Glut(s) expression PC-3 cell line-derived xenograft model.

Gene Set Enrichment Analysis (GSEA) from RNA sequencing data of PC3 cells stimulated with fructose or glucose.

Biological/clinical parameters (patient age, PSA levels, Gleason score) and glucose and fructose levels in benign and PCa patients.

Downregulation of Glut-5 expression in PC-3 cells using the siRNA methodology.

AbstractClinical localization of primary tumors and sites of metastasis by PET is based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer, however, PET-FDG imaging has shown limited clinical applicability, suggesting that prostate cancer cells may utilize hexoses other than glucose, such as fructose, as the preferred energy source. Our previous studies suggested that prostate cancer cells overexpress fructose transporters, but not glucose transporters, compared with benign cells. Here, we focused on validating the functional expression of fructose transporters and determining whether fructose can modulate the biology of prostate cancer cells in vitro and in vivo. Fructose transporters, Glut5 and Glut9, were significantly upregulated in clinical specimens of prostate cancer when compared with their benign counterparts. Fructose levels in the serum of patients with prostate cancer were significantly higher than healthy subjects. Functional expression of fructose transporters was confirmed in prostate cancer cell lines. A detailed kinetic characterization indicated that Glut5 represents the main functional contributor in mediating fructose transport in prostate cancer cells. Fructose stimulated proliferation and invasion of prostate cancer cells in vitro. In addition, dietary fructose increased the growth of prostate cancer cell line–derived xenograft tumors and promoted prostate cancer cell proliferation in patient-derived xenografts. Gene set enrichment analysis confirmed that fructose stimulation enriched for proliferation-related pathways in prostate cancer cells. These results demonstrate that fructose promotes prostate cancer cell growth and aggressiveness in vitro and in vivo and may represent an alternative energy source for prostate cancer cells.Significance:This study identifies increased expression of fructose transporters in prostate cancer and demonstrates a role for fructose as a key metabolic substrate supporting prostate cancer cells, revealing potential therapeutic targets and biomarkers.

Activation of glucose transporter-1 (Glut-1) gene expression is a molecular feature of cancer cells that increases glucose uptake and metabolism. Increased glucose uptake is the basis for the clinical localization of primary tumors using positron emission tomography (PET) and 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) as a radiotracer. However, previous studies have demonstrated that a considerable number of cancers, which include prostate cancer (CaP), express low to undetectable levels of Glut-1 and that FDG-PET has limited clinical applicability in CaP. This observation could be explained by a low metabolic activity of CaP cells that may be overcome using different hexoses, such as fructose, as the preferred energy source. However, these hypotheses have not been examined critically in CaP. This review article summarizes what is currently known about transport and metabolism of hexoses, and more specifically fructose, in CaP and provides experimental evidences indicating that CaP cells may have increased capacity to transport and metabolize fructose in vitro and in vivo. Moreover, this review highlights recent findings that allow better understanding of how metabolism of fructose may regulate cancer cell proliferation and how fructose uptake and metabolism, through the de novo lipogenesis pathway, may provide new opportunities for CaP early diagnosis, staging, and treatment.

Introduction PET-scanning can detect primary tumours relaying on their high glucose uptake ability through overexpression of the glucose transporter Glut-1. However, this method has shown limited clinical applicability for prostate cancer (PCa) diagnosis, suggesting that PCa cells do not use glucose as its primary source of energy. Preliminary data from our laboratory has shown overexpression of the fructose transporter Glut-5 in PCa cell lines and in clinical specimens of PCa, which suggest that fructose could play an important role in PCa biology. Material and methods We analysed the effect of fructose on aggressiveness and metabolic reprogramming in benign prostate epithelial and PCa cell lines. Cells were incubated with glucose or fructose in the media for 24, 48, and 72 hour and then we evaluated: 1) the proliferative rate of benign and PCa cells, 2) the invasion and migration capacity of PCa cells, and 3) the mRNA levels of the enzymes involved in glycolysis, pentose phosphate and de novo lipogenesis pathways using real-time PCR in PCa cells. The effect of fructose on tumour growth was analysed by a PC3 cell line xenograft in immunosuppressed NSG mice. 15% fructose was added to the drinking water for 8 weeks. Tumour weight and the expression of glycolytic enzymes by qPCR was evaluated with respect to the control (water without additives). Results and discussions PCa cells incubated with fructose or glucose, showed similar proliferative rate, invasion and migration capacities. However, fructose evokes a different expression profile of the enzymes involve in glycolysis, pentose phosphate and de novo lipogenesis pathways. Fructose promotes tumour growth of PC3 cells in the NSG mice. Conclusion Our data suggest that fructose could play an important role in PCa pathophysiology, promoting proliferation and migration of PCa cells in vitro , and increasing tumour growth in vivo thanks to a reprogram in their metabolism that allows PCa cells to use fructose as effectively as glucose.

