Search

Additional file 1. Figure 1. Controls for AZD7762 inhibitor in MCF-7 and Y537S cells. Western blot analyses of S296 phosphorylated CHK1, S516 phosphorylated CHK2, CHK1, and CHK2 expression levels in (A) MCF-7 and (B) Y537S cells treated for 2 hours with etoposide (ETO) (10 μM) both in the presence or in the absence of 1 μM AZD7762 (AZD). The loading control was done by evaluating vinculin expression in the same filter. (C, D) Western blot analyses of CHK1 and CHK2 intracellular levels after siRNA transfection procedure. These blots are the controls for the experiments described in Fig. 4O and 4P. The loading control was done by evaluating vinculin expression in the same filter. Figure 2. Controls for ATR, ATM, CHK1, and CHK2 inhibitors in MCF-7 and Y537S cells. Western blot analyses in MCF-7 (A-D) and Y537S (E-H) cells treated with the indicated doses of for 24 hours with the indicated doses of the specific inhibitors of either CHK1 (i.e., MK8776 - MK) (D and H), CHK2 (i.e., CCT241533 - CCT) (C and G), ATR (i.e., VE822 - VE) (B and F) or ATM (i.e., KU60019 - KU) (A and E) both in the presence and in the absence of etoposide (ETO – 10 µM 2 hours) of the phosphorylated forms of CHK1 and CHK2. Total CHK1 and CHK2 as well as vinculin expression was evaluated as loading controls in the same filters. The experiments were performed twice. Densitometric analyses are available upon request. Figure 3. Impact of clinically relevant CHK1 inhibitor in inducing ERα degradation and preventing proliferation in MCF-7 and Y537S cells. Western blot (A) and relative densitometric analyses (A’) of ERα expression levels in MCF-7 (yellow) and Y537S (red) cells treated for 24 hours with the indicated doses of GDC-0575 (GDC). The loading control was done by evaluating vinculin expression in the same filter. Significant differences with respect to control (0) were obtained by unpaired two-tailed Student’s t-test. Data show the mean ± the standard deviations, **** p < 0.0001; ** p < 0.01; * p < 0.05. The number of replicates is given as solid dots in the bar graph. Growth curve analyses in MCF-7 (B) and Y537S (C) cells were performed as indicated in the material and method section for 5 days with different doses of GDC-0575 (GDC). The graph shows the normalized cell index (i.e., cell number), which is detected with the xCelligence DP device and calculated at each time point with respect to the control sample. Each sample was measured in a quadruplicate. For details, please see the material and methods section. Figure 4. The impact of ATR:CHK1 and ATM:CHK2 pathways on hyperactive ERα transcriptional activity. (A) Estrogen response element promoter activity in Y537S ERE-NLuc cells pre-treated the specific inhibitors of either CHK1 (i.e., MK8776 – MK 1 μM), CHK2 (i.e., CCT241533 – CCT 0.5 μM), ATR (i.e., VE822 – VE 1 μM) or ATM (i.e., KU60019 – KU 1 μM) as well as with AZD7762 (AZD 1 μM) for 24 hours at the indicated doses. The experiments were performed three times in quintuplicate duplicate. Significant differences with respect to control (CTR) sample are calculated by Student t-test and indicated by **** p-value < 0.0001 and ** p-value < 0.01. (B) Western blot analyses of pS2 levels treated with the indicated inhibitors as described in figure 9. This histogram represents the densitometric analyses of the blots indicated in Fig. 9C. Figure 5. GDC-0575 synergic effects with abemaciclib in Y537S cells and synergy studies in BC cell lines. Synergy map of 5 days-treated Y537S cells with different doses of GDC-0575 (GDC) and abemaciclib (Abe) (A). Growth curves in Y537S cells showing the synergic effect of each combination of compounds with selected doses are shown in (A’). For details, please see the material and methods section. Significant differences with respect to untreated (i.e., -,-) samples are calculated by Student t-test and indicated by **** p-value < 0.0001. Significant differences between GDC+Abe with respect to GDC alone or Abe alone are calculated by Student t-test and indicated by ° or ^ p-value < 0.05, respectively. Number of MCF-7 (B, B’), BT-474 (C, C’) and MDA-MB-361 (D, D’) cells treated for 12 days with the indicated doses of 4OH-Tamoxifen (Tam) in combination with the indicated doses of MK8776 (MK) (B), GDC-0575 (GDC) (B’), AZD7762 (AZD) (C, D) or prexasertib (Prexa) (C’, D’). Figure 6. Synergy studies in Y537S cells. The number of Y537S cells treated for 5 days with the indicated doses of AZD7762 (AZD) (A, B) or GDC-0575 (GDC) (C) in combination with the indicated doses of abemaciclib (Abe) (A) or palbociclib (Palbo) (B, C). Figure 7. Inhibitor-dependent effect on DNA damage and replication stress and replication stress and X-rays dependent effect on ERα levels in MCF-7 and Y537S cells. (A, B, C, E, F, and H) Histograms relative to the heatmaps shown in main Fig. 7G and 7H for the densitometric analyses of ERα (A, E), RPA2 (B, F) and phosphorylated γH2AX (C, G) expression levels in MCF-7 (A, B, and C) and Y537S (E, F, and G) cells. Description of the treatments have been given in the figure caption of the main Fig. 7. (D and H) Histograms relative to the heatmaps shown in main Fig. 8C and 8F for the densitometric analyses of ERα, RPA2 and phosphorylated γH2AX expression levels in MCF-7 (D) and Y537S (H) cells. Description of the treatments has been given in the figure caption of the main Fig. 8.

