Alessandro Bolli

Flavonoid binding to human serum albumin.

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlows site I range between 3.3x10(-6) and 3.9x10(-5)M, at pH 7.0 and 20.0 degrees C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.

A novel mechanism of natural vitamin E tocotrienol activity: involvement of ERβ signal transduction

Vitamin E is a generic term used to indicate all tocopherol (TOC) and tocotrienol (TT) derivates. In the last few years, several papers have shown that a TT-rich fraction (TTRF) extracted from palm oil inhibits proliferation and induces apoptosis in a large number of cancer cells. However, the molecular mechanism(s) involved in TT action is still unclear. In the present study, we proposed for the first time a novel mechanism for TT activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicated a high affinity of TTs for ERbeta but not for ERalpha. In addition, in ERbeta-containing MDA-MB-231 breast cancer cells, we demonstrated that TTs increase the ERbeta translocation into the nucleus, which in turn activates estrogen-responsive genes (MIC-1, EGR-1 and cathepsin D), as demonstrated by cell preincubation with the ER inhibitor ICI-182,780. Finally, we observed that TT treatment is associated with alteration of cell morphology, DNA fragmentation, and caspase-3 activation. Altogether, these experiments elucidated the molecular mechanism underling gamma- and delta-TT effects.

Structural Determinants in the Group III Truncated Hemoglobin from Campylobacter jejuni

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 α-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-Å resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III.

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.

Laccase treatment impairs bisphenol A-induced cancer cell proliferation affecting estrogen receptor α-dependent rapid signals

A wide variety of environmental contaminants exert estrogenic actions in wildlife, laboratory animals, and in human beings through binding to nuclear estrogen receptors (ERs). Here, the mechanism(s) of bisphenol A (BPA) to induce cell proliferation and the occurrence of its bioremediation by treatment with laccase are reported. BPA, highly present in natural world and considered as a model of environmental estrogen action complexity, promotes human cancer cell proliferation via ERα‐dependent signal transduction pathways. Similar to 17β‐estradiol, BPA increases the phosphorylation of both extracellular regulated kinase and AKT. Specific inhibitors of these kinase completely block the BPA effect on cancer cell proliferation. Notably, high BPA concentrations (i.e., 0.1 and 1 mM) are cytotoxic even in ERα‐devoid cancer cells, indicating that an ERα‐independent mechanism participates to BPA‐induced cytotoxicity. On the other hand, BPA oxidation by laccase impairs the binding of this environmental estrogen to ERα loosing at all ERα‐dependent effect on cancer cell proliferation. Moreover, the laccase‐catalyzed oxidation of BPA reduces the BPA cytotoxic effect. Thus, laccase appears to impair BPA action(s), representing an invaluable bioremediation enzyme.

Binding of anti-HIV drugs to human serum albumin

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand (i.e., drug) binding capacity. Here, values of the dissociation equilibrium constant (Kd) for the binding of HIV protease and reverse transcriptase inhibitors to HSA are reported. The binding of abacavir, atazanavir, didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, zalcitabine, and zidovudine to the Sudlow site I (i.e., the warfarin cleft) located in the subdomain IIA involves the alteration of the HSA structure around Trp214 and induces intrinsic tryptophan fluorescence quenching. Accordingly, ibuprofen that primarily binds to the Sudlow site II located in the subdomain IIIA does not affect the HSA intrinsic tryptophan fluorescence and the binding of anti‐HIV drugs to the Sudlow site I. Accounting for the physiological concentration of HSA (= 7.0 × 10‐4 M), the average anti‐HIV drug concentration in plasma (= 1.0 × 10‐4 M), and Kd values for the binding of anti‐HIV drugs to HSA (ranging between 4.4 × 10‐5 M and 3.8 × 10‐4 M), it appears that the fraction of HIV protease and reverse transcriptase inhibitors bound to HSA ranges between 63% and 91%. This represents a significant drawback in the anti‐HIV therapy and management, the anti‐HIV drug concentration required to achieve 90% protease and reverse transcriptase inhibition in the presence of plasma proteins appears to be at least one order of magnitude higher than that required in their absence. IUBMB Life, 56: 609‐614, 2004

Hise11 and Hisf8 Provide Bis-Histidyl Heme Hexa-Coordination in the Globin Domain of Geobacter Sulfurreducens Globin-Coupled Sensor.

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS(162)) is reported. A combination of X-ray crystallography (crystal structure at 1.5 A resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS(162) as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS(162) is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS(162) is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS(162) is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.

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.

Effect of Bisphenol A with or without enzyme treatment on the proliferation and viability of MCF-7 cells

Recently, aqueous solutions polluted by BPA have been bioremediated by us using laccase immobilized on hydrophobic membranes in non-isothermal bioreactors. BPA degradation was checked using analytical methods. To assess in vitro the occurred bioremediation, the proliferation and viability indexes of MCF-7 cells incubated in the presence of aqueous solutions of BPA, or of enzyme-treated BPA solutions, have been measured as a function of the initial BPA concentration. The results demonstrated that: i) at each initial BPA concentration used, both the proliferation and viability indexes are a function of the duration of enzyme treatment; ii) proliferation and viability are uncoupled biological processes with respect to BPA enzyme treatment. Non-isothermal bioreactors are a useful tool for the bioremediation of aqueous solutions polluted by BPA, which is an example of an endocrine disruptor that belongs to the alkyl phenol family.

Bisphenol A Impairs Estradiol-induced Protective Effects Against DLD-1 Colon Cancer Cell Growth

Bisphenol A (BPA), a prototype of endocrine disruptors, mimics 17β‐estradiol (E2)‐induced proliferation in several cancer cells by binding to estrogen receptor α (ERα). However, scarce and conflicting data are available concerning the effect of BPA on estrogen receptor β (ERβ)‐mediated functions. Here, the detailed analysis of the effect of BPA, alone or in combination with E2, on ERβ‐mediated cellular functions is reported in ERβ‐expressing colon cancer cell line. BPA binds to ERβ without activating any receptor activities. On the other hand, BPA inhibits E2‐induced genomic activity of ERβ as well as ERβ extra‐nuclear activities (i.e., ERβ:p38 association and p38 activation). As a consequence, BPA impairs the E2‐induced activation of the apoptotic cascade which is at the root of the protective role played by the hormone against colon cancer growth. Thus, women may be considered a highly susceptible population with an increased risk of colon cancers after BPA exposures.