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(1) Background: The acidosis of the tumor micro-environment may have profound impact on cancer progression and on the efficacy of treatments. In the present study, we evaluated the impact of a treatment with UK-5099, a mitochondrial pyruvate carrier (MPC) inhibitor on tumor extracellular pH (pHe), (2) Methods: glucose consumption, lactate secretion and extracellular acidification rate (ECAR) were measured in vitro after exposure of cervix cancer SiHa cells and breast cancer 4T1 cells to UK-5099 (10 µM). Mice bearing the 4T1 tumor model were treated daily during four days with UK-5099 (3 mg/kg). The pHe was evaluated in vivo using either chemical exchange saturation transfer (CEST)-MRI with iopamidol as pHe reporter probe or 31P-NMR spectroscopy with 3-aminopropylphosphonate (3-APP). MR protocols were applied before and after 4 days of treatment, (3) Results: glucose consumption, lactate release and ECAR were increased in both cell lines after UK-5099 exposure. CEST-MRI showed a significant decrease in tumor pHe of 0.22 units in UK-5099-treated mice while there was no change over time for mice treated with the vehicle. Parametric images showed a large heterogeneity in response with 16% of voxels shifting to pHe values under 7.0. In contrast, 31P-NMR spectroscopy was unable to detect any significant variation in pHe, (4) Conclusions: MPC inhibition led to a moderate acidification of the extracellular medium in vivo. CEST-MRI provided high resolution parametric images (0.44 µL/voxel) of pHe highlighting the heterogeneity of response within the tumor when exposed to UK-5099.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze–thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKβ1−/− mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

Caspases are well known for their role in apoptosis. Recently, nonapoptotic roles of caspases have been identified, however, these noncanonical roles are not well documented and the mechanisms involved are not fully understood. Here, we studied the role of cleaved caspase-3 using human- and mouse-proficient caspase-3 cancer cell lines and human-deficient caspase-3 cancer cells. Cleaved caspase-3 functioned as a transcription factor and directly bound to DNA. A DNA-binding domain was identified in the small subunit of caspase-3 and an active conformation was essential for caspase-3 transcriptional activity. Caspase-3 DNA binding enhanced angiogenesis by upregulating the expression of proangiogenic genes and by activating pathways that promoted endothelial cell activation. Some proapoptotic genes were downregulated in caspase-3–proficient cells. Inhibiting caspase-3 increased the efficacy of chemotherapy and decreased spontaneous tumor development. These data highlight a novel nonapoptotic role of caspase-3 and suggest that cleaved caspase-3 could be a new therapeutic target in cancer. Significance: These findings report a noncanonical function of caspase-3 by demonstrating its ability to transcriptionally regulate the VEGFR pathway.

Although saturated fatty acid (FA) (SFA) and monounsaturated FA (MUFA) are synthesized in cancer cells from acetyl-CoA, polyunsaturated FAs (PUFAs) are necessarily obtained from diet. Depending on concentrations and metabolism, these different FAs may support tumor proliferation but also exert growth inhibitory effects. The mutual interplay between them also requires to integrate the FA oxidation component that may be concomitant with FA synthesis is cancer cells. New molecular mechanisms driving FA synthesis, lipotoxicity and anti-inflammatory activity of eicosanoids in mouse and human cancers were recently elicited. To block or take advantage of the above represent attractive perspectives of treatments to fight cancer progression. The various enzymatic reactions leading to SFA synthesis represent as many targets to prevent tumor growth. Ironically excess SFAs are per-se toxic for cancer cells and the introduction of a double bound to form MUFA is actually limiting lipotoxicity in cancer cells. Blocking stearoyl-CoA desaturase therefore represents another attractive modality. By contrast, dietary PUFAs may exert direct cytotoxic effects by promoting apoptosis or by generating anti-inflammatory eicosanoids. Altogether, these data point out the intricate relationship between SFA, MUFA and PUFA at the heart of the metabolism of proliferating cancer cells.

Background: Energy metabolism is described to be deregulated in cancer, and the Warburg effect is considered to be a major hallmark. Recently, cellular heterogeneity in tumors and the tumor microenvironment has been recognized to play an important role in several metabolic pathways in cancer. However, its contribution to papillary thyroid cancer (PTC) development and metabolism is still poorly understood. Methods: A proteomic analysis of five PTC was performed, and the cellular distribution of several upregulated metabolic proteins was investigated in the cancerous and stromal cells of these tumors. Results: Tandem mass spectrometry analysis revealed the upregulation of many metabolism-related proteins, among them pyruvate carboxylase (PC). PC knockdown in thyroid cell lines alters their proliferative and motility capacities, and measurements of oxygen consumption rates show that this enzyme is involved in the replenishment of the tricarboxylic acid cycle. Immunostainings of several upregulated metabolic proteins show that thyroid cancer cells have an increased mitochondrial oxidative metabolism compared to stromal cells. Conclusions: PTC has a very active tricarboxylic acid cycle, continuously replenished by a PC-mediated anaplerosis. This is specifically observed in the tumor cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Due to rising costs and the difficulty to identify new targets, drug repurposing appears as a viable strategy for the development of new anti-cancer treatments. Although the interest of disulfiram (DSF), an anti-alcohol drug, to treat cancer was reported for many years, it is only very recently that one anticancer mechanism-of-action was highlighted. This would involve the inhibition of the p97 segregase adaptor NPL4, which is essential for the turnover of proteins involved in multiple regulatory and stress-response intracellular pathways. However, recently DSF was also reported as one of the first phosphoglycerate dehydrogenase (PHGDH) inhibitors, a tetrameric enzyme catalyzing the initial step of the serine synthetic pathway that is highly expressed in numerous cancer types. Here, we investigated the structure-activity relationships (SAR) of PHGDH inhibition by disulfiram analogues as well as the mechanism of action of DSF on PHGDH via enzymatic and cell-based evaluation, mass spectrometric and mutagenesis experiments.

