Biological activities of the aerial and undergound parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) from the Italian Alps.

This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 µg/µL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright's staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied.

Curcumin nanoformulations for antimicrobial and wound healing purposes.

The development and spread of resistance to antimicrobial drugs is hampering the management of microbial infectious and wound healing processes. Curcumin is the most active and effective constituent of Curcuma longa L., also known as turmeric, and has a very long and strong history of medicinal value for human health and skincare. Curcumin has been proposed as strong antimicrobial potentialities and many attempts have been made to determine its ability to conjointly control bacterial growth and promote wound healing. However, low aqueous solubility, poor tissue absorption and short plasma half-life due its rapid metabolism needs to be solved for made curcumin formulations as suitable treatment for wound healing. New curcumin nanoformulations have been designed to solve the low bioavailability problem of curcumin. Thus, in the present review, the therapeutic applications of curcumin nanoformulations for antimicrobial and wound healing purposes is described.

Preclinical Pharmacological Activities of Epigallocatechin-3-gallate in Signaling Pathways: An Update on Cancer.

Epigallocatechin gallate (EGCG) is the main bioactive component of catechins predominantly present in svarious types of teas. EGCG is well known for a wide spectrum of biological activity as an anti-oxidative, anti-inflammatory, and anti-tumor agent. The effect of EGCG on cell death mechanisms via the induction of apoptosis, necrosis, and autophagy has been documented. Moreover, its anti-proliferative and chemopreventive action has been demonstrated in many cancer cell lines. It was also involved in the modulation of cyclooxygenase-2, in oxidative stress and inflammation of different cell processes. EGCG has been reported as a promising target for plasma membrane proteins, such as epidermal growth factor receptor (EGFR). In addition, it has been demonstrated a mechanism of action relying on the inhibition of ERK1/2, p38 MAPK, NF-κB, and vascular endothelial growth factor (VEGF). EGCG and its derivatives were used in proteasome inhibition and they were involved in epigenetic mechanisms. In summary, EGCG is the most predominant and bioactive constituent of teas and it has a pivotal role in cancer prevention. Its preclinical pharmacological activities are associated with complex molecular mechanisms that involve numerous signaling pathways.

Liposomal Cytarabine as Cancer Therapy: From Chemistry to Medicine.

Cancer is the second leading cause of death worldwide. The main modality to fight against cancer is surgery, radiotherapy, and chemotherapy, and more recently targeted therapy, gene therapy and immunotherapy, which play important roles in treating cancer patients. In the last decades, chemotherapy has been well developed. Nonetheless, administration of the drug is not always successful, as limited drug dosage can reach the tumor cells.. In this context, the possibility to use an encapsulated anti-cancer drug may potentially solve the problem. Liposomal cytarabine is a formulation with pronounced effectiveness in lymphomatous meningitis and reduced cardiotoxicity if compared to liposomal anthracyclines. Thus, the future liposomal cytarabine use could be extended to other diseases given its reduction in cytotoxic side effects compared to the free formulation. This review summarizes the chemistry and biology of liposomal cytarabine, with exploration of its clinical implications.

Biological Effects of EF24, a Curcumin Derivative, Alone or Combined with Mitotane in Adrenocortical Tumor Cell Lines.

BACKGROUND: Curcumin has numerous properties and is used in many preclinical conditions, including cancer. It has low bioavailability, while its derivative EF24 shows enhanced solubility. However, its effects have never been explored in adrenocortical tumor cell models. The efficacy of EF24 alone or combined with mitotane (reference drug for adrenocortical cancer) was evaluated in two adrenocortical tumor cell lines, SW13 and H295R. METHOD AND RESULTS: EF24 reduced cell viability with an IC50 (half maximal inhibitory concentration) of 6.5 ± 2.4 µM and 4.9 ± 2.8 µM for SW13 and H295R cells, respectively. Combination index (EF24 associated with mitotane) suggested an additivity effect in both cell lines. Cell cycle analysis revealed an increase in subG0/G1 phase, while motility assay showed a decrease in migratory cell capacity, and similarly, clonogenic assay indicated that EF24 could reduce colony numbers. Furthermore, Wnt/ß-catenin, NF-κB, MAPK, and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with EF24 alone or combined with mitotane. In addition, intracellular reactive oxygen species levels increased in both cell lines. CONCLUSION: This work analyzed EF24 in adrenocortical tumor cell lines for the first time. These results suggest that EF24 could potentially impact on adrenocortical tumors, laying the foundation for further research in animal models.

