Intrinsic Proteolytic Activities from Cancer Cells Are Sufficient to Activate Alkoxyamine Prodrugs and Induce Cell Death.

In search of better specificity and lower chances of resistance, protease-activatable alkoxyamine prodrugs to fight cancer have been proposed. These molecules are made of a peptide linked to an alkoxyamine. Proteolysis of the peptide converts the stable prodrug at 37 °C to a metastable alkoxyamine that spontaneously homolyzes into two free radicals: a stable nitroxide and a very reactive alkyl radical. The alkyl radical induces apoptosis in the surrounding cells by inducing random chemical alterations. Here, we show that varying the peptide moiety from succinyl-Ala-Ala-Pro-Val- to PyroGlu-Gly-Arg- or PyroGlu-Gly-Lys- is effective in switching the activating enzyme from elastase to urokinase. Furthermore, these prodrugs induce the death of HT-1080 cells, a cell line that secretes several active proteases in culture. This cytotoxic activity can be suppressed by protease inhibitors and does not affect cell lines devoid of active urokinase. We thus provide examples of alkoxyamine prodrugs that are efficiently activated by the limited intrinsic protease activity and that succeed in the destruction of cancer cell lines and cancer cells from tumor explants.

Neutrophil Elastase-Activatable Prodrugs Based on an Alkoxyamine Platform to Deliver Alkyl Radicals Cytotoxic to Tumor Cells.

Current chemotherapies suffer low specificity and sometimes drug resistance. Neutrophil elastase activity in cancer is associated with poor prognosis and metastasis settlement. More generally, tumors harbor various and persistent protease activities unseen in healthy tissues. In an attempt to be more specific, we designed prodrugs that are activatable by neutrophil elastase. Upon activation, these alkoxyamine-based drugs release cytotoxic alkyl radicals that act randomly to prevent drug resistance. As a result, U87 glioblastoma cells displayed high level caspase 3/7 activation during the first hour of exposure in the presence of human neutrophil elastase and the prodrug in vitro. The apoptosis process and cell death occurred between 24 and 48 h after exposure with a half lethal concentration of 150 µM. These prodrugs are versatile and easy to synthetize and can be adapted to many enzymes.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

The potential impact of salivary peptides in periodontitis.

Periodontitis is a complex immune-inflammatory condition characterized by the disruption of the periodontal ligament and subsequent formation of periodontal pockets, and by alveolar bone loss, often resulting in tooth loss. A myriad of factors, namely, genetic, metabolic, immunological, and inflammatory, is associated with progression of periodontitis. Periodontitis is also associated with systemic conditions such as neoplastic disorders, obesity, and diabetes. The current diagnosis of this disease relies on clinical measurements such as clinical attachment loss and probing depth, which have poor precision due to patient, operator and probe-related factors. Thus, there is a need to develop reliable, objective, and reproducible biomarkers for early diagnosis of periodontitis. In this regard, saliva, with contributions from the gingival crevicular fluid, holds great potential. However, most of the information on biomarkers of periodontium-related salivary proteins has come from studies on the molecular pathogenesis of periodontitis. In periodontitis, a more holistic approach, such as the use of -omics technologies, for biomarker discovery, is needed. Herein, we review the biomarkers proposed to date for the assessment of periodontitis, with emphasis on the role of salivary peptides in periodontitis and their assessment by high-throughput saliva proteomics. We also discuss the challenges pertaining to the identification of new periodontitis biomarkers in saliva.

Ghrelin and adipokines: An overview of their physiological role, antimicrobial activity and impact on cardiovascular conditions.

The human body has many different hormones that interact with each other and with other factors such as proteins, cell receptors and metabolites. There is still a limited understanding of some of the underlying biological mechanisms of some hormones. In the past decades, science and technology have made major advancements in regard to innovation and knowledge in fields such as medicine. However, some conditions are complex and have many variables that their full picture is still unclear, even though some of these conditions have an alarming rate of incidence and serious health consequences. Conditions such as type 2 diabetes, obesity, nonalcoholic liver disease (NAFLD), cancer in its different forms and even mental conditions, such as Alzheimer's disease, are some of the most common diseases in the 21st century. These conditions are relevant not only because of their high incidence on the general population, but also because of their severity. In this chapter, we present an overview of cardiovascular (CV) diseases. According to the World Health Organization (WHO), cardiovascular diseases, such as coronary artery disease (CAD), heart attack, cardiomyopathy and heart failure (among others), are the number one cause of death worldwide. In 2016, it was estimated that 17.9 million people died from CV diseases, representing more than 30% of all global deaths. Approximately 95% of people who died from CV diseases were so-called "premature deaths" because were referenced to individuals under the age of 70 years old. In this chapter we described some of the hormones that may have an impact on CV diseases, including ghrelin, a peptide that is mostly produced in the stomach, known to induce hunger. Ghrelin is linked to an increase in body fat, i.e., adipose tissue in animals. For this reason, we also included the adipokines leptin, adiponectin and resistin. The main objectives of this chapter are to present the state of the art knowledge concerning the mechanisms of each hormone relevant to CV diseases; to compile data and results that further elucidate the relevance of these peptides for several physiological events, conditions and diseases; and to discuss the metabolic impact of each hormone. We established connections between multiple peptides and the underlying condition/disease with tools such as STRING, referring to research using databases, such as UniProt, DisGeNET and Proteomics DB. Fig. 1 shows a network that summarizes the information presented in this chapter, which serves as a visual representation.

