Altered RBP1 Gene Expression Impacts Epithelial Cell Retinoic Acid, Proliferation, and Microenvironment.

Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA.

Dysbiosis in intestinal microbiome linked to fecal blood determined by direct hybridization.

The important physiological and pathophysiological roles of intestinal human microbiome (HMB) in human health have been emerging, owing to the access to molecular biology techniques. Herein we evaluated, for the first time, the intestinal HMB through direct hybridization approach using n-counter flex DX technology which bypasses the amplification procedure currently applied by other technologies to study the human microbiome. To this purpose, a clinical study was carried out on fecal samples, recruiting both healthy volunteers (N-FOB) and subjects positive for occult blood (P-FOB). A relevant custom panel of 79 16S rRNA target gene was engineered and 32 of them displayed a variation between the two clusters of subjects. Our findings revealed that bacteria belonging to Proteobacteria have higher distribution in P-FOB describing dysbiosis. Similarly, Bacteroidetes and Firmicutes phylum display high distribution in P-FOB. Of interest, the presence of Clostridium difficile that belongs to Firmicutes phylum displayed about 70% of low presence in N-FOB compared to P-FOB subjects. Only one bacterium belonging to the Actinobacteria phylum, the Bifidobacterium bifidum, was present.

Quercetin-3-oleoyl derivatives as new GPR40 agonists: Molecular docking studies and functional evaluation.

The G-protein-coupled receptor 40 (GPR40) is an attractive molecular target for the treatment of type 2 diabetes mellitus. Previously, based on the natural oleic acid substrate, an exogenous ligand for this receptor, named AV1, was synthesized. In this context, here we validated the activity of AV1 as a full agonist, while the corresponding catechol analogue, named AV2, was investigated for the first time. The ligand-protein interaction between this new molecule and the receptor was highlighted in the lower portion of the GPR40 groove that generally accommodates DC260126. The functional assays performed have demonstrated that AV2 is a suitable GPR40 partial agonist, showing a therapeutic potential and representing a useful tool in the management of type 2 diabetes.

Capsaicin Analogues Derived from n-3 Polyunsaturated Fatty Acids (PUFAs) Reduce Inflammatory Activity of Macrophages and Stimulate Insulin Secretion by ß-Cells In Vitro.

In this study, two capsaicin analogues, N-eicosapentaenoyl vanillylamine (EPVA) and N-docosahexaenoyl vanillylamine (DHVA), were enzymatically synthesized from their corresponding n-3 long chain polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both dietary relevant components. The compounds significantly reduced the production of some lipopolysaccharide (LPS)-induced inflammatory mediators, including nitric oxide (NO), macrophage-inflammatory protein-3α (CCL20) and monocyte chemoattractant protein-1 (MCP-1 or CCL2), by RAW264.7 macrophages. Next to this, only EPVA increased insulin secretion by pancreatic INS-1 832/13 ß-cells, while raising intracellular Ca2+ and ATP concentrations. This suggests that the stimulation of insulin release occurs through an increase in the intracellular ATP/ADP ratio in the first phase, while is calcium-mediated in the second phase. Although it is not yet known whether EPVA is endogenously produced, its potential therapeutic value for diabetes treatment merits further investigation.

Investigation of TNBC in vitro Antiproliferative Effects of Versatile Pirrolo[1,2-a]quinoxaline Compounds.

The triple-negative breast cancer (TNBC) is characterized by a more aggressive nature and poorer prognosis, nowadays none pharmaceutical approach is still available. For this reason, the research of new active compounds and attractive targets represents an interesting field. In this context MDA- MB-231 cell line was selected to evaluate the antiproliferative effects of new [1,2-a]-pyrroloquinoxaline derivatives. The MTT assay revealed that the amine forms of synthesized molecules were more active compared to iminic ones at 72 h of incubation. The antiproliferative effect of the most promising compounds highlighted the formation of autophagic vacuoles.

Ketogenic Diet Acts on Body Remodeling and MicroRNAs Expression Profile.