Investigation of the molecular reactivity of bioactive oxiranylmethyloxy anthraquinones.

The design of a multitarget and multifunctional small molecule containing two functional groups reacting through different mechanisms represents an attractive goal for the medicinal chemist. The preparation of two bifunctional oxiranylmethyloxy anthraquinones, previously investigated as anticancer agents, is described here. These compounds combine a planar, DNA-intercalating and pro-oxidant anthraquinone scaffold and the alkylating epoxide functions which can covalently react with the nucleic acid. Their multilevel molecular reactivity was studied through a combination of analytical techniques: The DNA-binding properties were investigated using a mass spectrometry-based binding assay and by nuclear magnetic resonance, highlighting the formation of a covalent adduct with a nucleobase. Moreover, the contribution of the pro-oxidant redox cycling was evaluated.

Crude extract of Origanum vulgare L. induced cell death and suppressed MAPK and PI3/Akt signaling pathways in SW13 and H295R cell lines.

Oregano (Origanum vulgare L.) is a common aromatic plant used in Mediterranean and Asian Regions for treating respiratory diseases, painful menstruation, rheumatoid arthritis, etc. Recently its role as an anticancer plant has been suggested, although oregano has been never evaluated into adrenocortical tumour cell models. This study analysed for the first time the anticancer effects of a crude extract of wild mountain oregano (Origanum vulgare L.) in SW13 and H295R cell lines. The crude extract was characterised by GC/MS and the toxic effects of oregano were first analysed by brine shrimp lethality assay. Our findings demonstrated that oregano decreased cell viability, survival, modified cell cycle and induced cell death (through necrotic process) and that the effects can be attributed to a blockade of MAPK and PI3 K/Akt pathways. These results suggest that oregano extract exerts anticancer activities in adrenocortical tumour cell lines, providing evidence for further research in higher models.

Anticancer Effects of Wild Mountain Mentha longifolia Extract in Adrenocortical Tumor Cell Models.

Mint [Mentha longifolia (L.) Hudson] is an aromatic plant that belongs to Lamiaceae family. It is traditionally used as herbal tea in Europe, Australia and North Africa and shows numerous pharmacological effects, such as spasmolytic, antioxidant, antimicrobial and anti-hemolytic. Recently, its antiproliferative role has been suggested in a small number of tumor cell models, but no data are available on adrenocortical carcinoma, a malignancy with a survival rate at 5 years of 20%-30% which frequently metastasize. This work aimed to study the effects of Mentha longifolia L. crude extract (ME) on two adrenocortical tumor cell models (H295R and SW13 cells). Chemical composition of ME was assessed by gas-chromatography/mass spectrometry and NMR spectroscopy analysis. Brine shrimp lethality assay showed ME effects at >0.5 µg/µl (p < 0.05). Cell viability and vitality were determined by MTT, SRB, and trypan blue assays in H295R and SW13 cells. The anti-proliferative effects of ME were more evident in SW13 cells at 72 h (ME > 0.5 µg/µl, p < 0.05). Combination of ME with mitotane (approved drug for adrenocortical carcinoma) seemed not to reinforce the efficacy of the herb. As control, human fibroblasts were treated with ME with no effect on cell viability. Clonogenic assay was concordant with previous cell viability tests (ME > 0.5 µg/µl, p < 0.05), while Wright staining demonstrated the presence of both necrotic and apoptotic cells. Cell cycle analysis showed a strong increase in subG0/G1 phase, related to cell death. Furthermore, MAPK and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with ME alone or combined with mitotane. The crude methanolic extract of wild mountain mint can decrease cell viability, vitality and survival of adrenocortical tumor cell models, in particular of SW13 cells. These data show the potential anticancer effects of ME, still more work is needed to corroborate these findings.

