Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network

Significance Statement

In the manuscript “Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network” a combination therapy comprising chemotherapeutics and death receptor agonists was analyzed in an in vitro model of pancreatic cancer.  This co-treatment led to a strong synergistic effect, killing a high amount of cancer cells. More detailed analysis revealed a sensitization mechanism via down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, which are typically overexpressed in cancer cells thereby causing defects in the cell death machinery. This gemcitabine-mediated decrease of c-FLIP protein levels changed the composition of the death-inducing signaling complex (DISC) after CD95L-treatment towards a higher amount of the apoptosis-inducing proteins Caspase-8 and Caspase-10. This modified protein complex composition is linked to the higher rate of cell death induction. Cell death assays in presence of inhibitors of apoptosis (zVAD-fmk) and necroptosis (Necrostatin-1) indicated that the combination of gemcitabine and CD95L induced both cell death modes, apoptosis and necroptosis, while the stimulation with CD95L alone led solely to apoptosis. Additionally, the usage of the cutting edge technology “imaging flow cytometry” confirmed that the combinatorial treatment not only sensitized cells to death, but also switched them to an alternative cell death pathway – necroptosis via RIPK1. These results demonstrate that this cell death network is regulated via caspase-dependent and caspase-independent pathways. The combined therapy of chemotherapeutics and death receptor agonists might offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-mediated cell death.  These findings might improve treatment strategies and are important for the development of the systems biology-based approaches for the personalized medicine.

 

About The Author

Dr. Sabine Pietkiewicz studied Biology at the Heinrich-Heine-University in Düsseldorf, Germany and already started focusing on apoptosis and necroptosis after death receptor stimulation during her diploma thesis in the Institute of Molecular Medicine, under the supervision of Prof. Schulze-Osthoff. In Düsseldorf she obtained her Dr. rer. nat. at the Laboratory of Molecular Radiooncology, Clinic and Policlinic for Radiation Therapy and Radiooncology, and has characterized the role of JNK isoforms in cell death pathways after treatment with the proteasomal inhibitor and chemotherapeutic Bortezomib. She currently conducts research in the department Translational Inflammation Research of Prof. Inna N. Lavrik at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany, analyzing the CD95/APO-1/Fas signaling networks.  

About The Author

Prof. Inna N. Lavrik is a head of department Translational Inflammation Research at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany. She did her Postdoctoral training in Heidelberg, at German Cancer Research Center, at the Division of Immunogenetics, Head Prof. Dr. Peter H. Krammer.  Afterwards she had a group leader position at DKFZ/Bioquant, Heidelberg, Germany, which she held before moving to Magdeburg.

Journal Reference

Exp Cell Res. 2015;339(1):1-9.

Pietkiewicz S¹, Eils R², Krammer PH³, Giese N⁴, Lavrik IN⁵. 

Show Affiliations

1. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany.
2. Bioquant, Heidelberg University, 69120 Heidelberg, Germany; Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, 69120 Heidelberg, Germany; Division of Theoretical Bioinformatics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
3. Division of Immunogenetics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
4. Department of General Surgery, University of Heidelberg, Germany (g)Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia.
5. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany; Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia. Electronic address: inna.lavrik@med.ovgu.de.

Abstract

Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Electrospun Polycaprolactone 3D Nanofibrous Scaffold with Interconnected and Hierarchically Structured Pores for Bone Tissue Engineering.

Significance Statement

Ultrathin fibers (with diameters from tens to hundreds of nanometers, commonly known as electrospun nanofibers) prepared via the electrospinning method are of great interests for tissue engineering applications, because electrospun nanofibers have diameters similar to those of fibrous structures in natural extracellular matrix (ECM).  The major limitation of electrospun scaffolds is owing to their morphological structure of overlaid nanofiber mats with apparent/equivalent pore sizes in sub-micrometers; in other words, electrospun nanofibrous mats lack the needed macropores (with sizes from tens to hundreds of micrometers) for cell growth and tissue formation.

