Inhibition of Glutamine Cellular Uptake Contributes to the Cytotoxic Effect of Xanthohumol in Triple-Negative Breast Cancer Cells.

Breast cancer constitutes the most incident cancer and one of the most common causes of cancer-related death. "Glutamine addiction", an important metabolic feature of cancer cells, is dependent on supply of this amino acid from external sources. In this study, the effect of several polyphenols (catechin, epicatechin, EGCG, catechin:lysine, naringenin, hesperidin, malvidin, delphinidin, kaempferol, quercetin, rutin, myricetin, resveratrol, xanthohumol, and chrysin) upon glutamine (3H-GLN) uptake by human breast epithelial adenocarcinoma cell lines with distinct characteristics (MCF-7 and MDA-MB-231) was assessed.Several polyphenols interfere with 3H-GLN uptake by both cell lines. Xanthohumol markedly decreases total and Na+-dependent 3H-GLN uptake and showed a cytotoxic and anti-proliferative effect in MDA-MB-231 cells. Xanthohumol is as an uncompetitive inhibitor of Na+-dependent 3H-GLN uptake and inhibits GPNA (L-γ-glutamyl-p-nitroanilide)-sensitive, both ASCT2 (alanine, serine, cysteine transporter 2)-mediated and non-ASCT2-mediated 3H-GLN uptake. Xanthohumol does not interfere with the transcription rates of ASCT2. The cytotoxic effect of xanthohumol, but not its anti-proliferative effect, is GPNA-sensitive and related to ASCT2 inhibition. Combination of xanthohumol with the breast cancer chemotherapeutic agent doxorubicin results in an additive anti-proliferative, but not cytotoxic effect.We conclude that targeting glutamine uptake might constitute a potential interesting strategy for triple-negative breast cancer.

Metformin and Breast Cancer: Molecular Targets.

Metformin has been the first-line drug for the treatment of type II diabetes mellitus for decades, being presently the most widely prescribed antihyperglycemic drug. Retrospective studies associate the use of metformin with a reduction in cancer incidence and cancer-related death. However, despite extensive research about the molecular effects of metformin in cancer cells, its mode of action remains controversial. The major molecular targets of metformin include complex I of the mitochondrial electron transport chain, adenosine monophosphate (AMP)-activated protein kinase (AMPK), and mechanistic target of rapamycin complex 1 (mTORC1), but AMPK-independent effects of metformin have also been described. Breast cancer is one of the leading causes of cancer-related morbidity and mortality among women worldwide. Several studies have reinforced a link between breast cancer risk and diabetes. Moreover, metformin significantly reduces breast cancer risk, compared to patients who are not using metformin and is independent of diabetes status. In this review, we summarize the current molecular evidence to elucidate metformin's mode of action against breast cancer cells.

Effect of polyphenols on glucose and lactate transport by breast cancer cells.

One of the cancer molecular hallmarks is a deviant energetic metabolism, known as the Warburg effect, whereby the rate of glucose uptake is significantly increased and a high rate of glycolysis and lactic acid production occurs even when oxygen is present-"aerobic lactatogenesis". Accordingly, GLUT1 and MCT1, which are the main glucose and lactate transporters in cancer cells, respectively, have been proposed as oncogenes and are currently seen as potential therapeutic targets in cancer treatment. Polyphenols, commonly contained in fruits and vegetables, have long been associated with a protective role against cancer. Generally considered as nontoxic, dietary polyphenols are considered ideal chemopreventive and possibly chemotherapeutic agents. Several mechanisms of action of polyphenols in breast cancer cells have been proposed including modulation of intracellular signaling, induction of apoptosis through redox regulation or modulation of epigenetic alterations. Additionally, in vitro studies have shown that several polyphenols act as specific inhibitors of glucose transport in breast cancer cell lines and an association between their anticarcinogenic effect and inhibition of glucose cellular uptake has been described. Also, some polyphenols were found to inhibit lactate transport. Importantly, some polyphenols behave as inhibitors of both glucose and lactate cellular uptake by breast cancer cells and these compounds are thus very interesting in the context of a chemopreventive effect, because they deplete breast cancer cells of their two most important energy suppliers. So, the antimetabolic effect of polyphenols should be regarded as a mechanism of action contributing to their chemopreventive/chemotherapeutic potential in relation to breast cancer.