BACKGROUND: The Ketogenic Diet (KD) promotes metabolic changes and optimizes energy metabolism. It is unknown if microRNAs (miRs) are influenced by KD in obese subjects. The screening of circulating miRs was performed with the FDA approved platform n-counter flex and blood biochemical parameters were dosed by ADVIA 1800. OBJECTIVES: The aim of this study was to evaluate mir profile under 6 weeks of biphasic KD in obese subjects. We enrolled 36 obese subjects (18 females and 18 males) in stage 1 of Edmonton Obesity Staging System (EOSS) parameter. RESULT: Any correlation was found between biochemical parameter and three miRs, hsa-let-7b-5p, hsa-miR-143-3p and hsa-miR-504-5p influenced in an equal manner in both sexes. The KD resulted safe and ameliorate both biochemical and anthropometric factors in obese subjects re-collocating them into stage 0 of EOSS parameters. CONCLUSION: The miRs herein identified under KD might be a useful tool to monitor low carbohydrate nutritional regimens which reflect indirectly the regulatory biochemical mechanisms and cell signaling that orchestrate metabolic and signaling pathways.

Erratum: Disruption of Myc-Max Heterodimerization with Improved Cell-Penetrating Analogs of the Small Molecule 10074-G5.

[This corrects the article DOI: 10.18632/oncotarget.1108.].

Therapeutic potential of Bcl-xL/Mcl-1 synthetic inhibitor JY-1-106 and retinoids for human triple-negative breast cancer treatment.

Overexpression of anti-apoptotic proteins belonging to the B cell lymphoma (Bcl)-2 family is observed in numerous cancer types and has been postulated to promote cancer cell survival and chemotherapy resistance. Bcl-extra large (xL)/myeloid cell leukemia sequence (Mcl)-1 was demonstrated to be expressed at relatively high levels in clinically aggressive basal-like cancers and inhibiting Bcl-xL overexpression could potentially provoke cell death. A molecule able to target Bcl-xL/Mcl-1, JY-1-106, is herein under investigation. It is also known that vitamin A-derived compounds exhibit antitumor activity in a variety of in vitro experimental models, promoting their effects via nuclear receptor isoforms including retinoic acid receptors (RARs). Pre-clinical observation highlighted that triple negative (estrogen receptor/progesterone receptor/human epidermal growth factor receptor)-breast cancer cells displayed resistance to retinoids due to the RARγ high expression profile. The present study used the triple-negative human breast cancer cell line, MDA-MB-231, to analyze the effects of the Bcl-xL/Mcl-1 synthetic inhibitor, JY-1-106, alone or in combination with retinoids on cell viability. The results revealed a synergistic effect in reducing cell viability primarily by using JY-1-106 with the selective RARγ antagonist SR11253, which induces massive autophagy and necrosis. Furthermore, the results highlighted that JY-1-106 alone is able to positively influence the gene expression profile of p53 and RARα, providing a therapeutic advantage in human triple-negative breast cancer treatment.

Variation in Immune-Related microRNAs Profile in Human Milk Amongst Lactating Women.

BACKGROUND: Human Milk (HM) is a biological fluid representing the first nutrient for newborns. It directly impacts the development of the infant's immune system. In this concern, specific microRNAs (miRNAs) such as hsa-miR-21, hsa-miR-181a, hsa-miR-150 and hsa-miR-223 are known to be involved in the innate and acquired immune response. OBJECTIVE: Herein, these miRNAs were evaluated in frozen and pasteurized samples of human colostrum and HM in order to elucidate the distribution and the expression profile of these biological mediators in both biological fluids. METHODS: Using quantitative approach qRT-PCR, we analyzed immune-related microRNAs in both, colostrum and HM. RESULTS: Our study provided evidence of a comparable profile of immune specific miRNAs in colostrum and HM. Although we detected all the four miRNAs tested, we point out the prevalence of hsamiR- 181a and hsa-miR-223 indicative to act on T and granulocytes cell populations as selective targets. Therefore, these biomolecules could affect newborn's immune homeostasis at early stages of life. While, variation in immune-related miRNAs was found in HM amongst lactating women, it was not evidenced in colostrum. Of interest, pasteurization procedure did not alter the distribution or the expression profile of the miRNAs tested in both colostrum and HM. Herein, we also proposed a simple method to determine the quantity of these biomolecules in biological fluids. CONCLUSION: Considering, this evidence the variation in immune-related miRNAs should be take into account and could be relevant for preterm and hospitalized infants who usually received pasteurized HM from donors.

Novel Gold and Silver Carbene Complexes Exert Antitumor Effects Triggering the Reactive Oxygen Species Dependent Intrinsic Apoptotic Pathway.