In the reported studies, novel electrospun three-dimensional (3D) nanofibrous scaffold has been developed by an innovative and convenient approach (i.e., thermally induced nanofiber self-agglomeration followed by freeze drying) from a biopolymer of polycaprolactone (PCL) for the first time, and the scaffold possesses interconnected and hierarchically structured pores (in specific, the macropores with sizes in hundreds of micrometers would maintain the structural stability of scaffold, support cell proliferation, ECM deposition, and tissue formation; the medium pores with sizes in tens of micrometers or smaller would facilitate the diffusion of nutrients and promote the formation of vascularization, while the small pores with sizes in micrometers or smaller would have impacts on some cell behaviors such as seeding and genes expressions).  The novel polycaprolactone 3D scaffold is soft and elastic with very high porosity of ~96.4%, thus it is morphologically/structurally similar to natural ECM and well-suited for cell functions (e.g., adhesion, proliferation, migration, and differentiation) and tissue formation.

The in vitro studies reveal that the novel scaffold can lead to high cell viability; more importantly, it is able to promote more potent bone morphogenetic protein 2 (BMP2) induced chondrogenic (rather than osteogenic) differentiation of mouse bone marrow mesenchymal stem cells (mBMSCs).  Consistent to the in vitro findings, the in vivo results indicate that electrospun polycaprolactone 3D scaffold acts as a favorable synthetic ECM for functional bone regeneration through the physiological endochondral ossification process (i.e., through formation of cartilage intermediate), which is a new strategy of “developmental tissue engineering” to mimic natural endochondral bone repair process.  Hence, it is envisioned that the developed electrospun polycaprolactone 3D nanofibrous scaffold would be very promising for tissue engineering applications including the regenerations of bone, cartilage, and their composite tissue of osteochondral (an especially challenged tissue repair in clinics).

About The Author

Dr. Hongli Sun is an Assistant Professor in the Biomedical Engineering program at the University of South Dakota (USD).  Prior to joining the faculty at the USD, he worked as a Research Fellow in the Department of Biologic and Materials Sciences at the University of Michigan.  Dr. Sun earned his Ph.D. in Cell Biology from the Chinese Academy of Sciences (Shanghai, China) in 2007.  Inspired by the fundamental findings in stem cell and developmental biology, Dr. Sun’s research has been focusing on the development of novel stem cell/nano-biomaterials based translational strategies for challenged bone regeneration.  Dr. Sun has authored many articles in peer-reviewed journals including Biomaterials, Stem Cells, and Tissue Engineering.  In addition, he has been serving as a manuscript/grant reviewer in the fields related to bone, stem cells, and tissue engineering.

 

About The Author

Dr. Hao Fong is one of the pioneers and renowned scientists worldwide in the field of “Electrospinning and Nanofibers”.  Presently, he is a tenured Full Professor in the Department of Chemistry and Applied Biological Sciences at the South Dakota School of Mines and Technology (SDSM&T); and he is also an important faculty member in the SDSM&T’s multidisciplinary graduate programs of Materials Engineering and Science (MES), Nanoscience and Nanoengineering (NANO), and Biomedical Engineering (BME).  His highest degree is a Ph.D. earned in 1999 from the Department of Polymer Science at the University of Akron (in Ohio, USA).  Prior to joining the faculty at the SDSM&T in 2003, he worked as a guest research scientist in the Polymer Branch of the Air Force Research Laboratory (AFRL) in the Wright-Patterson Air Force Base, and as a staff research scientist in the Paffenbarger Research Center of the American Dental Association (PRC-ADA) and/or the Polymer Division at the National Institute of Standards and Technology (NIST) in Maryland, for a total of three years.  In the recent years, Dr. Hao Fong’s research interests have been focused on “The Materials-processing Technique of Electrospinning and Various Applications of Electrospun Polymer, Ceramic, Carbon/Graphite, Metallic, Composite, and Hierarchically-structured Nanofibers and/or Nanofibrous Materials”.  The applications include, but not limited to, (1) filtration/separation applications (e.g., separation of biopharmaceutical therapeutics such as proteins, purification of air/water, microfiltration, ultrafiltration, nanofiltration, and reverse osmosis), (2) energy-related applications (e.g., solar cells, batteries, fuel cells, and supercapacitors), (3) biomedical applications (e.g., tissue engineering, drug delivery, and antimicrobial wound dressing), (4) microelectronics-related applications (e.g., sensors/detectors and transistors), (5) composite applications (e.g., hybrid multi-scale composites and dental restorative composites).

