Effects of curcumin and its adjuvant on TPC1 thyroid cell line.

Previous studies have demonstrated that different curcumin extracts are able to influence cell metabolic activity vitality in human papillary thyroid carcinoma TPC-1 cells. We continued the study using the most effective extract and adding other nutraceuticals such as piperine and vitamin E, in order to define the possible role of these in modulating the genetic expression of cell markers and to understand the effectiveness in modulating the regression of cancer phenotype. Cells were treated with one extract of curcumin (Naturex® Ultimate Botanical Benefits), with Piperine (Piper Longum, A.C.E.F.) and Vitamin E (Dry Vitamin E-Acetate 50% DC, BASF) alone and in combination, dissolved in the culture medium, for 48 h. Treatment with the different nutraceuticals is able to influence cell cycle regulators (cyclin D1, ß-catenin, p21, p53) and activators or inhibitors of apoptosis (BAX, pro-caspase3, Bcl-2). They are able to influence cell cycle distribution and metabolic activity vitality. The inhibitory effect of curcumin, piperine and vitamin E on cell proliferation involves different markers, and in particular inhibits ß-catenin, cyclinD1 and p53, making them candidates for a possible use in alternative therapies although further studies are needed.

Effects of different extracts of curcumin on TPC1 papillary thyroid cancer cell line.

BACKGROUND: The thyroid gland is one of the largest endocrine glands in the body. The vast majority of TCs (> 90%) originate from follicular cells and are defined as differentiated thyroid cancers (DTC) and the two histological subtypes are the papillary TC with its variants and the follicular TC. Curcumin possesses a wide variety of biological functions, and thanks to its properties, it has gained considerable attention due to its profound medicinal values (Prasad, Gupta, Tyagi, and Aggarwal, Biotechnol Adv 32:1053-1064, 2014). We have undertaken the present work in order to define the possible role of curcumin in modulating the genetic expression of cell markers and to understand the effectiveness of this nutraceutical in modulating the regression of cancer phenotype. METHODS: As a template we used the TPC-1 cells treated with the different extracts of turmeric, and examined the levels of expression of different markers (proliferative, inflammatory, antioxidant, apoptotic). RESULTS: Treatment with the three different curcumin extracts displays anti-inflammatory, antioxidant properties and it is able to influence cell cycle with slightly different effects upon the extracts. Furthermore curcumin is able to influence cell metabolic activity vitality. CONCLUSIONS: In conclusion curcumin has the potential to be developed as a safe therapeutic but further studies are needed to verify its antitumor ability in vivo.

Synergistic Interplay between Curcumin and Polyphenol-Rich Foods in the Mediterranean Diet: Therapeutic Prospects for Neurofibromatosis 1 Patients.

Neurofibromas are the hallmark lesions in Neurofibromatosis 1 (NF1); these tumors are classified as cutaneous, subcutaneous and plexiform. In contrast to cutaneous and subcutaneous neurofibromas, plexiform neurofibromas can grow quickly and progress to malignancy. Curcumin, a turmeric-derived polyphenol, has been shown to interact with several molecular targets implicated in carcinogenesis. Here, we describe the impact of different dietary patterns, namely Mediterranean diet (MedDiet) compared to the Western diet (WesDiet), both with or without curcumin, on NF1 patients' health. After six months, patients adopting a traditional MedDiet enriched with 1200 mg curcumin per day (MedDietCurcumin) presented a significant reduction in the number and volume of cutaneous neurofibromas; these results were confirmed in subsequent evaluations. Notably, in one patient, a large cranial plexiform neurofibroma exhibited a reduction in volume (28%) confirmed by Magnetic Resonance Imaging. Conversely, neither unenriched MedDiet nor WesDiet enriched with curcumin exhibited any significant positive effect. We hypothesize that the combination of a polyphenol-rich Mediterranean diet and curcumin was responsible for the beneficial effect observed on NF1. This is, to the best of our knowledge, the first experience with curcumin supplementation in NF1 patients. Our report suggests that an integrated nutritional approach may effectively aid in the management of NF1.