Although paclitaxel (Taxol) is an active chemotherapeutic agent for the treatment of breast cancer, not all breast tumors are sensitive to this drug. In particular, there is a wide agreement on the low sensitivity of estrogen receptor (ER) α-positive breast cancer to paclitaxel treatment. However, the ERα-based insensitivity to paclitaxel is still elusive. Here, the effect of the E2/ERα-dependent upregulation of neuroglobin (NGB), an antiapoptotic globin, on the reduced sensitivity of breast cancer cells to paclitaxel-induced apoptosis has been evaluated in ERα-containing MCF-7 cells. The E2 pretreatment enhances the ERα activity and significantly impairs paclitaxel-induced apoptosis as evaluated by Annexin V assay and PARP-1 cleavage. NGB displays a pivotal role in the E2/ERα-induced antiapoptotic pathway to abrogate paclitaxel-induced cell death in stable NGB-silenced MCF-7 cell clones. Moreover, in the absence of the active ERα, paclitaxel significantly reduces the NGB cell content. In conclusion, these results highlight the involvement of ERα activation and of E2/ERα-dependent NGB upregulation in the insensitivity of MCF-7 to paclitaxel. These novel findings could have important implications in the development of targeted therapeutics for overcoming paclitaxel insensitivity in ERα-positive human breast cancer

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17β-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs.

Previously, we found that telaprevir (Tel), the inhibitor of hepatitis C virus NS3/4A serine protease, reduces estrogen receptor α (ERα) content at the transcriptional level without binding to the receptor, prevents ERα transcriptional activity, and inhibits basal and 17β-estradiol (E2)-dependent cell proliferation in different breast cancer (BC) cell lines. Here, we further characterize the Tel action mechanisms on ERα levels and function, identify a possible molecular target of Tel in BC cells, and evaluate Tel as an antiproliferative agent for BC treatment. Tel-dependent reduction in ERα levels and function depends on a Tel-dependent decrease in FOXA1 levels and activity. The effect of Tel is transduced by the IGF1-R/AKT/FOXA1 pathway, with the antiviral compound interacting with IGF1-R. Tel prevents the proliferation of several BC cell lines, while it does not affect the proliferation of normal nontransformed cell lines, and its antiproliferative effect is correlated with the ratio of FOXA1/IGF1-R expression. In conclusion, Tel interferes with the IGF1-R/AKT/FOXA1 pathway and induces cell death in ERα-expressing BC cells. Thus, we propose that this antiviral could be repurposed for the treatment of ERα-expressing BC.

Background Challenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs. Methods ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan–Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17β-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed. Results A reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC. Conclusions CHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs.

Neuroglobin (NGB), an antiapoptotic protein upregulated by 17β-estradiol (E2), is part of E2/estrogen receptor α (ERα) pathway pointed to preserve cancer cell survival in presence of microenvironmental stressors including chemotherapeutic drugs. Here, the possibility that resveratrol (Res), an anticancer plant polyphenol, could increase the susceptibility of breast cancer cells to paclitaxel (Pacl) by affecting E2/ERα/NGB pathway has been evaluated. In MCF-7 and T47D (ERα-positive), but not in MDA-MB 231 (ERα-negative) nor in SK-N-BE (ERα and ERβ positive), Res decreases NGB levels interfering with E2/ERα-induced NGB upregulation and with E2-induced ERα and protein kinase B phosphorylation. Although Res treatment does not reduce cell viability by itself, this compound potentiates Pacl proapoptotic effects. Notably, the increase of NGB levels by NGB expression vector transfection prevents Pacl or Res/Pacl effects. Taken together, these findings indicate a new Res-based mechanism that acts on tumor cells impairing the E2/ERα/NGB signaling pathways and increasing cancer cell susceptibility to chemotherapeutic agent.

