Giovanni Vanni Frajese

The Inhibition of the Highly Expressed Mir-221 and Mir-222 Impairs the Growth of Prostate Carcinoma Xenografts in Mice

Background MiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity. Methodology/Principal Findings Here we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression. Conclusions/Significance These findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.

In Vitro and in Vivo Antitumoral Effects of Combinations of Polyphenols, or Polyphenols and Anticancer Drugs: Perspectives on Cancer Treatment

Carcinogenesis is a multistep process triggered by genetic alterations that activate different signal transduction pathways and cause the progressive transformation of a normal cell into a cancer cell. Polyphenols, compounds ubiquitously expressed in plants, have anti-inflammatory, antimicrobial, antiviral, anticancer, and immunomodulatory properties, all of which are beneficial to human health. Due to their ability to modulate the activity of multiple targets involved in carcinogenesis through direct interaction or modulation of gene expression, polyphenols can be employed to inhibit the growth of cancer cells. However, the main problem related to the use of polyphenols as anticancer agents is their poor bioavailability, which might hinder the in vivo effects of the single compound. In fact, polyphenols have a poor absorption and biodistribution, but also a fast metabolism and excretion in the human body. The poor bioavailability of a polyphenol will affect the effective dose delivered to cancer cells. One way to counteract this drawback could be combination treatment with different polyphenols or with polyphenols and other anti-cancer drugs, which can lead to more effective antitumor effects than treatment using only one of the compounds. This report reviews current knowledge on the anticancer effects of combinations of polyphenols or polyphenols and anticancer drugs, with a focus on their ability to modulate multiple signaling transduction pathways involved in cancer.

Neurosteroid biosynthetic pathways changes in prefrontal cortex in Alzheimer's disease

Expression of the genes for enzymes involved in neurosteroid biosynthesis was studied in human prefrontal cortex (PFC) in the course of Alzheimers disease (AD) (n=49). Quantitative RT-PCR (qPCR) revealed that mRNA levels of diazepam binding inhibitor (DBI), which is involved in the first step of steroidogenesis and in GABAergic transmission, were increased, as were mRNA levels for several neurosteroid biosynthetic enzymes. Aromatase, 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and aldo-keto reductase 1C2 (AKR1C2), were all increased in the late stages of AD. Several GABA-A subunits were significantly reduced in AD. Increased expression of aromatase in the PFC was confirmed by immunohistochemistry and was found to be localized predominantly in astrocytes. These data suggest a role for estrogens and allopregnanolone produced by astrocytes in the PFC in AD, possibly as part of a rescue program. The reduced gene expression of some synaptic and extra-synaptic GABA-A subunits may indicate a deficit of modulation of GABA-A receptors by neuroactive steroids, which may contribute to the neuropsychiatric characteristics of this disease.

Ontogenesis of Leptin Receptor in Rat Leydig Cells

Abstract There are still many controversies about the role of leptin in reproductive function and sexual development. We recently demonstrated that leptin receptors are expressed in rodent Leydig cells and that leptin has inhibitory effects on hCG-stimulated testosterone production by adult rat Leydig cells in culture. In this study, we evaluated the expression of leptin receptor (Ob-R) in rat testes from gestational to adult age in comparison with the pattern of expression of relaxin-like factor (RLF), a specific marker of Leydig cell differentiation status. Immunohistochemical analysis showed that, in prenatal life, Ob-R immunoreactivity was absent at early embryonic ages (E14.5) and appeared at a late embryonic age (E19.5); in postnatal life, immunoreactivity was evident only after sexual maturation (35-, 60-, and 90-days old), whereas it was absent in testes from sexually immature rats (7-, 14-, and 21-days old). Immunoreaction was always confined to Leydig cells and no signal of Ob-R was detected within the tubules. The pattern of expression of Ob-R during testicular development was similar with that of RLF immunoreactivity, which was present in mature fetal as well as adult-type Leydig cells. In contrast with the findings in the testis, in the hypothalamus, the immunohistochemical pattern of Ob-R was very similar between pre- and postpubertal life. Reverse transcription-polymerase chain reaction studies showed that Ob-R expression was present in embryonic, prepubertal, and adult rat testes; semiquantitative analysis showed that mRNA levels were much higher in late versus early embryonic testes, as well as in mature adults versus sexually immature testes, with a gradual increase from younger to older ages. Functional studies showed that, while leptin (150 ng/ml) significantly inhibited hCG-stimulated testosterone production in adult rat Leydig cells (46% reduction; P > 0.01), it did not modify prepubertal rat Leydig cells steroidogenic function in vitro. In conclusion, we showed that, in rat testis, Ob-R expression is characteristic of mature Leydig cells (fetal and adult type) and it is functional in adult but not prepubertal life.