Journal Reference

Adv Healthc Mater. 2015;4(15):2238-46.

Xu T¹, Miszuk JM², Zhao Y¹, Sun H², Fong H¹.

Show Affiliations

1. Program of BiomedicalEngineering, South Dakota School of Mines and Technology, Rapid City, SD, 57701, USA.
2. Program of BiomedicalEngineering, University of South Dakota, Sioux Falls, SD, 57107, USA.

Abstract

For the first time, electrospun polycaprolactone (PCL) 3D nanofibrous scaffold has been developed by an innovative and convenient approach (i.e., thermally induced nanofiber self-agglomeration followed by freeze drying), and the scaffold possesses interconnected and hierarchically structured pores including macropores with sizes up to ≈300 μm. The novel polycaprolactone 3D scaffold is soft and elastic with very high porosity of ≈96.4%, thus it is morphologically/structurally similar to natural extracellular matrix and well suited for cell functions and tissue formation. The in vitro studies reveal that the scaffold can lead to high cell viability; more importantly, it is able to promote more potent BMP2-induced chondrogenic than osteogenic differentiation of mouse bone marrow mesenchymal stem cells. Consistent to the in vitro findings, the in vivo results indicate that the electrospun polycaprolactone 3D scaffold acts as a favorable synthetic extracellular matrix for functional bone regeneration through the physiological endochondral ossification process.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Design and synthesis of 2-phenylnaphthalenoids and 2-phenylbenzofuranoids as DNA topoisomerase inhibitors and antitumor agents

Significance Statement

Human DNA topoisomerase IIα (TopoIIα), an Achilles’ heel of cancer, is a well-validated target for antitumor drugs. The inhibitors of TopoIIα are among the most effective and commonly used antitumor drugs in clinic, e. g. etoposide and doxorubicin. However, clinically used v inhibitors exhibited serious side effects including cardiotoxicity, development of secondary malignancies and multidrug resistance, thus that it is urgent to explore novel DNA topoisomerase IIα inhibitors for the development of antitumor drugs.

In the recent work of Shen laboratory, a group of 2-phenylnaphthalenoids (2PNs) and 2-phenylbenzofuranoids (2PBFs) were designed and synthesized for the sake of innovation of DNA topoisomerase IIα inhibitors. These 2PNs and 2PBFs were proved to display favorable TopoIIα inhibitory as well as antiproliferative activities. In particular, 2PBFs possess divergent mechanism of action on inhibiting DNA topoisomerase IIα from 2PNs, switching Topo poisons 2PNs to Topo catalytic inhibitors 2PBFs. These results suggest that the chromophore scaffold replacement drug design strategy used in this study may result in a change of the binding sites of inhibitors to TopoIIα. This study provides insights to the structure optimization of DNA topoisomerase IIα inhibitors.