Cisplatin and other platinum-based drugs are well-known valid anticancer drugs. However, during chemotherapy, the presence of numerous side effects and the onset of frequent phenomena of resistance has pushed many research groups to devise new metal-based compounds holding improved anticancer properties and fewer undesired effects. Amongst the variety of synthesized compounds, significant antiproliferative effects have been obtained by employing organometallic compounds, particularly those based on silver and gold. With this in mind, we synthesized four compounds, two silver complexes and two gold complexes, with good inhibitory effects on the in vitro proliferation of breast and ovarian cancer-cell models. The antitumor activity of the most active compound, that is, AuL4, was found to be ninefold higher than that of cisplatin, and this compound induced dramatic morphological changes in HeLa cells. AuL4 induced PARP-1 cleavage, caspases 3/7 and 9 activation, mitochondria disruption, cytochrome c release in cancer-cell cytoplasm, and the intracellular production of reactive oxygen species. Thus, AuL4 treatment caused cancer-cell death by the intrinsic apoptotic pathway, whereas no cytotoxic effects were recorded upon treating non-tumor cell lines. The reported outcomes may be an important contribution to the expanding knowledge of medicinal bio-organometallic chemistry and enlarge the available anticancer toolbox, offering improved features, such as higher activity and/or selectivity, and opening the way to new discoveries and applications.

Quercetin/oleic acid-based G-protein-coupled receptor 40 ligands as new insulin secretion modulators.

AIM: Management of Type 2 diabetes mellitus by diet is achievable at the early stage of the disease; patients usually underestimate this approach and an appropriate drug therapy is required. RESULTS: Starting from quercetin and oleic acid, that have effect on insulin secretion, a small set of hybrid molecules was synthesized. Insulin secretion was evaluated in both in vitro and ex vivo models.  AV1  was able to enhance insulin secretion dose dependently, behaving as a conceivable agonist of G-protein-coupled receptor 40. CONCLUSION: AV1 represents an interesting tool that interacts with G-protein-coupled receptor 40. Further studies will be carried out to evaluate the exact binding mode, and also to enlarge the library of these antidiabetic agents. [Formula: see text].

9-cis Retinoic acid modulates myotrophin expression and its miR in physiological and pathophysiological cell models.

Functional studies indicate that essential cellular processes are controlled by Vitamin A derivatives. Among these the retinoic acid isoforms, all-trans- and 9-cis (9cRA), regulate the expression of various genes in both physiological and pathological conditions. Using several in vitro experimental models such as pancreatic ß-cells, pre-adipocytes and breast cancer cells with different phenotypes, we demonstrated the capability of 9cRA to modulate myotrophin (Mtpn) and miR-375 expressions. The 9cRA effect in pancreatic ß-cells line INS-1 832/13 point out a decreased expression of Mptn at both mRNA and protein levels associated to a concomitant increase of miR-375. We also studied the effect of this molecule on 3T3-L1 pre-adipocytes cells demonstrating a down-regulation of Mtpn and a dramatic increase of miR-375. Moreover, in the in vitro breast cancer model such as MDA-MB-231 and MCF-7 cells, 9cRA showed different effect on both Mtpn and miR-375 expression. In INS-1 832/13, 3T3-L1 pre-adipocytes and MCF-7 but not in MDA-MB-231, the effect of 9cRA on Mptn gene expression and its miR was under the control of RARs and RXRs receptors, as revealed by the exposure of these cell line to LE540 or HX603 receptor antagonists. In our findings 9cRA emerges has a hormone with a regulatory action on miR-375 that in most cases interfere with Mtpn expression.

Biochemical and chemical characterization of Cynara cardunculus L. extract and its potential use as co-adjuvant therapy of chronic myeloid leukemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Ancient mediterranean diet was characterized by consuming the spontaneous forms of Cynara cardunculus L. (CCL), commonly called artichoke. Cultivated and/or spontaneous forms of CC studies have demonstrated that methanol extract of CCL flower and/or cynaropicrin showed remarkable anti-proliferative activity in vitro models of leukocyte cancer cell. AIM OF THE STUDY: Chronic myeloid leukemia (CML) is associated with a reciprocal translocation of the long arms of chromosomes 9 and 22 generating the BCR/ABL fusion gene, translated in the p210BCR/ABL oncoprotein kinase. This chimeric protein is the target of a kinase inhibitor, imatinib, but the development of mutations in the ABL kinase domain resulting in drug resistance and several approaches to overcoming resistance have been study. In this concern, we investigated the effect of CCL extract on human K562 CML and K562 imatinib resistant (IMAR) cell proliferation and on p210BCR/ABL expression. MATERIALS AND METHODS: Chemical characterization of the CCL extracts was performed by GC/MS analysis and semipreparative RP-HPLC chromatography. Structural characterization of compounds was assessed by 1H-13C NMR and LC/MS analysis. The effects of CCL extracts on the proliferation of K562 CML human cell line and K562 IMAR were screened by MTT assay. The p210BCR/ABL mRNA and protein expressions were analyzed by qRT-PCR and Western blot techniques respectively. RESULTS: We demonstrate that CCL extract affect cell viability of both K562 CML human cell line and K562 IMAR. The biocomponents of CCL were chemical characterized and we identify cynaropicrin and its deacyl derivative having the capability to down-regulate the p210BCR/ABL oncoprotein. CONCLUSIONS: Our study suggests that the use of those molecules could represent a novel and promising strategy to potentiate the ability of imatinib or of its analogues to induce cancer growth arrest in CML and to delay or overcome the resistance of CML to chemotherapy.