Dipeptidyl peptidase 4 (DPP-4) inhibitors and their role in Type 2 diabetes management

Dipeptidyl peptidase 4 (DPP-4) inhibitors are a new pharmacological class of drugs for treating Type 2 diabetes. They improve the capacity of the organism to control glycemia by increasing the levels of active incretins. Their mechanism of action is thus radically different from those of other anti-diabetic drugs currently available. DDP-4 inhibitors use a physiological mechanism to control hyperglycemia, by stimulating the secretion of insulin from β-cells, decreasing the secretion of glucagon from pancreatic α-cells, and at the same time reducing the production of glucose by the liver. DDP-4 inhibitors have shown significant efficacy in maintaining reduced levels of glycosylated hemoglobin for up to 1 year. In vitro and animal studies have shown that they can inhibit apoptosis of β-cells and favor their regeneration and differentiation. The oral DPP-4 inhibitors vildagliptin, sitagliptin, and saxagliptin are efficacious both alone and in association with other oral anti-diabetic agents and may be administered in a single daily dose. Lastly, they have substantial advantages with respect to other anti-diabetic drugs, since they involve a low risk of hypoglycemia and do not affect body weight.

Neurosteroid Biosynthetic Pathway Changes in Substantia Nigra and Caudate Nucleus in Parkinson's Disease

There is emerging evidence from animal studies for a neuroprotective role of sex steroids in neurodegenerative diseases, but studies in human brain are lacking. We have carried out an extensive study of the neurosteroid biosynthetic pathways in substantia nigra (SN), caudate nucleus (CN) and putamen (PU) of 7 Parkinsons disease (PD) patients and 7 matched controls. The mRNA levels of 37 genes including neurosteroid biosynthetic enzymes, hormone receptors and the neurosteroid‐modulated γ‐amino‐butyric acid ‐A (GABA‐A) receptor subunits were analyzed by quantitative PCR (qPCR). In the SN, we found downregulation of 5α‐reductase type 1 (5α‐R1), sulfotransferase 2B1 (SULT2B1) and some GABA‐A receptor subunits (α4, β1) while in the CN, upregulation of 3α‐hydroxysteroid dehydrogenase type 3 (3α‐HSD3) and α4 GABA‐A receptor subunit (22‐fold) was observed. No significant differences were found in the PU. These data imply an involvement of pregnane steroids and changes in GABAergic neurotransmission in the neurodegenerative process and suggest that neurosteroids may deserve further investigation as potential therapeutic agents in PD.

Growth hormone-releasing hormone and pituitary adenylate cyclase-activating polypeptide in the reproductive system

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) are both members of the glucagon superfamily that, with gonadotropins, act at central and peripheral levels as paracrine and autocrine coregulators of reproductive function. GHRH and PACAP are ancient peptides. Their original forms (both 27 amino acids long) were encoded by a single ancestral gene, several duplications of which led to the genes that encode the neuropeptides of the glucagon superfamily. In the male and female reproductive tracts, GHRH and PACAP interact with a subset of G protein-coupled receptors that are structurally similar to the PACAP receptor and variants of the vasoactive intestinal peptide receptor, and share several biological actions. These are related mainly to the modulation of cAMP-dependent and other signal transduction pathways in several cells of the pituitary-gonadal axis. The recent discovery that antagonists of GHRH and PACAP suppress the growth of human cancer cell lines that are derived from reproductive tissues indicates the potential importance of these peptides as local regulators of cell division, cell cycle arrest, differentiation and cell death.

