Targeting Glucose Transporters Research Articles

Synthesis and In Vitro Anti-tumor Evaluation of Novel Glycoconjugated SN38 Derivatives

Abstract Five types of novel glycoconjugated 7-ethyl-10-hydroxycamptothecin (SN38) derivatives were synthesized to evaluate their anti-tumor activity against HCT116 human colon cancer cells, and the results showed that all of them demonstrated potent anti-tumor activity. Notably, glucose and galactose conjugates exhibited the better anti-tumor activity, while the mannose and allose conjugates had slightly lower activity. Our findings suggested that the anti-tumor activity of glycoconjugated SN38 derivatives was affected by the type of modifying molecule used and the position of the modification. These findings could have implications for the development of novel anti-tumor agents targeting glucose transporters.

ROS-removing nano-medicine for navigating inflammatory microenvironment to enhance anti-epileptic therapy.

As a neurological disorder in the brain, epilepsy is not only associated with abnormal synchronized discharging of neurons, but also inseparable from non-neuronal elements in the altered microenvironment. Anti-epileptic drugs (AEDs) merely focusing on neuronal circuits frequently turn out deficient, which is necessitating comprehensive strategies of medications to cover over-exciting neurons, activated glial cells, oxidative stress and chronic inflammation synchronously. Therefore, we would report the design of a polymeric micelle drug delivery system that was functioned with brain targeting and cerebral microenvironment modulation. In brief, reactive oxygen species (ROS)-sensitive phenylboronic ester was conjugated with poly-ethylene glycol (PEG) to form amphiphilic copolymers. Additionally, dehydroascorbic acid (DHAA), an analogue of glucose, was applied to target glucose transporter 1 (GLUT1) and facilitate micelle penetration across the blood‒brain barrier (BBB). A classic hydrophobic AED, lamotrigine (LTG), was encapsulated in the micelles via self-assembly. When administrated and transferred across the BBB, ROS-scavenging polymers were expected to integrate anti-oxidation, anti-inflammation and neuro-electric modulation into one strategy. Moreover, micelles would alter LTG distribution invivo with improved efficacy. Overall, the combined anti-epileptic therapy might provide effective opinions on how to maximize neuroprotection during early epileptogenesis.

Inhibition of androgen receptor enhanced the anticancer effects of everolimus through targeting glucose transporter 12.

Everolimus was designed as a mammalian target of rapamycin (mTOR) inhibitor. It has been proven as a targeted drug for gastric cancer (GC) therapy. However, long-term treatment with everolimus may cause severe side effects for recipients. Decreasing the dosage and attenuating the associated risks are feasible to promote clinical translation of everolimus. This study aimed to identify the underlying mechanisms of responses to everolimus and develop novel regimens for GC treatment. Our findings proved that there was a significant dose-dependent relationship of everolimus-induced GC cell apoptosis and glycolysis inhibition. Then, we found that a member of glucose transporter (GLUT12) family, GLUT12, was actively upregulated to counteract the anticancer effects of everolimus. GLUT12 might be overexpressed in GC. High expression of GLUT12 might be correlated with tumor progression and short survival time of GC patients. Bioinformatic analysis suggested that GLUT12 might be involved in regulating cancer development and metabolism. The experiments proved that GLUT12 significantly promoted GC growth, glycolysis and impaired the anticancer effects of everolimus. Androgen receptor (AR) is a classical oncogenic factor in many types of cancer. Everolimus elevated GLUT12 expression in an AR-dependent manner. Inhibition of AR activity abrogated the promotive effects on GLUT12 expression. Both in-vitro and in-vivo experiments demonstrated that GLUT12 knockdown augmented anticancer effects of everolimus. Enzalutamide, an AR inhibitor, or AR knockdown was comparable to GLUT12 suppression. This study identified the role of the AR/GLUT12 pathway in the development of poor responses to everolimus. Interference with AR/GLUT12 pathway may serve as a promising approach to promoting the translational application of everolimus in GC therapy.

A new strategy for osteoarthritis therapy: Inhibition of glycolysis.