Antiproliferative activity of Juglone derivatives on rat glioma.

Malignant gliomas are aggressive and life-threatening tumours that still show a poor prognosis: the current therapeutic approach based on surgical resection and chemotherapy combined with radiotherapy does not provide a satisfactory chance of long-term survival to patients. Natural bioactive compounds represent a precious source of molecules with antiproliferative activity, potentially effective also against glioma cells. Among these, Juglone is a known allelopathic compound extracted from the eastern black walnut (Juglans nigra) whose antimitotic effect has been extensively described in mammalian cells. We investigated the antiproliferative effect of a synthetic derivative of this natural compound, 2-(2,4-dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone (DiNAF), in rat glioma cells. We compared this molecule and its effect with the natural reference compound and with newly synthesised derivatives to build a preliminar structure-activity relationship. Biological assays and NMR-based redox experiments confirmed that DiNAF is a promising lead and supported the hypothesis of a redox mechanism underlying its cytotoxic activity.

Investigation of N-cadherin/ß-catenin expression in adrenocortical tumors.

ß-catenin is a multifunctional protein; it is a key component of the Wnt signaling, and it plays a central role in cadherin-based adhesions. Cadherin loss promotes tumorigenesis by releasing membrane-bound ß-catenin, hence stimulating Wnt signaling. Cadherins seem to be involved in tumor development, but these findings are limited in adrenocortical tumors (ACTs). The objective of this study was to evaluate alterations in key components of cadherin/catenin adhesion system and of Wnt pathway. This study included eight normal adrenal samples (NA) and 95 ACT: 24 adrenocortical carcinomas (ACCs) and 71 adrenocortical adenomas (ACAs). ß-catenin mutations were evaluated by sequencing, and ß-catenin and cadherin (E-cadherin and N-cadherin) expression was analyzed by quantitative reverse transcription PCR (qRT-PCR) and by immunohistochemistry (IHC). We identified 18 genetic alterations in ß-catenin gene. qRT-PCR showed overexpression of ß-catenin in 50 % of ACC (12/24) and in 48 % of ACA (21/44). IHC data were in accordance with qRT-PCR results: 47 % of ACC (7/15) and 33 % of ACA (11/33) showed increased cytoplasmic or nuclear ß-catenin accumulation. N-cadherin downregulation has been found in 83 % of ACC (20/24) and in 59 % of ACA (26/44). Similar results were obtained by IHC: N-cadherin downregulation was observed in 100 % (15/15) of ACC and in 55 % (18/33) of ACA. ß-catenin overexpression together with the aberrant expression of N-cadherin may play important role in ACT tumorigenesis. The study of differentially expressed genes (such as N-cadherin and ß-catenin) may enhance our understanding of the biology of ACT and may contribute to the discovery of new diagnostic and prognostic tools.

The aurora kinase inhibitor VX-680 shows anti-cancer effects in primary metastatic cells and the SW13 cell line.