About The Author

Dr. Yuemao Shen received his B. Sc. degree in Chemistry from Anhui Normal University in 1986, M.Sc. in natural products chemistry from Kunming Institute of Botany (KIB) of Chinese Academy of Sciences in 1989. He went to Floss Lab at the University of Washington (UW) as a visiting Scientist and became a joint Ph. D. student of KIB and UW in 1995, and received his Ph. D. degree in Botany from KIB of Chinese Academy of Sciences in 1999. Before joining the faculty of Shandong University in 2010, he was an intern Researcher (1989-1991), assistant Researcher (1991-1995), associate Researcher (1998-1999) and Researcher (1999-2004) in KIB, and Professor in Xiamen University (2004-2010). Being a professor of Shandong University, Dr. Shen’s research interests focus on isolation, structure elucidation, and biosynthesis of natural products with anti-infective or antitumor activities.

About The Author

Miss Huilin Hao received her B. Sc. degree in Pharmacy from Shandong University in 2013. She is currently a graduate student under the supervision of Prof. Yuemao Shen at Shandong University School of Pharmaceutical Sciences. Her research interest includes antitumor mechanisms of topoisomerase inhibitors, endocytic vesicle trafficking and cancer cell metabolism.

About The Author

Dr. Wang Chen received his B. Sc. degree in Pharmacy from Xi’an Jiaotong University Health Sciences Center in 2010, and obtained his Ph. D. degree in Medicine from Shandong University School of Pharmaceutical Sciences in 2015. He is currently a lecturer in Shaanxi University of Technology Vitamin D Research Institute. His research interest is the synthesis of Vitamin D derivatives.

Journal Reference

Eur J Med Chem. 2015;102:277-87.

Hao H¹, Chen W¹, Zhu J², Lu C¹, Shen Y³.

Show Affiliations

1. Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, 250012, PR China.
2. State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong, 250100, PR China.
3. Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, 250012, PR China; State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong, 250100, PR China. Electronic address: yshen@sdu.edu.cn.

Abstract

Eight 2-phenylnaphthalenoids (2PNs) (3a-h) and twenty four 2-phenylbenzofuranoids (2PBFs) (4a–4j, 5a-5j, 6a, 6f-6h) were successfully designed, synthesized and their antiproliferative and in vitro DNA topoisomerase inhibitory activities were evaluated. Nine compounds (four 2PNs and five 2PBFs) showed either TopoI or TopoIIα inhibitory activities. Six compounds (four 2PNs and two 2PBFs) exhibited potent cytotoxicity with IC50 values for 72 h exposure ranging from 0.3 to above 20 μM against MDA-MB-231, MDA-MB-435, HepG2 and PC3 cell lines. The two 2PBFs displayed comparable and even better antiproliferative as well as TopoIIα inhibitory activities than 2PNs. Interestingly, the active 2PBFs displayed different mechanisms of TopoIIα inhibition from that of 2PNs, suggesting that the chromophore scaffold replacement may result in a change of the binding site of inhibitors to TopoIIα. Furthermore, the mechanisms of antiproliferation on MDA-MB-231 cells indicate that compounds 5a and 5f are promising for further development of anticancer agents. The results of this study reveal that the evolutionary strategy of medicinal chemistry through scaffold hopping is a promising strategy for structure optimization of TopoIIα inhibitors.

Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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The membrane anchor of the transcriptional activator SREBP is characterized by intrinsic conformational flexibility

Significance Statement

SREBP signaling regulates transcriptions of genes necessary for the homeostasis of fatty acids and cholesterol in the cell, which is a key factor for cellular wellbeing and proliferation. Accordingly, the abrogation of SREBP signaling is associated with numerous pathological conditions ranging from cardiometabolic disorders to metabolic syndrome and cancer. Representing many other signaling processes from homeostasis to proliferation, compartmentalization, and differentiation like Wnt and Notch, the signal cascade is elicited via a regulated intramembrane proteolysis (RIP), i. e. the proteolytic cleavage of a membrane-integral transcription factor. In this work, Linser et al. find that the membrane-spanning part of the SREBP transcriptional activator precursor molecule, which is the substrate in the RIP process, comprises interrupted structural stability. This enables conformational flexibility and maybe one of the factors explaining the enigmatic specificity and selectivity of the eminent and evolutionarily conserved RIP process, which is poorly understood to-date.