Osteoarthritis (OA) is a common degenerative disease of the joints. It is primarily caused by age, obesity, mechanical damage, genetics, and other factors, leading to cartilage degradation, synovial inflammation, and subchondral sclerosis with osteophyte formation. Many recent studies have reported that glycolysis disorders are related lead to OA. There is a close relationship between glycolysis and OA. Because of their hypoxic environment, chondrocytes are highly dependent on glycolysis, their primary energy source for chondrocytes. Glycolysis plays a vital role in OA development. In this paper, we comprehensively summarized the abnormal expression of related glycolytic enzymes in OA, including Hexokinase 2 (HK2), Pyruvate kinase 2 (PKM2), Phosphofructokinase-2/fructose-2, 6-Bisphosphatase 3 (PFKFB3), lactate dehydrogenase A (LDHA), and discussed the potential application of glycolysis in treating OA. Finally, the natural products that can regulate the glycolytic pathway were summarized. Targeting glucose transporters and rate-limiting enzymes to glycolysis may play an essential role in treating OA.

Adrenal functional imaging – which marker for which indication?

In recent years, a broad spectrum of molecular image biomarkers for assessment of adrenal functional imaging have penetrated the clinical arena. Those include positron emission tomography and single photon emission computed tomography radiotracers, which either target glucose transporter, CYP11B enzymes, C-X-C motif chemokine receptor 4, norepinephrine transporter or somatostatin receptors. We will provide an overview of key radiopharmaceuticals and determine their most relevant clinical applications, thereby providing a roadmap for the right image biomarker at the right time for the right patient. Numerous radiotracers for assessment of adrenal incidentalomas ([ 18 F]FDG; [ 123 I]IMTO/IMAZA), ACC ([ 123 I]IMTO/IMAZA; [ 18 F]FDG; [ 68 Ga]PentixaFor), pheochromocytomas and paragangliomas ([ 123 I]mIBG; [ 18 F]flubrobenguane; [ 18 F]AF78; [ 68 Ga]DOTATOC/-TATE), or primary aldosteronism ([ 11 C]MTO, [ 68 Ga]PentixaFor) are currently available and have been extensively investigated in recent years. In addition, the field is currently evolving from adrenal functional imaging to a patient-centered adrenal theranostics approach, as some of those radiotracers can also be labeled with ß-emitters for therapeutic purposes. The herein reviewed functional image biomarkers may not only allow to increase diagnostic accuracy for adrenal gland diseases but may also enable for achieving substantial antitumor effects in patients with adrenocortical carcinoma, pheochromocytoma or paraganglioma.

Conjugation of glucosylated polymer chains to checkpoint blockade antibodies augments their efficacy and specificity for glioblastoma.

Because of the blood-tumour barrier and cross-reactivity with healthy tissues, immune checkpoint blockade therapy against glioblastoma has inadequate efficacy and is associated with a high risk of immune-related adverse events. Here we show that anti-programmed death-ligand 1 antibodies conjugated with multiple poly(ethylene glycol) (PEG) chains functionalized to target glucose transporter 1 (which is overexpressed in brain capillaries) and detaching in the reductive tumour microenvironment augment the potency and safety of checkpoint blockade therapy against glioblastoma. In mice bearing orthotopic glioblastoma tumours, a single dose of glucosylated and multi-PEGylated antibodies reinvigorated antitumour immune responses, induced immunological memory that protected the animals against rechallenge with tumour cells, and suppressed autoimmune responses in the animals' healthy tissues. Drug-delivery formulations leveraging multivalent ligand interactions and the properties of the tumour microenvironment to facilitate the crossing of blood-tumour barriers and increase drug specificity may enhance the efficacy and safety of other antibody-based therapies.

Cancer Cells' Metabolism Dynamics in Renal Cell Carcinoma Patients' Outcome: Influence of GLUT-1-Related hsa-miR-144 and hsa-miR-186.