AIM: This study aimed at developing antibody-functionalized gold nanoparticles (AuNPs) to selectively target cancer cells and probing their potential radiosensitizing effects under proton irradiation.MATERIALS & METHODS: AuNPs were conjugated with cetuximab (Ctxb-AuNPs). Ctxb-AuNP uptake was evaluated by transmission electron microscopy and atomic absorption spectroscopy. Radioenhancing effect was assessed using conventional clonogenic assay.RESULTS & CONCLUSION: Ctxb-AuNPs specifically bound to and accumulated in EGFR-overexpressing A431 cells, compared with EGFR-negative MDA-MB-453 cells. Ctxb-AuNPs enhanced the effect of proton irradiation in A431 cells but not in MDA-MB-453 cells. These data indicate, for the first time, that combining enhanced uptake by specific targeting and radioenhancing effect, using conjugated AuNPs, is a promising strategy to increase cell killing by protontherapy.

The multidisciplinary field of photodynamic therapy (PDT) is a combination of photochemistry and photophysics sciences, which has shown tremendous potential for cancer therapy application. PDT employs a photosensitizing agent (PS) and light to form cytotoxic reactive oxygen species and subsequently oxidize light-exposed tissue. Despite numerous advantages of PDT and enormous progress in this field, common PSs are still far from ideal treatment because of their poor permeability, non-specific phototoxicity, side effects, hydrophobicity, weak bioavailability, and tendency to self-aggregation. To circumvent these limitations, PS can be encapsulated in liposomes, an advanced drug delivery system that has demonstrated the ability to enhance drug permeability into biological membranes and loading both hydrophobic and lipophilic agents. Moreover, liposomes can also be coated by targeting agents to improve delivery efficiency. The present review aims to summarize the principles of PDT, various PS generations, PS-loaded nanoparticles, liposomes, and their impact on PDT, then discuss recent photodynamic cancer therapy strategies using liposomes as PS-loaded vectors, and highlight future possibilities and perspectives.

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways can potentially sensitize tumors with limited treatment options, such as triple-negative breast cancer (TNBC), to chemo- and/or radiotherapy. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced reactive oxygen species (ROS) production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. In this study, we evaluated the effects of DCA and radiotherapy on two TNBC cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as an oxidative phosphorylation (OXPHOS)-promoting drug, DCA can also increase hypoxic radioresponses. This study therefore paves the way for the targeting of mitochondrial metabolism of hypoxic cancer cells, in particular to combat radioresistance.

Specific diets for cancer patients have the potential to offer an adjuvant modality to conventional anticancer therapy. If the concept of starving cancer cells from nutrients to inhibit tumor growth is quite simple, the translation into the clinics is not straightforward. Several diets have been described including the Calorie-restricted diet based on a reduction in carbohydrate intake and the Ketogenic diet wherein the low carbohydrate content is compensated by a high fat intake. As for other diets that deviate from normal composition only by one or two amino acids, these diets most often revealed a reduction in tumor growth in mice, in particular when associated with chemo- or radiotherapy. By contrast, in cancer patients, the interest of these diets is almost exclusively supported by case reports precluding any conclusions on their real capacity to influence disease outcome. In parallel, the field of tumor lipid metabolism has emerged in the last decade offering a better understanding of how fatty acids are captured, synthesized or stored as lipid droplets in cancers. Fatty acids participate to cancer cell survival in the hypoxic and acidic tumor microenvironment and also support proliferation and invasiveness. Interestingly, while such addiction for fatty acids may account for cancer progression associated with high fat diet, it could also represent an Achilles heel for tumors. In particular n-3 polyunsaturated fatty acids represent a class of lipids that can exert potent cytotoxic effects in tumors and therefore represent an attractive diet supplementation to improve cancer patient outcomes.

The fundamental unit of chromatin is the nucleosome which is composed of a histone octamer and the DNA that wraps around it. Histones are globular proteins subject to various reversible covalent modifications that primarily occur on their flexible N-terminal ends (the so-called tail). Histone acetylation is one of these major alterations that may influence the chromatin conformation and consecutively influence gene expression (1). Histone acetylation is usually associated with an increase in transcriptional activity. Indeed, since acetylation occurs on positively charged lysines residues, the addition of an acetyl group on these residues changes the overall charge of the histone tail thereby leading to weaker binding of the nucleosomal components (2). As a direct consequence of histone acetylation, DNA becomes more accessible to transcription factors. Enzymes named histone acetyltransferases (HATs) catalyzed this mode of post-translational modifications (3). Other enzymes termed histone deacetylases (HDACs) are involved in the reverse process of histone deacetylation that restores the ionic interactions between positively charged histones and negatively charged DNA, thereby yielding a more compact chromatin structure (making it harder for transcription factors to bind to the DNA) (2,3).

Acidosis, a common characteristic of the tumor microenvironment, is associated with alterations in metabolic preferences of cancer cells and progression of the disease. Here we identify the TGF-β2 isoform at the interface between these observations. We document that acidic pH promotes autocrine TGF-β2 signaling, which in turn favors the formation of lipid droplets (LD) that represent energy stores readily available to support anoikis resistance and cancer cell invasiveness. We find that, in cancer cells of various origins, acidosis-induced TGF-β2 activation promotes both partial epithelial-to-mesenchymal transition (EMT) and fatty acid metabolism, the latter supporting Smad2 acetylation. We show that upon TGF-β2 stimulation, PKC-zeta-mediated translocation of CD36 facilitates the uptake of fatty acids that are either stored as triglycerides in LD through DGAT1 or oxidized to generate ATP to fulfill immediate cellular needs. We also address how, by preventing fatty acid mobilization from LD, distant metastatic spreading may be inhibited., The tumour microenvironment is known to have an acidic pH but how this influences cancer cell phenotype is unclear. Here, the authors show that tumour cells upregulate TGF-β2 under acidosis, which leads to the increased formation of lipid droplets allowing for invasiveness and metastases.