Discovery of the short gamma-ray burst GRB 050709.

Gamma-ray bursts (GRBs) fall into two classes: short-hard and long-soft bursts. The latter are now known to have X-ray and optical afterglows, to occur at cosmological distances in star-forming galaxies, and to be associated with the explosion of massive stars. In contrast, the distance scale, the energy scale and the progenitors of the short bursts have remained a mystery. Here we report the discovery of a short-hard burst whose accurate localization has led to follow-up observations that have identified the X-ray afterglow and (for the first time) the optical afterglow of a short-hard burst; this in turn led to the identification of the host galaxy of the burst as a late-type galaxy at z = 0.16 (ref. 10). These results show that at least some short-hard bursts occur at cosmological distances in the outskirts of galaxies, and are likely to be caused by the merging of compact binaries.

Hermitian splines for modeling biological soft tissue systems that exhibit nonlinear force-elongation curves.

Numerical simulation of soft tissue mechanical properties is a critical step in developing valuable biomechanical models of live organisms. A cubic Hermitian spline optimization routine is proposed in this paper to model nonlinear experimental force-elongation curves of soft tissues, in particular when modeled as lumped elements. Boundary conditions are introduced to account for the positive definiteness and the particular curvature of the experimental curve to be fitted. The constrained least-square routine minimizes user intervention and optimizes fitting of the experimental data across the whole fitting range. The routine provides coefficients of a Hermitian spline or corresponding knots that are compatible with a number of constraints that are suitable for modeling soft tissue tensile curves. These coefficients or knots may become inputs to user-defined component properties of various modeling software. Splines are particularly advantageous over the well-known exponential model to account for the traction curve flatness at low elongations and to allow for more flexibility in the fitting process. This is desirable as soft tissue models begin to include more complex physical phenomena.

Nanostructured functionalized magnetic platforms for the sustained delivery of cisplatin: Synthesis, characterization and in vitro cytotoxicity evaluation.

Cisplatin has demonstrated extraordinary anticancer activity against a variety of solid tumors. However, its clinical efficacy is contrasted by its toxicity profile. Having in mind the need to reduce the toxicity, promote a sustained release and enhance the body-circulation time of cisplatin, herein novel nanocarriers consisting of core-shell silica-coated iron oxide nanoparticles functionalized with dicarboxylic acid groups were prepared and characterized. Cisplatin was conjugated with the functionalized nanoparticles by surface tethering. Controlled release of cisplatin was observed without burst effect and in a sustained profile for up to 3 days. In vitro studies showed cytotoxic and antiproliferative effects of the cisplatin nanoconjugates against a human pancreatic cancer cell line. Importantly, when compared with free cisplatin, nanoconjugates exhibited lower cytotoxic effects regarding nonmalignant human duct pancreatic cells.

Large neutral amino acids supplementation in phenylketonuric patients.

Phenylketonuria is an inborn error of amino acid metabolism that results in severe mental retardation if not treated early and appropriately. The traditional treatment, consisting of a low-phenylalanine diet, is usually difficult to maintain throughout adolescence and adulthood, resulting in undesirable levels of blood phenylalanine and consequent neurotoxicity. The neurotoxicity of phenylalanine is enhanced by its transport mechanism across the blood-brain barrier, which has the highest affinity for phenylalanine compared with the other large neutral amino acids that share the same carrier. The supplementation of large neutral amino acids in phenylketonuric patients has been showing interesting results. Plasma phenylalanine levels can be reduced, which may guarantee important metabolic and clinical benefits to these patients. Although long-term studies are needed to determine the efficacy and safety of large neutral amino acids supplements, the present state of knowledge seems to recommend their prescription to all phenylketonuric adult patients who are non-compliant with the low-phenylalanine diet.