Simple SummaryRenal cell carcinoma (RCC) is a metabolic associated cancer and the most common and lethal neoplasia in the adult kidney. This study aimed to understand the potential role of hsa-miR-144-5p and hsa-miR-186-3p (which target Glucose Transporter 1—GLUT-1) in clear cell RCC (ccRCC) glycolysis status, as well as their potential as biomarkers. A decrease of intracellular levels of these miRNAs and increase of their excretion was associated with an increase of GLUT-1’s levels and glycolysis’ markers. RCC patients presented higher plasmatic levels of hsa-miR-186-3p than healthy individuals and hsa-miR144-5p’s higher levels were associated with early clinical stages of RCC. Additionally, patients with low plasmatic levels of hsa-miR-144-5p and high plasmatic levels of hsa-miR-186-3p (high-risk group) showed a worse overall survival. Overall, these results indicate that circulating hsa-miR-144-5p and hsa-miR-186-3p may be potential biomarkers of ccRCC prognosis.The cancer cells’ metabolism is altered due to deregulation of key proteins, including glucose transporter 1 (GLUT-1), whose mRNA levels are influenced by microRNAs (miRNAs). Renal cell carcinoma (RCC) is the most common and lethal neoplasia in the adult kidney, mostly due to the lack of accurate diagnosis and follow-up biomarkers. Being a metabolic associated cancer, this study aimed to understand the hsa-miR-144-5p and hsa-miR-186-3p’s potential as biomarkers of clear cell RCC (ccRCC), establishing their role in its glycolysis status. Using three ccRCC lines, the intra- and extracellular levels of both miRNAs, GLUT-1’s mRNA expression and protein levels were assessed. Glucose consumption and lactate production were evaluated as glycolysis markers. A decrease of intracellular levels of these miRNAs and increase of their excretion was observed, associated with an increase of GLUT-1’s levels and glycolysis’ markers. Through a liquid biopsy approach, we found that RCC patients present higher plasmatic levels of hsa-miR-186-3p than healthy individuals. The Hsa-miR144-5p’s higher levels were associated with early clinical stages. When patients were stratified according to miRNAs plasmatic levels, low plasmatic levels of hsa-miR-144-5p and high plasmatic levels of hsa-miR-186-3p (high-risk group) showed the worst overall survival. Thus, circulating levels of these miRNAs may be potential biomarkers of ccRCC prognosis.

Glucosamine-conjugated nanoseeds for chemo-magneto hyperthermia therapy of cancer

Nowadays, magnetic hyperthermia is being extensively used in the treatment of cancer therapy due to its ability to kill cancer cells through the thermal effect. This study reports the synthesis of multifunctional Doxorubicin (DOX) loaded Gadolinium/Cobalt@Iron oxide-Dendrimer-Glucosamine-Nanoseed (Gd/Co@IO-D-G-NS) for the treatment of prostate cancer. Furthermore, glucosamine ligand was attached to enhance the effectiveness of nanoseeds via targeting glucose transporters. The release profile of DOX from Gd/Co@IO-D-G-NS nanoseeds was observed to be pH-dependent. The developed nanosseds showed acceptable hemocompatibility, and were able to produce combined hyperthermia and chemotherapeutic effect in vitro in PC3 cells. Moreover, cellular uptake studies revealed that nanoseeds were effectively uptaken by cancer cells through receptor-mediated endocytosis. It is expected that the outcome of this study will assist in progressing the know-how towards the development of nanoseeds for chemo-photothermal therapy of resectable cancers, including prostate and breast, to naming a few.

Glucose transporters in cardiovascular system in health and disease.

Glucose transporters are essential for the heart to sustain its function. Due to its nature as a high energy-consuming organ, the heart needs to catabolize a huge quantity of metabolic substrates. For optimized energy production, the healthy heart constantly switches between various metabolites in accordance with substrate availability and hormonal status. This metabolic flexibility is essential for the maintenance of cardiac function. Glucose is part of the main substrates catabolized by the heart and its use is fine-tuned via complex molecular mechanisms that include the regulation of the glucose transporters GLUTs, mainly GLUT4 and GLUT1. Besides GLUTs, glucose can also be transported by cotransporters of the sodium-glucose cotransporter (SGLT) (SLC5 gene) family, in which SGLT1 and SMIT1 were shown to be expressed in the heart. This SGLT-mediated uptake does not seem to be directly linked to energy production but is rather associated with intracellular signalling triggering important processes such as the production of reactive oxygen species. Glucose transport is markedly affected in cardiac diseases such as cardiac hypertrophy, diabetic cardiomyopathy and heart failure. These alterations are not only fingerprints of these diseases but are involved in their onset and progression. The present review will depict the importance of glucose transport in healthy and diseased heart, as well as proposed therapies targeting glucose transporters.