Introduction The clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET) is based on the enhanced cellular uptake of 2-deoxy-2-[ 18 F]-fluoro-d-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilise hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells. Material and methods We determined the glucose and fructose concentration in serum from benign and CaP patients, the protein expression of Glut1, Glut5 and Glut9 and mRNA expression of Glut1, Glut5, Glut7, Glut9 and Glut11 in clinical specimens of benign and malignant prostate tissue using immunostaining and qRT-PCR. Moreover, mRNA and protein expression for the glucose transporters was analysed in vitro in benign (RWPE-1) and malignant (LNCaP, LNCaP-C4-2, DU-145, and PC3) CaP cell lines using qRT-PCR and western blot. Fructose and glucose uptake was measured in vitro in prostate cell lines using radiolabelled d-[U- 14 C]-fructose or 2-[1,2–3 hour]-deoxy-d-[ 3 H]-glucose, respectively. The effect of fructose and glucose on mitochondrial metabolism was analysed in prostate cell lines using seahorse analysis and the effect of fructose on tumour growth was analysed in vivo using a PC3 cell line xenograft model using immunosuppressed NSG mice. Results and discussions Fructose concentration was elevated in CaP patients compared to benign and no difference in glucose levels was observed. The expression of the fructose transporters, Glut5 and Glut9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels, basal and maximal respiration in bening and CaP cells were similar in the presence of fructose or glucose. Lastly, fructose was able to increase the growth rate, size and weight of the tumours in the NSG mice compared to controls (without fructose). Conclusion Our results indicate that fructose may represent an alternative energy source for CaP cells and may promote CaP tumour growth of CaP cells in vivo .

Abstract The elevated level of glucose uptake and metabolism in cancers is the basis for the clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET scanning), based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilize hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells. mRNA and protein expression for the glucose transporter Glut-1 and fructose transporters Glut-2, Glut-5, Glut-7, Glut-9 and Glut-11 was analyzed in benign (PWR-1E, RWPE-1) and malignant (LNCaP, vCaP, LNCaP-C4-2, DU-145, and PC-3) human prostate cell lines using qRT-PCR and western blot, respectively. In addition, Glut(s) protein expression was analyzed on a tissue microarray containing 200 formalin-fixed paraffin-embedded benign and malignant human prostate tissues using immunohistochemistry. Fructose and glucose uptake was measured in vitro in benign and malignant human prostate cell lines using radiolabelled D-[U-14C]-fructose or 2-[1,2-3H]-deoxy-D-[3H]-glucose, respectively. Lastly, the effect of fructose or glucose on the levels of ATP, mitochondrial metabolism, and expression of the enzymes hexokinase-2 (HK2), type-C fructokinase (KHK-C), pyruvate kinase M2 (PKM2) and type-A lactate dehydrogenase (LDH-A) was analyzed in benign and malignant human prostate cell lines using chemiluminescence, seahorse, and qRT-PCR analyses, respectively. Our results indicated that expression of the fructose transporters, Glut-5 and Glut-9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut-1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels in CaP cells were similar in the presence of fructose or glucose. Fructose, but not glucose, significantly altered mRNA expression of HK2, KHK-C, PKM2, and LDH-A in malignant human prostate cells. Taken together, our results suggest that fructose may represent an alternative energy source and may reprogram hexose metabolism in CaP cells. Citation Format: Daniela Carreño, Nestor Corro, Marcia Arredondo, Carmen Navarro, Verónica Torres, Viviana Montecinos, Paula Sotomayor, Francisco Nualart, Julio Cesar Cárdenas, Alejandro S. Godoy. Role of fructose in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2017-448

Key points Extracellular ATP, in association with [Ca 2+ ] i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Abstract Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca 2+ ] i ‐dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca 2+ ] i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml −1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1–100 μ m ) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 m m ), associated with inflammatory conditions, lowered basal CBF by reducing [Ca 2+ ] i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.