New therapeutic targets are needed to fight cancer. Aurora kinases (AK) were recently identified as vital key regulators of cell mitosis and have consequently been investigated as therapeutic targets in preclinical and clinical studies. Aurora kinase inhibitors (AKI) have been studied in many cancer types, but their potential capacity to limit or delay metastases has rarely been considered, and never in adrenal tissue. Given the lack of an effective pharmacological therapy for adrenal metastasis and adrenocortical carcinoma, we assessed AKI (VX-680, SNS314, ZM447439) in 2 cell lines (H295R and SW13 cells), 3 cell cultures of primary adrenocortical metastases (from lung cancer), and 4 primary adrenocortical tumor cell cultures. We also tested reversan, which is a P-gp inhibitor (a fundamental efflux pump that can extrude drugs), and we measured AK expression levels in 66 adrenocortical tumor tissue samples. Biomolecular and cellular tests were performed (such as MTT, thymidine assay, Wright's staining, cell cycle and apoptosis analysis, Western blot, qRT-PCR, and mutation analysis). Our results are the first to document AK overexpression in adrenocortical carcinoma as well as in H295R and SW13 cell lines, thus proving the efficacy of AKI against adrenal metastases and in the SW13 cancer cell model. We also demonstrated that reversan and AKI Vx-680 are useless in the H295R cell model, and therefore should not be considered as potential treatments for ACC. Serine/threonine AK inhibition, essentially with VX-680, could be a promising, specific therapeutic tool for eradicating metastases in adrenocortical tissue.

Mitogen-Activated Protein Kinase Pathway: Genetic Analysis of 95 Adrenocortical Tumors.

Mitogen-activated protein kinase (MAPK) pathway is often deregulated in adrenocortical tumors (ACT) but with no concrete data confirming alteration rate. The objective of this study was to evaluate genetic alterations in key components of MAPK pathway. We found one BRAF mutation (p.V600E) and four HRAS silent mutations. No alteration was found in NRAS, KRAS, EGFR genes. The patient carrying BRAF mutation was further characterized by investigating his biomolecular and clinico-pathological findings. Therefore, even if MAPK signaling is activated in ACT, our results suggest that genetic alterations do not seem to represent a frequent mechanism of ACT tumorigenesis.

Semi-synthetic derivatives of natural isoflavones from Maclura pomifera as a novel class of PDE-5A inhibitors.

Natural (iso)flavonoids have been recently reported to inhibit cyclic nucleotide phosphodiesterases (PDEs) and induce vasorelaxation, albeit the results described in the literature are discordant. The cGMP-selective isoform PDE-5A, in particular, represents the target of sildenafil and its analogues in the treatment of erectile dysfunction (ED) and pulmonary hypertension by promoting relaxation in vascular smooth muscle through the activation of the NO/cGMP pathway. We undertook this study to verify if osajin and pomiferin, two natural prenylated isoflavones and major constituents of Maclura pomifera extracts previously investigated for their anticancer, antibacterial and antidiabetic properties, show inhibitory activity on PDE-5A. These two isoflavones were isolated from the plant extracts and then synthetically modified to obtain a set of semi-synthetic derivatives with slight and focused modifications on the natural scaffold. The compounds were at first screened against PDE-5A in vitro and, based on the encouraging results, further tested for their relaxant effect on isolated rat artery rings. Computational docking studies were also carried out to explore the mode of interaction with the target protein. The obtained data were compared to the behaviour of the well-known PDE-5A inhibitor sildenafil. Our results demonstrate that semi-synthetic derivatives of osajin and pomiferin show an inhibitory effect on the isolated enzyme that, for some of the compounds, is accompanied by a vasorelaxant activity. Based on our findings, we propose the here described isoflavones as potential lead compounds for the development, starting from natural scaffolds, of a new class of PDE-5A inhibitors with vasorelaxant properties.

Molecular targets in glioblastoma.

Glioblastoma is the most lethal brain tumor. The poor prognosis results from lack of defined tumor margins, critical location of the tumor mass and presence of chemo- and radio-resistant tumor stem cells. The current treatment for glioblastoma consists of neurosurgery, followed by radiotherapy and temozolomide chemotherapy. A better understanding of the role of molecular and genetic heterogeneity in glioblastoma pathogenesis allowed the design of novel targeted therapies. New targets include different key-role signaling molecules and specifically altered pathways. The new approaches include interference through small molecules or monoclonal antibodies and RNA-based strategies mediated by siRNA, antisense oligonucleotides and ribozymes. Most of these treatments are still being tested yet they stay as solid promises for a clinically relevant success.

The Old Made New: Natural Compounds against Erectile Dysfunction.