Effects of low-carbohydrate diet therapy in overweight subjects with autoimmune thyroiditis: possible synergism with ChREBP.

The thyroid is one of the metabolism regulating glands. Its function is to determine the amount of calories that the body has to burn to maintain normal weight. Thyroiditides are inflammatory processes that mainly result in autoimmune diseases. We have conducted the present study in order to have a clear picture of both autoimmune status and the control of body weight. We have evaluated the amount of either thyroid hormones, or antithyroid, or anti-microsomal, or anti-peroxidase antibodies (Abs) in patients with high amounts of Abs. In a diet devoid of carbohydrates (bread, pasta, fruit, and rice), free from goitrogenic food, and based on body mass index, the distribution of body mass and intracellular and extracellular water conducted for 3 weeks gives the following results: patients treated as above showed a significant reduction of antithyroid (-40%, P<0.013), anti-microsomal (-57%, P<0.003), and anti-peroxidase (-44%, P<0,029) Abs. Untreated patients had a significant increase in antithyroid (+9%, P<0.017) and anti-microsomal (+30%, P<0.028) Abs. Even the level of anti-peroxidase Abs increased without reaching statistical significance (+16%, P>0064). With regard to the body parameters measured in patients who followed this diet, reduction in body weight (-5%, P<0.000) and body mass index (-4%, P<0.000) were observed. Since 83% of patients with high levels of autoantibodies are breath test positive to lactase with a lactase deficit higher than 50%, this fact led us to hypothesize a correlation with carbohydrate-responsive element-binding protein and therefore a possible role of carbohydrate metabolism in the development and maintenance of autoimmune thyroiditis associated with body weight increase and slower basic metabolism.

A novel diagnostic method to detect truncated neurofibromin in neurofibromatosis 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic condition caused by dominant loss-of-function mutations of the tumor suppressor gene NF1 that encodes neurofibromin, a negative regulator of RAS activity. Mutation analysis of NF1 located at 17q11.2 has been hampered by the large size of the gene, the high rate of new mutations, the lack of mutational clustering, and the presence of several homologous loci. To date, about 80% of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the protein level. We evaluated a novel diagnostic method to detect truncated forms of neurofibromin in a large cohort of unrelated subjects suspected of having NF1, according to the NIH consensus criteria. Western blot analysis was carried out on protein extracts from patients' leukocytes to highlight the possible presence of altered neurofibromin as a result of mutations in NF1. Truncated neurofibromin was identified in 274/336 patients (81%), confirming the usefulness and reproducibility of the proposed diagnostic approach. Our methodology can be routinely applied in the diagnostic setting, thanks to its simplicity and reliability. Combined with molecular approaches, it may increase the accuracy and efficiency of NF1 genetic testing. We evaluated a novel diagnostic method to detect truncated forms of neurofibromin in patients fulfilling the clinical criteria for Neurofibromatosis 1. Western blot analysis identified truncated neurofibromin in 274/336 patients (81%). Our results indicate that the proposed technique is cheap and reliable, and could ideally be performed as a preliminary biochemical screening before molecular analysis of the NF1 gene.

Regulation of flavin-containing mono-oxygenase (Fmo3) gene expression by steroids in mice and humans.

Abstract Flavin-containing mono-oxygenases (FMOs) are a family of microsomal chemical- and drug-metabolizing enzymes. FMO3 is a major FMO form in adult mouse and human liver. FMO3 mutations have been associated with the incidence and severity of trimethylaminuria (TMAU), a metabolic disorder characterized by the inability of the affected individual to metabolize the odorous trimethylamine to its non-odorous N-oxide. In addition to this primary genetic form, there are other forms of TMAU that support the hypothesis that FMO3 activity may be modulated by steroid hormones. To understand the molecular mechanism involved in the regulation of Fmo3 gene expression by steroid hormones, we performed this study in an in vitro cellular system, mouse liver cells, and on the human FMO3 gene. Dexamethasone, 5α-dihydrotestosterone, thyroid hormone, and progesterone had no effect on the accumulation of Fmo3 mRNA. The use of increased concentration of theophylline inhibited estrogen receptor α (ERα)-mediated transcription of Fmo3 mRNA. 17ß-Estradiol inhibited Fmo3 mRNA accumulation. The use of ICI 164,384 abolished the inhibitory effect induced by estrogen. Gel-shift analyses showed a binding in the 5' region of the Fmo3 gene. This binding was abrogated by an excess of a cDNA containing an estrogen-responsive element. An estrogen-binding site was also present in the first intron of the human gene, as demonstrated by the gel-shift assay. Supershift experiments confirmed the binding of ERα in both mouse and human samples. Furthermore, chromatin immunoprecipitation assay confirmed the binding of ERα in the promoter region of mouse Fmo3 and in the first intron of the human FMO3 gene. Thus, 17ß-estradiol plays a fundamental role in the regulation of Fmo3 gene transcription.