Estimate of the digestibility, assimilability and intestinal permeability of butyltins occurring in wine.

An estimate of the digestibility and assimilability of butyltins occurring in contaminated wines (Port, red and white) was obtained by means of in vitro studies of gastrointestinal digestion. The influence of the wine matrix on the intestinal permeability was explored by studying the accumulation of butyltins in Caco-2 monolayers either when these species are dissolved in buffer only or in the dialysates of digested wines. Some important information about the fate of the butyltin compounds ingested from contaminated wines could be achieved. Only a very small fraction of the ingested DBT and TBT, the two most toxic forms, appear to be able to reach the epithelium as judged by the small dialyzable fraction found (<2%). This is probably independent from the food/drink matrix introducing these contaminants, since the influence of the involved enzymes appear to be dominant, especially for DBT and TBT. Additionally, the intestinal permeability of the three butyltins was also very low, the wine matrix possibly having a hindrance effect in a few cases.

Lack of effect of the procarcinogenic 17ß-estradiol on nutrient uptake by the MCF-7 breast cancer cell line.

Breast cancer is one of the most frequent cancers in the population, especially in older women. Estrogen is known to be a key hormone in the development and progression of mammary carcinogenesis. In this study, we investigated if the procarcinogenic effect of 17ß-estradiol (E2) in breast cancer MCF-7 cells is dependent on changes in glucose or folic acid cellular uptake. The effect of E2 on uptake of 3H-deoxy-d-glucose, 3H-folic acid, cell proliferation (3-thymidine incorporation assay), culture growth (sulforhodamine B assay), viability (lactate dehydrogenase activity assay), lactate production and migration capacity (injury assay) was evaluated. E2 (48h; 100nM) increased culture growth (16%), proliferation rate (24%), cellular viability (36%) and lactate production (38%). In contrast, E2 did not significantly affect the migration capacity of MCF-7 cells. The pro-proliferative, but not the cytoprotective effect of E2 was found to be ERß-dependent. The polyphenols rutin and caffeic acid were not able to counteract the effect of E2 upon cell proliferation and viability. Uptake of 3H-deoxy-d-glucose was not affected by E2, either in the absence or presence of GLUT inhibitors (cytochalasin B plus phloridzin). Moreover, E2 did not change GLUT1 mRNA levels. Finally, 3H-folic acid uptake was also not affected by E2, both in the absence and presence of the RFC1 inhibitor, methotrexate. The pro-proliferative and cytoprotective effects of E2 are not dependent neither of stimulation of glucose cellular uptake (both GLUT and non-GLUT-mediated) nor of stimulation of folic acid uptake (both RFC1-and non-RFC1-mediated).

Modulation of MPP+ uptake by tea and some of its components in Caco-2 cells.

The entry of most xeno/endobiotics into the organism is limited by their intestinal absorption. The interference of certain foods with the therapeutic efficacy of drugs or with chemical toxicity is becoming evident and growing attention is being given to these subjects. The aim of this work was to study the effect of green tea (GT) and black tea (BT), as well as some of their components, on the transport of organic cation molecules. For this purpose, 3H-MPP+ (radiolabeled 1-methyl-4-phenylpyridinium) was used as a model organic cation and Caco-2 cells were used as an intestinal epithelial model. Our results showed that both GT and BT significantly increased 3H-MPP+ absorption in these cells. Additionally, we studied the effect of epigallocatechin-3-gallate (EGCG), myricetin, caffeine, and theophylline. Whereas EGCG (2 mM) increased, myricetin (50 microM) and caffeine (1 mM) decreased, and theophylline (1 mM) had no effect on the uptake of 3H-MPP+ into Caco-2 cells. When GT was supplemented with caffeine or theophylline, we observed a partial loss of its effect. When BT was supplemented with EGCG, its ability to increase 3H-MPP+ uptake was much more pronounced than that observed with BT alone. In conclusion, this study showed that GT and BT might interfere with the absorption of the model organic cation MPP+ by the intestinal epithelium. Since important compounds are organic cations, the consequences of this interference may have an impact on human health. Although this constitutes only preliminary work and further studies are needed, tea should be included in the growing list of foodstuffs that have the potential to be involved in food-drug interactions.