Discovery and Optimization of Glucose Uptake Inhibitors.

Aerobic glycolysis, originally identified by Warburg as a hallmark of cancer, has recently been implicated in immune cell activation and growth. Glucose, the starting material for glycolysis, is transported through the cellular membrane by a family of glucose transporters (GLUTs). Therefore, targeting glucose transporters to regulate aerobic glycolysis is an attractive approach to identify potential therapeutic agents for cancers and autoimmune diseases. Herein, we describe the discovery and optimization of a class of potent, orally bioavailable inhibitors of glucose transporters, targeting both GLUT1 and GLUT3.

Decreasing the survival ability of brain metastatic breast cancer cell by targeting glucose transporter 3

Breast cancer brain metastases (BBMs) are found in 15–20 % of breast cancer patients. And it results in a high mortality once the brain metastases are diagnosed. Therefore, making it more comprehensive about the mechanisms of BBMs is emergently necessary. Several studies have demonstrated that tumor microenvironment has potent roles in the tumor progression and metastasis. Besides, according to the former studies, cells in different organs using glucose might be various; as well as the dominant glucose transporters used are distinct. Here, we analyzed the brain metastatic breast cancer (BR) cells collected from intracardiac injection of nude mice and found the utilization of glucose transporters in the BR cells were different from the parental breast cancer cells; the elevated expressions of glucose transporter 3 were observed. In addition, the higher anaerobic glycolysis ability was found in BR cells. And after GLUT3 knockdown, the glucose reprogramming in BR cells was significantly decreased as well as the cancer malignant behaviors both in vitro and in vivo. Besides manipulating GLUT3 expressions, we also provide a potential mechanism that CREB could be a potent regulator of GLUT3 in BR cells by chromatin immunoprecipitation (ChIP) and knockdown assay. After decreasing the expressions of CREB in BR cells, the expressions of GLUT3 were also significantly reduced. In sum, our results reveal that GLUT3 plays an important role in provoking breast cancer cells surviving in brain by mediating glucose utilizations. And these results could provide a potential therapeutic way to target BBMs.Support or Funding Information1. Ministry of Science and Technology, R.O.C 2. Molecular Medicine Taiwan International Graduate Program, Academia Sinica

Targeting Glucose Transporters for Breast Cancer Therapy: The Effect of Natural and Synthetic Compounds.

Reprogramming of cellular energy metabolism is widely accepted to be a cancer hallmark. The deviant energetic metabolism of cancer cells-known as the Warburg effect-consists in much higher rates of glucose uptake and glycolytic oxidation coupled with the production of lactic acid, even in the presence of oxygen. Consequently, cancer cells have higher glucose needs and thus display a higher sensitivity to glucose deprivation-induced death than normal cells. So, inhibitors of glucose uptake are potential therapeutic targets in cancer. Breast cancer is the most commonly diagnosed cancer and a leading cause of cancer death in women worldwide. Overexpression of facilitative glucose transporters (GLUT), mainly GLUT1, in breast cancer cells is firmly established, and the consequences of GLUT inhibition and/or knockout are under investigation. Herein we review the compounds, both of natural and synthetic origin, found to interfere with uptake of glucose by breast cancer cells, and the consequences of interference with that mechanism on breast cancer cell biology. We will also present data where the interaction with GLUT is exploited in order to increase the efficiency or selectivity of anticancer agents, in breast cancer cells.

Chromopynones are pseudo natural product glucose uptake inhibitors targeting glucose transporters GLUT-1 and -3.

The principles guiding the design and synthesis of bioactive compounds based on natural product (NP) structure, such as biology-oriented synthesis (BIOS), are limited by their partial coverage of the NP-like chemical space of existing NPs and retainment of bioactivity in the corresponding compound collections. Here we propose and validate a concept to overcome these limitations by de novo combination of NP-derived fragments to structurally unprecedented 'pseudo natural products'. Pseudo NPs inherit characteristic elements of NP structure yet enable the efficient exploration of areas of chemical space not covered by NP-derived chemotypes, and may possess novel bioactivities. We provide a proof of principle by designing, synthesizing and investigating the biological properties of chromopynone pseudo NPs that combine biosynthetically unrelated chromane- and tetrahydropyrimidinone NP fragments. We show that chromopynones define a glucose uptake inhibitor chemotype that selectively targets glucose transporters GLUT-1 and -3, inhibits cancer cell growth and promises to inspire new drug discovery programmes aimed at tumour metabolism.