Phthalates comprise a group of synthetic chemicals present in the environment because of their wide use as plasticizers and as additives in products for personal care. Among others, diethyl phthalate (DEP) is largely used in products for infants, children, and adults, in which its exposure has been correlated with an increased risk of breast cancer. The adverse health outcomes deriving from phthalate exposure have been associated with their activity as endocrine disruptors (EDCs) of the steroid and thyroid hormone signaling by affecting developmental and reproductive health, and even carcinogenicity. However, the estrogen disruptor activities of DEP are still controversial, and the mechanism at the root of the estrogenic-disrupting action of DEP remains to be clarified. Here, we evaluated the DEP mechanism of action on the activation status of estrogen receptor alpha (ER alpha) by analyzing the receptor's phosphorylation as well as both nuclear and extra-nuclear pathways triggered by the receptor to modulate the proliferation of breast cancer cells. Although DEP does not bind to ER alpha, our results suggest that this phthalate ester exerts multiple parallel interactions with ER alpha signaling and emphasize the importance to determine an appropriate battery of in vitro methods that will include specific molecular mechanisms involved in the endocrine disruption.

Estrogen receptor &alpha, expressing breast cancers (BC) are classically treated with endocrine therapy. Prolonged endocrine therapy often results in a metastatic disease (MBC), for which a standardized effective therapy is still lacking. Thus, new drugs are required for primary and metastatic BC treatment. Here, we report that the Food and Drug Administration (FDA)-approved drugs, ouabain and digoxin, induce ER&alpha, degradation and prevent proliferation in cells modeling primary and metastatic BC. Ouabain and digoxin activate the cellular proteasome, instigating ER&alpha, degradation, which causes the inhibition of 17&beta, estradiol signaling, induces the cell cycle blockade in the G2 phase, and triggers apoptosis. Remarkably, these effects are independent of the inhibition of the Na/K pump. The antiproliferative effects of ouabain and digoxin occur also in diverse cancer models (i.e., tumor spheroids and xenografts). Additionally, gene profiling analysis reveals that these drugs downregulate the expression of genes related to endocrine therapy resistance. Therefore, ouabain and digoxin behave as &lsquo, anti-estrogen&rsquo, like drugs, and are appealing candidates for the treatment of primary and metastatic BCs.

Introduction: The influence of sex and gender is particularly relevant in cardiovascular diseases (CVD) as well as in several aspects of drug pharmacodynamics and pharmacokinetics. Anatomical and physiological differences between the sexes may influence the activity of many drugs, including the possibility of their interaction with other drugs, bioactive compounds, foods and beverages. Phenolic compounds could interact with our organism at organ, cellular, and molecular levels triggering a preventive action against chronic diseases, including CVD. Results: This article will review the role of sex on the activity of these bioactive molecules, considering the existence of sex differences in oxidative stress. It describes the pharmacokinetics of phenolic compounds, their effects on vessels, on cardiovascular system, and during development, including the role of nuclear receptors and microbiota. Conclusions: Although there is a large gap between the knowledge of the sex differences in the phenolic compounds’ activity and safety, and the urgent need for more research, available data underlie the possibility that plant-derived phenolic compounds could differently influence the health of male and female subjects.

In the year 2000, the third member of the globin family was discovered in human and mouse brain and named neuroglobin (Ngb). Neuroglobin overexpression significantly protects both heart and brain from hypoxic/ischemic and oxidative stress-related insults, whereas decreased Ngb levels lead to an exacerbation of tissue injuries. Moreover, Ngb overexpression protects neurons from mitochondrial dysfunctions and neurodegenerative disorders such as Alzheimer disease; however, it facilitates the survival of cancer cells. Neuroglobin, representing a switch point for cell death and survival, has been reported to recognize a number of proteins involved in several metabolic pathways including ionic homeostasis maintenance, energy metabolism, mitochondrial function, and cell signaling. Here, the recognition properties of Ngb are reviewed to highlight its roles in health and disease.