Micron-sized iron oxide particles for both MRI cell tracking and magnetic fluid hyperthermia treatment.

Iron oxide particles (IOP) are commonly used for Cellular Magnetic Resonance Imaging (MRI) and in combination with several treatments, like Magnetic Fluid Hyperthermia (MFH), due to the rise in temperature they provoke under an Alternating Magnetic Field (AMF). Micrometric IOP have a high sensitivity of detection. Nevertheless, little is known about their internalization processes or their potential heat power. Two micrometric commercial IOP (from Bangs Laboratories and Chemicell) were characterized by Transmission Electron Microscopy (TEM) and their endocytic pathways into glioma cells were analyzed. Their Specific Absorption Rate (SAR) and cytotoxicity were evaluated using a commercial AMF inductor. T2-weighted imaging was used to monitor tumor growth in vivo after MFH treatment in mice. The two micron-sized IOP had similar structures and r2 relaxivities (100 mM-1 s-1) but involved different endocytic pathways. Only ScreenMAG particles generated a significant rise in temperature following AMF (SAR = 113 W g-1 Fe). After 1 h of AMF exposure, 60% of ScreenMAG-labeled cells died. Translated to a glioma model, 89% of mice responded to the treatment with smaller tumor volume 42 days post-implantation. Micrometric particles were investigated from their characterization to their intracellular internalization pathways and applied in one in vivo cancer treatment, i.e. MFH.

Amino acid starvation inhibits autophagy in lipid droplet-deficient cells through mitochondrial dysfunction.

Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions, they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast. It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy.

Chitin-glucan complex - Based biopolymeric structures using biocompatible ionic liquids.

This work explores the novelty of dissolving chitin-glucan complex (CGC), from two fungal strains, Komagataella pastoris (CGCP) and Aspergillus niger (CGCKZ) (KiOnutrime-CG™), using biocompatible ionic liquids (ILs). Three cholinium-based ILs were tested, choline acetate, choline propionate and choline hexanoate. Although all tested ILs resulted in the dissolution of the co-polymer at a concentration of 5 % (w/w), distinct polymeric structures, films or gels, were obtained from CGCP and CGCKZ, respectively. CGCP films were dense, flexible and elastic, with high swelling capacity (> 200 %). The IL anion alkyl chain length influenced the polymeric structures' properties, namely, the CGCP films elongation at break and swelling degree. CGCKZ resulted in weak gels. For both polymeric structures, exposure to the ILs under the dissolution conditions caused significant changes in the co-polymers' chemical structure, namely, reduction of their glucan moiety and reduction of the degree of acetylation, thus yielding chitosan-glucan complexes (ChGC) enriched in glucosamine (53.4 ± 0.3-60.8 ± 0.3 %).

Neutrophil Elastase Activity Imaging: Recent Approaches in the Design and Applications of Activity-Based Probes and Substrate-Based Probes.

The last few decades of protease research has confirmed that a number of important biological processes are strictly dependent on proteolysis. Neutrophil elastase (NE) is a critical protease in immune response and host defense mechanisms in both physiological and disease-associated conditions. Particularly, NE has been identified as a promising biomarker for early diagnosis of lung inflammation. Recent studies have shown an increasing interest in developing methods for NE activity imaging both in vitro and in vivo. Unlike anatomical imaging modalities, functional molecular imaging, including enzymatic activities, enables disease detection at a very early stage and thus constitutes a much more accurate approach. When combined with advanced imaging technologies, opportunities arise for measuring imbalanced proteolytic activities with unprecedented details. Such technologies consist in building the highest resolved and sensitive instruments as well as the most specific probes based either on peptide substrates or on covalent inhibitors. This review outlines strengths and weaknesses of these technologies and discuss their applications to investigate NE activity as biomarker of pulmonary inflammatory diseases by imaging.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide.

Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ±â€¯2.9 µM, kcat/Km = 930,000 s-1 M-1 and Km = 25 ±â€¯5.4 µM, kcat/Km = 640,000 s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.

In vivo MEMRI characterization of brain metastases using a 3D Look-Locker T1-mapping sequence.