Glycoconjugated Site-Selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells.

DNA-alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings because of the indiscriminate DNA damage that they cause and their inability to specifically target cancer cells. We have developed a strategy for overcoming the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells because of its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T-rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin is, a Food and Drug Administration-approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analogue of our molecule provide evidence of uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side effects resulting from off-target damage.

Abstract B49: Metabolic stress within tumors lead to exhaustion of melanoma antigen-specific CD8+ T cells

Abstract The 5-year survival rate of late-stage melanoma patients is less than 5%. Adoptive immune transfer has demonstrated the potency of melanoma-associated antigen (MAA)-specific T cells in inducing clinical responses. Nevertheless, cancer vaccines against melanoma that aim to induce such T cells have largely been ineffective in patients with advanced disease. One major barrier of cancer vaccine efficacy is immunoinhibition within the tumor microenvironment (TME). Vaccine-induced MAA-specific tumor infiltrating T cells (TILs) commonly show signs of T cell exhaustion within TME by upregulating co-inhibitory molecules combined with loss of functions. This has traditionally been viewed as the result of chronic antigen stimulation. However, our data show that TILs directed to an antigen that is not expressed by the tumor also become exhausted, which led us to hypothesize that exhaustion of TILs is linked to metabolic stress within the TME. Upon activation the energy production of T cells switches from oxidative phosphorylation (OXPHOS) to glycolysis, which requires enhanced uptake of glucose for ATP production. Glycolysis is also used by tumor cells, which may lead to glucose deprivation within the TME, forcing MAA-specific TILs to rely on OXPHOS. OXPHOS requires oxygen, which due to lack of blood supply can also be limiting within TME. TILs therefore face dual metabolic jeopardy, i.e., lack of glucose and oxygen, which may affect their functions and thereby impair the efficacy of cancer vaccines. Using a B16 mouse melanoma model and an adenoviral vector based polyepitope melanoma vaccine, we tested if vaccine-induced TILs experience metabolic stress within the TME and whether this contributes to T cell exhaustion. Our results indicate that tumors recruit vaccine-induced tyrosinase-related protein (Trp)-1-specific CD8+ T cells, which proliferate within TME, upregulate co-inhibitory markers and become functionally impaired. Activated CD8+ T cells directed to an unrelated antigen expressed by another vaccine were also recruited to the tumors; although they fail to proliferation they also increased co-inhibitors programmed death (PD)-1 and lymphocyte activation gene (LAG)-3 expression with downregulation of T-bet, a critical transcription factor regulating T cell effector functions. Comparing markers of mitochondrial functions and oxidative deficiencies between TILs and CD8+ T cells stimulated in vitro under conditions producing energy through OXPHOS or glycolysis and under normal or reduced oxygen levels, we showed that activated CD8+ TILs undergo oxidative stress evidenced by loss of mitochondrial membrane potential (MMP), increases of mitochondrial reactive oxygen species (MROS) and enhanced levels of hypoxia inducible factor 1 subunit α (HIF-1α) and its downstream target glucose transporter (Glut)1. This metabolic profile was largely mirrored by T cells activated in vitro under hypoxia. Furthermore, our data demonstrated links between the T cells metabolic stress and their exhaustion status. Enhanced expression of PD-1 was linked to lack of glucose as cells cultured in galactose significantly increased PD-1 compared to cells with access to glucose. Increased expression of LAG-3 was most pronounced on cells cultured under hypoxia or in medium with drug that stabilizes HIF-1α. Knocking down HIF-1α in activated CD8+ T cells cultured under hypoxia significantly reduced their LAG-3 expression and increased their T-bet levels, implying a direct impact of hypoxia-induced HIF-1α on the expression of co-inhibitor LAG-3 and overall exhaustion status of T cells. In summary, our data indicate that within the TME metabolic stress due to lack of glucose and oxygen rather than sustained activation causes T cell exhaustion. These findings suggest that lifting the metabolic stress of MAA-specific TILs may improve the therapeutic efficacy of cancer vaccines. Citation Format: Ying Zhang, Xiangyang Zhou, Raj Kurupati, Flavia Filisio, Abeer Hudaihed, Wynetta Giles-Davis, Hildegund CJ Ertl. Metabolic stress within tumors lead to exhaustion of melanoma antigen-specific CD8+ T cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B49.