Immunity to Extracellular Matrix Antigens is Associated with Ultrastructural Alterations of the Stroma and Stratified Epithelium Basement Membrane in the Skin of Hashimoto's Thyroiditis Patients:

Employing purified extracellular matrix (ECM) proteins, i.e. type I, III, IV and V collagens (CI, CHI, CIV, CV), laminin (LM) and fibronectin (FN), as antigen sources we detected autoantibodies to conformational and/or denatured ECM antigens among 34 of 50 sera obtained from Hashimotos thyroiditis (HT) patients and 6 of 51 control sera obtained from non-autoimmune thyroid disease patients and healthy donors (HT sera vs. control sera p=4×10−9). Reactivity to conformational antigens, mostly due to autoantibodies of the IgG isotype, was observed in 30/50 HT sera and in 6/51 control sera (p=3.5×10−7) and was not always concomitant with that to linear antigens, found in 23/50 HT and in 6/51 control sera (p=1.6×10−4). Ultrastructural analysis of skin biopsies obtained from 18 HT patients without symptomatic cutaneous diseases revealed defects of the stratified squamous epithelium basement membrane in 11/18, alterations of the stroma in 13/18 and both basement membrane and stromal defects in 9/18. Interestingly, 13/13 (p=0.012) and 9/11 (p=0.012) patients with stromal and basement membrane defects respectively, exhibited serum antibodies to at least one ECM antigen involved in the organization of the altered tissue compartment. Lastly, 10/18 skin biopsies presented immunoglobulin (Ig) and/or complement (C3) deposits along the cutaneous basement membrane zone (BMZ) or in the papillary dermis and 9/10 sera from the same patients simultaneously showed antibodies to at least one ECM antigen involved in the organization of these two skin compartments. Besides, 8/11 HT patients with basement membrane defects exhibited Ig or C3 deposits along the BMZ. Our findings suggest that autoantibodies to ECM molecules might contribute to the development of asymptomatic extra-thyroid skin diseases in HT patients.

Vitamin D (Calcifediol) Supplementation Modulates NGF and BDNF and Improves Memory Function in Postmenopausal Women: a Pilot Study

1,25-dihydroxy vitamin D (vitamin D), a steroid hormone essential for calcium metabolism and various extra skeletal functions, is an in vitro inducer of Nerve Growth Factor (NGF) and modulates Brain-Derived Neurotrophic Factor (BDNF) in neurons, while its receptor is expressed in central nervous system. Due to restricted sunlight exposure and/or dietary intake many people are vitamin D-deficient and need supplementation. Vitamin D status has been associated with impairment in cognitive functions, but up to now no clinical studies have been conducted to verify its effect on neurotrophins and memory in humans. 20 postmenopausal women with low vitamin D levels were treated by calcifediol (25-OH D3) and followed in open for three months. At baseline and after three months NGF and BDNF together with Wechsler Memory Scale (WMS) were assessed. Mean plasma vitamin D levels were 14.52 (+/-5.65 SD) ng/ml at baseline and 47.58 (+/-15.05 SD) ng/ml after three months of supplementation. At the end of the study a statistical significant decrease in both neurotrophins was observed. NGF plasma levels were respectively: 451.13 (+/-243.20 SD) pg/ml and 323.68 (+/-195.60 SD) pg/ml (p=0.05); BDNF levels were 431.10 (+/-182.21 SD) pg/ml and 366.25 (+/-142.75 SD) pg/ml (p=0.02). WMS scored meanly 97.00 (+/-13.54 SD) at baseline and 107.82 (+/-16.19 SD) after three months (p=0.003). Considering the role of vitamin D in calcium metabolism and neurotrophic factors regulation, it seems indispensable in brain function and its deficiency must be considered in cognitive diseases.

The crossroads between cancer immunity and autoimmunity: antibodies to self antigens

The production of autoantibodies to self antigens is dependent on the failure of immune tolerance. Cancer cells express antigens which elicit a spontaneous immune response in cancer patients. The repertoire of autoantibodies found in cancer patients partly covers that of patients with autoimmune diseases. Biological activities of autoantibodies to self antigens may induce paraneoplastic syndromes which reflect the attempt of cancer patients to counteract tumor growth. Autoantibodies with similar specificities may have different effects in cancer and autoimmune disease patients due to different immunological microenvironments. Tregs dysfunction has been observed in patients with paraneoplastic syndromes and/or with autoimmune diseases, while the increase of Tregs has been associated with poor cancer patients prognosis. Novel therapies have employed antibodies against Tregs immune-checkpoint receptors with the aim to boost immune response in cancer patients. The presence of autoantibodies to tumors antigens has also been investigated as a marker for cancer detection and cancer patients prognosis. This report reviews the current knowledge on the analysis and meaning of autoantibodies to self antigens detected in cancer and autoimmune disease patients.