New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

In vitro anti-tumoral and immunomodulatory effects of acidic polysaccharides isolated from the fungus Gloeosoma mirabile.

Polysaccharides from wood-rooting fungi have attracted attention due to their broad pharmacological properties. Herein, we report the antitumor and immunomodulatory activities of acid polysaccharides isolated from fungi Gloeosoma mirabile. The polysaccharide extracts displayed significant antiproliferative activity against cancer cell lines (MCF-7, HCT-116, U-937) in a dose-dependent manner and induction of IL-6 in macrophage RAW 264.7. Furthermore, flow cytometry analysis showed that high polysaccharide concentrations induced apoptosis by 83% in HL-60 cells. Based on gas chromatography-mass spectrometry (GC-MS) and Fourier transform infra-red (FT-IR) spectroscopy studies, acidic polysaccharides from G. mirabile were mainly composed of arabinose, α-D-galactopyranose and methyl ß-D-galactopyranoside.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

Immunomodulatory role of vasoactive intestinal peptide and ghrelin in Oncorhynchus mykiss.

Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In Oncorhynchus mykiss, vasoactive intestinal peptide (VIP) mRNA sequences have not been cloned, and the role of VIP in modulating the immune system has not been studied. Furthermore, in relation to other neuropeptides with possible immunomodulatory function, such as ghrelin, there are also few studies. Therefore, in this work, we performed molecular cloning, identification, and phylogenetic analysis of three VIP precursor sequences (prepro-VIP1, VIP2 and VIP3) in rainbow trout. In addition, the immunomodulatory function of both neuropeptides was evaluated in an in vitro model using the VIP1 sequence identified in this work and a ghrelin sequence already studied in O. mykiss. The results suggest that the prepro-VIP2 sequence has the lowest percentage of identity with respect to the other homologous sequences and is more closely related to mammalian orthologous sequences. VIP1 induces significant expression of both pro-inflammatory (IFN-γ, IL-1ß) and anti-inflammatory (IL-10 and TGF-ß) cytokines, whereas ghrelin only induces significant expression of proinflammatory cytokines such as IL-6 and TNF-α.

New strategy for the design, production and pre-purification of chimeric peptide with immunomodulatory activity in Salmosalar.

The intensive salmon farming is associated with massive outbreaks of infections. The use of antibiotics for their prevention and control is related to damage to the environment and human health. Antimicrobial peptides (AMPs) have been proposed as an alternative to the use of antibiotics for their antimicrobial and immunomodulatory activities. However, one of the main challenges for its massive clinical application is the high production cost and the complexity of chemical synthesis. Thus, recombinant DNA technology offers a more sustainable, scalable, and profitable option. In the present study, using an AMPs function prediction methodology, we designed a chimeric peptide consisting of sequences derived from cathelicidin fused with the immunomodulatory peptide derived from flagellin. The designed peptide, CATH-FLA was produced by recombinant expression using an easy pre-purification system. The chimeric peptide was able to induce IL-1ß and IL-8 expression in Salmo salar head kidney leukocytes, and prevented Piscirickettsia salmonis-induced cytotoxicity in SHK-1 cells. These results suggest that pre-purification of a recombinant AMP-based chimeric peptide designed in silico allow obtaining a peptide with immunomodulatory activity in vitro. This could solve the main obstacle of AMPs for massive clinical applications.

Antiproliferative activity of carotenoid pigments produced by extremophile bacteria.

Various microorganisms are able to synthesize pigments, which usually present antioxidant properties. The aim of this work was to evaluate the antiproliferative activity of bacterial pigments against cancer cells Neuro-2a, Saos-2 and MCF-7. Pigments were obtained from Deinococcus sp. UDEC-P1 and Arthrobacter sp. UDEC-A13. Both bacterial strains were isolated from cold environments (Patagonia and Antarctica, respectively). Pigments were purified and analyzed by HPLC. Antiproliferative activity was evaluated by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) assay. Deinoxanthin carotenoid obtained from Deinococcus sp. UDEC-P1 was able to reduce significatively the viability of Saos-2 (37.1%), while no effect was observed against MCF-7 and Neuro-2a. Pigments obtained from Arthrobacter sp. UDEC-A13 showed a significant viability reduction of three tumour cells (20.6% Neuro-2a, 26.3% Saos-2 and 13.2% MCF-7). Therefore, carotenoid pigments produced by extremophilic bacteria Deinococcus sp. UDEC-P1 and Arthrobacter sp. UDEC-A13 could be proposed as novel complementary compounds in anticancer chemotherapy.

Neuro-2a cell-based assay for toxicity equivalency factor - proposal and evaluation in Chilean contaminated shellfish samples.