Abstract Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Androgen deprivation therapy (ADT) is the standard treatment for advanced or metastatic CaP. However, during ADT, CaP progresses from an androgen-sensitive (AS-CaP) to a more aggressive, and eventually lethal, castration-resistant (CRPC) phenotype. There is evidence to suggest that the prostate tumor mass is under tight control of endothelial microvasculature due to an increase in angiogenesis by tumor cells. Nevertheless, now there is evidence to support that this influence is not one-directional and that the endothelial cells secrete a large number of active substances (angiocrine factors), which may directly or indirectly influence tumor growth and progression. However, the direct impacts of the endothelium on prostate tumor progression or the molecular mechanisms that are involved in this communication remain unclear. Here we investigated the potential influence of endothelium-derived paracrine factors on prostate cancer biology and the role of connexins in these interactions, since connexins play a major role in cell-cell communication and form a bidirectional signaling pathway to assemble gap junctions and alter cell behaviors. We measured the effect of conditioned media (CM) obtained from a primary culture of human endothelial cells isolated from umbilical vein (HUVEC) on viability, proliferation, migration and invasion of CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) and in the metastatic potential by in vivo assays using co-injection of CaP cell with HUVEC or injection of CaP cells pre-incubated with CM from HUVEC in a zebrafish embryo model. Finally, we studied the expression and the role of connexins on this stimulation using pharmacological (GJIC inhibitors) approaches. All together, our results showed that CM from endothelial cell induces an increases in the viability and proliferation in all CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) but only increases migration of the CRPC cell lines (LNCaP-C4-2 and PC3). We also observed in our in vivo model that endothelial cells either through cell-cell interaction or by paracrine communication increases the metastatic ability of the CaP cells. Moreover, the increase in viability and migration of CaP cells observed with CM from endothelial cells was blocked using inhibitors of gap junctions. Real-time PCR analyses detected an up-regulation of Cx43 mRNA after exposition to CM from endothelial cells. Our data suggest that angiocrine communication between endothelial cells and CaP cells increases proliferation and migration of more aggressive CaP cells which could be important for the acquisition of the aggressive phenotype of the disease, and this interaction could be mediated by Cx43. Delineation of such critical players may culminate in identifying therapeutic targets or biomarkers to counteract CaP, especially advanced CaP. Citation Format: Verónica Torres-Estay, Patricia Fuenzalida, Catalina Ascencio, Carla Cembrano, Daniela Carreño, Néstor Corro, Viviana Montecinos, Gareth Owen, Xavier Figueroa, Julio Amigo, Juan Carlos Saéz, Alejandro Godoy. Paracrine effect of the endothelium on prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2017-2969

BACKGROUND:Sex-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).RESULTS:Immunohistochemical 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.

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.

Prostaglandins (PGs) have been reported to be present in the seminal fluid and cervical mucus, affecting different stages of sperm maturation from spermatogenesis to the acrosome reaction. This study assessed the effects of low physiological PGE2 and PGF2α concentrations on human sperm motility and on the ability of the spermatozoa to bind to the zona pellucida (ZP). Human spermatozoa were isolated from seminal samples with normal concentration and motility parameters and incubated with 1μM PGE2, 1μM PGF2α or control solution to determine sperm motility and the ability to bind to human ZP. The effects of both PGs on intracellular calcium levels were determined. Incubation for 2 or 18h with PGE2 or PGF2α resulted in a significant (P<0.05) increase in the percentage of spermatozoa with progressive motility. In contrast with PGF2α, PGE2 alone induced an increase in sperm intracellular calcium levels; however, the percentage of sperm bound to the human ZP was doubled for both PGs. These results indicate that incubation of human spermatozoa with low physiological levels of PGE2 or PGF2α increases sperm functions and could improve conditions for assisted reproduction protocols.