Plant-derived conjugated linolenic acids (CLnA) have been widely studied for their preventive and therapeutic properties against diverse diseases such as cancer. In particular, punicic acid (PunA), a conjugated linolenic acid isomer (C18:3 c9t11c13) present at up to 83% in pomegranate seed oil, has been shown to exert anti-cancer effects, although the mechanism behind its cytotoxicity remains unclear. Ferroptosis, a cell death triggered by an overwhelming accumulation of lipid peroxides, has recently arisen as a potential mechanism underlying CLnA cytotoxicity. In the present study, we show that PunA is highly cytotoxic to HCT-116 colorectal and FaDu hypopharyngeal carcinoma cells grown either in monolayers or as three-dimensional spheroids. Moreover, our data indicate that PunA triggers ferroptosis in carcinoma cells. It induces significant lipid peroxidation and its effects are prevented by the addition of ferroptosis inhibitors. A combination with docosahexaenoic acid (DHA), a known polyunsaturated fatty acid with anticancer properties, synergistically increases PunA cytotoxicity. Our findings highlight the potential of using PunA as a ferroptosis-sensitizing phytochemical for the prevention and treatment of cancer.

Tumor acidosis promotes disease progression through a stimulation of fatty acid (FA) metabolism in cancer cells. Instead of blocking the use of FAs by acidic cancer cells, we examined whether excess uptake of specific FAs could lead to antitumor effects. We found that n-3 but also remarkably n-6 polyunsaturated FA (PUFA) selectively induced ferroptosis in cancer cells under ambient acidosis. Upon exceeding buffering capacity of triglyceride storage into lipid droplets, n-3 and n-6 PUFA peroxidation led to cytotoxic effects in proportion to the number of double bonds and even more so in the presence of diacylglycerol acyltransferase inhibitors (DGATi). Finally, an n-3 long-chain PUFA-rich diet significantly delayed mouse tumor growth when compared with a monounsaturated FA-rich diet, an effect further accentuated by administration of DGATi or ferroptosis inducers. These data point out dietary PUFA as a selective adjuvant antitumor modality that may efficiently complement pharmacological approaches.

Warburg's hypothesis that cancer cells take up a lot of glucose in the presence of ambient oxygen but convert pyruvate into lactate due to impaired mitochondrial function led to the misconception that cancer cells rely on glycolysis as their major source of energy. Most recent 13C-based metabolomic studies, including in cancer patients, indicate that cancer cells may also fully oxidize glucose. In addition to glucose-derived pyruvate, lactate, fatty acids and amino acids supply substrates to the TCA cycle to sustain mitochondrial metabolism. Here, we discuss how the metabolic flexibility afforded by these multiple mitochondrial inputs allows cancer cells to adapt according to the availability of the different fuels and the microenvironmental conditions such as hypoxia and acidosis. In particular, we focused on the role of the TCA cycle in interconnecting numerous metabolic routes in order to highlight metabolic vulnerabilities that represent attractive targets for a new generation of anticancer drugs.

The aim of this work is to develop the first validated HPLC-UV method quantification in blood serum for a new endoplasmic reticulum (ER)-specific benzophenazine photosensitizer (OR-141). A fast solid phase extraction (SPE) cleaning sample procedure was achieved on C18 encapped (ec) SPE cartridges and the separation was performed on a RP-18e column (5μM) using an isocratic elution with methanol. The method has been fully validated according to accuracy profiles based on total error and tolerance intervals. Calibration was performed in the matrix and trueness (

Deubiquitinating enzymes are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors with a shared α,β-unsaturated carbonyl substructure motif. Initially taken forward into a phase I/II clinical trial for refractory multiple myeloma, VLX1570 has since been put on full clinical hold due to dose limiting toxicity. Through a proteomic approach, here we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight complexes. Activity-based proteome profiling identified CIAPIN1 as a sub-micromolar covalent target of VLX1570, and further analysis demonstrated that high molecular weight complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce non-specific protein aggregation through cross-linking, providing a molecular explanation for general cellular toxicity.

Among all molecules developed for anticancer therapies, photodynamic therapeutic agents have a unique profile. Their maximal activity is specifically triggered in tumors by light, and toxicity of even systemically delivered drug is prevented in nonilluminated parts of the body. Photosensitizers exert their therapeutic effect by producing reactive oxygen species via a light-activated reaction with molecular oxygen. Consequently, the lowering of pO2 deep in solid tumors limits their treatment and makes essential the design of oxygen-independent sensitizers. In this perspective, we have recently developed Ir(III)-based molecules able to oxidize biomolecules by type I processes under oxygen-free conditions. We examine here their phototoxicity in relevant biological models. We show that drugs, which are mitochondria-accumulated, induce upon light irradiation a dramatic decrease of the cell viability, even under low oxygen conditions. Finally, assays on 3D tumor spheroids highlight the importance of the light-activation step and the oxygen consumption rate on the drug activity.

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness.

Piperlongumine (PL), naturally synthesized in long pepper, is known to selectively kill tumor cells via perturbation of reactive oxygen species (ROS) homeostasis. ROS are the primary effector molecules of radiation, and increase of ROS production by pharmacological modulation is known to enhance radioresponse. We therefore investigated the radiosensitizing effect of PL in colorectal cancer cells (CT26 and DLD-1) and CT26 tumor-bearing mice. Firstly, we found that PL induced excessive production of ROS due to depletion of glutathione and inhibition of thioredoxin reductase. Secondly, PL enhanced both the intrinsic and hypoxic radiosensitivity of tumor cells, linked to ROS-mediated increase of DNA damage, G2/M cell cycle arrest, and inhibition of cellular respiration. Finally, the radiosensitizing effect of PL was verified in vivo. PL improved the tumor response to both single and fractionated radiation, resulting in a significant increase of survival rate of tumor-bearing mice, while it was ineffective on its own. In line with in vitro findings, enhanced radioresponse is associated with inhibition of antioxidant systems. In conclusion, our results suggest that PL could be a potential radiosensitizer in colorectal cancer.

The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the &alpha, ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative &alpha, ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original &alpha, ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of &alpha, ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action.

Recent advances in the understanding of the relationship between cancer and metabolism have highlighted the relevance of the serine synthetic pathway (SSP), which consists of three successive enzymatic reactions. Enzymes of the SSP, such as phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT-1), were recently highlighted because they are amplified in a significant subset of human tumors, and their suppression by RNAi caused a decrease in cancer cell survival and growth. Currently, the discovery of drugs that inhibit these enzymes is still in its infancy, and the identification of suitable inhibitors could serve to understand the emerging biology of these metabolic enzymes. In this review, we present the SSP as a significant and novel emerging area for medicinal chemistry and we provide an overview of one of the key enzymes of the pathway, PHGDH.