There are two official PSP detection methods (mouse bioassay and HLPC-FLD) and a number of alternative methods. Ethical considerations have led to regulations being adopted in some countries that limit or prohibit the application of mouse bioassay. Analytical methodologies (e.g. HPLC-FLD or LC-MSMS) have the disadvantages of not being able to detect new toxins or analogues or reflecting the overall toxicity of the sample. In addition, they require highly trained personnel and expensive equipment, which are not always available. In this work, we have evaluated a method based on the Neuro-2a cell-based assay to detect substances that inhibit voltage-dependent sodium channels (Manger's method). We tested PSP standards and natural samples contaminated with PSP. Here we demonstrate that the adapted Manger's method is suitable for calculating Toxicity Equivalency Factors (TEF) for STX-analogues. The method was shown to be useful for screening contaminated natural samples in concentrations above the regulatory limit for these toxins (80 µg STX equivalents/100 g shellfish). We were able to detect PSP in 19 natural mollusc samples from South Chile despite the presence of other marine toxins. These preliminary results suggest that the method could be used as a first step in screening programmes.

Talaromyces australis and Penicillium murcianum pigment production in optimized liquid cultures and evaluation of their cytotoxicity in textile applications.

In this work Talaromyces australis and Penicillium murcianum pigment production in liquid cultures and the cytotoxic effect of such pigments on skin model cells were studied. Response surface methodology (RSM) was used to optimize culture conditions aiming to increase pigment production in malt extract and peptone-glucose-yeast extract medium. Cytotoxicity of fungal pigments and also from lixiviates of wool fabrics dyed with T. australis and P. murcianum pigment was evaluated on mammalian cell lines HEK293 and NIH/3T3. Results showed that variations on initial pH, NaCl and peptone, resulted in increments up to 188.2% for red pigment of T. australis and 107.4% for yellow pigment of P. murcianum, regarding non-optimized conditions. Tested fungi also showed great differences in culture conditions for the maximum pigment production, with P. murcianum requiring an alkaline medium (initial pH 9) supplemented with NaCl and T. australis an acidic medium (initial pH 5) without addition of salt. The cytotoxicity assays provided evidences on the safe nature of these natural pigments when used for textile applications. The cytotoxicity assay showed that the threshold of toxicity, given by the lowest IC50 value (0.21 g L-1) was more than double of the concentration of pigment required to dye the wool samples. In addition, cytotoxicity of lixiviates depicted no toxic effect over tested cells.

Polyphenols obtained from Didymosphenia geminata (Lyngbye) Schmith altered the viability and proliferation of salmonids cells lines SHK-1 and CHSE-214.

Didymosphenia geminata (Lyngbye) Schmidt, also referred to as Didymo, is an invasive diatom that forms nuisance mats. Since it was first reported in our country in approximately 2010, Didymo has expanded and colonized different rivers in the Zona Austral region of Chile. Its biology and effects on ecosystems are still being studied because Didymo is an invasive algal mat that forms in a range of systems from oligotrophic austral rivers to more subtropical systems. We aimed to evaluate the viability of two salmonid cell lines, CHSE-214 and SHK-1 (somatic and embryonic cell lines, respectively), in dilutions of river water alone and in river water contaminated with Didymo or polyphenols extracted from Didymo under controlled conditions. We developed an artificial river system (2 aquariums/replicate) from five different rivers from the central area (Bio-Bio) and Patagonia area (Futaleufú) of Chile to maintain Didymo in the benthic phase. The Didymo populations were maintained for six months in the water from the rivers, after which samples were obtained. Following the extraction of polyphenols from the Didymo samples maintained in the artificial rivers, toxicity assays (10 assays) were performed to determine cell viability. Our results indicated that the CHSE-214 cells were highly sensitive to increasing concentrations of Didymo extracts. We observed a 50% reduction in cell viability after 24 h of exposure to a 0.01 V/V dilution, and this treatment further reduced the proliferative capacity by 70% after 120 h. The SHK-1 cells were less responsive, showing only a 20% decrease in viability at 24 h and a lower cell proliferation rate (45%) after 120 h, which remained higher than that of the CHSE-214 cells. We conclude that certain cell types are sensitive to Didymo in rivers, suggesting that there are chronic effects on several aquatic species following exposure to these diatom substances. These effects should be further studied using this laboratory model to understand the full impact of Didymo on river ecosystems.

Study of the ichthyotoxic microalga Heterosigma akashiwo by transcriptional activation of sublethal marker Hsp70b in Transwell co-culture assays.