The interest toward sex-related diseases keeps growing through the years. In this review, we focus our attention on erectile dysfunction (ED), a condition that caught much attention especially after the introduction on the market of phosphodiesterase 5 inhibitors such as the well-known sildenafil. Here, we briefly describe both the etiology of ED and the available treatments, examining then extensively some natural derivatives that, coming from traditional medicine, could represent promising starting points for the development of alternative remedies. In fact, herbal remedies from several parts of the world have been traditionally known for long, and were recently reconsidered and are now being studied to demonstrate their eventual potential in the treatment of ED. Among the various examples reported in the literature and reviewed here, plants and extracts containing polyphenols­especially a class of compounds called kraussianones­appear to be particularly effective and promising against ED.

New naphthoquinone derivatives against glioma cells.

This work was aimed to the development of a set of new naphtoquinone derivatives that can act against glioma. The compounds were tested in order to find out their ability to inhibit the growth of glioma cells, and the results of these assays were correlated with electrochemical analysis and NMR-based reoxidation kinetic studies, suggesting that a redox mechanism underlies and may explain the observed biological behavior. In addition to a full description of the synthetic pathways, electrochemistry, NMR and single crystal X-ray diffraction data are provided.

Serotonin and noradrenaline reuptake inhibitors improve micturition control in mice.

Poor micturition control may cause profound distress, because proper voiding is mandatory for an active social life. Micturition results from the subtle interplay of central and peripheral components. It involves the coordination of autonomic and neuromuscular activity at the brainstem level, under the executive control of the prefrontal cortex. We tested the hypothesis that administration of molecules acting as reuptake inhibitors of serotonin, noradrenaline or both may exert a strong effect on the control of urine release, in a mouse model of overactive bladder. Mice were injected with cyclophosphamide (40 mg/kg), to increase micturition acts. Mice were then given one of four molecules: the serotonin reuptake inhibitor imipramine, its metabolite desipramine that acts on noradrenaline reuptake, the serotonin and noradrenaline reuptake inhibitor duloxetine or its active metabolite 4-hydroxy-duloxetine. Cyclophosphamide increased urine release without inducing overt toxicity or inflammation, except for increase in urothelium thickness. All the antidepressants were able to decrease the cyclophosphamide effects, as apparent from longer latency to the first micturition act, decreased number of urine spots and volume of released urine. These results suggest that serotonin and noradrenaline reuptake inhibitors exert a strong and effective modulatory effect on the control of urine release and prompt to additional studies on their central effects on brain areas involved in the social and behavioral control of micturition.

Age and isolation influence steroids release and chemical signaling in male mice.

Social interactions in mice involve olfactory signals, which convey information about the emitter. In turn, the mouse social and physiological status may modify the release of chemical cues. In this study, the influences of age and social isolation on the endocrine response and the release of chemical signals were investigated in male CD1 mice, allocated into four groups: Young Isolated (from weaning till 60days; N=6), Adult Isolated (till 180days; N=6), Young Grouped (6 mice/cage; till 60days; N=18), Adult Grouped (6 mice/cage; till 180days; N=18). Mice were transferred in a clean cage to observe the micturition pattern and then sacrificed. Body and organs weights, serum testosterone, dehydroepiandrosterone, corticosterone and the ratio Major Urinary Protein/creatinine were measured. Urinary volatile molecules potentially involved in pheromonal communication were identified. Androgen secretion was greater in isolated mice (P<0.05), suggesting a greater reactivity of the Hypothalamic-Pituitary-Gonadal axis. Grouped mice presented a higher degree of adrenal activity, and young mice showed a higher serum corticosterone (P<0.05) suggesting a greater stimulation of the Hypothalamic-Pituitary-Adrenal axis. The micturition pattern typical of dominant male, consisting in voiding numerous droplets, was observed in Young Isolated mice only, which showed a higher protein/creatinine ratio (P<0.05). Urinary 2-s-butyl-thiazoline was higher in both Young and Adult Isolated mice (P<0.005). Young Isolated mice showed the most prominent difference in both micturition pattern and potentially active substance emission, while long term isolation resulted in a less extreme phenotype; therefore social isolation had a higher impact on young mice hormone and pheromone release.