FMO3 allelic variants in Sicilian and Sardinian populations: trimethylaminuria and absence of fish-like body odor.

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria. The aim of this study was to determine the allelic and genotypic distributions of these four FMO3 variants in 528 healthy individuals collected from the Sicilian and Sardinian populations together with haplotype and linkage analyses. Finally, we present data on the genotype-phenotype correlation by ESI-MS/MS TMA/TMAO urinary determination in 158KK/308EG individuals. Variant 158K shows the same frequency in Sicilian and Sardinian populations while variant 257M was not observed in the Sardinian sampling. No significant differences were found for 308G and 475D variants among two populations. Cis-linkage between 158K and 308G was confirmed with the compound variant (158K-308G) being found in a proportion of 0.9% and 0.3% of Sicilian subjects, and 0.01% and 0.5% in Sardinian population. Urinary determination of TMA/TMAO ratio in 158KK/308EG individuals showed a considerable reduction in FMO3 activity although they do not show the classical features of trimethylaminuria as a strong body odor and breath. Our data support the conclusion that trimethylaminuria is not always accompanied by a fish-like odor, despite the coexistence in the same individual of the two variants 158K and 308G, and other factors account for the expression of that phenotype.

Hormonal regulation and characterisation of the aldehyde oxidase-like gene of hamster Harderian gland.

The HG is a compound tubulo-alveolar gland located in the orbital cavity of the majority of vertebrates. In the golden hamster it shows a clear cut sexual dimorphism in both morphological and biochemical parameters such as cell types, protein pattern, lipid metabolism, porphyrin content, steroid hormone receptor expression. In a previous study we found that in primary culture of male hamster Harderian gland (HG), androgens (A) increase the MHG07 (male Harderian gland) expression and this effect is abrogated by both flutamide and cycloheximide. The present study represents a deeper analysis on MHG07 regulation by other members of steroid/thyroid hormone superfamily. Estrogens (E) impair the stimulatory effect of A and after the addition of a pure anti-estrogen, ICI 164,384, the negative effect of E is abrogated. Dexamethasone (Dex), used alone or in combination with A negatively affect the MHG07 expression. Also T(3) increases the expression of MHG07 mRNA. Progesterone (P) does not affect the expression of MHG07 mRNA. The use of cycloheximide abrogates the effect of steroids, suggesting that the latter act through their own receptors. Dose-response experiments show that low steroid concentrations (10(-12)M) are sufficient to affect the MHG07 expression. It is argued that the expression of MHG07 is under a highly coordinate relationship between androgen, estrogen, glucocorticoid, retinoic acid and thyroid hormones.

A classical Mendelian cross-breeding study of the Naples high and low excitability rat lines.