Bioenergetic preferences of cancer cells foster tumor acidosis that in turn leads to dramatic reduction in glycolysis and glucose-derived acetyl-coenzyme A (acetyl-CoA). Here, we show that the main source of this critical two-carbon intermediate becomes fatty acid (FA) oxidation in acidic pH-adapted cancer cells. FA-derived acetyl-CoA not only fuels the tricarboxylic acid (TCA) cycle and supports tumor cell respiration under acidosis, but also contributes to non-enzymatic mitochondrial protein hyperacetylation, thereby restraining complex I activity and ROS production. Also, while oxidative metabolism of glutamine supports the canonical TCA cycle in acidic conditions, reductive carboxylation of glutamine-derived α-ketoglutarate sustains FA synthesis. Concomitance of FA oxidation and synthesis is enabled upon sirtuin-mediated histone deacetylation and consecutive downregulation of acetyl-CoA carboxylase ACC2 making mitochondrial fatty acyl-CoA degradation compatible with cytosolic lipogenesis. Perturbations of these regulatory processes lead to tumor growth inhibitory effects further identifying FA metabolism as a critical determinant of tumor cell proliferation under acidosis.

// Florence Polet 1 , Ruben Martherus 1 , Cyril Corbet 1 , Adan Pinto 1 , Olivier Feron 1 1 Pole of Pharmacology and Therapeutics, Institut de Recherche Experimentale et Clinique (IREC), Universite Catholique de Louvain, B-1200 Brussels, Belgium Correspondence to: Olivier Feron, email: olivier.feron@uclouvain.be Keywords: leukemia, ASCT2, LAT1, glycosylation, metabolism Received: April 07, 2016 Accepted: May 29, 2016 Published: June 17, 2016 ABSTRACT Leukemia cells are highly dependent on glucose and glutamine as bioenergetic and biosynthetic fuels. Inhibition of the metabolism of glucose but also of glutamine is thus proposed as a therapeutic modality to block leukemia cell growth. Since glucose also supports protein glycosylation, we wondered whether part of the growth inhibitory effects resulting from glycolysis inhibition could indirectly result from a defect in glycosylation of glutamine transporters. We found that ASCT2/SLC1A5, a major glutamine transporter, was indeed deglycosylated upon glucose deprivation and 2-deoxyglucose exposure in HL-60 and K-562 leukemia cells. Inhibition of glycosylation by these modalities as well as by the bona fide glycosylation inhibitor tunicamycin however marginally influenced glutamine transport and did not impact on ASCT2 subcellular location. This work eventually unraveled the dispensability of ASCT2 to support HL-60 and K-562 leukemia cell growth and identified the upregulation of the neutral amino acid antiporter LAT1/SLC7A5 as a mechanism counteracting the inhibition of glycosylation. Pharmacological inhibition of LAT1 increased the growth inhibitory effects and the inactivation of the mTOR pathway resulting from glycosylation defects, an effect further emphasized during the regrowth period post-treatment with tunicamycin. In conclusion, this study points towards the underestimated impact of glycosylation inhibition in the interpretation of metabolic alterations resulting from glycolysis inhibition, and identifies LAT1 as a therapeutic target to prevent compensatory mechanisms induced by alterations in the glycosylating process.

Crosstalk between cancer-associated fibroblasts (CAFs) and colorectal cancer cells promotes tumor growth and contributes to chemoresistance. In this study, we assessed the sensitivity of a primary CAF cell line, CT5.3hTERT, to standard-of-care and alternative cytotoxic treatments. Paclitaxel (PTX) and acriflavine (ACF) were identified as the most promising molecules to inhibit CAF development. To allow the translational use of both drugs, we developed lipid nanocapsule (LNC) formulations for PTX and ACF. Finally, we mixed CAFs and tumor cell lines in a cocultured spheroid, and the effect of both drugs was investigated by histological analyses. We demonstrated CAF inhibition by LNC-ACF and whole tumor inhibition by LNC-PTX. Altogether, we proposed a new strategy to reduce CAF populations in the colorectal microenvironment that should be tested in vivo.

For many years now, targeting deregulation within cancer cells&rsquo, metabolism has appeared as a promising strategy for the development of more specific and efficient cancer treatments. Recently, numerous reports highlighted the crucial role of the serine synthetic pathway, and particularly of the phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the pathway, to sustain cancer progression. Yet, because of very weak potencies usually in cell-based settings, the inhibitors reported so far failed to lay ground on the potential of this approach. In this paper, we report a structure&ndash, activity relationship study of a series of &alpha, ketothioamides that we have recently identified. Interestingly, this study led to a deeper understanding of the structure&ndash, activity relationship (SAR) in this series and to the identification of new PHGDH inhibitors. The activity of the more potent compounds was confirmed by cellular thermal shift assays and in cell-based experiments. We hope that this research will eventually provide a new entry point, based on this promising chemical scaffold, for the development of therapeutic agents targeting PHGDH.

Cycling hypoxia (cyH), also called intermittent hypoxia, occurs in solid tumors and affects different cell types in the tumor microenvironment and in particular the tumor-associated macrophages (TAMs). As cyH and TAMs both favor tumor progression, we investigated whether cyH could drive the pro-tumoral phenotype of macrophages. Here, the effects of cyH on human THP-1 macrophages and murine bone marrow-derived macrophages (BMDM), either unpolarized M0, or polarized in M1 or M2 phenotype were studied. In M0 macrophages, cyH induced a pro-inflammatory phenotype characterized by an increase in TNFα and IL-8/MIP-2 secretion. CyH amplified the pro-inflammatory phenotype of M1 macrophages evidenced by an increased pro-inflammatory cytokine secretion and pro-inflammatory gene expression. Furthermore, cyH increased c-jun activation in human M0 macrophages and highly increased c-jun and NF-κB activation in M1 macrophages. C-jun and p65 are implicated in the effects of cyH on M0 and M1 macrophages since inhibition of their activation prevented the cyH pro-inflammatory effects. In conclusion, we demonstrated that cyH induces or amplifies a pro-inflammatory phenotype in M0 and M1 macrophages by activating JNK/p65 signaling pathway. These results highlight a specific role of cyH in the amplification of tumor-related inflammation by modulating the inflammatory phenotype of macrophages.