Although MEMRI (Manganese Enhanced MRI) informations were obtained on primary tumors in small animals, MEMRI data on metastases are lacking. Thus, our goal was to determine if 3D Look-Locker T1 mapping was an efficient method to evaluate Mn ions transport in brain metastases in vivo. The high spatial resolution in 3D (156 × 156 × 218 µm) of the sequence enabled to detect metastases of 0.3 mm3. In parallel, the T1 quantitation enabled to distinguish three populations of MDA-MB-231 derived brain metastases after MnCl2 intravenous injection: one with a healthy blood-tumor barrier that did not internalize Mn2+ ions, and two others, which T1 shortened drastically by 54.2% or 24%. Subsequent scans of the mice, enabled by the fast acquisition (23 min), demonstrated that these T1 reached back their pre-injection values in 24 h. Contrarily to metastases, the T1 of U87-MG glioma remained 26.2% shorter for one week. In vitro results supported the involvement of the Transient Receptor Potential channels and the Calcium-Sensing Receptor in the uptake and efflux of Mn2+ ions, respectively. This study highlights the ability of the 3D Look-Locker T1 mapping sequence to study heterogeneities (i) amongst brain metastases and (ii) between metastases and glioma regarding Mn transport.

Identification of Atg8 from Acanthamoeba castellanii by genetic complementation in Saccharomyces cerevisiae.

Autophagy is a eukaryotic process responsible for the degradation of intracellular content such as damaged organelles. Several putative autophagy-related genes have been identified within the annotated genome of the free-living amoeba Acanthamoeba castellanii. However, the involvement of the corresponding proteins in the autophagy pathway had not been formerly established. Here, we report that AcAtg8 cDNA can complement ATG8-deficient Saccharomyces cerevisiae.

Increased fatty acid synthesis inhibits nitrogen starvation-induced autophagy in lipid droplet-deficient yeast.

Macroautophagy is a degradative pathway whereby cells encapsulate and degrade cytoplasmic material within endogenously-built membranes. Previous studies have suggested that autophagosome membranes originate from lipid droplets. However, it was recently shown that rapamycin could induce autophagy in cells lacking these organelles. Here we show that lipid droplet-deprived cells are unable to perform autophagy in response to nitrogen-starvation because of an accelerated lipid synthesis that is not observed with rapamycin. Using cerulenin, a potent inhibitor of fatty acid synthase, and exogenous addition of palmitic acid we could restore nitrogen-starvation induced autophagy in the absence of lipid droplets.

Orotracheal administration of contrast agents: a new protocol for brain tumor targeting.

The development of new non-invasive diagnostic and therapeutic approaches is of paramount importance in order to improve the outcome of patients with glioblastoma (GBM). In this work we investigated a completely non-invasive pre-clinical protocol to effectively target and detect brain tumors through the orotracheal route, using ultra-small nanoparticles (USRPs) and MRI. A mouse model of GBM was developed. In vivo MRI acquisitions were performed before and after intravenous or orotracheal administration of the nanoparticles to identify and segment the tumor. The accumulation of the nanoparticles in neoplastic lesions was assessed ex vivo through fluorescence microscopy. Before the administration of contrast agents, MR images allowed the identification of the presence of abnormal brain tissue in 73% of animals. After orotracheal or intravenous administration of USRPs, in all the mice an excellent co-localization of the position of the tumor with MRI and histology was observed. The elimination time of the USRPs from the tumor after the orotracheal administration was approximately 70% longer compared with intravenous injection. MRI and USRPs were shown to be powerful imaging tools able to detect, quantify and longitudinally monitor the development of GBMs. The absence of ionizing radiation and high resolution of MRI, along with the complete non-invasiveness and good reproducibility of the proposed protocol, make this technique potentially translatable to humans. To our knowledge, this is the first time that the advantages of a needle-free orotracheal administration route have been demonstrated for the investigation of the pathomorphological changes due to GBMs.

Canthin-6-one displays antiproliferative activity and causes accumulation of cancer cells in the G2/M phase.

Canthinones are natural substances with a wide range of biological activities, including antipyretic, antiparasitic, and antimicrobial. Antiproliferative and/or cytotoxic effects of canthinones on cancer cells have also been described, although their mechanism of action remains ill defined. To gain better insight into this mechanism, the antiproliferative effect of a commercially available canthin-6-one (1) was examined dose-dependently on six cancer cell lines (human prostate, PC-3; human colon, HT-29; human lymphocyte, Jurkat; human cervix, HeLa; rat glioma, C6; and mouse embryonic fibroblasts, NIH-3T3). Cytotoxic effects of 1 were investigated on the same cancer cell lines by procaspase-3 cleavage and on normal human skin fibroblasts. Strong antiproliferative effects of the compound were observed in all cell lines, whereas cytotoxic effects were very dependent on cell type. A better definition of the mechanism of action of 1 was obtained on PC-3 cells, by showing that it decreases BrdU incorporation into DNA by 60% to 80% and mitotic spindle formation by 70% and that it causes a 2-fold accumulation of cells in the G2/M phase of the cell cycle. Together, the data suggest that the primary effect of canthin-6-one (1) is antiproliferative, possibly by interfering with the G2/M transition. Proapoptotic effects might result from this disturbance of the cell cycle.