About The Author

Dr. Rasmus Linser obtained his PhD in Biophysics at the Leibniz Institute for Molecular Pharmacology in Berlin, Germany, in 2010. During his postdoctoral studies, he worked at the University of New South Wales in Sydney, Australia, and Harvard Medical School, Boston, MA, developing and using Nuclear Magnetic Resonance (NMR) spectroscopic methods both in the solution and solid state. His independent group, which he started in 2014 at the Max Planck Institute for biophysical chemistry in Göttingen, is concerned with the development and application of NMR methods mostly for solid proteins. With these, the molecular mechanisms of disease-related or functional amyloids, membrane proteins, and enzymes are characterized from a structural point of view and concerning protein dynamics. This helps to shed light on the various mechanisms that cells have invented to survive. Dr. Linser’s lab is currently funded by the German Research Association (DFG, Emmy Noether program and SFB 803, project A03), the Association of the Chemical Industries (VCI), and the Michael J. Fox Foundation. 

 

About The Author

 Prof. Dr. Gerhard Wagner obtained his PhD in Biophysics at the Eidgenössische Technische Hochschule (ETH) in Zürich, Switzerland, in 1977. After postdoctoral work at the Massachusetts Institute of Technology (MIT) in Cambridge, MA and the ETH, he accepted an Associate Professor position at the University of Michigan in 1987. Since 1990, he has been a Professor (since 1992 Elkan Rogers Blout Professor) at Harvard Medical School, Boston, MA, in the Department of Biological Chemistry and Molecular Pharmacology. He has been awarded with numerous high-rank honors and has been elected a member of various associations, including the German Academy of Sciences (Leopoldina), the National Academy of Sciences of the United States and the American Academy of Arts and Sciences. His lab has been pivotal regarding the development and application of solution NMR methods. The approaches developed in the Wagner lab have been leading the way to characterization of structure and function of proteins in solution  by NMR spectroscopy, for example with regard to the elucidation of eukaryotic translation initiation. Dr. Wagner’s lab is funded by the National Institute of Health (NIH) with grants GM046476 and HL116391. 

Figure Legend:  Dynamics of the substrate, enabled by interrupted structural definition, may play a role for the specificity in Regulated Intramembrane Proteolysis. Many molecular details of this evolutionarily conserved mechanism are still enigmatic, even though it is a constituent of numerous signaling cascades. This work was pursued in the case of SREBP signaling, which is a hallmark of numerous pathological conditions from cardiometabolic disorders to cancer.

Journal Reference

Proc Natl Acad Sci U S A. 2015;112(40):12390-5.

Linser R¹, Salvi N², Briones R³, Rovó P⁴, de Groot BL³, Wagner G⁵.

Show Affiliations

1. Department NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; rali@nmr.mpibpc.mpg.de gerhard_wagner@hms.harvard.edu.
2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; Université Grenoble Alpes, Centre National de la Recherche Scientifique, and Commissariat à l’Énergie Atomique et aux Énergies Alternatives, Institut de Biologie Structurale, F-38044 Grenoble, France;
3. Biomolecular Dynamics Group, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
4. Department NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; rali@nmr.mpibpc.mpg.de gerhard_wagner@hms.harvard.edu. 