Background and Purpose: The anti-diabetic biguanide drugs metformin and phenformin exhibit antitumor activity in various models. However, their radiomodulatory effect under hypoxic conditions, particularly for phenformin, is largely unknown. This study therefore examines whether metformin and phenformin as mitochondrial complex I blockades could overcome hypoxic radioresistance through inhibition of oxygen consumption. Materials and Methods: A panel of colorectal cancer cells (HCT116, DLD-1, HT29, SW480, and CT26) was exposed to metformin or phenformin for 16 h at indicated concentrations. Afterward, cell viability was measured by MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) was examined by western blot. Mitochondria complexes activity and oxygen consumption rate (OCR) were measured by seahorse analyzer. The radiosensitivity of tumor cells was assessed by colony formation assay under aerobic and hypoxic conditions. The in vitro findings were further validated in colorectal CT26 tumor model. Results: Metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced OCR in a dose-dependent manner starting at 3 mM and 30 μM, respectively. As a result, the hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin with an enhancement ratio about 2 at 9 mM and 100 μM, respectively. Regarding intrinsic radioresistance, both of them did not exhibit any effect although there was an increase of phosphorylation of AMPK and ROS production. In tumor-bearing mice, metformin or phenformin alone did not show any anti-tumor effect. While in combination with radiation, both of them substantially delayed tumor growth and enhanced radioresponse, respectively, by 1.3 and 1.5-fold. Conclusion: Our results demonstrate that metformin and phenformin overcome hypoxic radioresistance through inhibition of mitochondrial respiration, and provide a rationale to explore metformin and phenformin as hypoxic radiosensitizers.

Acriflavine (ACF) hydrochloride is currently repurposed as multimodal drug, inhibiting hypoxia-inducible factors (HIF) pathways and exerting cytotoxic properties. The aim of this study was to encapsulate ACF in reverse micelles and to incorporate this suspension in lipid nanocapsules (LNC). Designs of experiments were used to work under quality by design conditions. LNC were formulated using a phase-inversion temperature method, leading to an encapsulation efficiency around 80%. In vitro, the encapsulated drug presented similar cytotoxic activity and decrease in HIF activity in 4T1 cells compared to the free drug. In vivo, ACF-loaded nanoparticles (ACF dose of 5 mg/kg) demonstrated a higher antitumor efficacy compared to free ACF on an orthotopic model of murine breast cancer (4T1 cells). Moreover, the use of LNC allowed to drastically decrease the number of administrations compared to the free drug (2 versus 12 injections), suppressing the ACF-induced toxicity.

Small cell lung cancer (SCLC) has a poor prognosis. Focal adhesion kinase (FAK) is a non–receptor tyrosine kinase regulating cell proliferation, survival, migration, and invasion, which is overexpressed and/or activated in several cancers, including SCLC. We wanted to determine whether FAK contributes to SCLC aggressive behavior. We first evaluated the effect of FAK small-molecule inhibitor PF-573,228 in NCI-H82, NCI-H146, NCI-H196, and NCI-H446 SCLC cell lines. PF-573,228 (0.1–5 μmol/L) inhibited FAK activity by decreasing phospho-FAK (Tyr397), without modifying total FAK expression. PF-573,228 decreased proliferation, decreased DNA synthesis, induced cell-cycle arrest in G2–M phases, and increased apoptosis in all cell lines. PF-573,228 also decreased motility in adherent cell lines. To make sure that these effects were not off-target, we then used a genetic method to inhibit FAK in NCI-H82 and NCI-H446, namely stable transduction with FAK shRNA and/or FAK-related nonkinase (FRNK), a splice variant lacking the N-terminal and kinase domains. Although FAK shRNA transduction decreased total and phospho-FAK (Tyr397) expression, it did not affect proliferation, DNA synthesis, or progression through cell cycle. However, restoration of FAK-targeting (FAT) domain (attached to focal adhesion complex where it inhibits pro-proliferative proteins such as Rac-1) by FRNK transduction inhibited proliferation, DNA synthesis, and induced apoptosis. Moreover, although FAK shRNA transduction increased active Rac1 level, FRNK reexpression in cells previously transduced with FAK shRNA decreased it. Therefore, FAK appears important in SCLC biology and targeting its kinase domain may have a therapeutic potential, while targeting its FAT domain should be avoided to prevent Rac1-mediated protumoral activity.

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition., Tumor cells can fuel their metabolism with lactate. Here the authors show that inhibition of mitochondrial pyruvate carrier (MPC) blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation and inhibits oxidative metabolism, ultimately resulting in cytotoxicity and radiosensitization.

Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvβ3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvβ3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems.

A new series of combretastatin analogues with B-ring modifications were synthesized and evaluated for their cytotoxicity against one endothelial (HUVEC) and three tumor cell lines, e.g., the LoVo colon, the PC-3 prostate, and the U373 glioma cancer models. These new combretastatin analogues showed differential cytotoxic activities, cis derivatives 13 5-(2-Z-trimethoxyphenylethenyl)- benzo[1,2-c]1,2,5-oxadiazole N1-oxide and 14 5-(2-Ztrimethoxyphenylethenyl) benzo[1,2,5]thiadiazole exhibiting interesting cytotoxicity both on endothelial and on tumor cells. Unlike the cis benzofurazan 12 5-(2-Ztrimethoxyphenylethenyl) benzo[1,2-c]1,2,5-oxadiazole, induction of apoptosis by 13 appeared to be through caspase-3 activation. Metabolic investigations showed a positive correlation between highly metabolized compounds and cytotoxic activity, suggesting that highly cytotoxic derivatives may act as pro-drug via a reductive metabolization to more active metabolites.

