Stefano Leone

Survival versus apoptotic 17β‐estradiol effect: Role of ERα and ERβ activated non‐genomic signaling

The capability of 17β‐estradiol (E2) to induce the non‐genomic activities of its receptors (ERα and ERβ) 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) ERα or ERβ. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2–ERα 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–ERβ 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–ERβ complex did not activate any of the E2–ERα‐activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ERβ 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 ERα non‐genomic signaling and cell death through ERβ non‐genomic signaling.

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

The capability of 17β‐estradiol (E2) to induce the non‐genomic activities of its receptors (ERα and ERβ) 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) ERα or ERβ. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2–ERα 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–ERβ 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–ERβ complex did not activate any of the E2–ERα‐activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ERβ 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 ERα non‐genomic signaling and cell death through ERβ non‐genomic signaling.

Mechanisms of Naringenin‐induced Apoptotic Cascade in Cancer Cells: Involvement of Estrogen Receptor a and ß 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., ERα or ERβ). Cancer cells containing transfected (human cervix epitheloid carcinoma HeLa cells) or endogenous ERα (human hepatoma HepG2 cells) or ERβ (human colon adenocarcinoma DLD‐1 cells) were used. Our results show that Nar exerts an anti‐proliferative effect only in the presence of ERα or ERβ. 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 ERα containing cells; whereas in ERβ containing cells, Nar mimics the 17β‐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. IUBMB Life, 56: 491‐499, 2004

Quercetin-induced apoptotic cascade in cancer cells: antioxidant versus estrogen receptor α-dependent mechanisms.

The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti-proliferative effects in many cancer cell lines. Antioxidant or pro-oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin-induced anti-proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor alpha (ERalpha; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H(2)O(2)-induced ROS production both in the presence and in the absence of ERalpha. However, this flavonoid induces the activation of p38/MAPK, leading to the pro-apoptotic caspase-3 activation and to the poly(ADP-ribose) polymerase cleavage only in the presence of ERalpha. Notably, no down-regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERalpha-dependent mechanism involving caspase- and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.

The pro-apoptotic effect of quercetin in cancer cell lines requires ERβ-dependent signals.

Quercetin has potentially beneficial effects on disease prevention, including cancer. An intriguing issue regarding the mechanisms of action of quercetin is the ability of this drug to modulate estrogen receptor (ER) activities. In a previous study, we demonstrated that quercetin elicited apoptosis through an ERα‐dependent mechanism. However, the contribution of ERβ in quercetin‐induced apoptosis remains elusive. Here, we report that quercetin, at nutritionally relevant concentrations, mimicked the 17β‐estradiol (E2)‐induced apoptotic effect in both ERβ1‐transfected HeLa and in ERβ1‐containing DLD‐1 colon cancer cell lines by inducing the activation of p38. p38 activation is responsible for pro‐apoptotic activation of caspase‐3 and the cleavage of poly(ADP‐ribose) polymerase. Notably, no inactivation or downregulation of the survival kinases (i.e., AKT and ERK1/2) or the antiapoptotic protein Bcl‐2 was observed after quercetin stimulation. On the contrary, quercetin acted similarly to E2 by increasing the levels of the oncosuppressor protein PTEN and by impeding ERβ‐dependent cyclin D1 promoter activity, which subsequently resulted in the transcription of the estrogen‐responsive element remaining unchanged. As a whole, these data indicate that quercetin mimics the E2 effects in the presence of ERβ1, thus maintaining its anti‐carcinogenic potential. In addition, the quercetin pro‐apoptotic action in the presence of ERα may render it as a dual‐sided protective agent against E2‐related cancer in the reduction of tumour growth in organs that express ERα and/or ERβ. J. Cell. Physiol. 227: 1891–1898, 2012.

Resveratrol induces DNA double-strand breaks through human topoisomerase II interaction

Resveratrol, a stilbene found in grapes and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to delay cell cycle progression and to induce apoptotic death in several cell lines. Here we report that resveratrol treatment of human glioblastoma cells induces a delay in cell cycle progression during S phase associated with an increase in histone H2AX phosphorylation. Furthermore, with an in vitro assay of topoisomerase IIalpha catalytic activity we show that resveratrol is able to inhibit the ability of recombinant human TOPO IIalpha to decatenate kDNA, so that it could be considered a TOPO II poison.

Oxidative stress induces persistent telomeric DNA damage responsible for nuclear morphology change in Mammalian cells.

One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.

Naringenin and 17β‐estradiol coadministration prevents hormone‐induced human cancer cell growth

Flavonoids have been described as health‐promoting, disease‐preventing dietary components. In vivo and in vitro experiments also support a protective effect of flavonoids to reduce the incidence of certain hormone‐responsive cancers. In particular, our previous results indicate that the flavanone naringenin (Nar), decoupling estrogen receptor α (ERα) action mechanisms, drives cancer cells to apoptosis. Because these studies were conducted in the absence of the endogenous hormone 17β‐estradiol (E2), the physiological relevance of these findings is not clear. We investigate whether the antiproliferative Nar effect persists in the presence of physiological E2 concentration (i.e. 10 nM), using both ERα‐transfected (HeLa cells) and ERα‐containing (HepG2 cells) cancer cell lines. Ligand saturation experiments indicate that Nar decreases the binding of E2 to ERα without impairing the estrogen response element (ERE)‐driven reporter plasmid activity. In contrast, Nar stimulation prevents E2‐induced extracellular regulated kinases (ERK1/2) and AKT activation and still induces the activation of p38, the proapoptotic member of mitogen‐activating protein kinase (MAPK) family. As a consequence, Nar stimulation impedes the E2‐induced transcription of cyclin D1 promoter and reverts the E2‐induced cell proliferation, driving cancer cell to apoptosis. Thus, these results suggest that coexposure to this low‐affinity, low‐potency ligand for ERα specifically antagonizes the E2‐induced ERα‐dependent rapid signals by reducing the effect of the endogenous hormone in promoting cellular proliferation. As a whole, these data indicate that Nar is an excellent candidate as a chemopreventive agent in E2‐dependent cancers.

Resveratrol acts as a topoisomerase II poison in human glioma cells

Recently, we demonstrated that Resveratrol (RSV), a well known natural stilbene, is able to induce a delay in S progression with a concomitant increase in γH2AX expression in U87 glioma cells. Furthermore, we showed that it inhibits the ability of recombinant human topoisomerase IIα to decatenate kDNA in vitro. Because proliferating tumor cells express topoisomerases at high levels and these enzymes are important targets of some of the most successful anticancer drugs, we tested whether RSV is able to poison topoisomerase IIα in glioma cells. Then, we monitored the increase of micronuclei in RSV treated U87 cells as a consequence of the conversion of TOPOII/DNA cleavable complexes to permanent DNA damage. Finally, we assayed the ability of RSV in modulating the expression of target proteins involved in DNA damage signalling, namely ATR, ATM, Chk1, Chk2 and γH2AX. Through a molecular modelling here we show that RSV binds at the TOPOII/DNA interface thus establishing several hydrogen bonds. Moreover, we show that RSV poisons TOPOIIα so inducing DNA damage; ATM, Chk2 and γH2AX are involved in the DNA damage signalling after RSV treatment.

HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

Background HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3. Methodology/Principal Findings Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. Conclusions Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.