Alejandro S. Godoy

Differential subcellular distribution of glucose transporters GLUT1–6 and GLUT9 in human cancer: Ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2–6 and 9. The classical expression of GLUT1–5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low‐to‐negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT‐PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over‐expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers. J. Cell. Physiol.

Expression of the hexose transporters GLUT1 and GLUT2 during the early development of the human brain

We used immunohistochemistry with anti-glucose transporter antibodies to document the presence of facilitative hexose transporters in the fetal human brain. GLUT1 is expressed in all regions of the fetal brain from ages 10 to 21 weeks. GLUT1 was present in the endothelial cells of the brain capillaries, the epithelial cells of the choroid plexus and neurons. High expression of GLUT2 was observed in the granular layer of the cerebellum in brains 21 weeks old, but GLUT2 immunoreactivity was absent at earlier stages. GLUT3 and GLUT4 immunoreactivities were absent at all stages studied. GLUT5 immunoreactivity was evident only in the cerebellar region of 21-week old fetal brains. We conclude that GLUT1 plays a fundamental role in early human brain development. The data also suggest that the cerebellum of the developing brain has the capacity to transport fructose, a substrate that has not been previously identified as a source of metabolic energy in the adult human brain.

Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2 ACTIVATION BY SODIUM AND ABSOLUTE DEPENDENCE ON BIVALENT CATIONS

We characterized the human Na+-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na+-dependent transporter. The properties of SVCT2 are modulated by Ca2+/Mg2+ and a reciprocal functional interaction between Na+ and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na+ increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport Km without affecting the Vmax, thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na+ cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na+:ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na+:ascorbic acid:Na+. However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca2+/Mg2+ for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport Vmax without affecting the transport Km or the Na+ cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca2+/Mg2+. At least three active states can be envisioned, including a low affinity conformation at Na+ concentrations below 20 mm and two high affinity conformations at elevated Na+ concentrations whose Na+ cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca2+/Mg2+-dependent transporter.

Stromal-Epithelial Cell Interactions and Androgen Receptor-Coregulator Recruitment Is Altered in the Tissue Microenvironment of Prostate Cancer

Tissue recombination experiments show that prostate mesenchyma directs prostate epithelial cell growth and development in an androgen-dependent manner, and that functional differentiation of prostate epithelium requires androgen-driven processes in both epithelia and stroma. The androgen induction of target genes in primary cultures of prostate stromal and epithelial cells was determined using an adenoviral expression system, which employed the MMTV-enhancer driven luciferase reporter as an androgen receptor (AR)-mediated transcription assay. These studies indicate that both cell types contain functional AR. Androgen induction of luciferase reporter activity is 3-fold in stromal cells compared with 10-fold in epithelial cells. AR-mediated transcription activity in stroma cells was enhanced by coculture with epithelial cells or epithelial cell-conditioned media. The elevated AR-mediated transcription activity in stromal cells that were exposed to epithelial factors correlated with increased recruitment of coactivators to the AR transcriptional complex. Epithelial cells facilitated interactions of AR with SRC-1 in an androgen-dependent manner. However, AR-mediated transcriptional activity in stromal cells isolated from prostate cancer was reduced compared with stromal cells isolated from benign prostate and continued to be reduced when cocultured with tumor-derived prostate epithelial cells. The occupancy of AR and coregulators on target genes showed that androgen-bound AR in prostate cancer stromal cells was associated with the corepressor silencing mediator for retinoid and thyroid hormone receptor. Thus, the ability of epithelial cells to modulate coregulator recruitment to the AR transcriptional complex on androgen-responsive genes seems altered in the stromal microenvironment of prostate cancer.

Vitamin C Is an Essential Antioxidant That Enhances Survival of Oxidatively Stressed Human Vascular Endothelial Cells in the Presence of a Vast Molar Excess of Glutathione

Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.

Elevated expression of glucose transporter‐1 in hypothalamic ependymal cells not involved in the formation of the brain–cerebrospinal fluid barrier

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter‐1 has been detected in endothelial cells of the blood–brain barrier and in choroid plexus cells of the blood–cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, α, β1 and β2. The β2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid–median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter‐1 in α and β1 tanycytes. Intense anti‐glucose transporter‐1 staining was observed in cell processes located throughout the arcuate nucleus, in the end‐feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter‐1 in β2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes α, β1, and β2 express similar levels of glucose transporter‐1 mRNA. Further analysis using anti‐glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing α and β1 tanycytes, but were abundant in regions containing the barrier‐forming β2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter‐1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in α and β1 tanycytes, we speculate whether the tanycytes might have astrocyte‐like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area. J. Cell. Biochem. 80:491–503, 2001.

Androgen receptor in human endothelial cells

Androgen receptor (AR) is a ligand-inducible transcription factor, and a member of the steroid-thyroid-retinoid receptor superfamily, that mediates the biological effects of androgens in a wide range of physiological and pathological processes. AR expression was identified in vascular cells nearly 20 years ago, and recent research has shown that AR mediates a variety of actions of androgens in endothelial and vascular smooth muscle cells. In this mini-review, we review evidence indicating the importance of AR in human endothelial cell (HUVEC) homeostatic and pathogenic processes. Although a role for AR in the modulation of HUVEC biology is evident, the molecular mechanisms by which AR regulates HUVEC homeostasis and disease processes are not fully understood. Understanding these mechanisms could provide critical insights into the processes of pathogenesis of diseases ranging from cardiovascular disease to cancer that are major causes of human morbidity and mortality.