Mucociliary clearence constitutes a barrier against contaminants and microorganisms inhaled from the environment. Its efficiency depends mainly on the frequency of ciliary beat (CBF). Airways contaminants like lyposaccharide (LPS) activate a specific toll like receptors (TLR4) of the immune system that produce an inflammatory response associated to the production of cytokines by the airway epithelium. However, the effect of LPS in the effectiveness of mucociliary clearance it is unknown so we studied the effect of LPS in frequency of CBF and the mechanism involved.Primary cultures of mouse (Balbc) tracheal epithelium were incubated with different concentrations of LPS and P2×7 and pannexin‐1 inhibitors,(oATP, KN‐62, Probenecid and Carbenoxolone), CBF was determined using a microphotodensitometric technique (n >; 3). ATP was measured in culture medium using luciferin‐luceferase reagent and. All these experiments were statistically analyzed with ANOVA, and post‐test Tukey.LPS induce an increase in CBF in a dose response manner that was blocked by the incubation of all inhibitors used. LPS produce a raise in ATP concentration in culture media after 20min of incubation with 10 μg/ml.These results indicate that LPS affect ciliary activity in the airways by releasing ATP to the extracellular where P2×7 and pannexin‐1 could be involved.

Mucociliary transport (MCT) is a mechanism that is responsible for the movement of gametes and embryos through the oviduct and serves as physical barrier in the epithelium, to prevent microorganisms invasion. The rate of MCT depends on the ciliary beat frequency (CBF) of ciliated cells of the epithelium. The CBF is regulated by several molecules that are secreted by gametes, embryos or even the epithelium itself, such as ATP, prostaglandins (PG) and nitric oxide. ATP and PG increased the CBF, while NO antagonized this effects, in this system. Other molecule that is secreted by the oviductal epithelium is Tumor Necrosis factor-a (TNFα). High concentrations of this molecule are produced in pathological conditions, causing sloughing of ciliated cells. By the other hand, in physiological conditions TNF is present at low concentrations in the oviductal fluid. Although, in the oviduct there is no evidence about control of CBF by TNFα and the impact on the MTC. In others ciliated epitheliums, TNFα modifies the CBF, effects that are mediated by the nitric oxide pathway. In the oviduct, however there is no evidence about NO production in response to TNFα, but it was established that this molecule induce the synthesis of PG by the epithelial cells. The main goal of this work was to determine the concentration-dependent effect of TNFα, over the CBF in the rat oviduct, and the involvement of prostaglandins and nitric oxide pathways in this effect. Methods: Using microphotodensitometric analysis of primary cultured rat oviductal ciliated cells, we determined changes in CBF in response to TNFα, (0,01-100 ng/ml), in the presence or absence of an inhibitor of arachidonic acid release (AACOCF3 50 μM), an inhibitor of prostaglandins (Indometacin 100 μM), and nitric oxide (L-NAME 100 μM) production. Results: TNFα, in a concentration range of 0,1–10 ng/ml increased the CBF, while concentrations of 50 and 100 ng/ml did not affect the CBF. The concentration-response curve fit to a biphasic curve, with an ascending EC50 of 6,123 × 10−12 M (0,1 ng/ml) and a descending EC50 of 2,03 × 10−9 M (36 ng/ml). The increase in CBF observed with TNFα, 1 ng/ml is inhibited in the presence of AACOCF3 and Indometacin, while TNFα 50 ng/ml in the presence of L-NAME, increased CBF in a similar way as TNFα, 1 ng/ml. Conclusion: TNFa show a biphasic effect on the CBF that is concentration-dependent. The ascending phase of the CBF, observed at low concentration of TNFα, seems to be PG–dependent, while the nitric oxide pathway seems to be involve in the descending phase of CBF, observed at higher concentrations of TNFα, suggesting that nitric oxide pathway, is a negative regulator of the CBF, at this conditions. Our results suggest that TNFα, may have dual effects on the CBF and the MCT in the oviduct. FONDECYT 1040804. (poster)