Intestinal uptake of MPP+ is differently affected by red and white wine.

It is becoming increasingly evident that ingested products, such as wine, may have profound effects on the therapeutic efficacy of certain drugs. As various xeno- and endobiotics are organic cations, the purpose of our study was to examine the modulation of organic cations intestinal apical uptake by red (RW) and white wine (WW). For this purpose, we used RW, WW, the same alcohol-free wines, phenolic compounds and ethanol. The uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP+) was evaluated in Caco-2 cells, an intestinal epithelial cell model. RW and alcohol-free RW increased 3H-MPP+ apical uptake, although the effect of alcohol-free RW was less pronounced. On the other hand, WW and alcohol-free WW decreased the organic cation uptake but the effect of alcohol-free WW was more pronounced. Our results show that the total content in phenolic compounds was 7 times higher, and the dialysis index was about 4 times higher in RW compared to WW. Ethanol, in the same concentration found in wine, caused a significant decrease in 3H-MPP+ apical uptake. The solution containing high molecular weight compounds from dialyzed RW increased 3H-MPP+ apical uptake. In conclusion, the results suggest that RW may increase and WW may reduce the intestinal absorption of organic cations present in the diet, such as drugs or vitamins (e.g. thiamine and riboflavin). As ethanol alone decreased the uptake of MPP+, and alcohol-free RW and WW had a lower potency than intact wine upon the transport, the presence of ethanol is probably important for the solubilisation/bioavailability of the components endowed with the transport modulating activity.

An update on the extraneuronal monoamine transporter (EMT): characteristics, distribution and regulation.

Biological membranes prevent transmembrane diffusion in the majority of organic molecules that bear net charges at physiological pH. Consequently, these compounds must use more or less specific membrane-bound transport systems to be imported into or exported from cells or organisms. The extraneuronal monoamine transporter (EMT) is a transmembranar transport system involved in the transfer of monoamine compounds across cell membranes. It was identified more than 30 years ago [1], its functional characteristics being thereafter described [review by 2]. The recent cloning of this transporter in man and rat reopened investigation and interest in this entity. EMT is a Na(+) and Cl(-)-independent, potential-dependent carrier, known to have a broad tissue distribution (eg. myocardium, vascular and non-vascular smooth muscle cells, glandular cells, placenta and CNS glial cells). According to its transport function and primary structure, EMT is included in the amphiphilic solute facilitator (ASF) family of transporters. Physiological substrates for EMT include the monoamines serotonin, dopamine, noradrenaline, adrenaline and histamine. Moreover, several xenobiotics including the neurotoxin 1-methyl-4-phenylpyridinium, clonidine, cimetidine and the K(+)-channel blocker tetraethylammonium interact with this transporter. The aim of this work is to review knowledge concerning EMT, making an update on its functional characteristics, physiological importance and regulation. A special emphasis will be given to very recent investigations concerning regulation of EMT by intracellular second messenger systems and the interaction of modulators of P-glycoprotein, the product of the multidrug resistance gene MDR1, with EMT.

Molecular cloning and characterization of two novel transport proteins from rat kidney.

The recent cloning of renal transport systems for organic anions and cations (OAT1, OCT1, and OCT2) opened the possibility to search, via polymerase chain reaction (PCR) homology screening, for novel transport proteins. Two integral membrane proteins, UST1 and UST2, were cloned from rat kidney. RT-PCR revealed that UST1 is confined to the kidney whereas UST2 mRNA was detected in all tested tissues. Sequence analyses suggest that UST1 and UST2, together with four related transporters, comprise, within the major facilitator superfamily, a so far unrecognized transporter family, termed amphiphilic solute facilitator (ASF) family. Characteristic signatures for the ASF family were identified.

Primate brain evolution: genetic and functional considerations.