Theophylline action on primary human bronchial epithelial cells under proinflammatory stimuli and steroidal drugs: a therapeutic rationale approach.

Theophylline is a natural compound present in tea. Because of its property to relax smooth muscle it is used in pharmacology for the treatment of airway diseases (ie, chronic obstructive pulmonary disease, asthma). However, this effect on smooth muscle is dose dependent and it is related to the development of side effects. Recently, an increasing body of evidence suggests that theophylline, at low concentrations, also has anti-inflammatory effects related to the activation of histone deacetylases. In this study, we evaluated the effects of theophylline alone and in combination with corticosteroids on human bronchial epithelial cells under inflammatory stimuli. Theophylline administrated alone was not able to reduce growth-stimulating signaling via extracellular signal-regulated kinases activation and matrix metalloproteases release, whereas it strongly counteracts this biochemical behavior when administered in the presence of corticosteroids. These data provide scientific evidence for supporting the rationale for the pharmacological use of theophylline and corticosteroid combined drug.

When Nutraceuticals Reinforce Drugs Side Effects: A Case Report.

INTRODUCTION: Nutraceutical is a term applied for a plethora of products ranging from isolated nutrients, herbal products to dietary supplements and recently, the interest for a nutraceutical approach to lipid and metabolic disorders is growing. Patients with metabolic conditions seem to appreciate a therapeutic management that does not involve drug treatment, particularly for the side effects due to statins, a class of drug used for lipid disorders. Statins directly induce skeletal muscle injury and in the elderly patients, under polytherapy treatments, this risk relies to an increase in adverse drug reactions due to drug interactions. CASE DESCRIPTION: Herein we report a 70-year-old woman under polytherapy who developed rhabdomyolysis after starting the administration of a dietary supplement containing monacolin K. Using the Drug Interaction Probability Scale, we postulated that rhabdomyolysis was possibly related to a drug interaction between sertraline, rosuvastatin and monacolin K. These treatments were discontinued leading to a remission of both clinical symptoms and biochemical parameters. CONCLUSION: This case report highlights how pharmacological treatment must be periodically reassessed, since elderly people could take drugs by themselves when they donot need.

Different Omega-3 Formulations Yield to Diverse Clinical Response: A Case-Report.

Treatment guidelines recommend omega-3 with Docosahexaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) content not above 85% in patients with high plasma levels of triglycerides. Since the different up to date formulation of omega-3 available in commerce must be similar to clinical efficacy and safety, herein, we report the case a 52-year-old woman who presented clinical inefficacy using Olevia(®) omega-3 treatment. Clinical evaluation excluded the presence of intestinal or systemic diseases able to reduce the drug absorption. Switching the therapy from (Olevia(®)) to an equivalent omega-3 formulation (Esapent(®)), we documented a decrease in her plasma triglycerides levels. In order to evaluate a possible difference between these formulations we performed a single blind in vitro dissolution test using three pills for each formulation of omega-3 (Olevia(®), Esapent® and another one chosen between the several formulations available in commerce: DOC Generic(®)) that revealed a significant difference (>20%) in the dissolution time of three different omega- 3 commercially available drug formulation.

Vitamin A and Diabesity: New Insight for Drug Discovery.