Activation of rhodopsin gene transcription in cultured retinal precursors of chicken embryo: role of Ca(2+) signaling and hyperpolarization-activated cation channels.

This study reports that the spontaneous 50-fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13-day chicken embryo, relies on a Ca(2+)-dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant-negative form of the cAMP-responsive element-binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca(2+)/calmodulin-dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca(2+) rather than cAMP. This was confirmed by the inhibitory effect of general and T-type selective Ca(2+) channel blockers. Oscillations in Ca(2+) fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization-activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage-gated Ca(2+)-channels, activation of Ca(2+)/calmodulin-dependent kinase II and phosphorylation of cAMP-responsive element-binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+-dependent mechanism whereby hyperpolarization-activated cyclic nucleotide-gated channels (HCN) activate T-type voltage-dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin-dependent kinase II (CaMKII) to phosphorylate the cAMP-responsive element-binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo.

Alkoxyamines: toward a new family of theranostic agents against cancer.

Theranostics combines therapeutic and diagnostic or drug deposition monitoring abilities of suitable molecules. Here we describe the first steps of building an alkoxyamine-based theranostic agent against cancer. The labile alkoxyamine ALK-1 (t(1/2) = 50 min at 37 °C) cleaves spontaneously to generate (1) a highly reactive free alkyl radical used as therapeutic agents to induce cell damages leading to cell death and (2) a stable nitroxide used as contrast agent for Overhauser-enhanced magnetic resonance imaging (OMRI). The ALK-1 toxicity was studied extensively in vitro on the glioblastoma cell line U87-MG. Cell viability appeared to be dependent on ALK-1 concentration and on the time of the observation following alkoxyamine treatment. For instance, the LC50 at 72 h was 250 µM. Data showed that cell toxicity was specifically due to the in situ released alkyl radical. This radical induced oxidative stress, mitochondrial changes, and ultimately the U87 cell apoptosis. The nitroxide production, during the alkoxyamine homolysis, was monitored by OMRI, showing a progressive MRI signal enhancement to 6-fold concomitant to the ALK-1 homolysis. In conclusion, we have demonstrated for the first time that the alkoxyamines are promising molecules to build theranostic tools against solid tumors.

The MFS-type efflux pump Flr1 induced by Yap1 promotes canthin-6-one resistance in yeast.

Screening for suppressors of canthin-6-one toxicity in yeast identified Yap1, a transcription factor involved in cell response to a broad range of injuries. Although canthin-6-one did not promote a significant oxidative stress, overexpression of YAP1 gene clearly increased resistance to this drug. We demonstrated that Yap1-mediated resistance involves the plasma membrane major-facilitator-superfamily efflux pump Flr1 but not the vacuolar ATP-binding-cassette transporter Ycf1. FLR1 overexpression was sufficient to reduce sensitivity to the drug, but strictly dependent on a functional YAP1 gene.

Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

BACKGROUND: Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research. METHODOLOGY/PRINCIPAL FINDINGS: We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations. CONCLUSIONS/SIGNIFICANCE: These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

Observations on the viability of C6-glioma cells after sonoporation with low-intensity ultrasound and microbubbles.

Ultrasound (US) and microbubbles can be used to facilitate cellular uptake of drugs through a cavitationinduced enhancement of cell membrane permeability. The mechanism is, however, still incompletely understood. A direct contact between microbubbles and cell membrane is thought to be essential to create membrane perturbations lasting from seconds to minutes after US exposure of the cells. A recent study showed that the effect may even last up to 8 h after cavitation (with residual permeability up to 24 h after cavitation). In view of possible membrane damage, the purpose of this study was to further investigate the evolution of cell viability in the range of the 24-h temporal window. Furthermore, a description of the functional changes in tumor cells after US exposure was initiated to obtain a better understanding of the mechanism of membrane perturbation after sonication with microbubbles. Our results suggest that US does not reduce cell viability up to 24 h post-exposure. However, a perturbation of the entire cell population exposed to US was observed in terms of enzymatic activity and characteristics of the mitochondrial membrane. Furthermore, we demonstrated that US cavitation induces a transient loss of cell membrane asymmetry, resulting in phosphatidylserine exposure in the outer leaflet of the cell membrane.