We report the mass production of blue LEDs on dry-etched patterned sapphire substrates using the AIX R6 tool in a 31×4" configuration. The system was operated in a continuous run mode, i.e. cleaning the showerhead after a series of LED runs. Production stability was characterized by monitoring of wavelength, light output power (LOP), and electrostatic discharge (ESD) yields. We developed a dynamic multi-zone Topside Temperature Control and the TEQualizer function. The TEQualizer function is based on a 400nm pyrometry open loop wafer surface temperature control, using Inside P400 by Laytec. Combining this wafer-to-wafer and run -to-run temperature stability improvement with an optimized wafer carrier, we demonstrated an on-wafer uniformity of stdv of 1.1nm, a wafer-to-wafer uniformity of stdv 1.1nm and a run-to-run reproducibility of stdv 90% in a 6 nm bin. LOP stability was demonstrated within a 3% window with no visible run-to-run trend. An absolute buffer layer growth temperature window was defined through a Design of Experiment on buffer layers targeting best ESD yield - in particular looking into defect related morphology and its correlation with Inside P400 readings. We have demonstrated an ESD yield >90% in continuous run mode to be used in the mass production of InGaN based blue LEDs.

Given the putative role of PHGDH in cancer, development of inhibitors is required to explore its function. In this context, we established and validated a straightforward enzymatic assay suitable for high-throughput screening and we identified inhibitors with similar chemical scaffolds. Through a convergent pharmacophore approach, we synthesized α-ketothioamides that exhibit interesting in vitro PHGDH inhibition and encouraging cellular results. These novel probes may be used to understand the emerging biology of this metabolic target.

// Pierre Danhier 1 , Gladys Deumer 2 , Nicolas Joudiou 1 , Caroline Bouzin 3 , Philippe Leveque 1 , Vincent Haufroid 2 , Benedicte F. Jordan 1 , Olivier Feron 4 , Pierre Sonveaux 4 , Bernard Gallez 1 1 Louvain Drug Research Institute, Biomedical Magnetic Resonance Research Group, Universite Catholique de Louvain (UCL), Brussels, Belgium 2 Louvain Center for Toxicology and Applied Pharmacology, Universite Catholique de Louvain (UCL), Brussels, Belgium 3 Institut de Recherche Experimentale et Clinique (IREC), IREC Imaging Platform, Universite Catholique de Louvain (UCL), Brussels, Belgium 4 Institut de Recherche Experimentale et Clinique (IREC), Pole of Pharmacology, Universite Catholique de Louvain (UCL), Brussels, Belgium Correspondence to: Bernard Gallez, email: bernard.gallez@uclouvain.be Keywords: MRI, EPR, cell tracking, cancer metastasis, iron oxides Received: January 06, 2017 Accepted: March 29, 2017 Published: April 13, 2017 ABSTRACT Magnetic resonance imaging (MRI) cell tracking of cancer cells labeled with superparamagnetic iron oxides (SPIO) allows visualizing metastatic cells in preclinical models. However, previous works showed that the signal void induced by SPIO on T 2 (*)-weighted images decreased over time. Here, we aim at characterizing the fate of iron oxide nanoparticles used in cell tracking studies and the role of macrophages in SPIO metabolism. In vivo MRI cell tracking of SPIO positive 4T1 breast cancer cells revealed a quick loss of T 2 * contrast after injection. We next took advantage of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICP-MS) for characterizing the evolution of superparamagnetic and non-superparamagnetic iron pools in 4T1 breast cancer cells and J774 macrophages after SPIO labeling. These in vitro experiments and histology studies performed on 4T1 tumors highlighted the quick degradation of iron oxides by macrophages in SPIO-based cell tracking experiments. In conclusion, the release of SPIO by dying cancer cells and the subsequent uptake of iron oxides by tumor macrophages are limiting factors in MRI cell tracking experiments that plead for the use of (MR) reporter-gene based imaging methods for the long-term tracking of metastatic cells.

The purpose of this study was to develop vitamin E-based micelles loaded with Doxorubicin (DOX) (DOX-TOS-TPGS), taking advantages of the anti-cancer activity of vitamin E derivatives: Tocopherol succinate (TOS) and D- α-tocopherol polyethylene2000 succinate (TPGS). Therefore, we developed micelles consisting in a mixture of TOS (as solubilizer) and TPGS2000 (as stabilizer) (1:1). DOX-TOS-TPGS micelles exhibited a size of 78nm and a ζ potential of -7mV. High drug loading (40% w/w) was achieved. The critical micellar concentration was determined at 14μg/ml. In vitro, after 24h, DOX-TOS- TPGS micelles exhibited higher cytotoxicity than free-DOX (IC50 on MCF-7 cells, at 24h, 58.15 vs 4.89μg/ml). In vivo anti-tumor efficacy, performed on two tumor models (CT26 and MCF-7), demonstrated a 100% long-term survival of mice (- bearing MCF-7 tumors) when treated with DOX-TOS-TPGS compared to DOX-free (mean survival time=32 days). Interestingly, the survival time of mice treated with unloaded TOS-TPGS micelles was similar to DOX-free, suggesting the anti-cancer activity of vitamin E derivatives. Based on these results, it can be concluded that the formulations developed in this work may be considered as an effective DOX delivery system for cancer chemotherapy.

Dynamic contrast-enhanced (DCE)-MRI is useful to assess the early effects of drugs acting on tumor vasculature, namely anti-angiogenic and vascular disrupting agents. Ultra-high-field MRI allows higher-resolution scanning for DCE-MRI while maintaining an adequate signal-to-noise ratio. However, increases in susceptibility effects, combined with decreases in longitudinal relaxivity of gadolinium-based contrast agents (GdCAs), make DCE-MRI more challenging at high field. The aim of this work was to explore the feasibility of using DCE-MRI at 11.7 T to assess the tumor hemodynamics of mice. Three GdCAs possessing different molecular weights (gadoterate: 560 Da, 0.29 mmol Gd/kg; p846: 3.5 kDa, 0.10 mmol Gd/kg; and p792: 6.47 kDa, 0.15 mmol Gd/kg) were compared to see the influence of the molecular weight in the highlight of the biologic effects induced by combretastatin A4 (CA4). Mice bearing transplantable liver tumor (TLT) hepatocarcinoma were divided into two groups (n = 5-6 per group and per GdCA): a treated group receiving 100 mg/kg CA4, and a control group receiving vehicle. The mice were imaged at 11.7 T with a T1 -weighted FLASH sequence 2 h after the treatment. Individual arterial input functions (AIFs) were computed using phase imaging. These AIFs were used in the Extended Tofts Model to determine K(trans) and vp values. A separate immunohistochemistry study was performed to assess the vascular perfusion and the vascular density. Phase imaging was used successfully to measure the AIF for the three GdCAs. In control groups, an inverse relationship between the molecular weight of the GdCA and K(trans) and vp values was observed. K(trans) was significantly decreased in the treated group compared with the control group for each GdCA. DCE-MRI at 11.7 T is feasible to assess tumor hemodynamics in mice. With K(trans) , the three GdCAs were able to track the early vascular effects induced by CA4 treatment.