The antiproliferative effects of ouabain and everolimus on adrenocortical tumor cells.

Ouabain is a cardiotonic steroid obtained from Strophanthus. Recently its role as antiproliferative agent has been investigated in tumor cells. Everolimus is a derivative of rapamycin and acts as a signal transduction inhibitor. Adrenocortical carcinoma is a rare cancer, with poor prognosis. This research focuses on antineoplastic properties of ouabain and its association with everolimus. We analyzed the effects of drugs on cells by MTT assay, by [(3)H] thymidine assay, by Wright's staining, by homogeneous caspases assay, by flow cytometry analysis and by Western blot analysis on H295R and SW13 cells and on primary adrenocortical tumor cells. Ouabain induced cell viability reduction in SW13, H295R and 5 primary adrenocortical tumor cells. Combination of ouabain with everolimus produced a stronger cytotoxic effect on cell proliferation and viability. Marked morphological changes were observed in both SW13 and H295R cell lines after ouabain treatment, with an increase in necrosis. Cell cycle distribution was altered by ouabain in SW13. Analysis of apoptosis demonstrated an increase in caspase activity, clearly evident for SW13 at 72h. FACS analysis by Annexin V-FITC kit and propidium iodide confirmed an increased level of necrosis at higher concentrations. Western blot analysis showed that PI3k/Akt signaling pathway was modified after ouabain treatments in SW13. Ouabain exerts antiproliferative effects on SW13 and H295R cell lines and on primary adrenocortical tumor cells. These data suggest that ouabain or ouabain derivatives may be potential anticancer agents.

Prevalence, tumorigenic role, and biochemical implications of rare BRAF alterations.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland, accounting for 74-80% of all thyroid cancers. The 1799T>A transversion is an activating mutation of the BRAF oncogene that is common in and specific to conventional PTC. We studied the prevalence, tumorigenic role, and biochemical implications of rare BRAF variants in a large cohort of patients. METHODS: A total of 2131 fine-needle aspiration biopsy samples were collected and subjected to BRAF mutation analysis. BRAF genetic variants were analyzed by Western blot, immunofluorescence, and in silico analysis. RESULTS: BRAF mutations were found in 50% (347/700) of thyroid cancers (644 PTCs, 22 anaplastic thyroid carcinomas, 34 follicular thyroid carcinomas). They were the classic (c.1799T>A, p.V600E) mutation in 96.8% (336/347) and rare genetic variants in 3.2% (11/347). In all, five infrequent BRAF alterations were detected: (i) c.1795_1797dupACA (p.T599dup); (ii) c.1801A>G (p.K601E); (iii) c.1799_1801delTGA (p.V600_K601>E); (iv) c.1799_1814>A (p.V600_S605>D); and (v) c.1798_1810delinsA (p.V600_W604>R). The last BRAF variant has never been described in the literature. Western blot analysis and immunofluorescence both revealed a variegated reactivity pattern, again emphasizing the peculiar role of every specific BRAF genetic alteration. In silico analysis of the samples studied revealed a stabilization of the "active" geometrical conformation of the B-raf enzyme associated with the activated and productive state of the kinase domain. CONCLUSIONS: Rare BRAF variants were found in 1.6% of all thyroid malignancies, all clustered around the codon V600, in the binding pocket named A-loop, confirming its crucial role in the enzymatic activation of the B-Raf protein. These mutations were associated mainly with the activation of key effectors in the mitogen-activated protein kinase pathway, but a simultaneous stimulation of the PI3k/Akt cascade was demonstrated in some cases. The rare BRAF variants were not generally associated with an aggressive behavior of the PTC. To our knowledge, this is the largest series of thyroid cancers analyzed to identify and functionally characterize rare BRAF variants.