Functionally distinct regions of the brain to which maternal and paternal genomes contribute differentially (through genomic imprinting) have developed differentially over phylogenetic time. While certain regions of the primate forebrain (neocortex, striatum) have expanded relative to the rest of the brain, other forebrain regions have contracted in size (hypothalamus, septum). Areas of relative expansion are those to which the maternal genome makes a substantial developmental contribution. This may be significant with respect to the importance of primate forebrain expansion in the development of complex behavioural strategies and the way in which these are deployed, especially by the matriline. In many primate societies the maintenance of social cohesion and group continuity over successive generations is dependent on the matriline, with high ranking females producing high ranking daughters that stay within the group. Regions of relative contraction are those to which the paternal genome makes a differential contribution and these are target areas for gonadal hormones, which is congruent with the diminished role for gonadal hormones in the emancipation of primate reproductive behaviour.

Establishment of human cell lines producing anti-D monoclonal antibodies: identification of Rhesus D antigen.

Two cell lines producing monoclonal antibodies have been established from peripheral blood of a negative Rhesus blood donor which has been immunized with positive Rhesus red blood cells. Two monoclonal antibodies Co II 8.8 and Co II 7.12 have been selected. Both are IgG1 antibodies, but recognize different epitopes on the Rhesus D antigen, apparently associated with different subunits of the D antigen. Thus the Co II 8.8, like the positive serum, immunoprecipitates an antigen of a relative molecular weight of 33 kDa, while the Co II 7.12 recognizes an antigen of Mr 42 kDa.

Inward transport of [3H]-1-methyl-4-phenylpyridinium in rat isolated hepatocytes: putative involvement of a P-glycoprotein transporter.

1. The liver has an important role in the detoxification of organic cations from the circulation. [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+), a low molecular weight organic cation, is efficiently taken up and accumulated by rat hepatocytes through mechanisms partially unknown. 2. The aim of the present work was to characterize further the uptake of MPP+ by rat isolated hepatocytes. The putative interactions of a wide range of drugs, including inhibitors/substrates of P-glycoprotein, were studied. 3. The uptake of MPP+ was investigated in rat freshly isolated hepatocytes (incubated in Krebs-Henseleit medium with 200 nM [3H]-MPP+ for 5 min) and in the rat liver in situ (perfused with Krebs-Henseleit/BSA medium with 200 nM [3H]-MPP+ for 30 min). [3H]-MPP+ accumulation in the cells and in tissue was determined by liquid scintillation counting. 4. Verapamil (100 microM), quinidine (100 microM), amiloride (1 mM), (+)-tubocurarine (100 microM), vecuronium (45 microM), bilirubin (200 microM), progesterone (200 microM), daunomycin (100 microM), vinblastine (100 microM), cyclosporin A (100 microM) and cimetidine (100 microM) had a significant inhibitory effect on the accumulation of [3H]-MPP+ in isolated hepatocytes. Tetraethylammonium (100 microM) had no effect. 5. In the rat perfused liver, both cyclosporin A (100 microM) and verapamil (100 microM) had much less marked inhibitory effects as compared to their effects on isolated hepatocytes (0% against 35% and 45% against 96% of inhibition, respectively). 6. Inhibition of alkaline phosphatase activity by increasing or decreasing the pH of the incubation medium or by the presence of vanadate (1 mM) or homoarginine (500 microM) led to a significant increase in the accumulation of [3H]-MPP+ in isolated hepatocytes. 7. It was concluded that, in addition to the type I organic cation hepatic transporter, [3H]-MPP+ is taken up by rat hepatocytes through P-glycoprotein, a canalicular transport system that usually excretes endobiotics and xenobiotics. We proposed that the reversal of transport through P-glycoprotein may be related to the loss of efficacy of alkaline in isolated hepatocytes.

Uptake of 1-methyl-4-phenylpyridinium (MPP+) by the JAR human placental choriocarcinoma cell line: comparison with 5-hydroxytryptamine.