Altered corepressor SMRT expression and recruitment to target genes as a mechanism that change the response to androgens in prostate cancer progression

Androgen receptor (AR) is required for the development and progression of prostate cancer (CaP) from androgen-dependence to androgen-resistance. Both corepressors and coactivators regulate AR-mediated transcriptional activity, and aberrant expression or activity due to mutation(s) contributes to changes in AR function in the progression to androgen resistance acquired during hormonal ablation therapies. Primary culture of epithelial cells from androgen-dependent CWR22 and androgen-resistant CWR22R xenograft tumors were used to evaluate the effect of androgens on AR function, and the association with coactivators (SRC-1 and TIF-2) and corepressors (SMRT and NCoR). Both androgen-dependent CWR22 and androgen-resistant CWR22R cells expressed functional AR as the receptor bind ligand with high affinity and increased trafficking to the nuclei in the presence of androgens. However, in the presence of androgens, AR-mediated transcriptional activity in androgen-sensitive CWR22 cells was limited to a 2-fold increase, as compared to a 6-fold increase in androgen-resistance CWR22R cells. In androgen-sensitive CWR22 cells, immunoblot, confocal microscopy, and chromatin immunoprecipitation assays indicated that the androgen bound AR transcriptional initiation complex in the PSA promoter contained corepressor SMRT, resulting in limited receptor transcriptional activity. In contrast, increased AR-mediated transcriptional activity in the CWR22R cells was consistent with decreased expression and recruitment of the corepressors SMRT/NCoR, as well as increased recruitment of the coactivator TIF-2 to the receptor complex. Similar changes in the response to androgens were observed in the LNCaP/C4-2 model of androgen resistance prostate cancer. Thus, altered recruitment and loss of corepressors SMRT/NCoR may provide a mechanism that changes the response of AR function to ligands and contributes to the progression of the advanced stages of human prostate cancer.

Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms

BackgroundSex-related differences in the role of androgen have been reported in cardiovascular diseases and angiogenesis. Moreover, androgen receptor (AR) has been causally involved in the homeostasis of human prostate endothelial cells. However, levels of expression, functionality and biological role of AR in male- and female-derived human endothelial cells (ECs) remain poorly characterized. The objectives of this work were (1) to characterize the functional expression of AR in male- and female-derived human umbilical vein endothelial cell (HUVEC), and (2) to specifically analyze the biological effects of DHT, and the role of AR on these effects, in male-derived HUVECs (mHUVECs).ResultsImmunohistochemical analyses of tissue microarrays from benign human tissues confirmed expression of AR in ECs from several androgen-regulated and non-androgen-regulated human organs. Functional expression of AR was validated in vitro in male- and female-derived HUVECs using quantitative RT-PCR, immunoblotting and AR-mediated transcriptional activity assays. Our results indicated that functional expression of AR in male- and female-derived HUVECs was heterogeneous, but not sex dependent. In parallel, we analyzed in depth the biological effects of DHT, and the role of AR on these effects, on proliferation, survival and tube formation capacity in mHUVECs. Our results indicated that DHT did not affect mHUVEC survival; however, DHT stimulated mHUVEC proliferation and suppressed mHUVEC tube formation capacity. While the effect of DHT on proliferation was mediated through AR, the effect of DHT on tube formation did not depend on the presence of a functional AR, but rather depended on the ability of mHUVECs to further metabolize DHT.Conclusions(1) Heterogeneous expression of AR in male- and female-derived HUVEC could define the presence of functionally different subpopulations of ECs that may be affected differentially by androgens, which could explain, at least in part, the pleiotropic effects of androgen on vascular biology, and (2) DHT, and metabolites of DHT, generally thought to represent progressively more hydrophilic products along the path to elimination, may have differential roles in modulating the biology of human ECs through AR-dependent and AR-independent mechanisms, respectively.

Association of RNASEL and 8q24 variants with the presence and aggressiveness of hereditary and sporadic prostate cancer in a hispanic population

To study the association between the polymorphisms Arg462Gln and Asp541Glu from the RNASEL gene (1q25), and the polymorphisms rs620861, rs1447295, rs6983267, rs7837328 from the chromosome 8q24 with the risk of presenting prostate cancer (PCa) and its clinical characteristics in a Hispanic (Chilean) population. The study was performed on 21 control patients and 83 patients diagnosed with PCa. Polymorphisms were analysed from blood samples through real‐time PCR by using TaqMan probes, and the genetic analysis was performed with the SNPStats program. Also, a comparison was performed between clinical characteristics of PCa and the presence of the different polymorphism genotypes by using the Minitab software. There was a significant association between the genotype G/G from the polymorphism rs6983267 with an overall increased risk of PCa, in patients both with or without family history of PCa (OR = 4.47, 95% CI = 1.05–18.94, P = 0.034 and OR = 3.57, 95% CI = 0.96–13.35, P = 0.037, respectively). Regarding clinical parameters, patients carrying the genotype C/C from the polymorphism Asp541Glu had significantly higher prostate‐specific antigen (PSA) levels than patients carrying the other genotypes (P = 0.034). Moreover, patients with the genotype G/G of rs6983267 had higher PSA levels (P = 0.024). The polymorphism rs6983267 from region 3 of the chromosome 8q24 appears to be a prominent risk factor for PCa and a biomarker for cancer aggressiveness in the group of patients who presented higher levels of PSA at the time of diagnosis.