MRI cell tracking is a promising technique for tracking various cell types in living animals. Usually, cells are incubated with iron oxides so that the particles are taken up before the cells are injected in vivo. In the present study, we aimed to monitor migration of luciferase-expressing mouse renal cancer cells (RENCA-luc) after intrarenal or intrasplenic injection. These cells were labelled using Molday Ion Rhodamine B (MIRB) fluorescent superparamagnetic iron oxide particles. Their fate after injection was first assessed using ex vivo X-band electron paramagnetic resonance (EPR) spectroscopy. This biodistribution study showed that RENCA-luc cells quickly colonized the lungs and the liver after intrarenal and intrasplenic injection, respectively. Bioluminescence imaging (BLI) studies confirmed that this cell line preferentially metastasized to these organs. Early tracking of labelled RENCA-luc cells in the liver using high-field MRI (11.7 T) was not feasible because of a lack of sensitivity. MRI of MIRB-labelled RENCA-luc cells after injection in the left kidney was then performed. T2 - and T2 *-weighted images showed that the labelled cells induced hypointense signals at the injection site. Nevertheless, the hypointense regions tended to disappear after several days, mainly owing to dilution of the MIRB iron oxides with cell proliferation. In conclusion, EPR is well adapted to ex vivo analysis of tissues after cell tracking experiments and allows short-term monitoring of metastasizing cells. MRI is a suitable tool for checking labelled cells at their injection site, but dilution of the iron oxides owing to cell division remains a major limitation. BLI remains the most suitable technique for long-term monitoring of metastatic cells. Copyright © 2014 John Wiley & Sons, Ltd.

Background: Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy. Methods: Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin. Results: Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin. Conclusion: The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.

We hypothesized that vascular endothelial growth factor (VEGF)-containing hydrogels that gelify in situ after injection into a traumatized spinal cord, could stimulate spinal cord regeneration. Injectable hydrogels composed of 0.5% Pronova UPMVG MVG alginate, supplemented or not with fibrinogen, were used. The addition of fibrinogen to alginate had no effect on cell proliferation in vitro but supported neurite growth ex vivo. When injected into a rat spinal cord in a hemisection model, alginate supplemented with fibrinogen was well tolerated. The release of VEGF that was incorporated into the hydrogel was influenced by the VEGF formulation [encapsulated in microspheres or in nanoparticles or in solution (free)]. A combination of free VEGF and VEGF-loaded nanoparticles was mixed with alginate:fibrinogen and injected into the lesion of the spinal cord. Four weeks post injection, angiogenesis and neurite growth were increased compared to hydrogel alone. The local delivery of VEGF by injectable alginate:fibrinogen-based hydrogel induced some plasticity in the injured spinal cord involving fiber growth into the lesion site.

BACKGROUND: Preclinical studies have shown that PTEN loss enhances sensitivity to mammalian target of Rapamycin (mTOR) inhibitors because of facilitated PI3K (phosphatidylinositol-3 kinase)/Akt activation and consecutive stimulation of the mTOR pathway. In patients with advanced transitional cell carcinoma (TCC) treated with the mTOR inhibitor everolimus, PTEN loss was, however, associated with resistance to treatment. METHODS: Transitional cell carcinoma specimens, human bladder cancer cells and derived mouse xenografts were used to evaluate how the PTEN status influences the activity of mTOR inhibitors. RESULTS: Transitional cell carcinoma patients with a shorter progression-free survival under everolimus exhibited PTEN deficiency and increased Akt activation. Moreover, PTEN-deficient bladder cancer cells were less sensitive to rapamycin than cells expressing wild-type PTEN, and rapamycin strikingly induced Akt activation in the absence of functional PTEN. Inhibition of Akt activation by the PI3K inhibitor wortmannin interrupted this rapamycin-induced feedback loop, thereby enhancing the antiproliferative effects of the mTOR inhibitor both in vitro and in vivo. CONCLUSION: Facilitation of Akt activation upon PTEN loss can have a more prominent role in driving the feedback loop in response to mTOR inhibition than in promoting the mTOR pathway. These data support the use of both PI3K and mTOR inhibitors to treat urothelial carcinoma, in particular in the absence of functional PTEN.

Antibody-labeled gold nanoparticles represent a promising novel tool regarding cancer imaging and therapy. Nevertheless, the characterization of biodistribution of such immunonanocarriers has been poorly documented. In this study, the biodistribution of (89)Zr-labeled cetuximab before and after the coupling reaction to gold nanoparticles (AuNPs) was compared and the quantitative imaging performance of (89)Zr immuno-PET was evaluated. Cetuximab was functionalized with the desferal moiety and labeled with (89)Zr ((89)Zr-Df-Bz-NCS-cetuximab). AuNPs with a mean diameter of 5 nm were synthesized according a new method developed in the laboratory, and conjugated to (89)Zr-Df-Bz-NCS-cetuximab using carbodiimide chemistry (AuNPs-PPAA-cetuximab-(89)Zr). The two tracers were injected in A431 xenograft-bearing mice. Tumor and liver uptakes were assessed at different times after injection using quantitative PET imaging. The in vivo specificity of the binding was investigated using a saturating dose of unlabeled cetuximab. Radiolabeled cetuximab was conjugated to AuNPs with a coupling reaction yield >75%. All conjugates were stable in vitro and to a lesser extent in plasma. In vivo distribution studies revealed no significant difference in tumor uptake for cetuximab conjugated to nanoparticles up to 72 h after injection, compared with unconjugated cetuximab. Immuno-PET studies showed that AuNPs-PPAA-cetuximab-(89)Zr provided high tumor-to-background ratio. The liver uptake of AuNPs-PPAA-cetuximab-(89)Zr was higher, compared with (89)Zr-Df-Bz-NCS-cetuximab. In vivo blocking experiments demonstrated selective tumor targeting after coupling reaction. This study showed that the conjugation of AuNPs to cetuximab did not affect its tumor accumulation and that the efficacy of EGFR-targeted nanoparticles was unaltered. The (89)Zr-labeled cetuximab-targeted gold nanoparticles could be a valuable tool for theranostic purposes.