Targeting glucose uptake with siRNA-based nanomedicine for cancer therapy

Targeting cancer metabolism is emerging as a successful strategy for cancer therapy. However, most of the marketed anti-metabolism drugs in cancer therapy do not distinguish normal cells from cancer cells, leading to severe side effects. In this study, we report an effective strategy for cancer therapy through targeting glucose transporter 3 (GLUT3) with siRNA-based nanomedicine to simultaneously inhibit the self-renewal of glioma stem cells and bulk glioma cells in a glucose restricted tumor micro-environment. We have demonstrated that cationic lipid-assisted poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles can efficiently deliver siRNA into U87MG and U251 glioma stem cells and bulk glioma cells. Nanoparticles carrying specific siRNA targeting GLUT3 (NPsiGLUT3) were able to significantly reduce the expression of GLUT3 in glioma stem cells and bulk glioma cells, while GLUT3 knockdown results in obvious cell metabolism and proliferation inhibition, and further glioma stem cells percentage down-regulation. Moreover, systemic delivery of NPsiGLUT3, via intravenous injection, significantly inhibited tumor growth in a U87MG xenograft model, due to the reduced expression of GLUT3 and down-regulated stemness of glioma cells.

The Hypoxia-inducible Factor Renders Cancer Cells More Sensitive to Vitamin C-induced Toxicity

Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of the pharmacokinetics of Vc and recent reports using cancer cell lines have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL)-defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters. The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF-positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient glycolytic pathway for energy production. Thus, our experiments link Vc-induced toxicity and cancer metabolism, providing a new explanation for the preferential effect of Vc on cancer cells.

Hypoxia-inducible factor 1 contributes to N-acetylcysteine’s protection in stroke

Stroke is a leading cause of adult morbidity and mortality with very limited treatment options. Evidence from preclinical models of ischemic stroke has demonstrated that the antioxidant N-acetylcysteine (NAC) effectively protects the brain from ischemic injury. Here, we evaluated a new pathway through which NAC exerted its neuroprotection in a transient cerebral ischemia animal model. Our results demonstrated that pretreatment with NAC increased protein levels of hypoxia-inducible factor-1α (HIF-1α), the regulatable subunit of HIF-1, and its target proteins erythropoietin (EPO) and glucose transporter (GLUT)-3, in the ipsilateral hemispheres of rodents subjected to 90min middle cerebral artery occlusion (MCAO) and 24h reperfusion. Interestingly, after NAC pretreatment and stroke, the contralateral hemisphere also demonstrated increased levels of HIF-1α, EPO, and GLUT-3, but to a lesser extent. Suppressing HIF-1 activity with two widely used pharmacological inhibitors, YC-1 and 2ME2, and specific knockout of neuronal HIF-1α abolished NAC’s neuroprotective effects. The results also showed that YC-1 and 2ME2 massively enlarged infarcts, indicating that their toxic effect was larger than just abolishing NAC's neuroprotective effects. Furthermore, we determined the mechanism of NAC-mediated HIF-1α induction. We observed that NAC pretreatment upregulated heat-shock protein 90 (Hsp90) expression and increased the interaction of Hsp90 with HIF-1α in ischemic brains. The enhanced association of Hsp90 with HIF-1α increased HIF-1α stability. Moreover, Hsp90 inhibition attenuated NAC-induced HIF-1α protein accumulation and diminished NAC-induced neuroprotection in the MCAO model. These results strongly indicate that HIF-1 plays an important role in NAC-mediated neuroprotection and provide a new molecular mechanism involved in the antioxidant’s neuroprotection in ischemic stroke.