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.

Neuroglobin (NGB) is a relatively recent discovered monomeric heme-protein, which behave in neurons as a sensor of injuring stimuli including oxidative stress, hypoxia, and neurotoxicity. In addition, the anti-apoptotic activity of overexpressed NGB has been reported both in neurons and in cancer cell lines. We recently demonstrated that, NGB functions as a compensatory protein of the steroid hormone 17β-estradiol (E2) protecting cancer cells against the apoptotic death induced by oxidative stress. However, the E2-induced signaling pathways at the root of NGB over-expression and mitochondrial re-localization in breast cancer cells is still elusive. By using a kinase screening library, here, we report that: i) There is a strong positive correlation between NGB and ERα expression and activity in breast cancer cells; ii) The E2-activated phosphatidyl-inositol 3 kinase (PI3K)/protein kinase B (AKT) and protein kinase C (PKC) pathways are necessary to modulate the NGB protein levels; iii) The E2-induced persistent activation of AKT drive NGB to mitochondria; iv) Reactive oxygen species (ROS)-inducing compounds activating rapidly and transiently AKT does not affect the NGB mitochondrial level; and v) High level of NGB into mitochondria are necessary for the pro-survival and anti-apoptotic effect of this globin in cancer cells. As a whole, these results underline the E2 triggered pathways in E2-responsive breast cancer cells that involve NGB as a compensatory protein devoted to cancer cell survival.

Environmental factors or adverse growth conditions that may reduce cell function or viability are considered stress. The cell ability to sense and respond to environmental stresses determine its function and survival destiny. We recently defined Neuroglobin (NGB), a heme-protein, as a compensatory protein in the 17β-Estradiol (E2) anti-apoptotic activity and as a sensor of oxidative stress in both neurons and breast cancer cells. Here, the possibility that NGB levels could represent a pivotal regulator of integrated response of cancer cells to stress has been evaluated. Data obtained in neuroblastoma and in breast cancer cell lines evidence that nutrient deprivation significantly up-regulated NGB levels at different time points. However, the analysis of autophagy activation led to exclude any possible role of stress- or E2-induced NGB in the upstream regulation of general autophagy. However, the over-expression of Flag-NGB in ERα stable transfected HEK-293 cells completely affects nutrient deprivation-induced decrease in cell number. In addition, reported results indicate that modulation of the anti-apoptotic Bcl-2 level may play a key role in the protective NGB function against energetic stress. Overall, these data define a role of NGB as compensatory protein in the cell machinery activated in response to stress and as general stress adaptation marker of cancer cells susceptible to oxidative stress, oxygen and, as demonstrated here for the first time, even to nutrient willingness. Despite the lacking of any direct NGB role on autophagic flux activated by energetic stress, NGB upregulation appears functional in delaying stress-related cell death allowing an appropriate cell response and adaptation to the changing extracellular conditions.

The over-expression of human neuroglobin (NGB), a heme-protein preferentially expressed in the brain, displays anti-apoptotic effects against hypoxic/ischemic and oxidative stresses enhancing neuron survival. As hypoxic and oxidative stress injury frequently occurs in fast proliferating neoplastic tissues, here, the effect of these stressors on the level, localization, and anti-apoptotic function of NGB in wild type and NGB-stable-silenced MCF-7 breast cancer cells has been assessed. The well-known endogenous NGB inducer 17β-estradiol (E2) has been used as positive control. The median pO2 present in tumor microenvironment of breast cancer patients (i.e., 2% O2) does not affect the NGB level in breast cancer cells, whereas hydrogen peroxide and lead(IV) acetate, which increase intracellular reactive oxygen species (ROS) level, enhance the NGB levels outside the mitochondria and still activate apoptosis. However, E2-induced NGB up-regulation in mitochondria completely reverse lead(IV) acetate-induced PARP cleavage. These results indicate that the NGB level could represent a marker of oxidative-stress in MCF-7 breast cancer cells; however, the NGB ability to respond to injuring stimuli by preventing apoptosis requires its re-allocation into the mitochondria. As a whole, present data might lead to a new direction in understanding NGB function in cancer opening new avenues for the therapeutic intervention.

A semisynthetic approach to novel lipid A derivatives from Escherichia coli (E. coli) lipid A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipid A fraction from fed-batch fermentation of E. coli, followed by its structural modification through tailored, site-selective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipid A derivatives show that some of them stand out as promising, new immunoadjuvant candidates.