Abstract

Regulated intramembrane proteolysis (RIP) is a conserved mechanism crucial for numerous cellular processes, including signaling, transcriptional regulation, axon guidance, cell adhesion, cellular stress responses, and transmembrane protein fragment degradation. Importantly, it is relevant in various diseases including Alzheimer’s disease, cardiovascular diseases, and cancers. Even though a number of structures of different intramembrane proteases have been solved recently, fundamental questions concerning mechanistic underpinnings of RIP and therapeutic interventions remain. In particular, this includes substrate recognition, what properties render a given substrate amenable for RIP, and how the lipid environment affects the substrate cleavage. Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors are critical regulators of genes involved in cholesterol/lipid homeostasis. After site-1 protease cleavage of the inactive SREBP transmembrane precursor protein, RIP of the anchor intermediate by site-2 protease generates the mature transcription factor. In this work, we have investigated the labile anchor intermediate of SREBP-1 using NMR spectroscopy. Surprisingly, NMR chemical shifts, site-resolved solvent exposure, and relaxation studies show that the cleavage site of the lipid-signaling protein intermediate bears rigid α-helical topology. An evolutionary conserved motif, by contrast, interrupts the secondary structure ∼9-10 residues C-terminal of the scissile bond and acts as an inducer of conformational flexibility within the carboxyl-terminal transmembrane region. These results are consistent with molecular dynamics simulations. Topology, stability, and site-resolved dynamics data suggest that the cleavage of the α-helical substrate in the case of RIP may be associated with a hinge motion triggered by the molecular environment.

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The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells

Journal Reference

Oncotarget. 2015;6(33):34910-23.

Eike LM¹, Yang N², Rekdal Ø^1,3, Sveinbjørnsson B¹.

Show Affiliations

1. Department of Molecular Inflammation Research and Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway.
2. Department of Community Medicine, Faculty of Health University of Tromsø, Tromsø, Norway.
3. Lytix Biopharma, Oslo, Norway.

Abstract

Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models.

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Adipose-derived stromal cells for the reconstruction of a human vesical equivalent

Journal Reference

J Tissue Eng Regen Med. 2015;9(11):E135-43.

Rousseau A¹, Fradette J¹, Bernard G¹, Gauvin R¹, Laterreur V¹, Bolduc S^1,2

Show Affiliations

1. Centre LOEX de l’Université Laval, Génie Tissulaire et Régénération, LOEX du Centre de Recherche FRSQ du CHU de Québec, Département de Chirurgie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
2. CMDGT/LOEX-ÉquipeReconstruction Vésicale, Centre de Recherche FRQS du CHU de Québec, Aile-R, Hôpital de l’Enfant-Jésus, Centre Hospitalier Affilié Universitaire de Québec, QC, Canada.

Abstract

Despite a wide panel of tissue-engineering models available for vesical reconstruction, the lack of a differentiated urothelium remains their main common limitation. For the first time to our knowledge, an entirely human vesical equivalent, free of exogenous matrix, has been reconstructed using the self-assembly method. Moreover, we tested the contribution of adipose-derived stromal cells, an easily available source of mesenchymal cells featuring many potential advantages, by reconstructing three types of equivalent, named fibroblast vesical  equivalent, adipose-derived stromal cell vesical equivalent and hybrid vesical equivalent – the latter containing both adipose-derived  stromal  cells and fibroblasts. The new substitutes have been compared and characterized for matrix composition and organization, functionality and mechanical behaviour. Although all three vesical equivalents displayed adequate collagen type I and III expression, only two of them, fibroblast vesical equivalent and hybrid vesical equivalent, sustained the development of a differentiated and functional urothelium. The presence of uroplakins Ib, II and III and the tight junction marker ZO-1 was detected and correlated with impermeability. The mechanical resistance of these tissues was sufficient for use by surgeons. We present here in vitro tissue-engineered vesical equivalents, built without the use of any exogenous matrix, able to sustain mechanical stress and to support the formation of a functional urothelium, i.e. able to display a barrier function similar to that of native tissue. Copyright © 2013 John Wiley & Sons, Ltd.

Copyright © 2013 John Wiley & Sons, Ltd.

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Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons

Journal Reference

PLoS One. 2015;10(9):e0138215.

Yang B¹, Rajput PS², Kumar U², Sastry BR¹.

Show Affiliations

1. Neuroscience Research Laboratory, Department of Anesthesiology, Pharmacology & Therapeutics, Faculty of Medicine, The University of British Columbia, Vancouver, Canada.
2. Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, Canada.

Abstract

The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.

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