Despite the advance of knowledge about the factors and potential mechanisms triggering the ichthyotoxicity in microalgae, these remain unclear or are controversial for several species (e.g. Heterosigma). Neither typical toxicity tests carried out with cell extracts nor direct exposure to harmful species were proved suitable to unravel the mechanism of harm. Ichthyotoxic species show a complex harmful effect on fish, which is mediated through various mechanisms depending on the species. In this work, we present a method to study sub-lethal effects triggered by reactive oxygen species of a population of harmful algae in vivo over a fish cell line. To that end, Transwell co-cultures in which causative and target species are separated by a 0.4 µm pore membrane were carried out. This allowed the evaluation of the effect of the released molecules by cells in a rapid and compact test. In our method, the harmful effect was sensed through the transcriptional activation of sub-lethal marker Hsp70b in the CHSE214 salmon cell line. The method was tested with the raphidophyte Heterosigma akashiwo and Dunaliella tertiolecta (as negative control). It was shown that superoxide intracellular content and its release are not linked in these species. The methodology allowed proving that reactive oxygen species produced by H. akashiwo are able to induce the transcriptional activation of sub-lethal marker Hsp70b. However, neither loss of viability nor apoptosis was observed in CHSE214 salmon cell line except when exposed to direct contact with the raphidophyte cells (or their extract). Consequently, ROS was not concluded to be the main cause of ichthyotoxicity in H. akashiwo.

Steroidal composition and cytotoxic activity from fruiting body of Cortinarius xiphidipus.

From the fruiting body of ectomycorrhizal fungi Cortinarius xiphidipus, sterols were identified from the crude extract and the cytotoxic effect of ergosta-4, 6, 8(14), 22-tetraen-3-one (ergone) was evaluated. Ten sterols including ergosta-3,5,7,9(11),22-pentaene, (22E)-ergosta-5,7,9(11),22-tetraen-3b-ol, (3ß,22E)-ergosta-5,7,22-trien-3-ol, (22E)-ergosta-7,22-dien-3-ol, neoergosterol, (3ß)-ergosta-5,8-dien-3-ol, (3ß)-ergosta-7-en-3-ol, stigmasterol, stigmasterol 22,23-dihydro and (22E)-ergosta-4,6,8(14),22-tetraen-3-one were identified from the crude extract. The cytotoxic activity of the sterol fraction containing ergosta-4, 6, 8(14), 22-tetraen-3-one was assessed on four tumour cell lines (Neuro-2a, Saos-2, MCF7 and LNCaP-C42). The cytotoxic activity against the four tumour cell lines tested, being Neuro-2a and Saos-2 the most sensitive, with a half-maximal inhibitory concentration (IC50) of 20.8 ± 2.2 and 27.8 ± 1.0 µg/mL, respectively. This is the first report of this Antarctic fungi collected in the Magallanes and Chilean Antarctica Region. This work represents a potential source for the development of anticancer drugs.

Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.

Dehydroascorbic Acid Promotes Cell Death in Neurons Under Oxidative Stress: a Protective Role for Astrocytes.

Ascorbic acid (AA), the reduced form of vitamin C, is incorporated into neurons via the sodium ascorbate co-transporter SVCT2. However, this transporter is not expressed in astrocytes, which take up the oxidized form of vitamin C, dehydroascorbic acid (DHA), via the facilitative hexose transporter GLUT1. Therefore, neuron and astrocyte interactions are thought to mediate vitamin C recycling in the nervous system. Although astrocytes are essential for the antioxidant defense of neurons under oxidative stress, a condition in which a large amount of ROS is generated that may favor the extracellular oxidation of AA and the subsequent neuronal uptake of DHA via GLUT3, potentially increasing oxidative stress in neurons. This study analyzed the effects of oxidative stress and DHA uptake on neuronal cell death in vitro. Different analyses revealed the presence of the DHA transporters GLUT1 and GLUT3 in Neuro2a and HN33.11 cells and in cortical neurons. Kinetic analyses confirmed that all cells analyzed in this study possess functional GLUTs that take up 2-deoxyglucose and DHA. Thus, DHA promotes the death of stressed neuronal cells, which is reversed by incubating the cells with cytochalasin B, an inhibitor of DHA uptake by GLUT1 and GLUT3. Additionally, the presence of glial cells (U87 and astrocytes), which promote DHA recycling, reverses the observed cell death of stressed neurons. Taken together, these results indicate that DHA promotes the death of stressed neurons and that astrocytes are essential for the antioxidative defense of neurons. Thus, the astrocyte-neuron interaction may function as an essential mechanism for vitamin C recycling, participating in the antioxidative defense of the brain.

Saxitoxin Modulates Immunological Parameters and Gene Transcription in Mytilus chilensis Hemocytes.

