Micronutrients Involved in One-Carbon Metabolism and Risk of Breast Cancer Subtypes

Background

Vitamins involved in one-carbon metabolism are hypothesized to influence breast cancer (BC) risk. However, epidemiologic studies that examined associations between B vitamin intake and BC risk have provided inconsistent results. We prospectively examined, in the Italian ORDET cohort, whether B vitamin consumption was associated with risk of BC and BC subtypes.

Methods

After a mean follow-up of 16.5 years, 391 BCs were diagnosed among 10,786 cohort women. B vitamin intakes were estimated from food frequency questionnaires. Cox proportional hazard models adjusted for energy intake and confounders, estimated hazard ratios (HR) with 95% confidence intervals (CIs) for BC according to intake.

Results

RRs were 0.61 (95% CI 0.38–0.97 highest vs. lowest quartile; P trend 0.025) for thiamine; 0.48 (95% CI 0.32–0.71; P trend <0.001) for riboflavin; 0.59 (95% CI 0.39–0.90; P trend 0.008) for vitamin B6, and 0.65 (95% CI 0.44–0.95; P trend 0.021) for folate. As regards risk of BC subtypes, high riboflavin and folate were significantly associated with lower risk of estrogen receptor positive (ER+) and progesterone receptor positive (PR+) cancers, and high thiamine was associated with lower risk of ER-PR- cancers. High riboflavin was associated with lower risk of both HER2+ and HER2- cancers, high folate with lower risk of HER2- disease, and high thiamine with HER2+ disease.

Conclusions

These findings support protective effects of thiamine and one-carbon metabolism vitamins (folate, riboflavin, and vitamin B6) against BC in general; while folate may also protect against ER+PR+ and HER2- disease; and thiamine against ER-PR-, and HER2+ disease.     

Physical Interaction between MYCN Oncogene and Polycomb Repressive Complex 2 (PRC2) in Neuroblastoma: FUNCTIONAL AND THERAPEUTIC IMPLICATIONS*

CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.     

A phase II trial of vaccination with autologous, tumor-derived heat-shock protein peptide complexes Gp96, in combination with GM-CSF and interferon-α in metastatic melanoma patients

The aim of this study was to determine the immunogenicity and antitumor activity of autologous, tumor-derived heat shock protein gp96-peptide complex vaccine (HSPPC-96; Oncophage®) given with GM-CSF and IFN-α in pre-treated metastatic (AJCC stage IV) melanoma patients. Patients underwent surgical resection of metastatic lesions for HSPPC-96 production. HSPPC-96 was administered subcutaneously (s.c.) in four weekly intervals (first cycle). Patients with more available vaccine and absence of progressive disease received four additional injections in 2-week intervals (second cycle) or more. GM-CSF was given s.c. at the same site at days –1, 0 and +1, while IFN-α (3 MU) was administered s.c. at a different site at days +4 and +6. Antigen-specific anti-melanoma T and NK lymphocyte response was assessed by enzyme-linked immunospot assay on peripheral blood mononuclear cells obtained before and after vaccination. Thirty-eight patients were enrolled, 20 received at least four injections (one cycle) of HSPPC-96 and were considered assessable. Toxicity was mild and most treatment-related adverse events were local erythema and induration at the injection site. Patients receiving at least four injections of HSPPC-96 were considered evaluable for clinical response: of the 18 patients with measurable disease post surgery, 11 showed stable disease (SD). The ELISPOT assay revealed an increased class I HLA-restricted T and NK cell-mediated post-vaccination response in 5 out of 17 and 12 out of the 18 patients tested, respectively. Four of the five class I HLA-restricted T cell responses fall in the group of SD patients. Vaccination with autologous HSPPC-96 together with GM-CSF and IFN-α is feasible and accompanied by mild local and systemic toxicity. Both tumor-specific T cell-mediated and NK cell responses were generated in a proportion of patients. Clinical activity was limited to SD. However, both immunological and clinical responses were not improved as compared with those recorded in a previous study investigating HSPPC-96 monotherapy.L.P. and R.P. have equally contributed to the work.     

Analogues of both Leu- and Met-enkephalin containing a constrained dipeptide isostere prepared from a Baylis-Hillman adduct

An efficient route was developed for the synthesis of the Fmoc-protected dipeptide 4, isostere of Gly-Gly containing an α-methylene β-amino acid; the conformationally restricted analogues of Leu-enkephalin, 3a, and Met-enkephalin, 3b, respectively, were prepared by changing 4 for Gly²-Gly³ in the native compounds 3a and 3b whose biological activities were significantly lower than the parent compounds.     

Towards novel S-DABOC inhibitors: synthesis, biological investigation, and molecular modeling studies

A small family of S-DABO cytosine analogs (S-DABOCs) has been synthesized and biologically evaluated as HIV-1 inhibitor both on wild type (wt) and drug-resistant mutants leading to the identification of an interesting compound (5d). Molecular modeling studies have been finally performed in order to rationalize the results.     

Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system: effect of diabetes

The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol), 17alpha- and 17beta-estradiol (17alpha-E and 17beta-E) by liquid chromatography and tandem mass spectrometry (LC-MS/MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17alpha-E in cerebellum, 17alpha-E, 17beta-E, DHP and THP in cerebral cortex, and 17alpha-E, 17beta-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3alpha-diol in cerebral cortex, of PROG, DHT and 3alpha-diol in spinal cord and of PREG, DHP, THP, T, DHT and 3alpha-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC-MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy.     

Synthesis of novel N1-substituted bicyclic pyrazole amino acids and evaluation of their interaction with glutamate receptors

N1-substituted bicyclic pyrazole amino acids (S)-9a-9c and (R)-9a-9c, which are conformationally constrained analogues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested for activity at ionotropic and metabotropic glutamate receptors. Some of them turned out to be selective for the NMDA receptors, where they behaved as weak antagonists. The biological activity is mainly due to the interaction with the glutamate binding site, and not with the glycine co-agonist site.     

Viral replication-independent blockade of dendritic cell maturation and interleukin-12 production by human herpesvirus 6

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive CD4(+)-T-lymphotropic betaherpesvirus that causes severe human thymocyte depletion in heterochimeric SCID-hu thy/liv mice and has been implicated as a potential cofactor in the progression of AIDS. However, the mechanisms of HHV-6-mediated immunosuppression have not yet been fully elucidated. We investigated the phenotypic and functional alterations induced by HHV-6 on peripheral blood-derived human dendritic cells (DC). The infection of DC with HHV-6 A or B was nonproductive, as revealed by calibrated real-time PCR measuring the accumulation of viral genome equivalents over time. Nevertheless, preexposure to HHV-6 markedly impaired the maturation of DC driven by gamma interferon and lipopolysaccharide, as shown by the reduced surface expression of major histocompatibility complex class I molecules, HLA-DR, CD40, and CD80. Moreover, HHV-6, but not the closely related betaherpesvirus HHV-7, dramatically suppressed the secretion of interleukin-12 (IL-12) p70 by DC, while the production of other cytokines that influence DC maturation, i.e., IL-10 and tumor necrosis factor alpha, was not significantly modified. Likewise, the secretion of the CC chemokines macrophage inflammatory protein 1beta and RANTES was unaltered. Functionally, a pretreatment with HHV-6 impaired the ability of DC to stimulate allogeneic T-cell proliferation. Altogether, these data identify interference with the functional maturation of DC as a potential mechanism of HHV-6-mediated immunosuppression.     

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.