The aim of this work was to characterize the uptake of 1-methyl-4-phenylpyridinium (MPP(+)) in the JAR human choriocarcinoma cell line. As JAR cells, as well as the placenta, express the neuronal serotonin transporter (SERT), a comparison between the uptake of (3)H-MPP(+) and (3)H-serotonin ((3)H-5HT) was made. Specific uptake of (3)H-MPP(+) (0.2 microM ) was temperature-, Na(+)- and potential-dependent. 5HT and MPP(+) reduced (3)H-MPP(+) specific uptake (for 5HT, its IC(50) was found to be 4 microM ). The SERT inhibitors desipramine and fluoxetine also inhibited (3)H-MPP(+) specific uptake (with IC(50)s of 189 and 0.92 microM, respectively). The inhibitors of the extraneuronal monoamine transporter (EMT) and of the organic cation transporter type 2 (OCT2), corticosterone and decynium22, had no effect on (3)H-MPP(+) specific uptake, but cyanine863 concentration-dependently reduced it (with an IC(50) of 23 microM ). Specific uptake of (3)H-5HT (0.2 microM ) by JAR cells was temperature-, Na(+)- and potential-dependent. 5HT, MPP(+), desipramine and fluoxetine concentration-dependently inhibited (3)H-5HT specific uptake (with IC(50)s of 1.9 microM, 50 microM, 0.17 microM and 0.046 microM, respectively). Corticosterone showed no effect, but decynium22 and cyanine863 significantly reduced(3) H-5HT specific uptake. For cyanine863, its IC(50) was found to be 11 microM. In conclusion, the results suggest that: (1) uptake of (3)H-5HT by JAR cells occurs exclusively through SERT; (2) uptake of(3) H-MPP(+) by JAR cells involves SERT and also another transporter; (3) neither EMT nor OCT2 are functionally present in JAR cells.

Opioid receptor blockade reduces maternal affect and social grooming in rhesus monkeys.

Seven lactating female rhesus macaques, housed in social groups, were administered with low doses (0.5 mg/kg) of the opioid antagonist naloxone when their infants were 4, 6, 8, and 10 weeks old. A control group received saline. Mothers receiving naloxone were involved in less grooming with other group members, and were less protective towards their infants. By infant-age week 8 they also groomed their infants less, while other monkeys groomed the infants more. Other behavioural measures of mother-infant interactions were not altered. With time, from infant-age week 6 onwards, some short-lived dysphoric conditioned drug responses to naloxone became apparent, although these were not correlated with the decline in social interaction. These results are interpreted in terms of possible interference of naloxone with maternal affect.

Proactive interference effects on short-term memory in rats: I. Basic parameters and drug effects.

An operant delayed-matching task was used to assess the role of proactive interference (PI) effects on short-term memory capacity of rats. Task performance was analyzed in terms of the influence of the sample positions and response choices on previous trials. PI was predominantly attributable to the influence of the immediately previous trial but not preceding trials and was abolished by increasing the intertrial intervals from 5 to 15 s. Nicotine induced a decline in choice accuracy only on trials in which the previous response had been to the side opposite the current sample and correct response, suggesting an increased susceptibility to PI. Physostigmine induced a mild, relatively nonspecific decline in response accuracy. Clonidine induced delay-dependent impairments irrespective of responses on previous trials. None of these drugs enhanced choice accuracy at any dose tested.

Proactive interference effects on short-term memory in rats: II. Effects in young and aged rats.

Whether the short-term memory impairments of aged rats in an operant delayed-matching task is attributable to increased susceptibility to proactive interference (PI) was tested. Groups of young and aged rats were trained on the task, and the previously reported delay-dependent deficit of aged rats was replicated. The aged rats showed a significantly greater decline in performance on previous-response-opposite trials compared with previous-response-same trials than did young rats, when tested over the same range of delay intervals, suggesting a higher sensitivity to PI. However, this effect was established against a higher overall baseline performance of the young animals on both types of trial. When the young and aged animals were equated for performance on previous-response-same trials (by increasing the range of delay intervals used for testing the young animals), the interaction effect was abolished. Thus, the delay-dependent deficits of aged rats are independent of their sensitivity to PI.