Saxitoxin (STX) is a neurotoxin produced by dinoflagellates in diverse species, such as Alexandrium spp., and it causes paralytic shellfish poisoning (PSP) in humans after the ingestion of contaminated shellfish. Recent studies have suggested that the immune functions of bivalves could be affected by harmful algae and/or by their toxins. Herein, hemocytes are the main effector cells of the immune cellular response. In this study, we evaluated the response of hemocytes from the mussel Mytilus chilensis to STX exposure in a primary culture. Cell cultures were characterized according to size and complexity, while reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein diacetate (DCFH-DA) assay. Finally, phagocytic activity was measured using both flow cytometry and fluorescence microscopy assays. Additionally, gene transcription of candidate genes was evaluated by qPCR assays. The results evidenced that exposures to different concentrations of STX (1-100 nM) for 24 h did not affect cell viability, as determined by an MTT assay. However, when hemocytes were exposed for 4 or 16 h to STX (1-100 nM), there was a modulation of phagocytic activity and ROS production. Moreover, hemocytes exposed to 100 nM of STX for 4 or 16 h showed a significant increase in transcript levels of genes encoding for antioxidant enzymes (SOD, CAT), mitochondrial enzymes (COI, COIII, CYTB, ATP6, ND1) and ion channels (K+, Ca2+). Meanwhile, C-type lectin and toll-like receptor genes revealed a bi-phase transcriptional response after 16 and 24-48 h of exposure to STX. These results suggest that STX can negatively affect the immunocompetence of M. chilensis hemocytes, which were capable of responding to STX exposure in vitro by increasing the mRNA levels of antioxidant enzymes.

The Na+-dependent L-ascorbic acid transporter SVCT2 expressed in brainstem cells, neurons, and neuroblastoma cells is inhibited by flavonoids.

Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium-vitamin C co-transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na(+)-dependent L-AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT-PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT-PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose-dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.

Vitamin C uptake and recycling among normal and tumor cells from the central nervous system.

Specialized cells transport vitamin C in its reduced form using sodium-dependent cotransporters (SVCT1 and SVCT2). Additionally, different cells transport the oxidized form of vitamin C, dehydroascorbic acid, through glucose transporters (GLUTs). We have proposed recently a model for vitamin C uptake that resolves the apparent contradiction that although only ascorbic acid is detectable in vivo, there are cells that transport only dehydroascorbic acid. We carried out a detailed kinetic analysis to compare the mechanisms of vitamin C uptake in normal human melanocytes, neurons isolated from brain cortex, hypothalamic ependymal-glial cells, and astrocytes. Uptake of ascorbic acid was also analyzed in the human oligodendroglioma cell line TC620, in human choroid plexus papilloma cells (HCPPC-1), and in the neuroblastoma cell line Neuro-2a. Melanocytes were used to carry out a detailed analysis of vitamin C uptake. Analysis of the transport data by the Lineweaver-Burk plot revealed the presence of one functional component (K(m) 20 microM) involved in ascorbic acid transport by melanocytes. Vitamin C sodium-dependent saturable uptake was also observed in neurons and hypothalamic tanycytes. We confirmed SVCT2 expression in neurons by in situ hybridization; however, SVCT2 expression was not detected in astrocytes in situ. Functional data indicate that astrocytes transport mainly dehydroascorbic acid, using the glucose transporter GLUT1. Our functional uptake analyses support the hypothesis that astrocytes are involved in vitamin C recycling in the nervous system. This recycling model may work as an efficient system for the salvage of vitamin C by avoiding the hydrolysis of dehydroascorbic acid produced by antioxidative protection.

Estrogen and progesterone up-regulate glucose transporter expression in ZR-75-1 human breast cancer cells.

Breast cancer incidence increases in women receiving combined estrogen and progesterone therapy. Breast tumors show increased expression of the glucose transporter GLUT1. We determined the effect of these hormones on GLUT1-4 expression and deoxyglucose transport in ZR-75-1 breast cancer cells. Immunoblotting, immunocytochemistry, flow cytometry, and RT-PCR showed that GLUT1 expression is up-regulated by progesterone and, to a greater degree, combined therapy. GLUT2 expression is unaffected by hormonal treatment. GLUT3 protein and RNA is up-regulated by progesterone and combined therapy, and GLUT4 protein expression is up-regulated by all hormonal treatments. Deoxyglucose transport studies revealed the presence of three transport components with characteristics corresponding to GLUT1/4, GLUT2, and GLUT3. 17beta-Estradiol produced a slight increase in transport at the Michaelis constant (Km) corresponding to GLUT3. Progesterone produced a small increase in transport at the Km corresponding to GLUT1/4, and combined 17beta-estradiol and progesterone produced a small increase in transport at the Km corresponding to GLUT3 and a large increase in transport at the Km corresponding to GLUT1/4. This indicates that 17beta-estradiol and progesterone differentially regulate GLUT1-4 expression and that these changes correlate to changes in glucose uptake. We postulate that combined hormone replacement therapy provides a survival advantage to developing ZR-75 breast cancer cells.