflicting on whether E2 lowers AD risk. One potential contributing factor is APOE. The greatest genetic risk factor for AD is APOE4, a factor that is pronounced in female individuals post-menopause. Clinical data suggests that APOE impacts the response of AD patients to E2 replacement therapy. However, whether APOE4 prevents, is neutral, or promotes any positive effects of E2 is unclear. Therefore, our goal was to determine whether APOE modulates the impact of E2 on behavior and AD pathology in vivo. To that end, mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ42 were ovariectomized at either 4 months (early) or 8 months (late) and treated with vehicle or E2 for 4 months. In E3FAD mice, we found that E2 mitigated the detrimental effect of ovariectomy on memory, with no effect on Aβ in the early paradigm and only improved learning in the late paradigm. Although E2 lowered Aβ in E4FAD mice in the early paradigm, there was no impact on learning or memory, possibly due to higher Aβ pathology compared to E3FAD mice. In the late paradigm, there was no effect on learning/memory and Aβ pathology in E4FAD mice. Collectively, these data support the idea that, in the presence of Aβ pathology, APOE impacts the response to E2 supplementation post-menopause.</p

DataSheet_2_Estradiol improves behavior in FAD transgenic mice that express APOE3 but not APOE4 after ovariectomy.xlsx

Increasing evidence suggests that female individuals have a higher Alzheimer’s disease (AD) risk associated with post-menopausal loss of circulating estradiol (E2). However, clinical data are conflicting on whether E2 lowers AD risk. One potential contributing factor is APOE. The greatest genetic risk factor for AD is APOE4, a factor that is pronounced in female individuals post-menopause. Clinical data suggests that APOE impacts the response of AD patients to E2 replacement therapy. However, whether APOE4 prevents, is neutral, or promotes any positive effects of E2 is unclear. Therefore, our goal was to determine whether APOE modulates the impact of E2 on behavior and AD pathology in vivo. To that end, mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ42 were ovariectomized at either 4 months (early) or 8 months (late) and treated with vehicle or E2 for 4 months. In E3FAD mice, we found that E2 mitigated the detrimental effect of ovariectomy on memory, with no effect on Aβ in the early paradigm and only improved learning in the late paradigm. Although E2 lowered Aβ in E4FAD mice in the early paradigm, there was no impact on learning or memory, possibly due to higher Aβ pathology compared to E3FAD mice. In the late paradigm, there was no effect on learning/memory and Aβ pathology in E4FAD mice. Collectively, these data support the idea that, in the presence of Aβ pathology, APOE impacts the response to E2 supplementation post-menopause.</p

Nicotinamide Phosphoribosyltransferase Positive Allosteric Modulators Attenuate Neuronal Oxidative Stress

Evidence supports boosting nicotinamide adenine dinucleotide (NAD+) to counteract oxidative stress in aging and neurodegenerative disease. One approach is to enhance the activity of nicotinamide phosphoribosyltransferase (NAMPT). Novel NAMPT positive allosteric modulators (N-PAMs) were identified. A cocrystal structure confirmed N-PAM binding to the NAMPT rear channel. Early hit-to-lead efforts led to a 1.88-fold maximum increase in the level of NAD+ in human THP-1 cells. Select N-PAMs were assessed for mitigation of reactive oxygen species (ROS) in HT-22 neuronal cells subject to inflammatory stress using tumor necrosis factor alpha (TNFα). N-PAMs that increased NAD+ more effectively in THP-1 cells attenuated TNFα-induced ROS more effectively in HT-22 cells. The most efficacious N-PAM completely attenuated ROS elevation in glutamate-stressed HT-22 cells, a model of neuronal excitotoxicity. This work demonstrates for the first time that N-PAMs are capable of mitigating elevated ROS in neurons stressed with TNFα and glutamate and provides support for further N-PAM optimization for treatment of neurodegenerative diseases