The purpose of this study was to determine the value of different imaging modalities, that is, magnetic resonance imaging/spectroscopy (MRI/MRS) and positron emission tomography (PET), to assess early tumor response to sorafenib with or without radiotherapy. Diffusion-weighted (DW)-MRI, choline (1)H MRS at 11.7 T, and (18)F-FLT PET imaging were used to image fibrosarcoma (FSaII) tumor-bearing mice over time. The imaging markers were compared with apoptosis cell death and cell proliferation measurements assessed by histology. Anti-proliferative effects of sorafenib were evidenced by (1)H MRS and (18)F-FLT PET after 2 days of treatment with sorafenib, with no additional effect of the combination with radiation therapy, results that are in agreement with Ki67 staining. Apparent diffusion coefficient calculated using DW-MRI was not modified after 2 days of treatment with sorafenib, but showed significant increase 24 h after 2 days of sorafenib treatment combined with consecutive irradiation. The three imaging markers were able to show early tumor response as soon as 24 h after treatment initiation, with choline MRS and (18)F-FLT being sensitive to sorafenib in monotherapy as well as in combined therapy with irradiation, whereas DW-MRI was only sensitive to the combination of sorafenib with radiotherapy.

Eight tridentate bipyrazole derivatives with different side arms have been prepared in one step and with good yields. The products were screened for their cytotoxic activity against three tumor cell lines—human breast cancer cell line MDA-MB231, human prostate cancer cell line PC3, and human colorectal cell line LoVo, by use of colorimetric MTT assay. Structure–activity relationships reflected the effect of substituted drugs. Among this series, two compounds had remarkable in-vitro antiproliferative activity against the LoVo cell line with IC50 values ranging from 2.6 to 2.7 μg ml−1. All the compounds had suitable drug-like characteristics according to Lipinski’s rule.

// Hui Wang 1 , Soumaya Bouzakoura 1 , Sven de Mey 1 , Heng Jiang 1 , Kalun Law 1 , Ines Dufait 1, 2 , Cyril Corbet 3 , Valeri Verovski 1 , Thierry Gevaert 1 , Olivier Feron 3 , Dirk Van den Berge 1 , Guy Storme 1 , Mark De Ridder 1 1 Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium 2 Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium 3 Pole of Pharmacology and Therapeutics (FATH), Institut de Recherche Experimentale et Clinique (IREC), Universite Catholique de Louvain, Brussels, Belgium Correspondence to: Mark De Ridder, email: mark.deridder@uzbrussel.be Keywords: auranofin, radiosensitization, ROS, thioredoxin reductase, buthionine sulfoximine Received: September 30, 2016 Accepted: March 01, 2017 Published: March 10, 2017 ABSTRACT Auranofin (AF) is an anti-arthritic drug considered for combined chemotherapy due to its ability to impair the redox homeostasis in tumor cells. In this study, we asked whether AF may in addition radiosensitize tumor cells by targeting thioredoxin reductase (TrxR), a critical enzyme in the antioxidant defense system operating through the reductive protein thioredoxin. Our principal findings in murine 4T1 and EMT6 tumor cells are that AF at 3–10 μM is a potent radiosensitizer in vitro , and that at least two mechanisms are involved in TrxR-mediated radiosensitization. The first one is linked to an oxidative stress, as scavenging of reactive oxygen species (ROS) by N-acetyl cysteine counteracted radiosensitization. We also observed a decrease in mitochondrial oxygen consumption with spared oxygen acting as a radiosensitizer under hypoxic conditions. Overall, radiosensitization was accompanied by ROS overproduction, mitochondrial dysfunction, DNA damage and apoptosis, a common mechanism underlying both cytotoxic and antitumor effects of AF. In tumor-bearing mice, a simultaneous disruption of the thioredoxin and glutathione systems by the combination of AF and buthionine sulfoximine was shown to significantly improve tumor radioresponse. In conclusion, our findings illuminate TrxR in cancer cells as an exploitable radiobiological target and warrant further validation of AF in combination with radiotherapy.

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3uM, 1 hour) and chronic (10mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.

Purpose: Because of its paramagnetic properties, oxygen may act as an endogenous magnetic resonance imaging contrast agent by changing proton relaxation rates. Changes in tissue oxygen concentrations have been shown to produce changes in relaxation rate R1 of water. The aim of the study was to improve the sensitivity of oxygen enhanced R1 imaging by exploiting the higher solubility of oxygen in lipids (as compared with water) to sensitively monitor changes in tissue oxygen levels by selectively measuring the R1 of lipids. Methods: The method, with the acronym ‘‘MOBILE’’ (mapping of oxygen by imaging lipids relaxation enhancement), was applied in different mouse models of hypoxic processes on a 11.7 T magnetic resonance imaging system. MOBILE was compared with R*2, R1 of water, and with pO2 measurements (using electron paramagnetic resonance oximetry). MOBILE was also applied in the brain of healthy human volunteers exposed to an oxygen breathing challenge on a 3 T magnetic resonance imaging system. Results: MOBILE was shown to be able to monitor changes in oxygenation in tumor, peripheral, liver, and brain tissues. The clinical translation was demonstrated in human volunteers. Conclusion: MOBILE arises as a promising noninvasive and sensitive tool for diagnosis and therapeutic guidance in disorders involving hypoxia. Magn Reson Med 70:732–744, 2013. V C 2012 Wiley Periodicals, Inc.