The development of brain and behaviour is controlled by the interaction of genetic determinants and environmental factors. To study genetic determinants, model systems such as the Naples rat lines, i.e. Naples high (NHE) and low excitability (NLE), are useful. They have been selectively bred for divergent behaviour arousal to novelty. Aim of this study was to assess the extent of the genetic control of the selection trait. Thus adult albino rats of NHE and NLE lines have been used throughout. According to a classical Mendelian cross-breeding design, a first experiment was carried out with hybrids obtained from parental lines P1 (NHE) and P2 (NLE) as F1, F2 and related backcrosses B1 (F1xP1) and B2 (F1xP2). Young adults (60-80 days) offspring of both gender were exposed separately for two 10min tests to a spatial novelty (Lát-maze). To verify a possible sex link of the trait, a second experiment was carried out adding to the Mendelian cross design parental gender. Behavioural variables were horizontal (corner-crossings: HA), vertical (rearings on hindlimbs: VA) or total activity (HVA: HA+VA) scores. The heritability of HVA trait was estimated across the 20 generations of selection and Mendelian cross hybrids. Quantitative-genetic analysis on this trait and its HA and VA components, was applied by the Lynch and Walsh joint-scaling test procedure to evaluate underlying genetic mechanism. The correlation between experimental data of hybrids and estimated values from different heritability models were also computed. Results indicated that (i) the activity scores by Mendelian hybrids were intermediate between the two parental lines and were also graded; (ii) there was no sex effect on the heritability of trait but only a general tendency of females to higher activity levels; (iii) the heritability of HVA trait was very high (h2 index=0.824); (iv) heritability model of HVA and HA trait was polygenic with a marked epistatic control where as VA trait was fitted by simpler model with less genes and lower epistatic effect. In conclusion the Naples lines reveal strong genetic determinants for behavioural traits associated with polygenic pattern. Moreover, HA and VA activity components with prevailing cognitive and non-cognitive meaning, respectively, show differential genetic control.

Sequence analysis of retinoic acid receptor alpha, beta and gamma isoforms in the lizard, Podarcis sicula.

Vitamin A and its principal biologically active derivative, retinoic acid (RA), play a fundamental role in diverse processes, such as proliferation, differentiation, morphogenesis, metabolism and apoptosis of many types of cells. In addition, RA has been shown to be involved in the regulation of testicular function. These effects are mediated by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), each with three subtypes alpha, beta and gamma. The physiological involvement of retinoids in testicular function has been conducted mainly in mammals. Recently, we found that exogenous all-trans-retinoic acid impairs spermatogenesis and enhance testicular germ cell apoptosis in the lizard, Podarcis sicula, a seasonal breeder. To further investigate the role of retinoic acid in lizard, we focus this work principally on the characterization of lizard retinoic acid receptors (alpha, beta and gamma isoforms). RARalpha is 2720 bp long with a putative ORF between 699 and 2133. A Kozac sequence is present at 696 and a putative poly-adenilation site is present in position 2612. The RARalpha sequence shares 87% homology with mouse RARalpha mRNA while it has 76 and 80% homology with lizard RARbeta and gamma mRNAs. RARbeta is 2478 bp long showing a putative ORF between 196 and 1543. A canonical Kozac sequence is present at 193 and a putative poly-adenilation site is present at 2294. RARbeta shares 91% homology with mouse RARbeta mRNA and has 76% homology with both RARalpha and gamma. RARgamma is 2416bp long. With a putative ORF between 444 and 1818. A Kozac sequence is present at 441 and a putative poly-adenilation site is present at 2288. RARgamma shares 86% homology with mouse RARbeta mRNA and has 80 and 76% homology with both RARalpha and beta respectively. It is worth to note that, as in mouse, the 5'UTR of all isoforms is TATA and CAAT less. Both Northern blot and PCR analyses indicate that lizard testis expresses only RARalpha and RARbeta mRNAs, while RARgamma mRNA transcript was not found. In the period analysed, RARbeta was expressed during the gonadal full activity (May) and RARalpha was present in the post-reproductive period (August). During the autumnal recrudescence (October) RARalpha and RARbeta are co-expressed and, as indicated by quantitative PCR analysis, RARbeta mRNA levels are lower than RARalpha ones. Thus, the appearance and abundance of each receptor correspond to a specific phase of lizard reproductive cycle, allow us to hypothesize that each RAR subtype could play a specific role in the regulation of spermatogenetic activity. The results of the present study show, for the first time, the characterization of RAR mRNAs in the testis of lizard P. sicula, whose expression is related to the different phase of reproductive cycle. Moreover, the gamma form, is principally expressed in the skin during the March-July period, having probably a role in regulating skin homeostasis and colour livery, which are important factor in mating during the reproductive cycle.

Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula.

In mammals, retinoic acid is involved in the regulation of testicular function by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Among RAR isoforms, the testicular cells of the lizard were found to express only RARalpha (3.7 kb) and RARbeta (3.4 kb) mRNAs, as reported here. In this study, the effects of exogenous all-trans-retinoic acid (atRA) on spermatogenesis of a non-mammalian seasonal reproducer were investigated. Daily intraperitoneal injections of atRA or atRA plus testosterone (atRA+T) were given for 2 weeks to adult males of the lizard Podarcis sicula. In animals treated with atRA, the seminiferous tubules were markedly reduced in cross-area. The seminiferous epithelium collapse was responsible for a sensible reduction in the number of germ cells and disruption in normal epithelial organization. In comparison, in atRA+T-treated lizards the loss of germinal cells was significantly less. The loss of germ cells observed in both experimental groups results from an induction of apoptotic process, as revealed by TUNEL analysis. Although low in number, apoptotic germ cells were also observed in the control groups (saline- and T-treated lizard), where the main germ cells undergoing apoptosis are primary spermatocytes (most frequently) and some spermatogonia. In conclusion, it is shown here that retinoic acid has deleterious effects on lizard spermatogenesis, causing a severe depletion of seminiferous epithelium, probably via induction of apoptotic processes. These effects are not completely inhibited by simultaneous administration of testosterone, although this hormone, once injected, is able to stimulate spermatogenesis and protect germinal cells from apoptotic cell death.

The hamster androgen receptor promoter: a molecular analysis.

The steroid/thyroid hormone receptors are members of a very large family of nuclear-activated transcription factors. These receptors play a crucial role in most biological function, including regulation of development, metabolism, behaviour and reproduction. Among androgen receptor (AR), we have recently demonstrated that its expression in the Harderian gland (HG) of the male hamster is under a well-co-ordinated cross-talk between various steroid hormone receptors. Here, are presented data on the sequence of hamster AR promoter region (5'UTR) and the molecular tools of its regulation. The 5'UTR is 1585 bp. The promoter region shows various responsive elements. Two putative CREM elements are present at -71 and -1576 bp. A putative retinoic acid responsive element is present at -1476 bp. An androgen/glucocorticoid responsive element is present at -473 bp. A putative thyroid hormone-responsive element at -381 bp and an estrogen responsive element at -230 bp. Also, a homopurinic stretch is evident between -1199 and -1118. Furthermore, Sp1 sites are also spread along the sequence. As well as for human, mouse, rat and pig, the hamster lacks the canonical promoter TATA and CCAAT boxes. Gel retardation experiments confirm the presence of active responsive elements for AR, estrogen receptor, glucocorticoid receptor and thyroid hormone receptor. Previous data on the regulation of expression of AR by other members of steroid/thyroid hormone receptors well correlate with sequence analysis and gel retardation experiments. Thus, androgens, thyroid hormone, stimulate the AR transcription, while synthetic glucocorticoid (Dex) and estrogen are potent inhibitors of AR expression. The comparison of hamster AR promoter sequence with other AR promoter shows an 89, 82, 84 and 84% identity with human, rat, mouse and pig AR promoter, respectively. These results, in the light of the extreme plasticity of hamster HG, suggest that the comparative study of expression and regulation of AR gene in the HG of the hamster offers a useful tool to approach the normal and pathological phenotype in human.

Estrogen receptor beta localization in the lizard (Podarcis s. sicula) testis.

There is increasing evidence that 17beta-estradiol is necessary for normal male fertility. The aim of the present study was to characterize estrogen receptor beta (ERbeta) expression in a non-mammalian vertebrate model, the lizard (Podarcis s. sicula) testis. Immunocytochemical analysis shows that ERbeta proteins are present among germ cells in the nucleus of the spermatogonia, in primary spermatocytes and spermatids. Western blot analysis with antibodies against the ERbeta gene product revealed an isoform with a specific weight of 55 kDa. In conclusion, the widespread expression of ERbeta in the Podarcis s. sicula testis is consistent with a role for estrogens in modulating spermatogenesis in the male.