Therapeutic role of EF24 targeting glucose transporter 1‐mediated metabolism and metastasis in ovarian cancer cells

Cancer cells require glucose to support their rapid growth through a process known as aerobic glycolysis, or the Warburg effect. As in ovarian cancer cells, increased metabolic activity and glucose concentration has been linked to aggressiveness of cancer. However, it is unclear as to whether targeting the glycolytic pathway may kill the malignant cells and likely have broad therapeutic implications against ovarian cancer metastasis. In the present research, we found that EF24, a HIF-1α inhibitor, could significantly block glucose uptake, the rate of glycolysis, and lactate production compared with vehicle treatment in SKOV-3, A2780 and OVCAR-3 cells. These results might possibly contribute to the further observation that EF24 could inhibit ovarian cancer cell migration and invasion from wound healing and Transwell assays. Furthermore, as an important mediator of glucose metabolism, glucose transporter 1 (Glut1) was found to contribute to the function of EF24 in both energy metabolism and metastasis. To examine the effect of EF24 and the mediated role of Glut1 in vivo in a xenograph subcutaneous tumor model, intraperitoneal metastasis and lung metastasis model were introduced. Our results indicated that EF24 treatment could inhibit tumor growth, intraperitoneal metastasis and lung metastasis of SKOV-3 cells, and Glut1 is a possible mediator for the role of EF24. In conclusion, our results highlight that an anti-cancer reagent with an inhibiting effect on energy metabolism could inhibit metastasis, and EF24 is a possible candidate for anti-metastasis therapeutic applications for ovarian cancer.

Abstract 1274: Investigation into the influence of the H1047R PIK3CA mutation on the bioenergetic dependency of a cell

Abstract Background: During tumorigenesis, cells acquire mutations that confer selective advantages for growth. These mutations may reprogram cellular metabolism to meet the requirements of rapid proliferation and survival in an environment of fluctuating and scarce nutrients. Phosphoinositide-3-kinases (PI3Ks) play key roles in fundamental cellular processes and are frequently mutated in a broad range of cancers. The aim of this study was to investigate how a single endogenous activating mutation in PI3K, H1047R, affects the metabolic programming of human breast epithelial cells. Methods: We utilized X-MAN isogenic cells derived from MCF10a mammary epithelial cells, which harbour a single H1047R PIK3CA mutation introduced via AAV-mediated homologous recombination and compared them to matched parental cells with a wild type (WT) allele. This system allows use of endogenous promoters thereby more accurately recapitulating the genomic architecture in human tumours. Metabolic gene expression profiles were measured by RT-PCR. Results: We identified a multi-gene expression signature indicative of increased dependency on the glycolytic pathway. Hexokinase 2, which is involved at an early stage of the glycolytic pathway, was strongly up-regulated along with the lactate transporters MCT1 and 4. The fructose transporter GLUT5 was nearly undetectable in H1047R PIK3CA cells, suggesting a strong preference for glucose and elevated levels of aerobic glycolysis. PI3K mutant cells also down-regulated genes in the pentose phosphate pathway-shunt, tricarboxylic acid cycle and glutaminolysis pathways, indicating that aerobic glycolysis is heavily favoured. Treatment with the PI3K inhibitor GDC-0941 led to a general reversal of gene expression for these pathways, confirming the PI3K specificity of these metabolic alterations. We proceeded to investigate growth dependence of the H1047R PIK3CA and parental MCF10a cells on various nutrient sources (e.g. glucose, fructose, galactose and glutamine). While the growth rates of cells WT for PIK3CA were not significantly affected by glucose withdrawal and nutrient replacement, growth of mutant PIK3CA cells was dramatically compromised in the absence of glucose, revealing a specific dependency for this nutrient as an energy substrate. Consistent with PI3K activation mediating glucose dependency, treatment of H1047R PIK3CA cells with GDC-0941 promoted growth of the cells under low or no glucose conditions. Conclusion: Isogenic cells WT for PIK3CA are capable of utilizing a range of nutrient sources, while cells harboring H1047R PIK3CA appear hard-wired to exclusively utilize glucose. These data suggest that targeting glucose transporters or glycolytic enzymes may be a powerful therapeutic strategy for mutant PI3K-cancers. The overt glucose dependence of cells with this mutation may also offer routes to non-invasively image PI3K-pathway activated tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1274. doi:10.1158/1538-7445.AM2011-1274