Obesity, insulin resistance, metabolic syndrome and type 2 diabetes have reached epidemic proportions, from the term: diabesity. Vitamin A is delivered by a specific binding protein called retinol-binding protein 4 (RBP4) a soluble protein, emerging to have a role in insulin resistance, the major cause of diabetes is highly associated with adipose tissue inflammation and obesity with action. RBP4, interacts with two receptors, the Toll-like receptor 4 (TLR4) and the plasma membrane protein are stimulated by retinoic acid 6 (STRA6), leading to the activation of c-Jun N-terminal protein kinase (JNK) pathways and JAK2/STAT5 cascade, respectively. Both mechanisms sustain insulin resistance. Therefore, ablation of STRA6 protects mice from RBP4-induced suppression of insulin signaling. In addition, mice harboring deletion of a specific chaperon for retinol, show infiltration of α-cells in the core of pancreatic islets, where usually only ß-cells reside, showing a pre-diabetic like phenotype. Not only proteins in vitamin A shuttle and signaling are emerging in diabesity, recently, the discovery of 9cis retinoic acid (9cRA) with effects on controlling glucose levels have opened a new scenario. So far, only pancreas ß-cells have been shown to synthesize it, and high levels of 9cRA correlate with obesity mice models. In this article, we summarize the recent literature present on this topic raising the hypothesis.

Dual Effect of Ziconotide in Primary Erythromelalgia.

Erythromelalgia (EM) is a rare disabling clinical syndrome more commonly known to affect the lower extremities. There is no single effective treatment for this disease that often requires a multidisciplinary approach. Herein, we report the case of a 31-year-old woman affected by primary erythromelalgia who was successfully treated with intrathecal Ziconotide. We also observed an unexpected result following therapy with Ziconotide. The legs and feet of the patient that at the time of admission were swollen and tumefied dramatically improved after one week of the drug administration.

Crystallographic study and biological evaluation of 1,4-dimethyl-N-alkylcarbazoles.

The 9-(bromoalkyl)-1,4-dimethyl-9H-carbazole (2a-d) derivatives, characterized by the presence of five or seven methylenic spacer groups bonded to the carbazole nitrogen, have been synthesized from the corresponding 1,4- dimethyl-9H-carbazole and appropriate dibromoalkane following a general synthetic method. All the prepared species have been fully characterized by means of IR, and (1)H and (13)C NMR spectroscopy, GC-MS and Elemental analysis. Good crystals of the 2c have been obtained and the crystal structure has been solved by means of X-ray diffractometry. In order to study the cytotoxic effect of 2a, 2b, 2c, 2d carbazole derivatives on A2780 ovarian cancer cells, we performed MTT assay after exposure of this cell population to those compounds in a concentration range from 1 to 10µM. Finally, we want to verify whether the cytotoxic effect of the 2c carbazole is mediated by apoptotic mechanisms, by performing chromatin condensation assay on the A2780 cell cultures upon the carbazole treatment at concentration of 10 µM for 72h. All together our data demonstrate that carbazole derivatives exert inhibitory effects on ovarian cancer cell growth, highlighting a stronger and a dose-dependent anti proliferative activity displayed by 2c carbazole, designating this compound, as a better candidate in the treatment of human ovarian cancer.

Expression and Subcellular Localization of Retinoic Acid Receptor-α (RARα) in Healthy and Varicocele Human Spermatozoa: Its Possible Regulatory Role in Capacitation and Survival.

Varicocele, an abnormal tortuosity and dilation of veins of the pampiniform plexus, is the most common identifiable and correctable cause of male infertility. It is now becoming apparent that signaling through vitamin A metabolites, such as all-trans retinoic acid (ATRA), is indispensable for spermatogenesis and disruption of retinoic acid receptor-α (RARα) function may result in male sterility and aberrant spermatogenesis. Herein, we investigated by Western blot and immunogold electron microscopy the expression profiles and subcellular localization of RARα in healthy and varicocele human sperm; in addition, we analyzed the effects of ATRA on cholesterol efflux and sperm survival utilizing enzymatic colorimetric CHOD-PAP method and Eosin Y technique, respectively. In varicocele samples, a strong reduction of RARα expression was observed. Immunogold labeling evidenced cellular location of RARα also confirming its reduced expression in "varicocele" samples. Sperm responsiveness to ATRA treatment was reduced in varicocele sperm. Our study showed that RARα is expressed in human sperm probably with a dual role in promoting both cholesterol efflux and survival. RARα might be involved in the pathogenesis of varicocele as its expression is reduced in pathologic samples. Thus, ATRA administration in procedures for artificial insemination or dietary vitamin A supplementation might represent a promising therapeutic approach for the management of male infertility.